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The ecto-ATPase inhibitor ARL 67156 enhances parasympathetic neurotransmission in the guinea-pig urinary bladder
Abstract
The influence of enzymatic degradation on the neurotransmitter actions of ATP was studied using the ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL 67156). Field stimulation of the parasympathetic nerves innervating guinea-pig urinary bladder muscle strips (1-8 Hz for 20 s) produced characteristic biphasic contractions, the peak magnitudes of which were significantly increased by 29-32% by ARL 67156 (100 microM). A similar degree of enhancement was seen in the presence of atropine (1 microM), consistent with ARL 67156 acting to enhance the action of neuronally released ATP. The effects of ARL 67156 reversed rapidly on washout of the drug. Contractions evoked by exogenous ATP (100 microM) were also potentiated by ARL 67156 (100 microM), but those to the stable analogue alpha,beta-methyleneATP (5 microM) were unaffected. ARL 67156 (100 microM) also enhanced contractions to exogenous acetylcholine (1 microM) and histamine (3 microM), but this potentiation was abolished by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (100 mciroM). It is concluded that when ATP acts as a neurotransmitter its postjunctional actions are attenuated by enzymatic degradation. ARL 67156 inhibits this breakdown.
... There exist possible binding sites for NTPDase in Macrophages which may facilitate adherence and infection. Antibodies against the NTPDase when added to macrophages before Leishmania infantum chagasi infection reduced adherence and infection by the parasites [49]. ...
... One of the protein from Leishmania infantum chagasi named E-NTPDase-2 was expressed, purified, and characterized leading to in vitro infection, and its blockade leads to lower rate of infection of macrophages. It can be established as a good antigen for immunodiagnosis of canine visceral leishmaniasis and a target for drug development [49]. However, ecto-ATPDase from L. donovani may be further characterized as a good antigen for immunodiagnosis and as a target for drug development. ...
In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10(7) cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2 stimulated the ATP hydrolysis to a greater extent compared with CaCl2 and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10(7) cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg(2+)-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
... Some inhibitors of ecto-ATPase were identified, including suramin and ethacrynic acid, which were strongly effective in enhancing the response to ATP; difluorodinitrobenzene was less effective, while N-ethylmaleimide, ATPγS and Reactive blue 2 were without effect. In a later study, ARL 67 l56 was identified as a potent ATPase inhibitor [164] which potentiated the response to ATP (but not α,β-meATP) and responses to atropine-resistant responses to nerve stimulation [722] (Fig. 4a and b). Cyclopiazonic acid, an inhibitor of sarcoplasmic ATPase, potentiated the contractile response of the guinea-pig bladder to exogenous ATP and NANC excitatory nerve stimulation but this was non-specific [764]. ...
... **P <0.01, ***P <0.001. (a and b Reproduced from [722], with permission from Elsevier.) c Contractile responses of isolated strips of urinary bladder of adult and neonate (2-to 6-dayold) rabbits to nerve stimulation. ...
Purinergic signalling is involved in a number of physiological and pathophysiological activities in the lower urinary tract. In the bladder of laboratory animals there is parasympathetic excitatory cotransmission with the purinergic and cholinergic components being approximately equal, acting via P2X1 and muscarinic receptors, respectively. Purinergic mechanosensory transduction occurs where ATP, released from urothelial cells during distension of bladder and ureter, acts on P2X3 and P2X2/3 receptors on suburothelial sensory nerves to initiate the voiding reflex, via low threshold fibres, and nociception, via high threshold fibres. In human bladder the purinergic component of parasympathetic cotransmission is less than 3 %, but in pathological conditions, such as interstitial cystitis, obstructed and neuropathic bladder, the purinergic component is increased to 40 %. Other pathological conditions of the bladder have been shown to involve purinoceptor-mediated activities, including multiple sclerosis, ischaemia, diabetes, cancer and bacterial infections. In the ureter, P2X7 receptors have been implicated in inflammation and fibrosis. Purinergic therapeutic strategies are being explored that hopefully will be developed and bring benefit and relief to many patients with urinary tract disorders.
... In the presence of 4 U/ml apyrase, exogenously applied ATP during S 2 , but not the P2X 1,7 selective agonist Bz-ATP (p = 0.05; n = 5; Fig. 5 ARL67156. In the presence of this drug at a concentration which likely prevents, albeit not totally, ATP conversion without affecting adenosine levels [32], the neurogenic contraction was enhanced too (by 72 ± 7, n = 4; p = 0.004; Fig. 5). ...
... As of the matter of fact, in the presence of the potent ecto-ATPase inhibitor ARL67156, endogenous ATP released in response to nerve stimulation can significantly enhance [ 3 H]-ACh release and, in turn, smooth muscle contraction. In consideration of the use of ARL67156 at a fixed concentration, which prevents but not able to produce total inhibition of the ecto-ATPase activity [32], agents resistant to ecto-ATPases degradation would be expected to induce a more effective enhancement. Really, a marked potentiation (by about 250%) was produced in normal detrusor strips by the non-hydrolyzable ATP analog, ABMA. ...
... Analysing structural similarity along phenotypic similarity allows identification of potential off-target effects or the mechanism of action of tested compounds. In particular, drugs with similar chemical structure and similar phenotypic profile but different mechanisms of action can reflect off-target effects 25 . For example, we found a group of structurally similar drugs in PhenoCluster 7, many of which are antagonists of dopamine, including triflupromazine hydrochloride, chlorpromazine hydrochloride, prochlorperazine dimaleate, and fluphenazine dihydrochloride (Fig. 4D). ...
Angiogenesis plays a key role in several diseases including cancer, ischemic vascular disease, and Alzheimer’s disease. Chemical genetic screening of endothelial tube formation provides a robust approach for identifying signalling components that impact microvascular network morphology as well as endothelial cell biology. However, the analysis of the resulting imaging datasets has been limited to a few phenotypic features such as the total tube length or the number of branching points. Here we developed a high content analysis framework for detailed quantification of various aspects of network morphology including network complexity, symmetry and topology. By applying our approach to a high content screen of 1,280 characterised drugs, we found that drugs that result in a similar phenotype share the same mechanism of action or common downstream signalling pathways. Our multiparametric analysis revealed that a group of glutamate receptor antagonists enhances branching and network connectivity. Using an integrative meta-analysis approach, we validated the link between these receptors and angiogenesis. We further found that the expression of these genes is associated with the prognosis of Alzheimer’s patients. In conclusion, our work shows that detailed image analysis of complex endothelial phenotypes can reveal new insights into biological mechanisms modulating the morphogenesis of endothelial networks and identify potential therapeutics for angiogenesis-related diseases.
... Some inhibitors of ectoATPase including suramin (Hourani and Chown, 1988) and CPA (Ziganshin et a l, 1994b) have been shown to enhance the response o f guinea pig detrusor to ATP but these are non-specific also having antagonistic actions at P2 receptors. In a later study, ARL 67156 was identified as a potent ecto-ATPase inhibitor (Crack et al, 1995), which at a concentration of lOOpM, potentiates the response to ATP and atropine resistant nervemediated contractions but not to those evoked by a,p-Me-ATP (Westfall et a/., 1997). ...
Bladder instability is a prevalent condition, especially in the elderly. However, despite its recognition as the greatest cause of urinary incontinence the aetiology is poorly understood. Bladder smooth muscle (detrusor) obtained from some patients with bladder instability exhibit atropine-resistant nerve-mediated contractions, in contrast to detrusor from stable bladders. These contractions are blocked by purinoceptor antagonists suggesting the presence of a second neurotransmitter, ATP. This study was undertaken to characterise further purinergic neuromuscular transmission in human and guinea pig detrusor and its relationship to the development of detrusor instability in patients. The contractile sensitivity of muscle from stable and unstable bladders to P2- purinoceptor agonists was measured using ATP and the non-hydrolysable analogues α,β-methylene-ATP and β,γ-methylene-L-ATP. ATP was significantly more potent in detrusor from unstable or obstructed bladders than from stable bladders; α,β-methylene- ATP was more potent than ATP in all tissues, β,γ-methylene-L-ATP was also more potent in guinea-pig tissue. Ecto-ATPase activity, measured using the bioluminescent protein luciferase, was lower in detrusor samples obtained from unstable compared to stable bladders. The involvement of inhibitory P1-purinoceptors in modulating nerve-mediated contractions and agonist-induced contractures was also investigated. Detrusor preparations from unstable and obstructed bladders were less sensitive to the synthetic adenosine analogue, N-ethylenecycloadenosine, than those from stable bladders. The actions of adenosine were similar in all detrusor samples tested. In experiments using several P1-receptor subtype-selective agonists and the P1-receptor antagonist, 1,3- dipropyl-8-cyclopentylxanthine, in guinea pig detrusor the presence of a pre-synaptic A1 and a postjunctional A2B receptor was identified. In this study the role of purinergic agonists in regulating the contractility of detrusor muscle has been characterised. Differences were found in detrusor samples from patients with stable and unstable bladders. The implications of these findings in understanding neuromuscular transmission in the normal and unstable human bladder are discussed.
... However, as there is little, if any, atropine-resistant contractions in this group, this would suggest that ATP is released but is hydrolysed by ectoATPases prior to postsynaptic purinoceptor activation. This possibility is in keeping with Westfall et al's (1997) findings (Lluel et al, 2002) that ABMA did not diminish the force generated (except at the very high concentration of 300 pM) in an obstructed rat model. ...
In utero bladder outflow obstruction (BOO) of the human developing bladder, commonly caused by posterior urethral valves, produces significant bladder dysfunction with symptoms of urinary incontinence, poor bladder emptying and urinary tract infection. Furthermore, many of these boys suffer significant renal impairment. To investigate the antenatal events that lead to this postnatal dysfunction, I have used an experimental fetal ovine model of in utero BOO. I found that the obstructed fetal bladder became larger, heavier and had grown; this was accompanied by an increase in proliferation and apoptosis within the detrusor muscle layer and an increase of apoptosis with no increase in proliferation within the lamina propria layer. Also documented was a downregulation of the anti-death protein Bcl-2 and an upregulation of the pro-death protein Bax. Moreover, activated caspase-3, an effector of apoptotic death, was increased in obstructed bladders. I found the obstructed fetal bladder became hypocontractile and denervated and exhibited greater atropine-resistant contractions. Nitric oxide-mediated relaxations were also present. In addition, I observed increased compliance and reduced elasticity and viscoelasticity in the obstructed fetal bladder. My results suggest that enhanced apoptosis in detrusor smooth muscle cells appeared to be a part of a remodelling response during compensatory hyperplasia and hypertrophy. In the lamina propria, an imbalance between death and proliferation led to a relative depletion of cells. Furthermore, the obstructed fetal bladder became hypocontractile; in addition to denervation, this may result from a reduction in the elastic modulus that may prevent any extramuscular components from sustaining force. These discoveries provide insight into why the fetal bladder functions poorly postnatally when exposed to bladder outflow obstruction and suggests potential molecular avenues for fetal bladder manipulation. Future strategies will characterise the in vivo pressure changes after obstruction, contractile properties of isolated detrusor myocytes and changes in the 'deobstructed' obstructed fetal bladder.
... Analysing structural similarity along phenotypic similarity allows identification of potential offtarget effects or the mechanism of action of tested compounds. In particular, when a drug that is structurally similar to a group of drugs with a known mechanism of action, have also a similar phenotypic profile (Westfall et al., 1997). For example, we found a group of structurally similar drugs in PhenoCluster 7, many of which are antagonists of dopamine, including Triflupromazine hydrochloride, Chlorpromazine hydrochloride, Prochlorperazine dimaleate, Fluphenazine dihydrochloride (Fig. 2D). ...
Angiogenesis plays a key role in several diseases including cancer, ischemic vascular disease, and Alzheimer's disease. High throughput screening of endothelial tube formation provides a robust approach for identifying drugs that impact microvascular network formation and morphology. However, the analysis of resulting imaging datasets has been limited to a few phenotypic features such as the total tube length or the number of branching points. Here we developed a high content analysis framework for detailed quantification of various aspects of network morphology including network complexity, symmetry and topology. By applying our approach to a high content screen of 1,280 drugs, we found that many drugs that result in a similar phenotype share the same mechanism of action or common downstream signalling pathways. Our multiparametric analysis revealed a group of drugs, that target glutamate receptors, results in enhanced branching and network connectivity. Using an integrative meta-analysis approach, we validated the link between these receptors and angiogenesis. We further found that the expression of these genes correlates with the prognosis of Alzheimer's patients. In conclusion, our work shows that detailed image analysis of complex endothelial phenotypes can reveal new insights into biological mechanisms modulating the morphogenesis of endothelial networks and identify potential therapeutics for angiogenesis-related diseases.
... It relies on the proximity of ATP releasing cells to the effectors cells and on several different mechanisms [26]. For instance, AMP and adenosine, formed upon ATP degradation, act on pre-synaptic P1-purinoceptors and induce of a slow hyper polarization of parasympathetic neurons which significantly contributes to the reduction of bladder contractions [7,[27][28][29][30][31]. Adenosine may also inhibit contractions by acting on P1-purinoceptors present in detrusor smooth muscle cells [32,33]. ...
... As recombinant apyrase does not penetrate cellular PMs, we suspected that only extracellular ATP levels were selectively reduced. Instead, application of the specific ecto-ATPase inhibitor ARL 67156 (50 µM, [236][237][238]) to pre-washed MIN6 cells immediately started [Ca 2+ ]i oscillations ( Fig. 6 A+B v, H). All this already identified ATP as an extracellular endogenous (autocrine) signaling factor that is essential for β-cell activity. ...
Secretion of insulin in response to extracellular stimuli, such as elevated glucose levels and small molecules that act on G-protein coupled receptors (GPCRs), is the hallmark of β-cell physiology. Sufficiently high blood insulin levels are ensured by the coupling of the secretory activity within pancreatic islets. Intercellular and inter-islet coordination are partly mediated by small diffusible ligands of GPCRs within the extracellular space of pancreatic islets. Therefore, insulin release is considered a synchronized multi-cellular process. We show herein that β-cell activity and insulin secretion essentially rely on the presence of extracellular endogenous (autocrine) signaling factors, exemplified by two classes of small cellular metabolites. Trace amines (TAs) are small aromatic metabolites that were identified as low-abundant ligands of the trace amine-associated receptor 1 (TAAR1) in the central nervous system (CNS). In the presented work, we identify TAs as essential autocrine signaling factors that maintain and regulate oscillations of the intracellular Ca2+ concentration ([Ca2+]i oscillations) along with insulin secretion from β-cells via TAAR1. We found that the modulation of endogenous TA levels by the selective inhibition of TA biosynthetic pathways directly translated into changes of [Ca2+]i oscillations and insulin secretion. Application of aromatic amine-withdrawing β-cyclodextrin temporarily reduced [Ca2+]i oscillations. This demonstrates the essential role of TAs for β-cell activity as well as their high metabolic turnover rates. Notably, herein applied inhibitors and synthetic TAAR1 (ant-)agonists are partly approved for the therapeutic modulation of biogenic amine levels within the CNS, and hence for the treatment of common neurological disorders. According to our findings, these drugs even affect β-cell activity and insulin secretion through pancreatic TAAR1. With the discovery of the free fatty acid (FA) receptor GPR40 in β-cells, FAs have come into focus as exogenous insulin secretagogues. However, the role of FAs as endogenous (local) signaling factors of β-cells has not been considered so far. We show herein that lowering endogenous FAlevels in the presence of FA-free bovine serum albumin (FAF-BSA) immediately reduced [Ca2+]i oscillations and insulin secretion. [Ca2+]i oscillations resumed upon exchange of FAF-BSA by buffer or upon restoration of extracellular FA pools. The latter was accomplished by the photolysis of caged FAs on plasma membranes, by the addition of a recombinant lipase or of FA-loaded BSA. Our approach to subordinate β-cell activity and insulin secretion to the presence of autocrine signaling factors of the yet underestimated receptors TAAR1 and GPR40 in the pancreas contributes to a more detailed and complete understanding of the fundamental regulation of β-cell activity and insulin secretion.
... Purinergic dysfunction is a common phenomenon in human bladder diseases; elevated ATP release, impaired ATP hydrolysis, and dysregulated P2X expression have all been reported in patients with OAB, interstitial cystitis, and outlet obstruction (16)(17)(18)(43)(44)(45)(46)(47)(48)(49)(50). The role of P2Y 12 and A2b receptors in human bladder function is not well known. ...
Abnormalities in purine availability or purinergic receptor density are commonly seen in patients with lower urinary tract symptoms (LUTS), but the underlying mechanisms relating altered receptor function to LUTS are unknown. Here we provide extensive evidence for the reciprocal interplay of multiple receptors responding to ATP, ADP (adenosine diphosphate), and adenosine, agonists that regulate bladder function significantly. ADP stimulated P2Y12 receptors, causing bladder smooth muscle (BSM) contraction, whereas adenosine signaling through potentially newly defined A2b receptors, actively inhibited BSM purinergic contractility. The modulation of adenylyl cyclase-cAMP signaling via A2b and P2Y12 interaction actively regulated bladder contractility by modulating intracellular calcium levels. KO mice lacking the receptors display diametrically opposed bladder phenotypes, with P2Y12-KO mice exhibiting an underactive bladder (UAB) phenotype with increased bladder capacity and reduced voiding frequency, whereas A2b-KO mice have an overactive bladder (OAB), with decreased capacity and increased voiding frequency. The opposing phenotypes in P2Y12-KO and A2b-KO mice not only resulted from dysregulated BSM contractility, but also from abnormal BSM cell growth. Finally, we demonstrate that i.p. administration of drugs targeting P2Y12 or A2b receptor rescues these abnormal phenotypes in both KO mice. These findings strongly indicate that P2Y12 and A2b receptors are attractive therapeutic targets for human patients with LUTS.
... Ecto-ATPase activity was inversely associated with the magnitude of nerve-mediated purinergic contractions and this was corroborated in human tissue by reduced ecto-ATPase expression of the predominant enzyme E-NTPDase-1 in detrusor from overactive compared to stable bladders. E-NTPDase-1 is inhibited by ARL-67156 (Lévesque, Lavoie, Lecka, Bigonnesse, & Sévigny, 2007) which would account for the increase of nerve-mediated contractions in guinea pig detrusor (Westfall, Kennedy, & Sneddon, 1997) and validated its use to estimate ecto-ATPase activity in detrusor homogenates. Several arguments may favour variability of ecto-ATPase activity as an explanation for variable atropine resistance: firstly, nerve-mediated ATP release was similar in the three cohorts used in this study although atropine resistance varied greatly and secondly, the A 1 receptor agonist N6-cyclopentyladenosine (CPA) had no effect on humanstable detrusor but a significant action on guinea pig tissue (Pakzad et al., 2016) suggesting that adenosine-mediated suppression of ATP, via A 1 receptors , was not a feature of human-stable detrusor. ...
Background and Purpose
This study aims to characterise the molecular mechanisms that determine variability of atropine resistance of nerve‐mediated contractions in human and guinea pig detrusor smooth muscle.
Experimental Approach
Atropine resistance of nerve‐mediated contractions and the role of P2X1 receptors, were assessed in isolated preparations from guinea pigs and also humans with or without overactive bladder syndrome, from which the mucosa was removed. Nerve‐mediated ATP release was measured directly with amperometric ATP‐sensitive electrodes. Ecto‐ATPase activity of guinea pig and human detrusor samples was measured in vitro by measuring the concentration‐dependent rate of ATP breakdown. The transcription of ecto‐ATPase subtypes in human samples was measured by qPCR.
Key Results
Atropine resistance was greatest in guinea pig detrusor, absent in human tissue from normally functioning bladders, and intermediate in human overactive bladder. Greater atropine resistance correlated with reduction of contractions by the ATP‐diphosphohydrolase apyrase, directly implicating ATP in their generation. E‐NTPDase‐1 was the most abundantly transcribed ecto‐ATPase of those tested, and transcription was reduced in tissue from human overactive, compared to normal, bladders. E‐NTPDase‐1 enzymic activity was inversely related to the magnitude of atropine resistance. Nerve‐mediated ATP release was continually measured and varied with stimulation frequency over the range of 1–16 Hz.
Conclusion and Implications
Atropine resistance in nerve‐mediated detrusor contractions is due to ATP release and its magnitude is inversely related to E‐NTPDase‐1 activity. ATP is released under different stimulation conditions compared with ACh, implying different routes for their release.
... The non-hydrolyzable nucleotide analogues either have no or very little effect on P2 receptor signaling and act as competitive inhibitors with an estimated Ki values in lower micromolar ranges. Namely, ARL 67156 (6-N,N-diethyl-D-β,γ-dibromomethylene ATP), and 8-thiobutyladenosine-5′triphosphate are potent non-hydrolyzable analogues of nucleotides that inhibit the individual E-NTPDase isozymes with varying degree of inhibitory potentials [27][28][29][30] . ...
Ecto-nucleotidase enzymes catalyze the hydrolysis of extracellular nucleotides to their respective nucleosides. Herein, we place the focus on the elucidation of structural features of the cell surface located ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase1-3 and 8). The physiological role of these isozymes is crucially important as they control purinergic signaling by modulating the extracellular availability of nucleotides. Since, crystal or NMR structure of the human isozymes are not available - structures have been obtained by homology modeling. Refinement of the homology models with poor stereo-chemical quality is of utmost importance in order to derive reliable structures for subsequent studies. Therefore, the resultant models obtained by homology modelling were refined by running molecular dynamic simulation. Binding mode analysis of standard substrates and of competitive inhibitor was conducted to highlight important regions of the active site involved in hydrolysis of the substrates and possible mechanism of inhibition.
... NTPDase1 involvement in insulin secretion has been confirmed by the results of studies in which the administration of apyrase inhibitor ARL67156 resulted in increased insulin secretion [64][65][66]. Thus, apyrase reduces insulin secretion by extracellular degradation of ATP and ADP, but it also participates, together with 5'-nucleotidase, in formation of adenosine, which probably slightly inhibits insulin secretion through activation of P1 receptors. ...
Extracellular purines activate P1 adenosine receptors and P2 nucleotide receptors. These receptors are present on the pancreatic islet cells as well as on hepatocytes, adipocytes, pancreatic blood vessels and nerves. ATP is released together with insulin from b-cell granules in response to a rapid decrease in blood glucose levels. The ATP-dependent P2X receptor activation on pancreatic b-cells results in a positive autocrine signal and subsequent insulin secretion. Adenosine, through activation of P1 receptors present on adipocytes and pancreatic islet cells, inhibits the release of insulin. Adenosine activates A2B receptors thereby stimulating production of IL-6 and other cytokines, which increases insulin resistance. Interleukin-6 also plays an important role in diabetes. In type 2 diabetes and obesity, the long-Term increase of IL-6 concentration in blood above 5 pg/mL leads to the chronic and permanent increase in expression of SOCS3, contributing to the increase in insulin resistance in cells of the skeletal muscles, liver and adipose tissue. In diabetes there is an increased synthesis and release of pro-inflammatory cytokines, which cause the damage of the pancreatic islet cells, and in type 2 diabetes cause the development of insulin resistance. Ecto-enzymes metabolizing nucleotides are involved in the termination of the nucleotide signalling pathway and play the key role in regulation of extracellular ATP concentration. Ecto-NTPDases in cooperation with 5'-nucleotidase may significantly increase ecto-Adenosine concentration. NTPDase3 activity has only been demonstrated on Langerhans cells. NTPDase3 may influence the secretion of insulin by hydrolysing adenine nucleotides. In diabetes the pro-inflammatory cytokines such as interleukin 1b (IL-1b), tumour necrosis factor-A (TNF-A) and interferon-g (IFN-g), as well as pancreatic derived factor PANDER are involved in the apoptosis of pancreatic b-cells. This causes disturbance of the balance between pro-inflammatory and protective cytokines. We believe that neutralization of pro-inflammatory cytokines, especially interleukin 1b, with the IL-1 receptor antagonist (IL-1Ra) and/or IL-1b antibodies might cause the reduction of the inflammatory process in pancreas islets, normalize concentration of glucose in blood and decrease the insulin resistance. (Clin Diabetol 2017; 6, 3: 90-100).
... Treatments with ecto-ATPase and ecto-nucleotidase activity inhibitors ARL67156 selectively inhibits ecto-ATPase activity without activating purinergic receptors [51]. A volume of 0.6 μl saline solution containing 200 μM ARL67156 was injected within the vitreous (pIC 50 = 4.62 and 5.1 in the human blood and rat vas deferens, respectively) [52,53]. ARL67156 was administered inside the vitreous chamber during three different intervals: 1-7, 0-3, and 4-7 dpl. ...
Damage in fish activates retina repair that restores sight. The purinergic signalling system serves multiple homeostatic functions and has been implicated in cell cycle control of progenitor cells in the developing retina. We examined whether changes in the expression of purinergic molecules were instrumental in the proliferative phase after injury of adult zebrafish retinas with ouabain. P2RY1 messenger RNA (mRNA) increased early after injury and showed maximal levels at the time of peak progenitor cell proliferation. Extracellular nucleotides, mainly ADP, regulate P2RY1 transcriptional and protein expression. The injury-induced upregulation of P2RY1 is mediated by an autoregulated mechanism. After injury, the transcriptional expression of ecto-nucleotidases and ecto-ATPases also increased and ecto-ATPase activity inhibitors decreased Müller glia-derived progenitor cell amplification. Inhibition of P2RY1 endogenous activation prevented progenitor cell proliferation at two intervals after injury: one in which progenitor Müller glia mitotically activates and the second one in which Müller glia-derived progenitor cells amplify. ADPβS induced the expression of lin28a and ascl1a genes in mature regions of uninjured retinas. The expression of these genes, which regulate multipotent Müller glia reprogramming, was significantly inhibited by blocking the endogenous activation of P2RY1 early after injury. We consistently observed that the number of glial fibrillary acidic protein-BrdU-positive Müller cells after injury was larger in the absence than in the presence of the P2RY1 antagonist. Ecto-ATPase activity inhibitors or P2RY1-specific antagonists did not modify apoptotic cell death at the time of peak progenitor cell proliferation. The results suggested that ouabain injury upregulates specific purinergic signals which stimulates multipotent progenitor cell response.
... In the presence of atropine (a competitive muscarinic ACh receptor antagonist; 1 μM), nerve-evoked contractions of urinary bladder strips were reduced by half in wild type mice and nearly absent in P2X1 knockout animals (Heppner et al., 2009). In agreement with a postjunctional contractile effect of ATP, the inhibition of NTPDase activ- ity (obviously NTPDase1, see next section) on the detrusor smooth mus- cle ( Yu et al., 2011) by ARL 67156 (100 μM) increased nerve-evoked contractions of the guinea-pig urinary bladder ( Westfall et al., 1997). ...
Adenine and uracil nucleotides play key functions in the autonomic nervous system (ANS). For instance, ATP acts as a neurotransmitter, co-transmitter and neuromodulator in the ANS. The purinergic system encompasses (1) receptors that respond to extracellular purines, which are designated as P1 and P2 purinoceptors, (2) purine release and uptake, and (3) a cascade of enzymes that regulate the concentration of purines near the cell surface. Ectonucleotidases and adenosine deaminase (ADA) are enzymes responsible for the hydrolysis of ATP (and other nucleotides such as ADP, UTP, UDP, AMP) and adenosine, respectively. Accordingly, these enzymes are expected to play an important role in the control of neuro-effector transmission in tissues innervated by both the sympathetic and parasympathetic divisions of the ANS. Indeed, ectonucleotidases have the ability to either terminate P2 receptor responses initiated by nucleoside triphosphates (ATP and UTP), and/or to favor the activation of ADP (e.g. P2Y1,12,13) and UDP (e.g. P2Y6) and/or adenosine (P1) specific receptors. In addition, ectonucleotidases can also importantly protect some P2 receptors from desensitization (e.g. P2X1, P2Y1). In this review, we present the (putative) roles of ectonucleotidases and ADA in the ANS with a focus on their regulatory activity at neuro-effector junctions in the following tissues: heart, vas deferens, urinary bladder, salivary glands, blood vessels and the intestine. We also present their implication in nociceptive transmission.
Copyright © 2015. Published by Elsevier B.V.
... Participation of NTPDase1 (apyrase) in insulin secretion was experimentally confirmed by the results of studies, in which administration of the apyrase inhibitor -ARL67156 caused an increase in insulin secretion (Crack et al., 1995;Westfall et al., 1997;Levesque et al., 2007). NTPDase1 impairs insulin secretion both by hydrolysis of extracellular ATP and ADP as by providing AMP as a substrate for 5'-nucleotidase. ...
It is widely accepted that purinergic signaling is involved in the regulation of functions of all known tissues and organs. Extracellular purines activate two classes of receptors, P1-adenosine receptors and P2-nucleotide receptors, in a concentration-dependent manner. Ecto-enzymes metabolizing nucleotides outside the cell are involved in the termination of the nucleotide signaling pathway through the release of ligands from their receptors. The pancreas is a central organ in nutrient and energy homeostasis with endocrine, exocrine and immunoreactive functions. The disturbances in cellular metabolism in diabetes mellitus lead also to changes in concentrations of intra- and extracellular nucleotides. Purinergic receptors P1 and P2 are present on the pancreatic islet cells as well as on hepatocytes, adipocytes, pancreatic blood vessels and nerves. The ATP-dependent P2X receptor activation on pancreatic β-cells results in a positive autocrine signal and subsequent insulin secretion. Ecto-NTPDases play the key role in regulation of extracellular ATP concentration. These enzymes, in cooperation with 5'-nucleotidase can significantly increase ecto-adenosine concentration. It has been demonstrated that adenosine, through activation of P1 receptors present on adipocytes and pancreatic islets cells, inhibits the release of insulin. Even though we know for 50 years about the regulatory role of nucleotides in the secretion of insulin, an integrated understanding of the involvement of purinergic signaling in pancreas function is still required. This comprehensive review presents our current knowledge about purinergic signaling in physiology and pathology of the pancreas as well as its potential therapeutic relevance in diabetes.
... Further experiments were conducted to determine if a prejunctional role of  3 -adrenoceptors could also be observed in the rat. However, one important difference in rodent bladder physiology is the enhanced role of ATP in the neurogenic response [27][28][29]. Under the current experimental conditions neurogenic responses of rat bladder to 15 Hz were inhibited 35% by atropine, while PPADS reduced the response by an additional 30% indicating that Ach and ATP play approximately equal roles in the neurogenic response in the rat. ...
Activation of β3-adrenoceptors has been shown to have a direct relaxant effect on urinary bladder smooth muscle from both rats and humans, however there are very few studies investigating the effects of β3-adrenoceptor agonists on nerve-evoked bladder contractions. Therefore in the current study, the role of β3-adrenoceptors in modulating efferent neurotransmission was evaluated. The effects of β3-adrenoceptor agonism on neurogenic contractions induced by electrical field stimulation (EFS) were compared with effects on contractions induced by exogenous acetylcholine (Ach) and αβ-methylene adenosine triphosphate (αβ-meATP) in order to determine the site of action. Isoproterenol inhibited EFS-induced neurogenic contractions of human bladder (pD2=6.79; Emax=65%). The effect of isoproterenol was selectively inhibited by the β3-adrenoceptor antagonist L-748,337 (pKB=7.34). Contractions induced by exogenous Ach (0.5-1μM) were inhibited 25% by isoproterenol (3μM) while contractions to 10Hz in the same strip were inhibited 67%. The selective β3-adrenoceptor agonist CL-316,243 inhibited EFS-induced neurogenic contractions of rat bladder (pD2=7.83; Emax=65%). The effects of CL-316,243 were inhibited in a concentration dependent manner by L-748,337 (pA2=6.42). Contractions induced by exogenous Ach and αβ-meATP were significantly inhibited by CL-316,243, 29% and 40%, respectively. These results demonstrate that the activation of β3-adrenoceptors inhibits neurogenic contractions of both rat and human urinary bladder. Contractions induced by exogenously applied parasympathetic neurotransmitters are also inhibited by β3-agonism however the effect is clearly less than on neurogenic contractions (particularly in human), suggesting that in addition to a direct effect on smooth muscle, activation of prejunctional β3-adrenoceptors may inhibit neurotransmitter release.
... These enzyme activities largely aect P2 receptor-mediated physiological responses. Indeed, the ecto-ATPase inhibitor ARL67156 was reported to potentiate the neurotransmitter action of ATP in the guinea-pig vas deferens (Westfall et al., 1996) and urinary bladder (Westfall et al., 1997). Furthermore, it has been shown that rapid conversion of ATP to adenosine results in P1 receptor-mediated responses. ...
We previously demonstrated that extracellular adenine nucleotides induced cyclic AMP elevation through local adenosine production at the membrane surface and subsequent activation of adenosine A2A receptors in NG108-15 cells. Furthermore, the adenosine formation was found to be mediated by an ecto-enzyme distinct from the ecto-5′-nucleotidase (CD73). In this study, we investigated the properties of the ecto-AMP phosphohydrolase activity in NG108-15 cells.
NG108-15 cells hydrolyzed AMP to adenosine with the KM value of 18.8±2.2 μM and Vmax of 5.3±1.6 nmol min−1 106 cells−1. This activity was suppressed at pH 6.5, but markedly increased at pH 8.5.
The AMP hydrolysis was blocked by levamisole, an alkaline phosphatase (ALP) inhibitor. NG108-15 cells released orthophosphate from 2′- and 3′-AMP as well as from ribose-5-phosphate and β-glycerophosphate, indicating that NG108-15 cells express ecto-ALP.
The cyclic AMP accumulation induced by several adenine nucleotides was inhibited by levamisole, p-nitrophenylphosphate and β-glycerophosphate, with a parallel decrease in the extracellular adenosine formation.
Reverse transcriptase polymerase chain reaction analysis revealed that NG108-15 cells express mRNA for the tissue-nonspecific isozyme of ALP.
These results demonstrate that AMP phosphohydrolase activity in NG108-15 cells is due to ecto-ALP, and suggest that this enzyme plays an essential role for the P1 antagonist-sensitive ATP-induced cyclic AMP accumulation in NG108-15 cells.
British Journal of Pharmacology (2000) 131, 1667–1672; doi:10.1038/sj.bjp.0703750
... ARL67156, an ATP analogue, selectively inhibits ecto-ATPase activity without activating purinergic receptors (Robson et al., 2006). We injected 100 mM ARL67156 (Westfall et al., 1997;Newman, 2003) via the intravitreal route in 0.5 mL of saline solution (pIC 50 = 4.62 and 5.1 in human blood and rat vas deferens, respectively). Zebrafish were euthanized 22 h later. ...
Regeneration and growth that occur in the adult teleost retina have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. Here, it is reported that S-phase cell number, in the ciliary marginal zone (CMZ) of the adult zebrafish retina, exhibits day-night variations with a mid-light phase peak. Oscillations persist for 24 h in constant darkness (DD), suggesting control by a circadian component. However, variations in the S-phase nuclei number were rapidly dampened and not present during and after a second day in DD. An ADPβS treatment significantly enhanced S-phase activity at night to mid-light levels, as assessed by in vivo BrdU incorporation in a 2-h interval. Moreover, daylight increase in S-phase cell number was completely abolished when extracellular nucleotide levels or their extracellular hydrolysis by ectonucleoside triphosphate diphosphohydrolases (NTPDases) were significantly disrupted or when a selective antagonist of purinergic P2Y1 receptors was intraocularly injected before BrdU exposure. Extracellular nucleotides and NTPDase action were also important for maintaining nocturnal low levels of S-phase activity in the CMZ. Finally, we showed that mRNAs of NTPDases 1, 2 (3 isoforms), and 3 as well as of P2Y1 receptor are present in the neural retina of zebrafish. NTPDase mRNA expression exhibited a 2-fold increment in light versus dark conditions as assessed by quantitative RT-PCR, whereas P2Y1 receptor mRNA levels did not show significant day-night variations. This study demonstrates a key role for nucleotides, principally ADP as a paracrine signal, as well as for NTPDases, the plasma membrane-bound enzymes that control extracellular nucleotide concentration, for inducing S-phase cell entry in the CMZ-normally associated with retinal growth-throughout the light-dark cycle.
... NANC-and ATP-mediated detrusor smooth muscle contractions could also be suppressed by desensitization with a,b-meATP or by various nonselective purinergic antagonists, such as quinidine, reactive blue 2, suramin, and PPADS, without depressing responses to acetylcholine (Dean and Downie 1978;Kasakov and Burnstock 1983;Hoyle and Burnstock 1985;Brading and Williams 1990;Ziganshin et al. 1993;Tong et al. 1997;King et al. 1997). Ecto-ATPase inhibitors were shown to potentiate NANC and ATP responses in the guinea pig bladder (Hourani and Chown 1989;Westfall et al. 1997), and release of ATP in response to transmural stimulation of NANC nerves was demonstrated (Burnstock et al. 1978;Tong et al. 1997). Electrophysiological recordings from isolated detrusor smooth muscle cells from guinea pig, rabbit, and pig also showed that ATP and a,b-meATP elicited concentration-dependent, membrane depolarization and inward currents that showed rapid desensitization (Fujii 1988;Brading 1990, 1991). ...
The pharmacological concept of specifically targeting purinoceptors (receptors for ATP and related nucleotides) has emerged over the last two decades in the quest for novel, differentiated therapeutics. Investigations from many laboratories have established a prominent role for ATP in the functional regulation of most tissue and organ systems, including the urinary tract, under normal and pathophysiological conditions. In the particular case of the urinary tract, ATP signaling via P2X1 receptors participates in the efferent control of detrusor smooth muscle excitability, and this function may be heightened in disease and aging. Perhaps of greater interest, ATP also appears to be involved in bladder sensation, operating via activation of P2X3-containing receptors on sensory afferent neurones, both on peripheral terminals within the urinary tract tissues (e.g., ureters, bladder) and on central synapses in the dorsal horn of the spinal cord. Such findings are based on results from classical pharmacological and localization studies in nonhuman and human tissues, gene knockout mice, and studies using recently identified pharmacological antagonists - some of which have progressed as candidate drug molecules. Based on recent advances in this field, it is apparent that the development of selective antagonists for these receptors will occur that could lead to therapies offering better relief of storage, voiding, and sensory symptoms for patients, while minimizing the systemic side effects that curb the clinical effectiveness of current urologic medicines.
... Several hypotheses have been suggested for the physiological role of these enzymes. They include: (1) protection from the cytolytic effects of extracellular ATP (Fillipini et al., 1990;Steinberg & Di Virgilio, 1991); (2) termination of purinergic signaling (Weisman et al., 1996;Westfall et al., 1997); (3) involvement in signal transduction (Margolis et al., 1990;Dubyak & El-Moatassim, 1993); and (4) involvement in cellular adhesion (Kirley, 1997;Bisaggio et al., 2003;Pinheiro et al., 2006;Santos et al., 2009). ...
In this work, we describe the ability of intact cells of Candida parapsilosis to hydrolyze extracellular ATP. ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. The ecto-ATPase activity was increased in the presence of 5 mM MgCl(2), with values of V(max) and apparent K(m) for Mg-ATP(2-) increasing to 33.80 +/- 1.2 nmol Pi h(-1) 10(-8) cells and 0.6 +/- 0.06 mM, respectively. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases and Na(+)-ATPases had no effect on the C. parapsilosis Mg(2+)-stimulated ATPase activity, but extracellular impermeant compounds, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid and suramin, reduced enzyme activity in yeast living cells by 83.1% and 81.9%, respectively. ARL 67156 (6-N,N'-diethyl-d-beta-gamma-dibromomethylene ATP), a nucleotide analogue, also inhibited the ecto-ATPase activity in a dose-dependent manner. ATP was the best substrate for the yeast Mg(2+)-stimulated ecto-enzyme, but ADP, ITP, CTP, GTP and UTP were also hydrolyzed. A direct relationship between ecto-ATPase activity and adhesion to host cells was observed. In these assays, inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells. Based also on the differential expression of ecto-ATPase activities in the different isolates of C. parapsilosis, the possible role of this enzyme in fungal biology is discussed.
... The present results revealed that while bladder contractions and relaxations evoked by ATP and UTP were only slightly affected by the ecto-nucleoside triphosphate diphosphohydrolase ARL 67156, the presence of the inhibitor significantly reduced the phasic component of the EFS-evoked bladder contraction. These findings are in contrast to studies of the guinea-pig bladder where inhibition of ectonucleotidases leads to enhanced EFS-as well as ATP-evoked bladder contractions [32]. Species differences may, however, exist and neuronally released ATP in the rat bladder may also be metabolized faster than exogenously administered agonists. ...
The aim of the present study was to assess the purinoceptor functional responses of the urinary bladder by using isolated rat urinary bladder strip preparations. ATP elicited a transient bladder contraction followed by a sustained relaxation and ADP, UDP and UTP generated predominantly potent relaxations (relaxatory potencies: ADP = ATP > UDP = UTP). The ATP contractions were desensitized with the P2X(1/3) purinoceptor agonist/desensitizer alpha,beta-meATP and reduced by the P2 purinoceptor antagonist PPADS but unaffected by the P2 purinoceptor antagonist suramin. Electrical field stimulation (1-60 Hz) evoked frequency-dependent bladder contractions that were decreased by incubation with alpha,beta-meATP but not further decreased by PPADS. Suramin antagonized relaxations generated by UDP but not those by ADP, ATP or UTP. PPADS antagonized and tended to antagonize UTP and UDP relaxations, respectively, but did neither affect ADP nor ATP relaxations. ADP relaxations were insensitive to the P2Y(1) purinoceptor antagonist MRS 2179 and the ATP-sensitive potassium channel antagonist glibenclamide. The ATP relaxations were inhibited by the P1 purinoceptor antagonist 8-p-sulfophenyltheophylline but unaffected by the A2A adenosine receptor antagonist 8-(3-chlorostyryl)caffeine and glibenclamide. Adenosine evoked relaxations that were antagonized by the A2B adenosine receptor antagonist PSB 1115. Thus, in the rat urinary bladder purinergic contractions are elicited predominantly by stimulation of the P2X(1) purinoceptors, while UDP/UTP-sensitive P2Y purinoceptor(s) and P1 purinoceptors of the A2B adenosine receptor subtype are involved in bladder relaxation.
... Firstly, ATP is metabolised by ecto-enzymes in the extracellular space of Ž . smooth muscles Westfall et al., 1997 . Furthermore, in order for the P class of purinoceptors to be activated, 2X they must bind two ATP molecules, a condition that is Ž difficult to fulfil over long diffusion pathways Bennett et . ...
The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2X1 receptor clusters were observed on the smooth muscle cells of mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32+/-0.21 microm long and 0.96+/-0.10 microm in diameter; and a smaller spherical cluster with a diameter of 0.32+/-0.05 microm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.
... rAAV has been shown to be capable of stable, long-term transgene expression both in vitro and in vivo in a variety of tissues, although the transduction efficiency of rAAV is markedly variable in different cell types. For example, rAAV has been reported to transduce lung epithelial cells at low levels (6,11), while high-level, persistent transgene expression has been demonstrated in muscle cells, neurons, and other nondividing cells (2,8,12,13,15,(28)(29)(30). These tissue-specific differences in rAAV-mediated gene transfer may be due, in part, to variable levels of cellular factors affecting AAV infectivity (i.e., receptors and coreceptors such as heparan sulfate proteoglycan, FGFR-1, and ␣V5 integrin) (21,25,26), as well as the latent life cycle (i.e., nuclear trafficking of virus and/or the conversion of single-stranded genomes to expressible forms) (20,22). ...
Long-term recombinant AAV (rAAV) transgene expression in muscle has been associated with the molecular conversion of single-stranded rAAV genomes to high-molecular-weight head-to-tail circular concatamers. However, the mechanisms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermediates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase (Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular recombination of independent viral genomes. The GFP shuttle vector also encoded ampicillin resistance and contained a bacterial origin of replication to allow for bacterial rescue of circular intermediates from Hirt DNA of infected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which reached 33% of the total circular intermediates by 120 days postinfection. Furthermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in cell lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering multiple DNA segments at a single locus. Such developments will expand the utility of rAAV for splicing large gene inserts or large promoter-gene combinations carried by two or more independent rAAV vectors.
... This is close to the IC 50 value for inhibition of ATP breakdown by eNTPDases by ARL 67156 in human blood (25 mM; Crack et al., 1995) and by PPADS in rat vas deferens (40 mM; Khakh et al., 1995), macrophages (16 mM), C6 glioma cells (20 mM) and bovine pulmonary artery endothelial cells (100 mM) (Chen et al., 1996). At similar concentrations ARL 67156 also inhibits ATP metabolism and potentiates the neurotransmitter actions of ATP in the guinea-pig isolated vas deferens and urinary bladder (Westfall et al., 1996;1997a). A single concentration of PPADS (100 mM) has also been reported to inhibit recombinant rat CD39 and ecto-ATPase to a similar extent (Heine et al., 1999). ...
The release of ATPase activity evoked by electrical field stimulation (EFS) (8 Hz, 25 s) was investigated in several tissues in which adenosine 5′-triphosphate (ATP) acts as a neurotransmitter.
Superfusate collected during EFS of sympathetic nerves of the guinea-pig, rat and mouse isolated vas deferens and parasympathetic nerves of the guinea-pig isolated urinary bladder contained ATPase activity. ATP breakdown was fastest in superfusate collected from the guinea-pig isolated vas deferens. However, EFS of the enteric nerves of the guinea-pig isolated taenia coli did not release any detectable ATPase.
The ATPase released from the guinea-pig isolated vas deferens metabolized ATP at similar rates at incubation temperatures of 37°C and 20°C. Lineweaver–Burke analysis of the initial rates of ATP hydrolysis gave a KM of 39 μM and a Vmax of 1039 pmol ATP metabolized min−1 ml−1 superfusate.
6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156), pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and pyridoxal-5′-phosphate (P-5-P) all inhibited the ATPase activity in a concentration-dependent manner with a potency order of ARL 67156=PPADS>P-5-P.
In conclusion, EFS of several tissues in which ATP is a neurotransmitter causes the release of an ATPase and activity is greatest in the guinea-pig vas deferens. The enzyme has pharmacological and kinetic characteristics that are similar to ectonucleoside triphosphate diphosphohydrolases.
British Journal of Pharmacology (2000) 129, 1684–1688; doi:10.1038/sj.bjp.0703271
... Inhibition of ecto-ATPase adversely affects the actions of a number of cells of the immune system (Dombrowski et al, 1993;1998). Ecto-ATPase is also found in a variety of muscles and may play an important role in the modulation of nucleotide signaling via P 2 purinergic receptors (Westfall et al, 1997;Zinchuk et al, 1999). Sertoli cells have previously been reported to express ecto-ATPase activity on their surface (Barbacci et al, 1996), but its function is not clear. ...
A putative messenger RNA (mRNA) sequence, designated C8, that was up-regulated in Sertoli cells prepared from hypophysectomized rats treated with testosterone, was isolated from a Sertoli cell complementary DNA (cDNA) library. The coding region of C8 exhibited 99% identity with rat brain ecto-ATPase and expressed a 60-kilodalton protein following in vitro transcription/translation. Transfection of COS7 cells with C8 cDNA resulted in a marked increase in Ca2+- and Mg2+-dependent ATPase activity in both whole cells and cell homogenates, which is consistent with localization of this enzyme in the plasma membrane. C8 ecto-ATPase steady state mRNA levels were increased within 6 hours and for 3 day, by follicle-stimulating hormone (FSH) in Sertoli cells but not in peritubular cells. In contrast, dibutyryl-cyclic adenosine monophosphate (cAMP) increased ecto-ATPase in both Sertoli and peritubular cells. Testosterone had no significant effect under these conditions. These data indicate that ecto-ATPase mRNA is positively regulated by FSH in Sertoli cells and by cAMP in both Sertoli and peritubular cells. This enzyme may play a role in the control of extracellular signaling by ATP, adenosine, or both in the cells of the seminiferous epithelium.
Geoff Burnstock created an outstanding scientific legacy that includes identification of adenosine 5′-triphosphate (ATP) as an inhibitory neurotransmitter in the gut, the discovery and characterisation of a large family of purine and uridine nucleotide-sensitive ionotropic P2X and metabotropic P2Y receptors and the demonstration that ATP is as an excitatory cotransmitter in autonomic nerves. The evidence for cotransmission includes that: 1) ATP is costored with noradrenaline in synaptic vesicles in postganglionic sympathetic nerves innervating smooth muscle tissues, including the vas deferens and most arteries. 2) When coreleased with noradrenaline, ATP acts at postjunctional P2X1 receptors to elicit depolarisation, Ca²⁺ influx, Ca²⁺ sensitisation and contraction. 3) ATP is also coreleased with acetylcholine from postganglionic parasympathetic nerves innervating the urinary bladder, where it stimulates postjunctional P2X1 receptors, and a second, as yet unidentified site to evoke contraction of detrusor smooth muscle. In both systems membrane-bound ecto-enzymes and soluble nucleotidases released from postganglionic nerves dephosphorylate ATP and so terminate its neurotransmitter actions. Currently, the most promising potential area of therapeutic application relating to cotransmission is treatment of dysfunctional urinary bladder. This family of disorders is associated with the appearance of a purinergic component of neurogenic contractions. This component is an attractive target for drug development and targeting it may be a rewarding area of research.
Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation (IP) and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by IP, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by co-administration of inhibitory factor 1; limiting inorganic phosphate (Pi) in the media; and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-NTPDases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low Pi supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion.
The pioneering work on neurotransmission in the peripheral nervous system carried out earlier in the century led to the widely accepted idea that noradrenaline (NA) was the sole neurotransmitter released by postganglionic sympathetic nerves and that acetylcholine (ACh) was the sole neurotransmitter released by postganglionic parasympathetic nerves (Dale 1935). However, by the early 1970s evidence was accumulating to support the idea that adenosine 5′-triphosphate (ATP) was also a neurotransmitter from “purinergic” nerves in some parts of the autonomic nervous system (Burnstock 1972). Several years later Burnstock (1976) developed a further understanding of peripheral neurotransmission by suggesting that these nerves could in fact release more than one substance as a neurotransmitter. Since then, cotransmission has become accepted as the rule rather than the exception, with NA, ACh, ATP, nitric oxide (NO) and numerous peptides all having been shown to act as cotransmitters in numerous types of peripheral tissue.
Extracellular nucleotides such as ATP, or diadenosine polyphosphates and also adenosine play important receptor-mediated functions in the cardiovascular system by contributing to the regulation of vascular tone, myocardial contractility, and the function of blood cells. Both the inactivation of extracellular nucleotides and the extracellular production of adenosine arc key functional roles of ecto-nucleotidases. The final hydrolysis product adenosine may be salvaged by adjacent cells or become deaminated extracellularly to inosine by ecto-adenosine deaminase. The extracellular hydrolysis pathways have been identified by vascular perfusion or by analysis of isolated endothelial or smooth muscle cells or of cardiomyocytes. In principle, a considerable variety of enzymes can be involved in these catalytic functions. The diadenosine polyphosphatases have not yet been identified in molecular terms. Recent progress has led to the molecular cloning and functional expression of members of a new family of proteins that hydrolyze either nucleoside-5′-di- and/or triphosphates (ecto-ATP diphosphohydrolase, ecto-ATPase). The same compounds are substrates also of members of another family of ecto-nucleotidases, the phosphodiesterase/nucleotide pyrophosphatases. These enzymes have nucleotide pyrophosphatase as well as phosphodiesterase activity and can also cleave extracellular cAMP. The hydrolysis of extracellular AMP to adenosine is catalyzed by ecto-5′-nucleotidase. Finally, ecto-alkaline phosphatase is capable of releasing inorganic phosphate from a variety of organic compounds including the degradation of ATP, ADP, AMP. It should be noted that these enzymes have a broad substrate specificity hydrolyzing purine and pyrimidine nucleotides. The information concerning the distribution of the individual enzymes in the cardiovascular system and its specific cellular elements is still sparse. There is good support for a major role of ecto- ATP diphosphohydrolase in the degradation of ATP and ADP in the vascular system with an inhibitory effect on platelet activation. The expression of the individual enzymes may vary between cells in culture and their in situ location. Future studies need to unravel the cellular distribution of the enzymes by in situhybridization and immunocytological methods, analyze their functional role by intervention with specific inhibitors, reveal their regulation of expression, and evaluate the deletion of the various ecto-nucleotidase genes.
ARC 67156 is a structural analog of ATP, which selectively inhibits breakdown of ATP and related nucleotides by ecto-nucleotidases. At high concentrations, ARC 67156 can also act as an antagonist at P2X receptors, but no effect has been reported at P2Y receptors.
Ecto-nucleotidases hydrolyze extracellular nucleotides. Nucleotides are amongst the most ubiquitous messenger substances in the vertebrate body. Receptors for nucleotides are expressed on the surface of essentially every cell and many cells carry several types of nucleotide receptors. Several families of ecto-nucleotidases have been identified that differ in tissue distribution and functional properties. They modulate ligand availability at nucleotide and adenosine receptors. Ecto-nucleotidases were first identified in the 1940ies. Work of the past two decades has unraveled molecular identities and important functional properties. Using targeted gene deletion clear examples highlighting the importance of ecto-nucleotidases in nucleotide and adenosine signaling have been elaborated. These reach from the control of blood flow and angiogenesis to the modulation of immune functions and neural development. Specific ecto-nucleotidases are associated with stem cells in the adult mammalian brain, implicating a role of nucleotides and nucleosides in the control of adult neurogenesis. Ecto-nucleotidases represent important therapeutic targets to interfere with P2 or P1 receptor-mediated receptor signaling pathways. The development of high throughput assays promises a considerable acceleration in the development of subtype-specific ecto-nucleotidase inhibitors.
Ecto-nucleotidases hydrolyze extracellular nucleotides. Nucleotides are amongst the most ubiquitous messenger substances in the vertebrate body. Receptors for nucleotides are expressed on the surface of essentially every cell and many cells carry several types of nucleotide receptors. Several families of ecto-nucleotidases have been identified that differ in tissue distribution and functional properties. They modulate ligand availability at nucleotide and adenosine receptors. Ecto-nucleotidases were first identified in the 1940ies. Work of the past two decades has unraveled molecular identities and important functional properties. Using targeted gene deletion clear examples highlighting the importance of ecto-nucleotidases in Discurso de ingreso como Académico Correspondiente de la Real Academia Nacio-nal de Farmacia. Abbreviations: Ap n A, dinucleoside polyphosphate; ART, ADP-ribosyltransferase; NADase, NAD glycohydrolases; E-NTPDase, ecto-nucleoside triphosphate diphospho-hydrolase; E-NPP, ecto-nucleotide pyrophosphatase/phosphodiesterase; IAP, intestinal alkaline phosphatase; PC-1, plasma cell differentiation antigen-1; SVZ, subventricular zone; TNAP, tissue nonspecific form of alkaline phosphatase.
The role of adenosine 5'-triphosphate (ATP) as a major intracellular energy source is well-established. In addition, ATP and related nucleotides have widespread extracellular actions via the ionotropic P2X receptors (ligand-gated cation channels) and metabotropic P2Y receptors (G protein-coupled receptors). Numerous experimental techniques, including myography, electrophysiology and biochemical measurement of neurotransmitter release, have been used to show that ATP has two major roles as an excitatory cotransmitter from autonomic nerves. 1) It is costored with noradrenaline in synaptic vesicles in postganglionic sympathetic nerves innervating smooth muscle preparations, such as the vas deferens and most arteries. When coreleased with noradrenaline, ATP acts at postjunctional P2X1 receptors to evoke depolarisation, Ca2 + influx, Ca2 + sensitisation and contraction. 2) ATP is also coreleased with acetylcholine from postganglionic parasympathetic nerves innervating the urinary bladder and again acts at postjunctional P2X1 receptors, and possibly also a P2X1+4 heteromer, to elicit smooth muscle contraction. In both systems the neurotransmitter actions of ATP are terminated by dephosphorylation by extracellular, membrane-bound enzymes and soluble nucleotidases released from postganglionic nerves. There are indications of an increased contribution of ATP to control of blood pressure in hypertension, but further research is needed to clarify this possibility. More promising is the upregulation of P2X receptors in dysfunctional bladder, including interstitial cystitis, idiopathic detrusor instability and overactive bladder syndrome. Consequently, these roles of ATP are of great therapeutic interest and are increasingly being targeted by pharmaceutical companies.
In contrast to the well-known signaling role of urothelial ATP to control bladder function, the hypothesis that uracil nucleotides (UTP and/or UDP) also exert autocrine/paracrine actions only recently gained experimental support. Urothelial cells express UDP-sensitive P2Y6 receptors, yet their role in the control of bladder activity has been mostly neglected. This study was designed to investigate the ability of PSB0474, a stable UDP analogue which exhibits selectivity for P2Y6 receptors, to modulate urodynamic responses in the anaesthetized rat in vivo. Instillation of PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions. PSB0474-induced bladder overactivity was prevented by the selective P2Y6 antagonist, MRS2578. The increase in the VF produced by PSB0474 was also blocked by inhibitors of pannexin-1 hemichannels, (10)Panx or carbenoxolone, when these drugs were applied inside the bladder lumen but not when they were administered intravenously. Reduction of hemichannels pore permeability with H1152 also prevented PSB0474-induced bladder overactivity, but the exocytosis inhibitor, Exo-1, was inactive. PSB0474 increased by 3-fold the urinary ATP content. Implication of hemichannels permeability on PSB0474-induced ATP release was demonstrated by real-time fluorescence videomicroscopy measuring the uptake of propidium iodide by intact urothelial cells in the absence and in the presence of MRS2578 or carbenoxolone. Confocal microscopy studies confirmed the co-localization of pannexin-1 and P2Y6 receptors in the rat urothelium. Data indicate that activation of P2Y6 receptors causes bladder overactivity in the anaesthetized rat indirectly by releasing ATP from the urothelium via pannexin-1 hemichannels.
Extracellular nucleotides such as ATP, ADP, UTP, UDP, and also diadenosine polyphosphates act as signaling molecules and can be inactivated by hydrolysis via ectonucleotidases. A considerable number of surface-located enzymes can potentially be involved in the extracellular hydrolysis pathway. These include the E-NTPDase family (ectonucleoside triphosphate diphosphohydrolase family), the E-NPP family (ectonucleotide pyrophosphatase/phosphodiesterase family), ecto-5′-nucleotidase, and alkaline phosphatases. In addition, activity of ectonucleoside diphosphokinase can interconvert extracellular nucleotides, and ATP can be used as a cosubstrate of ectoprotein kinase in the phosphorylation of surface-located proteins. Members of the various ectonucleotidase families reveal overlapping substrate specificity and tissue distribution whose functional significance needs to be further elucidated. Considerable progress has been made in the past several years in characterizing novel enzyme species and their molecular and functional properties. First knock-out mice reveal insight into physiological processes governed by the activity of specific ectonucleotidases. Together this work has led to a deeper understanding of the pathways of extracellular nucleotide metabolism, including their interplay with P2 and P1 receptors or also other physiological mechanisms. Drug Dev. Res. 52:44–56, 2001. © 2001 Wiley-Liss, Inc.
Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ecto-nucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.
Defects in bone homeostasis are a major health problem. Osteoclast differentiation and activation have a crucial role in bone remodeling in health and disease. Osteoclasts are bone-resorbing cells derived from mononuclear phagocyte progenitors. The key event in osteoclast formation is fusion of mononucleate precursors to form mature multinucleated osteclasts. Here we provide evidence of an absolute requirement for the P2X7 receptor, ATP release, and adenosine signaling in human osteoclast formation, as shown by the following findings: macrophage-colony stimulating factor/receptor activator for nuclear factor-κB ligand (M-CSF/RANKL)-stimulated fusion of human monocytes is fully prevented by an anti-P2X7 mAb, by specific P2X7 pharmacological antagonists, or by inhibition of CD39/NTPDase; fusion-competent monocytes release ATP via the P2X7 receptor; accelerated degradation of released ATP by addition of either apyrase or hexokinase strongly increases fusion; removal of extracellular adenosine by adenosine deaminase blocks, while addition of exogenous adenosine strongly potentiates, fusion; and pharmacologic stimulation of the adenosine A2A receptor increases, while selective A2A blockade inhibits, fusion. These results show that the purinergic axis plays a crucial and as yet undescribed role in osteoclast formation and reconcile previous evidence advocating a key role for either ATP or adenosine receptors in multinucleated giant cell formation.
Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human beta cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i), and hormone release in vitro, we show that human beta cells express ionotropic ATP receptors of the P2X(3) type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X(3) receptors in the beta-cell plasma membrane, resulting in increased [Ca(2+)](i) and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the beta cell's secretory machinery. This may explain how the human pancreatic beta cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.
The lower urinary tract is a muscular system composed of the urinary bladder and the outflow tract. During filling with urine the bladder is relaxed and the outflow tract offers a high resistance; during emptying the outflow resistance falls and the bladder wall generates a high wall tension to raise intravesical pressure. The coordination of these responses is organized in the brainstem and sacral spinal cord to control the activity of autonomic and somatic efferents to the smooth muscle of the bladder (detrusor) and the smooth and skeletal muscle of the bladder base and urethra. Detrusor contraction is predominantly controlled by parasympathetic fibres releasing acetylcholine and ATP; the outflow tract is controlled by parasympathetic and sympathetic fibres to the bladder base (trigone) and urethral smooth muscle (including a nitregic component) and somatic fibres to the urethral rhabdosphincter. The smooth muscles also develop spontaneous contractions that determine the tone of the musculature. The cellular signaling pathways that evoke contraction due to neurotransmitter release and the origin of spontaneous activity are discussed, as well as the electrical properties of the smooth muscle relevant to the propagation of electrical signals. Finally the interaction of muscle cells with other cell types (epithelium and interstitial cells) is considered, relevant to their ability to regulate muscle contractility. Throughout, the basic physiological processes are considered in relation to pathological developments that are prevalent in the human lower urinary tract, in particular the overactive bladder and urinary incontinence, and the identification of drug targets to manage these conditions.
Ecto-nucleotidases hydrolyze extracellular nucleotides. Nucleotides are amongst the most ubiquitous messenger substances in the vertebrate body. Receptors for nucleotides are expressed on the surface of essentially every cell and many cells carry several types of nucleotide receptors. Several families of ecto-nucleotidases have been identified that differ in tissue distribution and functional properties. They modulate ligand availability at nucleotide and adenosine receptors. Ecto-nucleotidases were first identified in the 1940ies. Work of the past two decades has unraveled molecular identities and important functional properties. Using targeted gene deletion clear examples highlighting the importance of ecto-nucleotidases in Discurso de ingreso como Académico Correspondiente de la Real Academia Nacio-nal de Farmacia. Abbreviations: Ap n A, dinucleoside polyphosphate; ART, ADP-ribosyltransferase; NADase, NAD glycohydrolases; E-NTPDase, ecto-nucleoside triphosphate diphospho-hydrolase; E-NPP, ecto-nucleotide pyrophosphatase/phosphodiesterase; IAP, intestinal alkaline phosphatase; PC-1, plasma cell differentiation antigen-1; SVZ, subventricular zone; TNAP, tissue nonspecific form of alkaline phosphatase.
Overactive bladder (OAB) signifies the presence of urinary urgency and can have major effects on quality of life and social functioning. Standard antimuscarinic drugs have good initial response rates but substantial adverse effects and long-term compliance problems.
To review the complexities of the mechanisms underlying OAB and the current drugs available for treating its symptoms.
The literature was reviewed to define current therapies and drugs in clinical trials. Articles were identified by means of a computerised PubMed and Cochrane Library search (using the following keywords: overactive bladder, detrusor overactivity, urgency and bladder), supported by a search of the PharmaProjects database.
New drug classes, such as beta-3 adrenergic agonists, may work by reducing contractility or excitability of bladder muscle. Moderation of afferent activity may allow improved OAB symptoms, with lower risk of affecting voiding function. Agents acting on the CNS could influence OAB favourably, but target selection and adverse effects are an issue. The recognition of the functional contribution of the urothelium and the diversity of nerve transmitters has sparked interest in both peripheral and central modulation of OAB pathophysiology.
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The mechanism of action of P2 nucleotide receptor agonists that produce endothelium-independent relaxation and the influence of ecto-ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle.
At 25°C, ATP, 2-methylthioATP (2-MeSATP) and 2-chloroATP (2-ClATP), dose-dependently inhibited spontaneous contractile activity of endothelium-denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half-inhibitory concentrations was 2-ClATP (2.7±0.5 μM, n=7)>ATP (12.9±1.1 μM, n=9)2-MeSATP (21.9±4.8 μM, n=4). In the presence of αβ-methylene ATP (αβ-MeATP, 200 μM) which itself produced a transient contractile effect, the relaxing action of ATP and 2-MeSATP was completely abolished and that of 2-ClATP strongly inhibited.
The non-selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 100 μM) did not affect the relaxation induced by ATP, 2-MeSATP, and 2-ClATP.
The A2A-adenosine receptor antagonist ZM 241385 inhibited the ATP-induced relaxation in a concentration-dependent manner (1–100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2-MeSATP and 2-ClATP were also inhibited.
ADP, AMP and adenosine also produced concentration-dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to αβ-MeATP (200 μM) but were inhibited by ZM 241385 (100 nM).
Simultaneous measurements of contraction and ecto-ATPase activity estimated by the degradation of [γ-32P]-ATP showed that muscular strips rapidly (10–60 s) hydrolyzed ATP. This ecto-ATPase activity was abolished in the presence of EDTA and was inhibited by 57±11% (n=3) by 200 μM αβ-MeATP.
These results suggest that ATP and other P2-receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A2A-receptors.
British Journal of Pharmacology (1998) 123, 1732–1740; doi:10.1038/sj.bjp.0701773
The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
Suramin (1 μM–1 mM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi-effective concentrations of α,β-methyleneATP (α,β-meATP) (5 μM), 2-methylthioATP (2-meSATP) (100 μM) and adenosine 5′-triphosphate (ATP) (1 mM) in a concentration-dependent manner. Responses to α,β-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.
Contractions evoked by uridine 5′-triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM).
Desensitization of the P2X1-receptor by α,β-meATP abolished contractions evoked by 2-meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control.
Responses to α,β-meATP (5 μM) and 2-meSATP (100 μM) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response.
ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β-meATP (5 μM), 2-meSATP (100 μM) and ATP (1 mM) in a concentration-dependent manner.
These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X1-receptors and G-protein-coupled P2Y-receptors.
Electrical field stimulation (EFS) caused contraction of isolated tail arteries of rats. The EFS-induced contraction showed frequency-dependence and was entirely abolished by the sodium channel blocker tetrodotoxin (1 x 10(-7) M). The EFS-induced (at 20 Hz) contraction was reduced by about 60% in the presence of phentolamine (1 x 10(-6) M). Therefore, later experiments were carried out in the presence of phentolamine. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1 x 10(-8)-1 x 10(-6) M) and basilen blue E-3G (3 x 10(-5)-5 x 10(-5) M), P2-receptor antagonists, significantly inhibited the contraction evoked by EFS. In addition, PPADS significantly inhibited the contractions induced by ATP (1 x 10(-4) M) and a selective P2x-receptor agonist, alpha,beta-methylene ATP (1 x 10(-6) M). In contrast, basilen blue E-3G did not inhibit alpha,beta-methylene ATP-induced contraction. The ecto-ATPase activator apyrase (5 and 10 U/ml) significantly reduced the EFS-induced contractions. These findings suggest that endogenous ATP released by EFS causes contractions of rat tail artery via both the P2x-receptors and P2y-receptors.
Intracellular microelectrodes were used to record the transmembrane potential and excitatory junction potentials (e.j.p.s) produced by sympathetic nerve stimulation (1 Hz) in smooth muscle cells of the guinea-pig isolated vas deferens.
The symmetrical 3′-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid (NF023) produced a concentration-dependent inhibition of e.j.p. magnitude (IC50=4.8×10−6 M), but had no effect on the resting membrane potential of the smooth muscle cells.
Pyridoxal-5-phosphate (P-5-P) also depressed e.j.p. magnitude in a concentration-dependent manner, but was less potent than NF023 (IC50=2.2×10−5 M). At 10−4 M and above P-5-P significantly depolarized the smooth muscle cells.
The nucleoside triphosphatase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156) (5×10−5 M) significantly increased e.j.p. amplitude. ARL 67156 (10−4 M) further increased e.j.p. amplitude such that they often reached threshold for initiation of action potentials, causing muscle contraction and expulsion of the recording electrode.
After reduction of e.j.p.s by NF023 or P-5-P (both 10−5 M), subsequent co-addition of ARL 67156 (10−4 M) significantly increased their magnitude.
The overflow of endogenous ATP evoked by field stimulation of sympathetic nerves (8 Hz, 1 min) was measured by HPLC and flurometric detection. ARL 67156 (10−4 M) enhanced ATP overflow by almost 700% compared to control.
We conclude that for electrophysiological studies NF023 is preferable to other P2X receptor antagonists such as pyridoxalphosphate -6-azophenyl-2′,4′-disulphonic acid (PPADS), suramin or P-5-P. Furthermore, breakdown of endogenous ATP by nucleoside triphosphatases is an important modulator of purinergic neurotransmission in the guinea-pig vas deferens.
British Journal of Pharmacology (2000) 129, 1089–1094; doi:10.1038/sj.bjp.0703163
In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.
The properties of the ATPase released during electrical field stimulation (EFS) (8 Hz, 25 s) of the sympathetic nerves of the superfused rabbit isolated vas deferens were investigated.
Superfusate collected during EFS rapidly metabolised exogenous ATP (100 μM) and 50% was broken down in 5.67±0.65 min. The main metabolite was ADP, virtually no AMP was produced and adenosine was absent. No enzyme activity was seen in samples collected in the absence of EFS.
Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a KM of 40 μM and Vmax of 20.3 nmol ATP metabolized min−1 ml−1 superfusate. ATPase activity was unaffected by storage at room temperature for 24 h, but was abolished at pH4 or by heating at 80°C for 10 min.
ARL 67156 inhibited ATP breakdown in a concentration-dependent manner (IC50=25 μM (95% confidence limits=22–27 μM), Hill slope=−1.06±0.04).
When EFS was applied three times at 30 min intervals, ATP metabolism was 20–30% less in superfusate collected during the second and third stimulation periods compared with the first. ATPase activity was released in a frequency-dependent manner, with significantly greater activity seen after stimulation at 4 and 8 Hz than at 2 Hz.
In conclusion, EFS of the sympathetic nerves in the rabbit vas deferens causes release of substantial ATPase, but little ADPase activity into the extracellular space. This contrasts with the guinea-pig vas deferens, which releases enzymes that degrade ATP to adenosine. Thus, the complement of enzymes released by nerve stimulation is species-dependent.
British Journal of Pharmacology (2000) 131, 909–914; doi:10.1038/sj.bjp.0703662
The urinary bladder and the small intestine are presented as the principal models of purinergic cotransmission in the parasympathetic and enteric nervous systems, drawing upon evidence provided by functional, histochemical and ultrastructural studies. In the parasympathetic division ATP probably commonly transmits alongside acetylcholine, and in enteric nerves it is more likely to be transmitting alongside nitric oxide and VIP. Other organs, including some blood vessels and exocrine glands, in which there are hints that ATP might be involved as a parasympathetic cotransmitter are also given consideration.
Extracellular ATP can produce various effects acting via P2-purinoceptors. ATP is rapidly broken down by ecto-ATPase and other ecto-enzymes that limit its effect. Further, adenosine, a metabolite of ATP breakdown, can produce its own effect acting via P1-purinoceptors, sometimes masking the effects of ATP. An inhibitor of ATP degradation would be a useful pharmacological tool to discriminate between effects of ATP and its metabolites, as well as to potentiate its actions. Diverse compounds that have been claimed to be inhibitors of ATP-metabolising ectoenzymes are evaluated, but specific and selective Ca2+ /Mg2+ -dependent ecto-ATPase inhibitors still appear to be lacking.
The affinity and selectivity of racemic terodiline (N-tert-butyl-1-methyl-3,3-diphenylpropylamine HCl) for muscarinic receptor subtypes was determined from functional responses of rabbit vas deferens (M1), guinea pig atria (M2) and bladder detrusor muscle (M3). (+/-)-Terodiline was found to be about as potent as pirenzepine in the rabbit vas deferens (Kb = 15 and 31 nM, respectively) and at least as selective for M1 relative to M2 (11-fold) and M3 (19-fold) receptors. Like pirenzepine, (+/-)-terodiline does not distinguish between M2 and M3 receptors in vitro. The peripheral actions of (+/-)-terodiline were evaluated in vivo in terms of its ability to induce mydriasis, and to inhibit salivary secretion and urinary bladder contraction. (+/-)-Terodiline given s.c. was equipotent in inhibiting intravesical bladder pressure and carbachol-induced salivary secretion (ID50 = 24 and 35 mg/kg, respectively), and in increasing pupil diameter (ED50 = 59 mg/kg). These results suggest that the in vivo actions of racemic terodiline at (M3) receptors mediating bladder contraction may not be separable from its actions at receptors mediating mydriasis and salivation. Moreover, its effects on the pupil and salivary glands are apparently not mediated through M1 receptors. Together, these findings help clarify the action of (+/-)-terodiline in the treatment of neurogenic bladder.
In vitro contractile responses to electrical stimulation, ATP, histamine, and carbachol were measured in strips of guinea pig detrusor. The contractile responses were redetermined at intervals after replacement of the Krebs bicarbonate buffer with a nominally calcium-free medium. Agonist induced accumulation of [3H]-inositol phosphates was measured in a suspension of detrusor slices. Electrical stimulation (five second train; frequency 20 Hz; pulse width 100 microseconds) produced a contraction that was abolished by tetrodotoxin (10(-6) M) and reduced by approximately 50% in the presence of atropine (10(-8) M). This atropine resistant component was abolished by desensitization of the purine receptors with alpha, beta-methylene ATP, confirming that the response was mediated by nerves that released ATP and acetylcholine. Carbachol, ATP, and histamine produced concentration dependent contractions in guinea-pig detrusor strips. The response to ATP was much more dependent on extracellular calcium than the response to carbachol. Muscarinic, but not purine-receptor stimulation induced the accumulation of [3H]-inositol phosphates. These data suggest that ATP stimulates a purine receptor which opens a membrane ion channel and allows an influx of calcium while muscarinic receptor stimulation can mobilize intracellular calcium via hydrolysis of inositol phospholipid and production of the second messenger inositol triphosphate.
1. Suramin, an inhibitor of several types of ATPase, was investigated for its ability to antagonize responses mediated via P2X-purinoceptors in the guinea-pig urinary bladder and P2Y-purinoceptors in the guinea-pig taenia coli. 2. In isolated strips of bladder detrusor muscle, suramin (100 microM-1 mM) caused a non-competitive antagonism of responses to alpha, beta-methylene ATP with an estimated pA2 of approximately 4.7, and inhibited responses to stimulation of the intramural purinergic nerves, with a similar pA2 value. At a concentration of 10 microM, suramin had little effect, but at a concentration of 1 microM, suramin potentiated responses to alpha,beta-methylene ATP, and potentiated responses to electrical stimulation of intramural purinergic nerves. 3. In isolated strips of taenia coli, in which a standard tone had been induced by carbachol (100 nM), suramin at 100 microM and 1 mM significantly antagonized relaxant responses to ATP (at an EC50 concentration) with an estimated pA2 of 5.0 +/- 0.82 and relaxant responses to electrical stimulation of the intramural non-adrenergic, non-cholinergic inhibitory nerves, either single pulses or trains of 8 Hz for 10 s, with estimated pA2 values of 4.9 +/- 0.93 and 4.6 +/- 1.01, respectively. Suramin had no significant effect at 1 or 10 microM. 4. Suramin, at any of the concentrations tested, did not affect contractile responses to histamine (10 microM) or carbachol (10 microM) in the bladder detrusor preparations. In the taenia coli, suramin did not affect either the relaxant responses to noradrenaline (at an EC50 concentration) or the contractile responses to carbachol (100 nM). 5. Thus, suramin at concentrations above 10 microM blocked actions mediated via P2x- and P2y-purinoceptors in the guinea-pig urinary bladder and taenia coli respectively. Potentiation of purinoceptor-mediated activity was seen only at a low concentration of suramin (1 microM) and only in the urinary bladder (P2x-purinoceptor). For its antagonistic activity suramin did not discriminate between P2X- and P2y-purinoceptors, but it was selective for P2-purinoceptor-mediated activity rather than that mediated via cholinoceptors, adrenoceptors or histamine receptors.
Strength‐duration curves for threshold mechanical responses to single transmural stimuli were identical for rat and guinea‐pig detrusor. In both species atropine had no effect on the curves, but the curves were shifted to the right by nerve blockade with tetrodotoxin (TTX), and by blockade of P 2 ‐purinoceptors with α,β‐methylene ATP (α,β‐MeATP).
With short duration pulses of 50 V and less, the responses were nerve‐mediated. Increase in either the strength or duration of the stimulus caused direct muscle stimulation, resistant to blockade with atropine, TTX and α‐β‐MeATP.
The shape of the contractile response to a single nerve stimulus varied from tissue to tissue. The responses could be mono‐, bi‐, or multiphasic. Bi‐ or multiphasic responses were normally seen in tissues which were spontaneously active. The multiphasic nature of the response was enhanced by factors which increased the excitability of the cells and was reduced by factors which decreased the excitability.
The frequency‐response curves in the rat are similar to those previously obtained in the guinea‐pig. Atropine suppresses the high frequency response by 25%, with little effect at low frequencies, whereas desensitization of P 2 ‐purinoceptors with α,β‐MeATP suppresses the responses maximally at low frequencies but still by 75% at high frequencies. A combination of both drugs eliminates the nerve‐mediated responses.
It is concluded that the response to a single nerve stimulus is mediated by a non‐cholinergic transmitter, through activation of P 2 ‐purinoceptors. The possibility that simultaneous release of acetylcholine can modify the excitability of cells and thus the configuration of the response to a single stimulus is discussed.
1. The effects of some possible inhibitors of ectonucleotidases on the breakdown of extracellular ATP by strips of guinea-pig urinary bladder were investigated. 2. Suramin and ethacrynic acid (10 mM) both inhibited ATP breakdown significantly, and difluorodinitrobenzene (10 mM) inhibited it slightly whereas N-ethylmaleimide, adenosine 5'-(gamma-thiotriphosphate) (ATP-gamma-S) and reactive blue-2 (10 mM) were without effect. 3. The inhibitory effects of suramin on ATP breakdown were non-competitive. 4. Ethacrynic acid (1 mM) irreversibly inhibited contractions of the guinea-pig bladder induced by ATP, substance P, histamine, non-adrenergic, non-cholinergic nerve stimulation or KCl, whereas suramin (100 microM) had no inhibitory effect. 5. The results suggest that suramin might provide a starting point for the design of selective inhibitors of ectonucleotidases.
1 The electrical and mechanical responses to transmural stimulation of intrinsic nerves have been recorded from smooth muscle strips dissected from the dome of the guinea-pig bladder, by use of intracellular microelectrodes, and conventional tension recording techniques. 2 Stimulation of intrinsic nerves evoked action potentials in all cells studied. Hyperpolarization of the cells by extracellular current injection revealed subthreshold excitatory junction potentials (e.j.ps) in about a quarter of the cells studied. 3 Action potentials could still be evoked in the presence of atropine and neostigmine, but were abolished after desensitization of the cells to alpha, beta-methylene ATP, a stable analogue of ATP. 4 In the presence of neostigmine, the evoked action potential was followed by a slow depolarization of the membrane. The mechanical response increased in amplitude and duration. 5 The contractile response to transmural nerve stimulation was reduced but not abolished in the presence of either atropine or desensitizing doses of alpha, beta-methylene ATP. Atropine was more effective at high frequencies of stimulation (greater than or equal to 30 Hz), and alpha, beta-methylene ATP at low frequencies (less than or equal to 15 Hz). In combination the drugs abolished the response. 6 The results suggest that the mechanical response to excitatory nerve stimulation is biphasic. The early transient response is elicited by e.j.ps and evoked spikes, is resistant to atropine, but sensitive to desensitization of purinoceptors. The late response is mediated through muscarinic receptors, involves little membrane depolarization, and is unaffected by desensitization of purinoceptors. These responses are analogous to the responses seen in rabbit bladder, and in the sympathetically innervated rat tail artery and guinea-pig vas deferens.
The dephosphorylation of adenine nucleotides and their analogues by ectonucleotidases on the guinea-pig urinary bladder was studied using HPLC. The rate of dephosphorylation of each analogue was compared with its pharmacological potency at causing contraction. ATP, ADP and AMP were rapidly dephosphorylated, and substitution on the purine ring did not affect the rate of breakdown. The ectonucleotidases showed stereoselectivity towards the ribose moiety and towards the polyphosphate chain. In general, methylene isosteres of the nucleotides, and analogues in which one of the oxygen atoms on the terminal phosphate had been replaced, were resistant to degradation. None of the analogues that were readily dephosphorylated are more potent than ATP, and most but not all of the analogues resistant to degradation are more potent than ATP, suggesting that while resistance to degradation does not in itself confer high potency, susceptibility to degradation does limit the potency of ATP and its degradable analogues.
1. The effects of alpha,beta-methylene ATP (alpha,beta-MeATP) on membrane properties and excitatory junction potentials (EJPs) were examined in smooth muscle cells of the guinea-pig, rabbit and pig bladder. 2. Intracellular recording with microelectrodes was used to record membrane electrical activity from the guinea-pig bladder. ATP (10(-3) M) produced a rapid, large depolarization with a marked increase in spike frequency, while carbachol (10(-4) M) or acetylcholine (ACh; 10(-4) M) produced only a small or no depolarization with a smaller increase in spike frequency. alpha,beta-MeATP produced a similar response to that of ATP but at a much lower concentration (5 x 10(-6) M), and the response was transient even in the continuous presence of this agent. 3. Changes in the membrane potential and conductance elicited by alpha,beta-MeATP were also measured with the double sucrose-gap method. alpha,beta-MeATP (5 x 10(-6) M) depolarized the membrane and increased the membrane conductance in all three species, but both parameters returned to control values during continuous exposure to this agent. 4. Intracellular recording with microelectrodes showed that in the guinea-pig bladder treatment with alpha,beta-MeATP abolished the response to ATP, while the response to ACh was unchanged. 5. With the double sucrose-gap method, EJPs were elicited by transmural nerve stimulation of strips of the guinea-pig, rabbit and pig bladder and had spikes superimposed, leading to contractions. Desensitization of P2-purinoceptors by alpha,beta-MeATP (3-5 x 10(-6) M) abolished the EJPs and spikes, and reduced the contraction. Atropine (10(-6) M) alone did not alter the EJPs but reduced the contraction. Combined application of both agents abolished the contraction. 6. It is concluded that in the guinea-pig, rabbit and pig bladder ATP is an excitatory transmitter with ACh and EJPs are mediated by ATP.
The spontaneous and electrically evoked activity was examined in guinea-pig, ferret and marmoset urinary bladder. Electrical field stimulation of detrusor strips in vitro induced a rapid, frequency-dependent contraction with a maximum response at 40 Hz. This contraction was partly decreased by either atropine (0.29 microM) or desensitisation to alpha, beta-methylene ATP (alpha, beta-MeATP) (5 microM), and was totally blocked by a combination of the two. Atropine blocked responses to carbachol (30 microM) but not to ATP (80 microM), whereas desensitisation to alpha, beta-MeATP blocked those to ATP but not to carbachol. The nature of the excitatory neurotransmission mechanism in the bladder of those species examined in the present experiments was shown to be similar to that of other species described previously.
1. In mucosa‐free preparations of the detrusor muscle electrical stimulation with 0·1 msec pulses has been utilized to reveal the non‐cholinergic nature of most, if not all, of the post‐ganglionic motor neurones in the guinea‐pig bladder.
2. The twitches elicited by 0·1 msec pulses were abolished by tetrodotoxin, but were not reduced by dimethyltubocurarine or by hexamethonium. Hexamethonium was nevertheless present in all the experiments in order to restrict acetylcholine action to ‘muscarinic’ receptors in the muscle fibres.
3. There was little or no diminution in the twitches after prolonged exposure to atropine, 10 ⁻⁸ ‐10 ⁻⁵ g/ml., although the twitch‐matching dose of acetylcholine was raised 1000–2500 times. Hence, there was no anomalous refractoriness to atropine in these ‘muscarinic’ receptors.
4. Despite massive atropinization, the recruitment of unoccupied transmitter‐receptors by means of extra pulses remained unaffected.
5. Eserine failed to potentiate the atropine‐resistant twitches.
6. The twitches were not depressed by morphine.
7. Noradrenaline produced relaxation and twitch reduction. The twitches persisted after α + β adrenoceptor blockade with phentolamine + pronethalol and were unaffected by the monoamine oxidase and catecholamine‐ O ‐methyl transferase inhibitors, tranylcypromine and pyrogallol.
8. Rapid contractions were elicited by 5‐HT and by histamine but the twitches remained unaltered after antagonism of 5‐HT by methysergide and of histamine by mepyramine.
9. The twitches could be mimicked by injections of ATP, but after desensitization of the preparation to ATP the response to electrical stimulation remained unaltered.
10. Prostaglandins E 2 and F 2α , only in large doses, produced delayed, sluggish contractions which persisted after the wash; these contractions were quite different from the responses to electrical stimulation, which were immediate, sharp and brief.
11. The non‐cholinergic nature of the post‐ganglionic motor neurones was confirmed in the bladder of two other species, the cat and the rabbit.
alpha, beta-Methylene ATP has advantages over ATP in producing desensitisation of the P2-purinoceptor since it is degraded more slowly than ATP and does not initiate synthesis of prostaglandins. Following desensitisation of the excitatory P2-purinoceptors in the guinea-pig urinary bladder, the excitatory responses to non-adrenergic, non-cholinergic nerve stimulation were abolished, while those to acetylcholine and histamine were little affected. This result is consistent with the purinergic nerve hypothesis.
Adenosine 5′‐triphosphate (ATP) and adenylyl 5′‐(β,γ methylene)‐diphosphonate (AMP‐PCP) both contracted the guinea‐pig urinary bladder, but the response to AMP‐PCP was much greater. We synthesized the enantiomer of AMP‐PCP, L‐adenylyl 5′‐(β,γ‐methylene)‐diphosphonate ( L ‐AMP‐PCP), and tested it on the guinea‐pig bladder.
L ‐AMP‐PCP contracted the guinea‐pig bladder, and was more potent than AMP‐PCP and much more potent than ATP. The potential breakdown product of L ‐AMP‐PCP, L ‐adenosine, unlike adenosine (the breakdown product of AMP‐PCP), did not inhibit contractions of the guinea‐pig bladder.
ATP and its enantiomer L ‐adenosine 5′‐triphosphate ( L ‐ATP) were rapidly degraded by the muscle, and AMP‐PCP was also degraded, but more slowly. L ‐AMP‐PCP, however, was completely resistant to degradation.
L ‐AMP‐PCP would appear to be a useful ATP analogue, as it is potent and resistant to degradation, and its potential breakdown product, L ‐adenosine, is inactive.
FPL 67156 (6‐N,N‐diethyl‐β,γ‐dibromomethylene‐ d ‐ATP), is a newly synthesized analogue of ATP.
In a rabbit isolated tracheal epithelium preparation, measuring P 2U ‐purinoceptor‐dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP‐γ‐S. UTP agonist‐concentration effect (E/[A]) curves were shifted to the left by 5‐fold in the presence of 100 μ m FPL 67156. The differential effect of FPL 67156 on UTP and ATP‐γ‐S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process.
FPL 67156 was tested as an ecto‐ATPase inhibitor in a human blood cell assay, measuring [γ ³² P]‐ATP dephosphorylation. The compound inhibited [γ ³² P]‐ATP degradation with a pIC 50 of 4.6.
FPL 67156 was then tested for its effects on ATP and α,β‐methylene‐ATP responses at P 2X ‐purinoceptors in the rabbit isolated ear artery. In the concentration range 30 μ m ‐1 m m , the compound potentiated the contractile effects of ATP but not those of α,β‐methylene‐ATP. At 1 m m , FPL 67156 produced a 34‐fold leftward shift of ATP E/[A] curves.
The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analysed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the p K 1 for FPL 67156 as an ecto‐ATPase inhibitor of 5.2.
Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P 2X ‐ and P 2T ‐purinoceptors (p A 2 ≅ 3.3 and 3.5 respectively), and weak agonist effects at P 2U ‐purinoceptors (p[ A 50 ] ≅ 3.5).
The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto‐ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors.
This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.
Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.
To compare the electrical and mechanical activity, and assess the effect of suramin on strips of detrusor from various species.
Bladder muscle strips from guinea-pigs, rabbits, monkeys and sheep and detrusor strips from humans (obtained at operation) were studied. The mechanical responses to nerve stimulation were recorded with a force transducer and electrical activity was recorded with the double sucrose gap.
In all species acetylcholine was released from the nerves and a prolonged contraction was produced. A second transmitter, possibly adenosine triphosphate, produced a rapid transient contraction, the amplitude of which varied with the species. In the rabbit and guinea-pig the phasic contraction and accompanying depolarization were large, whereas in primates they were small and in sheep were intermediate. At high concentrations, suramin reduced the contraction and accompanying depolarization in rabbit and guinea-pig muscle but not in sheep. Suramin enhanced the late cholinergic responses and increased spontaneous mechanical activity in all species. These latter effects were not seen after desensitization of the receptors with the ATP analogue alpha, beta- methylene ATP.
Although suramin reduces the excitatory effect of nerve activity in some species, it would produce little beneficial effect in the human hyperexcitable bladder as any inhibitory effect might be offset by the increase in spontaneous activity.
In smooth muscles of the guinea-pig bladder, intramural nerve stimulation generated an excitatory junctional potential (e.j.p.), action potential and twitch contraction. Nicardipine inhibited the action potential but not the e.j.p. The e.j.p. amplitude was reduced by suramin, or desensitization of the ATP receptor with receptor agonists. The amplitude of the twitch contraction was reduced by atropine, and the remainder was blocked by nicardipine. In the presence of maximally effective concentrations of atropine, the threshold concentration of acetylcholine required to produce contraction was about 10(-7) M, whereas acetylcholine concentrations greater than 10(-6) M were required to cause depolarization. It is concluded that nerve stimulation releases acetylcholine and ATP, and the former produces contraction without change in the membrane potential, while the latter generates the e.j.p. which triggers an action potential and thus elicits contractions.
1. The effects of transmural nerve stimulation were examined on preparations of detrusor smooth muscle from guinea-pig urinary bladder using intracellular recording techniques. Most recordings were made from preparations in which spontaneous and evoked action potentials had been inhibited by nifedipine (10 microM), a dihydropyridine that blocks L-type Ca2+ channels. 2. Supramaximal stimuli evoked excitatory junction potentials (EJPs) which could be divided into three basic types. Type 1 EJPs had short latencies (< 30 ms) and fast rise times (< 60 ms). Type 2 EJPs consisted of two components: a small depolarization that was followed by a second depolarization with a faster rise time. In a third type of cell, at high strengths of stimulation, EJPs resembled type 1 EJPs but at lower strengths of stimulation were similar in time course to type 2 EJPs. 3. All EJPs were abolished by tetrodotoxin (1 microM) and reduced by omega-conotoxin (0.1 microM), but were unaffected by hexamethonium (0.1 mM), suggesting that they result from the release of transmitter from post-ganglionic nerve fibres. All responses persisted in the presence of atropine (1 microM) but were abolished following the desensitization of P2-purinoceptors with alpha, beta-methylene ATP (m-ATP; 10 microM). 4. Spontaneous excitatory junction potentials (SEJPs) were also recorded from most cells. SEJPs were similar in appearance to fast single-component EJPs; however, in general they had a briefer time course. SEJPs persisted in the presence of tetrodotoxin (1 microM). 5. The electrical properties of urinary bladder smooth muscle were also examined. Voltage changes induced by point current injection into cells had fast rates of rise and decay (time constant, 5-20 ms). The input resistance of cells ranged between 12 and 108 M omega. When recordings were taken from cells near the point of current injection, resultant electrotonic potentials could be detected in only a small proportions of cells. 6. The results are discussed in relation to the idea that transmural nerve stimulation in the guinea-pig urinary bladder causes the activation of at least two different membrane conductances. Cells appear to be electrically coupled with one another. However, it is likely that coupling exists within discrete bundles of the smooth muscle.
Field stimulation of the sympathetic nerves of the guinea‐pig isolated vas deferens with trains of pulses for 20 s at 1–8 Hz produced characteristic biphasic contractions. The effect of the novel ecto‐ATPase inhibitor, 6‐N,N‐diethyl‐D‐β,γ‐dibromomethyleneATP (ARL 67156, formerly known as FPL 67156), on the magnitude of the initial, predominantly purinergic peak of this response was studied in order to determine the influence of enzymatic degradation of adenosine 5′‐triphosphate (ATP) on its action as a neurotransmitter.
The peak magnitude of the response to nerve stimulation was significantly increased in a concentration‐dependent manner by ARL 67156 (5–100 μ m ) and the size of the neurogenic response at 4 Hz was approximately doubled in the presence of ARL 67156 (100 μ m ).
ARL 67156 (100 μ m ) has a rapid onset of action. The enhancing effect on neurogenic contractions was maximal after 10 min, was well maintained for at least 30 min and was rapidly reversed, with responses returning to control levels 10 min after washout.
The neurogenic contraction in the presence of prazosin (0.1 μ m ) was purely purinergic, as it was abolished by the P 2 ‐purinoceptor antagonist, PPADS (100 μ m ). ARL 67156 (100 μ m ) produced a similar degree of enhancement of neurogenic responses in the absence and presence of prazosin, supporting the view that the enhancing effects of ARL 67156 on neurogenic contractions result from potentiation of the action of ATP.
Exogenous ATP and α,β‐methyleneATP produced rapid transient contractions. Responses to ATP were increased in magnitude and duration in the presence of ARL 67156 (100 μ m ), whereas those to the stable analogue, α,β‐methyleneATP were not significantly affected.
Contractions to exogenous noradrenaline (10 μ m ) and KCl (40 mM) were significantly enhanced by ARL 67156 (100 μ m ), but this potentiation was abolished by PPADS (100 μ m ). Therefore, this effect of the ecto‐ATPase inhibitor may be due to a build up of endogenous ATP, increasing the sensitivity of the smooth muscle to other agonists.
It is concluded that ARL 67156 potentiates the action of ATP, and that when ATP acts as a neurotransmitter its postjunctional actions are greatly attenuated by enzymatic degradation.
Inhibition of ec-toATPase and Ca-ATPase in rat vas deferens by P: purinoceptor antagonists A comparative study of electrical field stinmlation of the guinea-pig, ferret and marmoset urinary bladder
- B S Khakh
- A D Michel
- P P A Humphrey
Khakh, B.S., Michel, A.D., Humphrey, P.P.A., 1995. Inhibition of ec-toATPase and Ca-ATPase in rat vas deferens by P: purinoceptor antagonists. Br. J. Pbarmacoh 115, 2P. Moss, H.E., Burnstock, G., 1985. A comparative study of electrical field stinmlation of the guinea-pig, ferret and marmoset urinary bladder. Eur. J. Pharmacol. 114, 311-316.
Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia-coli
- Hoyle
(±)-Terodiline: an M1-selective muscarinic receptor antagonist
- Noronha-Blob