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Atropine dissociates complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein in heart membranes
Abstract
Complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein (G protein) are formed in the presence of the agonist carbachol. The complexes remain stable after removal of agonist, and survive subsequent solubilization from cardiac membranes and purification. Dissociation of the receptor from the G protein occurs when the antagonist atropine is added following removal of agonist. This is the first direct demonstration of destabilization of receptor-G protein complexes by the binding of an antagonist.
... Thus, our results indicate true chamber-specific differences in coupling preferences of mAChRs. This finding in the human heart corresponds well to a previous report using a coimmunoprecipitation approach in rat heart [44]. In this study, carbachol facilitated the association of mAChRs with Gia-2 and Goa in the ventricle, but to Goa only in atria. ...
... In atrial myocardium, acetylcholine exerts two main effects, a direct negative inotropic effect mediated by activation of I KACh and an indirect negative inotropic effect mediated by inhibition of prestimulated I Ca . Given that Goa knockout animals exhibited normal regulation of I KACh [30] and that Matesic and Luthin [44] did not find co-immunoprecipitation of Gia-2 with mAChR in rat atria, Gia-3 is by exclusion the most likely candidate that mediates mAChR-induced activation of I KACh . The indirect negative inotropic effect seems to critically depend on Goa and is kinetically modulated, but in contrast to the ventricle, not dependent on Gia-2 [45]. ...
Muscarinic acetylcholine receptors (mAChRs) mediate their main cardiac effects via pertussis toxin-sensitive G-proteins. Physiological effects differ considerably between atrium and ventricle, and it is unknown to which extent these differences derive from selective receptor-G-protein coupling or further downstream events. We have characterized specific coupling between mAChRs and Gi/Go-protein isoforms in atrial and ventricular myocardium by agonist-dependent photoaffinity labeling with [(32)P]azidoanilido GTP (aaGTP) and immunoprecipitation in sarcolemmal membranes from terminally failing human hearts. The total amount of mAChRs, as determined by specific binding of [(3)H]QNB, was significantly higher in right-atrial (RA +/- SEM, 959 +/- 68 fmol/mg, n = 4) than in left-ventricular membranes (LV, 582 +/- 53 fmol/mg, n = 6). Standardized immunoblots revealed that Gialpha-2 was the predominant subtype in both regions. A 40-kDa splice variant of Goalpha (Goalpha-1 and/or Goalpha-3) was almost exclusively detectable in RA. Levels of Gialpha-3 and a 39-kDa splice variant of Goalpha (Goalpha-2) were also higher in RA. Basal aaGTP binding was higher in RA than in LV for all Gialpha/Goalpha subtypes. The carbachol (10 micromol/l)-induced increase in aaGTP binding was significantly higher in RA than in LV for Goalpha-1/3 (336 +/- 95% of LV, n = 4) and for Gialpha-3 (211 +/- 83%), lower for Gialpha-2 (42 +/- 5%), and was similar in both regions for Goalpha-2 (130 +/- 62%). The differential coupling of mAChRs in human RA and LV suggests that the initiation of different physiological responses to mAChR stimulation starts with signal sorting at the receptor-G-protein level.
... Uncoupling the unoccupied receptors from the pertussis toxin-sensitive G proteins by pertussis toxin pretreatment will, therefore, lead to a reduction in total [35S]GTPyS binding as was observed in the present study. In opposition to this view is the fact that Na + ions (and antagonists) are believed to uncouple G proteins from unoccupied receptors, thereby resulting in a reduction in basal [35S]GTPyS binding (Hilf et al., 1989;Gierschik et al., 1989;Tian et al., 1994;Matesic and Luthin, 1991). As NaC1 (100 mM) was present in the incubation buffer, it was surprising to see such a dramatic apparent constitutive activity of pertussis toxin-sensitive G proteincoupled receptors. ...
Chinese hamster ovary (CHO) cells expressing recombinant human m1 (CHO-m1 cells), m2 (CHO-m2 cells), or m3 (CHO-m3 cells) muscarinic receptors were characterised pharmacologically with [3H]N-methylscopolamine. Agonist-stimulated coupling of these receptors with guanine nucleotide-binding proteins (G proteins) was measured by guanine nucleotide- and pertussis toxin-modification of carbachol competition-binding curves, and pertussis toxin-sensitivity of agonist-stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) binding, in membrane preparations of the CHO cell clones. High affinity agonist binding and agonist-stimulated [35S]GTP gamma S binding was abolished in CHO-m2 cell membranes (expressing 99 +/- 25 fmol of [3H]N-methylscopolamine binding sites/mg protein) after pertussis toxin pretreatment of cells, suggesting that muscarinic m2 receptors expressed in these cell membranes couple predominantly with pertussis toxin-sensitive G proteins. CHO-m1 (713 +/- 102 fmol/mg protein) and CHO-m3 (1212 +/- 279 fmol/mg protein) cell membranes produced smaller elevations in agonist-stimulated [35S]GTP gamma S binding considering the higher receptor levels, compared with CHO-m2 cell membranes. Pertussis toxin pretreatment of these clones also resulted in a significant attenuation of agonist-stimulated [35S]GTP gamma S binding suggesting that, under these experimental conditions, muscarinic m1 and m3 receptors can couple with both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. Guanine nucleotide-modification of agonist binding in CHO-m1 and CHO-m3 cell membranes was comparatively smaller than in CHO-m2 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
In recent years our understanding of molecular mechanisms of drug action and interindividual variability in drug response has grown enormously. Meanwhile, the practice of anesthesiology has expanded to the preoperative environment and numerous locations outside the OR. Anesthetic Pharmacology: Basic Principles and Clinical Practice, 2nd edition, is an outstanding therapeutic resource in anesthesia and critical care: Section 1 introduces the principles of drug action, Section 2 presents the molecular, cellular and integrated physiology of the target organ/functional system and Section 3 reviews the pharmacology and toxicology of anesthetic drugs. The new Section 4, Therapeutics of Clinical Practice, provides integrated and comparative pharmacology and the practical application of drugs in daily clinical practice. Edited by three highly acclaimed academic anesthetic pharmacologists, with contributions from an international team of experts, and illustrated in full colour, this is a sophisticated, user-friendly resource for all practitioners providing care in the perioperative period.
Activation mechanism of the α-factor pheromone receptor of Saccharomyces cerevisiae was analyzed using biochemical and genetic techniques. An in vitro partial proteolysis assay was developed to determine the conformational change of the receptor that occurs upon binding of agonist. The activation specific cleavages were established by comparing cleavage products with antagonist versus agonist occupied receptor. Of the changes in peptide pattern that were revealed by trypsinization, the fragment resulting from the exposure of the third loop to the protease was found to be agonist specific and to be G-protein independent. A low-affinity binding receptor mutant was isolated which failed to undergo this agonist induced conformational change. Four intra-allelic suppressors of this receptor mutant were isolated and all were mapped to the ends of transmembrane helices 4, 5, 6 and 7; all were found to be replacements of non-polar residues by polar ones. The role of the suppressor mutations in conformational change was analyzed.
In this study we have investigated the functional activity of GR127935 (2-methyl-4-(5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-[4-carboxylic acid 4-methoxy-3-(4-methyl-piperazine-1-yl)-phenyl]-amide) at human 5-HT1Dα and 5-HT1Dβ receptors which have been expressed in a Chinese Hamster Ovary (CHO) cell line. Using [35S]GTPγS binding to cell membranes as a measure of receptor-G protein coupling, GR127935 showed partial agonist activity in both 5-HT1Dα and 5-HT1Dβ receptor expressing cells (Emax: 29 and 31% above basal control; pEC50: 8.6 and 9.7, respectively). GR127935 also acted as a potent antagonist at the 5-HT1Dα (app. pA2 8.5) and 5-HT1Dβ (app. pA2 9.1) receptors. From studies measuring cAMP accumulation in cultured CHO cells GR127935 also displayed partial agonism, as well as acting as a potent antagonist at the 5-HT1Dα receptors which stimulate cAMP levels and 5-HT1Dβ receptors which inhibit cAMP levels (app. pA2 8.6 and 9.7, respectively). The 5H̄T1-like receptor antagonist methiothepin showed negative intrinsic activity at both receptors in the [35S]GTPγS binding assay only. From studies using the receptor alkylating agent EEDQ (N-ethoxycarbonyl-2-methoxy-1,2-dihydroquinoline) the 5-HT1Dα cell line displayed a lack of receptor reserve but it was evident in the 5-HT1Dβ cell line. In previous studies we have also shown that agonist stimulation of 5-HT1Dα receptors increases cAMP levels which may be due to high receptor expression. Further investigation using up to 1 μM EEDQ to reduce 5-HT1Dα receptor number did not reveal an underlying inhibitory adenylyl cyclase response. In conclusion, GR127935 acts as a partial agonist, aswell as a potent antagonist, at the human 5-HT1Dα and 5-HT1Dβ receptors when expressed in CHO cells.
Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP(3)-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.
Muscarinic agonists regulate the L-type calcium current in isolated cardiac myocytes. The second messengers pathways involved in this regulation are discussed briefly, with particular emphasis on the involvement of cAMP and cGMP pathways.
The metabolism and pharmacology of naturally-occurring alkaloids are reviewed, with emphasis on work of the last ten years during which there have been many important advances. It is recognized that the final effects of alkaloids on animals may result from activity of their metabolic products rather than of the original substance. Therefore one section discusses how alkaloids are metabolized, first in terms of the general processes and then with many examples of the metabolism of particular types of alkaloid structures. The second section on pharmacology deals with detailed biochemistry of alkaloid action at the level of molecule-to-molecule, rather than describing behavioral or gross physiological effects. Modern molecular biological methods have revealed the intimate structures of neural receptors and other cellular molecules with which the alkaloids interact. In this section of the review the material is organized according to the processes being affected rather than according to types of alkaloid. Thus there are subsections for the various neural receptors, structural components of cells, enzymes, etc.
At least five muscarinic receptor genes have been cloned and expressed. Muscarinic receptors act via activation of G proteins: m1, m3 and m5 muscarinic receptors couple to stimulate phospholipase C, while m2 and m4 muscarinic receptors inhibit adenylyl cyclase. This review describes the localization, pharmacology and function of the five muscarinic receptor subtypes. The actions of muscarinic receptors on the heart, smooth muscle, glands and on neurons (both presynaptic and postsynaptic) in the autonomic nervous system and the central nervous system are analyzed in terms of subtypes, biochemical mechanisms and effects on ion channels, including K+ channels and Ca2+ channels.
Muscarinic receptors were solubilized from porcine atria in digitonin-cholate and were purified by chromatography on DEAE-Sepharose and 3-(2'-aminobenzhydryloxy)tropane-Sepharose. The product identified on Western blots migrated with an apparent molecular mass of 60-75 kDa, with additional bands indicative of homotrimers (190 kDa) and homotetramers (240 kDa). Receptor eluted from the affinity column was accompanied by a mixture of guanyl nucleotide-binding proteins (G-proteins) identified on Western blots as Gi1/2, G(o), Gq/11, and Gs (preparation M2G); the G-proteins were largely removed by further processing on hydroxyapatite (preparation M2). Solubilized purified receptors bound muscarinic ligands in an apparently cooperative manner. In studies at equilibrium, the antagonists [3H]AF-DX 384, N-[3H]methylscopolamine (NMS), and [3H]quinuclidinylbenzilate (QNB) revealed Hill coefficients between about 0.8 and 1.2. Also, the apparent capacity for [3H]QNB exceeded that for [3H]AF-DX 384 and [3H]NMS by about 1.5-fold in M2 and by 2-fold in M2G. Binding to M2G at high concentrations of [3H]QNB was fully inhibited by unlabeled NMS, which therefore affected sites not labeled at similar concentrations of [3H]NMS. Oxotremorine-M displayed a biphasic inhibitory effect on the binding of [3H]AF-DX 384 in M2 and M2G, suggesting that multiple states of affinity are intrinsic to the receptor; 5'-guanylylimidodiphosphate was without appreciable effect in M2 but resulted in a bell-shaped binding profile for the agonist in M2G. All of the data can be described in terms of cooperative interactions within a receptor that is at least tetravalent and presumably an oligomer. In the context of the model, copurifying G-proteins and guanyl nucleotides serve to regulate the degree of cooperativity between successive equivalents of muscarinic ligands.
Activation of muscarinic acetylcholine receptors is primarily responsible for urinary bladder emptying. Because multiple subtypes of muscarinic receptors exist, we wished to characterize those present in bladder and ultimately to attribute function to those that regulate bladder contractility, neurotransmitter release and perhaps other cholinergic functions in this tissue. Although the m2 and m3 subtypes could be immunoprecipitated after solubilization from human, rat, rabbit and guinea pig bladder membranes, the m1, m4 and m5 subtypes could not. The m2:m3 ratio was 9:1 in rat bladder but was only 3:1 in the other species examined. Immunoprecipitation of the m2 subtype correlated with the relative levels of high-affinity agonist binding sites measured by competition of carbachol for [3H]N-methylscopolamine binding or measured directly using [3H]oxotremorine-M. In the presence of agonist, but not antagonist, GTP binding proteins could be immunoprecipitated in concert with the m2 or m3 receptors using anti-receptor antibodies. These proteins were members of the Gi and Gq/11 subfamilies for both the m2 and the m3 receptor subtypes. In spite of the preponderance of the m2 receptor in all species studied, Schild analysis using somewhat selective antagonists showed that the pharmacologically defined m3 receptor mediated contractility in strips of rat and rabbit bladder. Thus acetylcholine activates bladder smooth muscle via the m3 receptor subtype, and subsequent contractility may be transduced by guanine nucleotide binding proteins such as the Gi and Gq/11 subfamilies.
We have used a luciferase reporter gene under the transcriptional control of a cAMP response element as a sensitive monitor of the regulation by muscarinic acetylcholine receptors (mAChRs) of intracellular cAMP levels and cAMP-regulated gene expression. Treatment with the muscarinic agonist carbachol results in an increase in luciferase activity in JEG-3 cells transiently transfected with mouse m1 (8-10-fold) and chick m4 (3-5-fold) mAChRs. Control experiments indicate that these responses are not due to a calcium-mediated pathway and are dependent upon a functional protein kinase A. The m1 and m4 responses are not sensitive to pertussus toxin and the m4 response was potentiated by it. Thus, these responses do not result from direct stimulation of adenylate cyclase by beta gamma subunits released from pertussis toxin-sensitive G-proteins. Atropine treatment of cells transfected with high levels of m4 mAChR, but not m1, causes an elevation in basal levels of cAMP response element-mediated luciferase expression in the absence of agonist. This suggests that the m4 receptor is spontaneously active and can cause constitutive inhibition of adenylyl cyclase that is relieved by atropine treatment. Surprisingly, the m4 receptor exhibits little if any agonist-induced inhibition of luciferase expression at either low or high levels of receptor expression. JEG-3 cells express Gi alpha-1 and Gi alpha-3 but not Gi alpha-2. Cotransfection of Gi alpha subunits with m4 demonstrates that the m4 receptor requires Gi alpha-2 for optimal agonist-mediated inhibition. Even in the presence of Gi alpha-2, high levels of receptor increased luciferase expression at high concentrations of agonist. Thus, the m4 mAChR can undergo a switch in functional coupling from inhibition to stimulation of adenylyl cyclase. This switch is dependent on the level of receptor expression, the subtypes of G-proteins coexpressed with the receptor, and the concentration of agonist. Furthermore, we demonstrate that the Gi alpha-2 G-protein alpha subunit preferentially couples the m4 mAChR to inhibition of adenylyl cyclase in JEG-3 cells.
1. The whole-cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L-type calcium currents (ICa) in frog and rat ventricular myocytes, and on the mAChR-activated K+ current (IK(ACh)) in frog atrial myocytes. 2. In frog ventricular myocytes, atropine (0.1 nM to 1 microM) reversed the inhibitory effect of acetylcholine (ACh, 1 nM) on ICa previously stimulated by isoprenaline (Iso, 2 microM), a beta-adrenergic agonist. However, in the concomitant presence of Iso, ACh and atropine, ICa was > 50% larger than in Iso alone. 3. The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of ICa with Iso (0.1 or 2 microM), atropine induced a dose-dependent stimulation of ICa. EC50 (i.e. the concentration of atropine at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of ICa expressed as percentage increase in ICa with respect to the level in Iso alone) were respectively 0.6 nM and 35%. The stimulatory effect of atropine on ICa was not voltage dependent. 4. Atropine (1 microM) had no effect on frog ICa (i) under basal conditions, (ii) upon stimulation of ICa by the dihydropyridine agonist (-)-Bay K 8644 (1 microM), or (iii) when ICa had been previously stimulated by intracellular perfusion with cyclic AMP (3 microM). However, atropine increased ICa after a stimulation by forskolin (0.3 microM). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on ICa. 5. The order of potency of mAChR antagonists to reverse the inhibitory effect of ACh on Iso elevated ICa in frog ventricle was atropine > AF-DX 116 > pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on Iso elevated ICa with the same order of potency. 6. Intracellular substitution of Gpp(NH)p (5'-guanylylimidiphosphate) for GTP (420 microM) induced a strong inhibition of frog ICa in the presence of Iso (2 microM). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP-binding regulatory protein (G protein), Gi, responsible for adenylyl cyclase inhibition. Atropine (1 microM) slowed down by a factor of 2 the rate of ICa inhibition induced by Gpp(NH)p. 7. In frog atrial myocytes, intracellular perfusion with 1 mM Gpp(NH)p induces spontaneous activation of IK(ACh). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, GK.(ABSTRACT TRUNCATED AT 400 WORDS)
It is currently accepted that occupancy of opioid receptors by agonists, but not antagonists, promotes the association of the receptors to guanine nucleotide binding proteins (G-proteins) and stimulates a high affinity GTPase as part of the mechanism that links the receptor-ligand complex to adenylate cyclase inhibition. In this work we report that in rat brain membranes selective delta-opioid antagonists, the peptides N,N-Diallyl-Tyr-D-Leu-Gly-Tyr-Leu-OH (Diallyl-G) and N-N-Diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174,864), inhibit the low Km GTPase activity in a concentration dependent way. On the other hand the delta-opioid agonists D-Ala2-D-Leu5-enkephalin (DADLE) and D-Ser2-Leu5-Thr6-enkephalin stimulate dose-dependently the low Km GTPase activity in rat brain membranes. This stimulation was blocked in the presence of Diallyl-G, and reciprocally the inhibition induced by Diallyl-G was reversed by DADLE. The inhibitory effect of Diallyl-G as well as the stimulation induced by DADLE were abolished when membranes were exposed to low concentrations of N-ethylmaleimide or by ADP ribosylation with pertussis toxin which interferes with the ability of the receptor to couple to G-proteins. These observations indicate that the inhibitory effect of Diallyl-G on GTPase requires a functional G-protein and suggest that certain delta-opioid antagonists exhibit negative intrinsic activity and may have the ability to inhibit the receptor-mediated activation of G-proteins.
In this study we have investigated the functional activity of GR127935 (2-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-[4-carboxylic acid 4-methoxy-3-(4-methyl-piperazine-1-yl]-amide) at human 5-HT1D alpha and 5-HT1D beta receptors which have been expressed in a Chinese Hamster Ovary (CHO) cell line. Using [35S] GTP gamma S binding to cell membranes as a measure of receptor-G protein coupling. GR127935 showed partial agonist activity in both 5-HT1D alpha and 5-HT1D beta receptor expressing cells (Emax: 29 and 31% above basal control; pEC50: 8.6 and 9.7, respectively). GR127935 also acted as a potent antagonist at the 5-HT1D alpha (app. pA2 8.5) and 5-HT1D beta (app. pA2 9.1) receptors. From studies measuring cAMP accumulation in cultured CHO cells GR127935 also displayed partial agonism, as well as acting as a potent antagonist at the 5-HT1D alpha receptors which stimulate cAMP levels and 5-HT1D beta receptors which inhibit cAMP levels (app. pA2 8.6 and 9.7, respectively). The 5\-HT1-like receptor antagonist methiothepin showed negative intrinsic activity at both receptors in the [35S]GTP gamma S binding assay only. From studies using the receptor alkylating agent EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) the 5-HT1D alpha cell line displayed a lack of receptor reserve but it was evident in the 5-HT1D beta cell line. In previous studies we have also shown that agonist stimulation of 5-HT1D alpha receptors increases cAMP levels which may be due to high receptor expression. Further investigation using up to 1 microM EEDQ to reduce 5-HT1D alpha receptor number did not reveal an underlying inhibitory adenylyl cyclase response. In conclusion, GR127935 acts as a partial agonist, as well as a potent antagonist, at the human 5-HT1D alpha and 5-HT1D beta receptors when expressed in CHO cells.
Mg2+-ions have been suspected to attenuate the inhibitory effect of allosteric modulators on the dissociation of orthosteric ligands from muscarinic M2 receptors. It was aimed to gain more insight into the molecular events underlying the effect of Mg2+. The interaction of Mg2+ with the allosteric model compounds W84 (hexane-1,6-bis [dimethyl-3'-phthalimidopropylammonium bromide]) and Chin3/6 (hexane-1 ,6-bis[dimethyl-3'-¿4-oxo-2-phenyl-3,4-dihydro-2H-quinazolin-1-yl propylammonium bromide]) was studied in porcine heart muscarinic receptors, the primary binding site of which was occupied by the ligand [3H]N-methylscopolamine ([3H]NMS). The incubation buffer was composed of 4 mM Na2HPO4 and 1 mM KH2PO4 (pH 7.4, 23 degrees C). The retardation of [3H]NMS dissociation (control t1/2=5.6 min) induced by the allosteric test compounds was diminished by 3 mM Mg2+ to a greater extent than to be expected with regard to its contribution to the ionic strength of the buffer solution. Concentration-effect curves for the allosteric retardation of [3H]NMS dissociation by W84 (half maximal effective concentration EC0.5=24 nM in the absence of Mg2+) and by Chin3/6 (EC0.5=28 nM) were shifted by Mg2+ to the right in a parallel fashion. The curve-shift was compatible with a competitive interplay between Mg2+ and the modulators. The pKb-values as a measure of the antagonistic potency of Mg2+, however, differed depending on the modulator, i.e. pKb=3.4 with W84 and pKb=2.8 with Chin3/6. Mg2+ itself was capable of slowing the dissociation of [3H]NMS; the maximal retardation of [3H]NMS dissociation was about 3 fold, the concentration-effect relationship was compatible with a two-site model using the above-mentioned pKb-values as affinity constants. Since the equilibrium-binding of [3H]NMS remained unchanged up to a Mg2+-concentration of 3 mM, the cation appears to inhibit the association and dissociation of [3H]NMS to the same extent in this concentration range. Taken together, the findings indicate that Mg2+ may bind to the allosteric region of muscarinic M2 receptors and that more than one site is involved in this interaction. The sites of action may represent divalent cation binding sites.
We review the literature describing constitutive activity of the five muscarinic acetylcholine receptors in native and recombinant systems and discuss the effect of constitutive activity on muscarinic pharmacology in the context of modern models of receptor activation. We include a summary of mutations found to cause constitutive activity and discuss the implications of these data for the structure, function, and activation mechanism of muscarinic receptors. Finally, we discuss the possible physiological significance of constitutive activity of muscarinic receptors, incorporating information provided by targeted deletion of each of the muscarinic subtypes.
Preincubation of human platelets with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) has been shown previously to attenuate the ability of agonists both to suppress formation of cAMP and to stimulate hydrolysis of phosphoinositides. In the present study, we have examined whether the attenuation caused by PMA can be attributed to the phosphorylation of the alpha subunit(s) of Gi, a GTP-binding regulatory protein implicated in several pathways of signal transduction. PMA was found to promote the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [gamma-32P]ATP and [32P]H3PO4, respectively. None of the phosphoproteins, however, was precipitated by either of two antisera containing antibodies differing in specificities for epitopes within Gi alpha, despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently described Gz alpha or both Gz alpha and Gi alpha, precipitated a 40-kDa phosphoprotein. Phosphorylation of this protein occurred not only in response to PMA, but to thrombin and the thromboxane A2 analog U46619. These data suggest that activators of protein kinase C lead to the phosphorylation within platelets of a select population of G alpha subunits. The identified phosphoprotein is not Gi alpha, but is similar or identical to Gz alpha. Because Gz alpha does not contain the consensus site for ADP-ribosylation by the Bordetella pertussis toxin islet-activating protein, the data also suggest that effects of PMA on processes otherwise sensitive to this toxin are not exerted at the level of G proteins responsible for transduction.
Complexes of agonist-bound muscarinic acetylcholine receptor (mAChR) and guanine nucleotide-binding protein (G protein) were solubilized and isolated from rat heart. Heart membranes were incubated with mAChR agonists or antagonists, solubilized using digitonin and cholate, and subjected to chromatography over wheat germ agglutinin-Affi-Gel. Eluted fractions were precipitated using a cardiac-selective anti-mAChR antibody (Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6898). Using samples obtained from membranes initially incubated with carbachol (10 nM, 100 nM, or 1 mM), G alpha immunoreactivity was detected on Western blots probed using antibodies with specificity for G alpha subunits. The G alpha immunoreactivity was not detected when atropine alone (10 nM or 1 microM) or when excess atropine (1 microM) plus carbachol (100 nM) was used during the membrane preincubation. G beta immunoreactivity, when detectable on Western blots, was present in substoichiometric amounts relative to that of G alpha. The G alpha immunoreactivity was not present if GTP was included during incubation of membranes with agonist and following membrane solubilization. Further results indicate that although agonist binding to receptors is rapidly reversed by GTP or GDP (t1/2 less than 10 min), the mAChR-G protein complex is reversed more slowly or not at all. It was also shown that at high agonist concentrations, the cardiac mAChR interacts with both Go and Gi-like proteins. Together, these results demonstrate the utility of an immunoaffinity approach to the purification and biochemical study of receptor-G protein interactions.
Since the reports elucidating the sequence of four subtypes of muscarinic cholinergic receptors appeared, it has been clear that pharmacological approaches to the study of subtypes of these receptors are inadequate to selectively detect one subtype in the presence of the others. One methodology that can provide more selective reagents with which to study these subtypes is immunology. Thus, using the information on the primary sequence of these receptors available in the literature, rabbits were injected with an oligopeptide, CRKIPKRPGSVHRTPSRQ, conjugated to keyhole limpet hemocyanin. This oligopeptide (m1 C-terminal peptide) corresponds to the 17-amino acid sequence of the carboxyl terminus of a rat m1 muscarinic receptor. This portion of the amino acid sequence of the muscarinic receptor protein has been shown to be unique to the m1 receptor and has not been found in the other subtypes of the receptor thus far sequenced. The antisera (anti-m1 antisera) had high titer against the m1 C-terminal peptide in a solid phase radioimmunoassay. The anti-m1 antisera were shown to immunoprecipitate [3H] quinuclidinyl benzilate ([ 3H]QNB) binding activity solubilized from rat forebrain. [3H]Pirenzepine ([ 3H]PZ) has been shown to interact with a subset of [3H]QNB binding sites in forebrain and heart. The anti-m1 antisera were shown to immunoprecipitate [3H]PZ binding sites in cerebral cortex, hippocampus, and corpus striatum, areas believed to be rich in the m1 subtype of the muscarinic receptor. Although [3H]PZ binding activity was present in receptor preparations solubilized from heart, neither [3H]PZ- nor [3H]QNB-binding activities could be immunoprecipitated from this tissue using the anti-m1 antisera. A monoclonal antibody raised against the porcine atrial muscarinic receptor was shown to immunoprecipitate both [3H]PZ- and [3H]QNB-binding activities solubilized from rat heart, but only [3H]QNB-binding activity could be immunoprecipitated from forebrain using this antibody. Immunoprecipitation of [3H]PZ- and [3H]QNB-binding activity by anti-m1 antisera could be inhibited by the m1 C-terminal peptide. Peptides corresponding to the C-terminal portions of the rat m3 and m4 muscarinic receptor were not inhibitory in the immunoprecipitation assay. This study provides further evidence for subtypes of muscarinic receptors in rat tissues and supports the hypothesis that receptor subtypes defined using PZ can be further subclassified on the basis of differences in primary structure.(ABSTRACT TRUNCATED AT 400 WORDS)
A number of monoclonal antibodies were raised against the purified porcine atrial muscarinic acetylcholine receptor. The antibodies were shown to exhibit a high degree of specificity for the receptor by their ability to recognize the purified receptor but not other porcine atrial glycoproteins in enzyme-linked solid-phase immunosorptive assays and by immunoblot analyses. Several of the antibodies were able to quantitatively precipitate the muscarinic receptor in both pig and rat heart and a portion of the receptor from rat cerebellum but little if any receptor from rat cerebral cortex. Thus, these monoclonal antibodies not only exhibit specificity for the muscarinic receptor but also are specific for the cardiac receptor subtype.
- C W Leutje
- C Brumwell
- M Gainer Norman
- G L Peterson
- M I Schimcrlik
- N M Nathanson
Leutje, C.W., Brumwell, C., Gainer Norman, M., Peterson,
G.L.,
Schimcrlik,
M.I.
and
Nathanson,
N.M.
(1987)
Biochemistry 26, 6892-6896.
- Jl D Matesic
- D R Manning
- B B Wolfe
- G R J Luthin
VI
[Jl
Matesic. D., Manning, D.R., Wolfe, B.B. and Luthin, G.R.J.
(1989) Biol. Chem. 264, 21638-21645.