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Estrogen Modulates a Large Conductance Chloride Channel in Cultured Porcine Aortic Endothelial Cells
Abstract
Estrogen is known to exert a protective effect on cardiovascular disease, but the mechanism for this effect is unclear. It has, however, been reported that estrogen and antiestrogen modify ionic currents and membrane potential in various cells. The aim of this study was to clarify whether the chloride channel of aortic endothelial cells was, in fact, modified by estrogen and antiestrogen with inside-out patch and cell-attached patch recording methods. Tamoxifen activated a large-conductance (368 +/- 23 pS, n = 6, in symmetric 150 mM Cl- solution) chloride channel of endothelial cells grown in the presence of 1 microg/ml colchicine. The channels were activated mainly between +/-40 mV, but were inactivated at more extreme potentials. The open probability of channels in cell-attached patches increased from <0.01 to 0.37 +/- 0.08 (n = 8) when cells were treated with 15 microM tamoxifen. This effect can be blocked by 17beta-estradiol, but not by progesterone. The results showed that tamoxifen increased chloride channel activity in the presence of colchicine in cultured endothelial cells, and this action was suppressed by 17beta-estradiol but not by progesterone. This rapid effect by estrogens suggests that these hormones exert nongenomic, short-term activity and do not appear to affect the nuclear estrogen receptor. With these effects, estrogen and antiestrogen bind to the endothelial cells plasma membrane site and subsequently may activate an intracellular second messenger pathway.
... In line with this, we have recently observed this channel in murine neuronal caveolae in a complex with estrogen receptor alpha (mERα), where it participates in the toxic effect provoked by Aβ exposure (Marin et al., 2007). The modulation of pl-VDAC is largely unknown, and only a few data in cellular models have demonstrated that, in general, Maxi Cl − channels can be modulated by phosphorylation (Liberatori et al., 2004), G-proteins (Schwiebert et al., 1990;McGill et al., 1993;Mitchell et al., 1997), fatty acids (Riquelme and Parra, 1999), amyloid beta (Jalonen et al., 1997), and estrogens and anti-estrogens (Li et al., 2000;Diaz et al., 2001;Valverde et al., 2002), the latter suggesting that estrogen receptors (ERs) may be involved in VDAC functionality at the plasma membrane. ...
... In the present work, pl-VDAC was found for the first time to be present in lipid rafts of human temporal cortex and hippocampus. In cortex, VDAC was shown to co-precipitate with ERα and caveolin-1, considered as the main protein responsible for the induction of caveolae formation (Li et al., 2000), a finding that is in agreement with our previous experiments demonstrating the interaction of pl-VDAC, ERα and caveolin-1 in murine cortical and hippocampal neurons (Marin et al., 2007(Marin et al., , 2008. In contrast, no coprecipitation was found in these samples with flotillin, another hallmark of lipid rafts in neurons (data not shown). ...
Membrane estrogen receptor Caveolae Caveolin-1 Human brain Alzheimer's disease Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane (pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models have evidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERα), which participates in neuroprotection against amyloid beta (Aβ), and whose integration into this hydrophobic domain remains unclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDAC and mERα, in a complex with scaffolding caveolin-1 which likely provides mERα stability at the plasma membrane. In Alzheimer's disease (AD) brains, VDAC was accumulated in caveolae, and it was observed in dystrophic neurites of senile plaques, whereas ERα was expressed in astrocytes surrounding the plaques. Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Aβ-induced neurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed in AD neuropathology.
... In line with this, we have recently observed this channel in murine neuronal caveolae in a complex with estrogen receptor alpha (mERα), where it participates in the toxic effect provoked by Aβ exposure (Marin et al., 2007). The modulation of pl-VDAC is largely unknown, and only a few data in cellular models have demonstrated that, in general, Maxi Cl − channels can be modulated by phosphorylation (Liberatori et al., 2004), G-proteins (Schwiebert et al., 1990;McGill et al., 1993;Mitchell et al., 1997), fatty acids (Riquelme and Parra, 1999), amyloid beta (Jalonen et al., 1997), and estrogens and anti-estrogens (Li et al., 2000;Diaz et al., 2001;Valverde et al., 2002), the latter suggesting that estrogen receptors (ERs) may be involved in VDAC functionality at the plasma membrane. ...
... In the present work, pl-VDAC was found for the first time to be present in lipid rafts of human temporal cortex and hippocampus. In cortex, VDAC was shown to co-precipitate with ERα and caveolin-1, considered as the main protein responsible for the induction of caveolae formation (Li et al., 2000), a finding that is in agreement with our previous experiments demonstrating the interaction of pl-VDAC, ERα and caveolin-1 in murine cortical and hippocampal neurons (Marin et al., 2007(Marin et al., , 2008. In contrast, no coprecipitation was found in these samples with flotillin, another hallmark of lipid rafts in neurons (data not shown). ...
Membrane estrogen receptor Caveolae Caveolin-1 Human brain Alzheimer's disease Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane (pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models have evidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERα), which participates in neuroprotection against amyloid beta (Aβ), and whose integration into this hydrophobic domain remains unclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDAC and mERα, in a complex with scaffolding caveolin-1 which likely provides mERα stability at the plasma membrane. In Alzheimer's disease (AD) brains, VDAC was accumulated in caveolae, and it was observed in dystrophic neurites of senile plaques, whereas ERα was expressed in astrocytes surrounding the plaques. Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Aβ-induced neurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed in AD neuropathology.
... The activation of Maxi Cl _ channels by antioestrogen can be prevented by pre-treatment with 17b-oestradiol in NIH3T3 fibroblasts (Hardy & Valverde, 1994) and endothelial cells (Li et al. 2000). This modulatory action of oestradiol was also investigated in C1300 neuroblastoma cells in the present study (Fig. 6A). ...
... While the channel in the fibroblasts shows marked inactivation at positive and negative potentials (Hardy & Valverde, 1994), the channel in the neuroblastoma cell line (this study) only presented inactivation at negative potentials, although the inactivation constant varied greatly from cell to cell. A Maxi Cl _ channel activated by antioestrogens has also been described in endothelial cells, although its voltage dependence was not reported (Li et al. 2000). Differences in the voltage-dependent inactivation of Maxi Cl _ channels have been observed among different cell types (Blatz & Magleby, 1983;Schwarze & Kolb, 1984;Kolb et al. 1985;Forshaw et al. 1993;Riquelme et al. 1995). ...
• The regulation of Maxi Cl− channels by 17-oestradiol and non-steroidal triphenylethylene antioestrogens represents a rapid, non-classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl− channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells. • Whole-cell Maxi Cl− currents were readily and reversibly activated by tamoxifen, toremifene and the membrane-impermeant ethyl-bromide tamoxifen, only when applied to the extracellular medium. • Pre-treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen-induced activation of Maxi Cl− channels. The inhibitory effect of 17-oestradiol and cAMP was abolished by the kinase inhibitor staurosporine. • Current activation was unaffected by the removal of intracellular Ca2+ and Mg2+, but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid-sensitive serine/threonine protein phosphatase in the activation of Maxi Cl− channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells. • Although the role of these Maxi Cl− channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways.
... In epithelia, the urinary bladder [53], gastric [54], pancreatic555657, colonic585960, airway616263, choroid plexus [64], bile duct [65, 66], ciliary676869, renal [9, 19,707172737475767778 , ves- tibular [79], placental80818283848586, ruminal [87] and ovarian [84] epithelial cells were also found to express the maxianion channel with properties similar to those of excitable cells. Resembling maxi-anion channels were also found in fibroblasts [14, 66, 88–92] and endothelial cells9394959697. In the immune system, the maxi-anion channel activity has been confirmed in B lymphocytes9899100101 , in T lym- phocytes102103104 and in peritoneal macrophages [17, 105]. ...
... In cell-attached patches, the maxi-anion channels were activated by an agonist of A1-adenosin receptor, N 6 -cyclohexyladenosine, in cortical collecting duct cells [75] and by bombesin, a Ca-mobilizing peptide mitogen, in Swiss 3T3 fibroblasts [90]. On NIH3T3 fibroblasts and porcine aortic endothelial cells, both grown in the presence of colchicines, the maxi-anion channels were activated by extracellularly added antiestrogens, toremifene [89] and tamoxifen [94]. The effect could be blocked by 17b-estradiol but not by progesterone. ...
The maxi-anion channel is widely expressed and found in almost every part of the body. The channel is activated in response to osmotic cell swelling, to excision of the membrane patch, and also to some other physiologically and pathophysiologically relevant stimuli, such as salt stress in kidney macula densa as well as ischemia/hypoxia in heart and brain. Biophysically, the maxi-anion channel is characterized by a large single-channel conductance of 300-400 pS, which saturates at 580-640 pS with increasing the Cl(-) concentration. The channel discriminates well between Na(+) and Cl(-), but is poorly selective to other halides exhibiting weak electric-field selectivity with an Eisenman's selectivity sequence I. The maxi-anion channel has a wide pore with an effective radius of approximately 1.3 nm and permits passage not only of Cl(-) but also of some intracellular large organic anions, thereby releasing major extracellular signals and gliotransmitters such as glutamate(-) and ATP(4-). The channel-mediated efflux of these signaling molecules is associated with kidney tubuloglomerular feedback, cardiac ischemia/hypoxia, as well as brain ischemia/hypoxia and excitotoxic neurodegeneration. Despite the ubiquitous expression, well-defined properties and physiological/pathophysiological significance of this classical channel, the molecular entity has not been identified. Molecular identification of the maxi-anion channel is an urgent task that would greatly promote investigation in the fields not only of anion channel but also of physiological/pathophysiological signaling in the brain, heart and kidney.
... However, after toremifene-activation, the estradiol administration appears to be ineffective [290]. In the presence of mitotic inhibitor colchicine, estradiol also rapidly and dose dependently reduces the open probability of tamoxifen activated largeconductance Cl − channels on the membranes of cultured porcine aortic endothelial cells (IC 50 ≈ 440 nM, E max ≈ 0.38, E min ≈ 0.009, n H ≈ −0.32) [291] and testosterone rapidly and dose dependently inhibits forskolin (10 M)-induced Cl − currents in rat testicular efferent duct epithelia (IC 50 ≈ 1.3 M, E max ≈ 1, E min ≈ 0, n H ≈ −1.4) [292]. ...
This review is focused on the physiological and pathophysiological relevance of steroids influencing the activities of the central and peripheral nervous systems with regard to their concentrations in body fluids and tissues in various stages of human life like the fetal development or pregnancy. The data summarized in this review shows that DHEA and its unconjugated and sulfated metabolites are physiologically and pathophysiologically relevant in modulating numerous ion channels and participate in vital functions of the human organism. DHEA and its unconjugated and sulfated metabolites including 5α/β-reduced androstane steroids participate in various physiological and pathophysiological processes like the management of GnRH cyclic release, regulation of glandular and neurotransmitter secretions, maintenance of glucose homeostasis on one hand and insulin insensitivity on the other hand, control of skeletal muscle and smooth muscle activities including vasoregulation, promotion of tolerance to ischemia and other neuroprotective effects. In respect of prevalence of steroid sulfates over unconjugated steroids in the periphery and the opposite situation in the CNS, the sulfated androgens and androgen metabolites reach relevance in peripheral organs. The unconjugated androgens and estrogens are relevant in periphery and so much the more in the CNS due to higher concentrations of most unconjugated steroids in the CNS tissues than in circulation and peripheral organs.
... Our results did not show any signi¢cant e¡ects of tamoxifen. By contrast, there are reports concerning nongenomic e¡ect of tamoxifen in cultured porcine aortic endothelial cells (Li et al., 2000) and lens of the eye (Zhang et al., 1994). ...
We investigated the nongenomic effects of female sex steroid hormones on the short circuit current (Isc,probe) across gerbil stria vascularis using the voltage-sensitive vibrating probe. The strial marginal cell epithelial layer produces Isc,probe by secreting K+ via IKs channels in the apical membrane. Application of 17β-estradiol (E2) caused a decrease of Isc,probe in a dose-dependent manner (10 nM–10 μM) within seconds. Tamoxifen, a competitive inhibitor of the intracellular estrogen receptor, did not change the inhibitory effect of E2. Activation of IKs channels by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid in the presence and absence of E2 was used to test the mechanism of action. The results were consistent with a direct inhibitory effect of E2 on the IKs channels. By contrast, progesterone caused a transient increase of Isc,probe. These results suggest that E2 decreases secretion of K+ by inhibition of IKs channels via a nongenomic mechanism at concentrations near those occurring under some physiologic conditions while progesterone caused only transient effects on Isc,probe.
... DRG neurons in vitro are a valuable preparation because adult primary sensory neurons can be studied without the interference of modulation by central or peripheral messengers. Visceral afferents are sensitive to ATP [3], and several indirect pieces of evidence suggest that visceral afferents are E 2 -sensitive: (i) visceral pain is affected by hormonal level in cycling females [4] ; (ii) there are gender differences in the prevalence of functional disorders involving the viscera [5]; (iii) putative visceral afferents [6] fit into the population of DRG neurons that are E 2 -sensitive. Although it is generally accepted that each primary afferent neuron is a single sensory channel, several studies have challenged that view and demonstrate that a population of DRG neuron can innervate both the viscera and somatic tissues. ...
Clinical studies suggest the comorbidity of functional pain syndromes such as irritable bowel syndrome, painful bladder syndrome, chronic pelvic pain, and somatoform disorders approaches 40% to 60%. The incidence of episodic or persistent visceral pain associated with these "functional" disorders is two to three times higher in women than in men. One of the possible explanations for this phenomenon is estrogen modulation of viscerovisceral cross-sensitization. While a central site of this modulation has been shown previously, our studies suggest a peripheral site, the dorsal root ganglion (DRG). Estrogens have remarkably wide range of functions including modulation of voltage-gated calcium channels (VGCCs) and purinoreceptors (P2Xs). Significantly, inflammation dramatically alters purinoception by causing a several fold increase in ATP-activated current, alters the voltage dependence of P2X receptors, and enhances the expression of P2X receptors increasing neuronal hypersensitivity. Gonadal hormones are thought as indispensable cornerstones of the normal development and function, but it appears that no body region, no neuronal circuit, and virtually no cell is unaffected by them. Thus, increasing awareness toward estrogens appears to be obligatory.
... Thus, vitamin D 3 has been shown to be a potent modulator of osteosarcoma calcium channels (Caffrey and Farach-Carson, 1989;Farach-Carson et al., 1991), and estrogens modulate potassium channels by a cell membrane-delimited cGMP-mediated pathway (White et al., 1995) or by direct interaction with the channels themselves (Valverde et al., 1999). Anion channels are similarly modulated by steroids, affecting chloride currents in tissues as diverse as neural tissue (Gee et al., 1987), osteoblasts (Zanello and Norman, 1997), and aortic endothelial cells (Li et al., 2000). Horwitz and coworkers Greenberger et al., 1990) also have demonstrated that progesterone binds P-glycoprotein and blocks P-glycoproteinmediated drug transport. ...
Cystic fibrosis (CF) is an autosomal genetic disease associated with impaired epithelial ion transport. Mutations in the CF gene alter the primary sequence of the CF transmembrane conductance regulator (CFTR). Several therapeutic modalities have been proposed for CF patients, including the phytoestrogen genistein. Experiments were completed in cellular and subcellular systems to evaluate the impact of naturally occurring and synthetic estrogens on epithelial ion transport, and specifically on the CF protein CFTR. 17beta-Estradiol, a naturally occurring estrogen, caused a rapid and reversible inhibition of forskolin-stimulated chloride secretion across T84 epithelial cell monolayers with a K(i) of 8 microM. In addition, 17alpha-estradiol, a stereoisomer that fails to bind and activate nuclear estrogen receptors was equipotent with 17beta-estradiol, arguing against a genomic-mediated mechanism of action. Synthetic estrogens, including diethylstilbesterol and the antiestrogen tamoxifen likewise inhibited forskolin-stimulated ion transport. Aldosterone, dexamethasone, and cholesterol were without effect at the highest concentrations tested (>/=1 mM). Studies indicated that diethylstilbesterol and other synthetic estrogens that inhibited anion secretion in intact monolayers likewise inhibited CFTR chloride channel activity with similar concentration dependencies in excised membrane patches. Experiments with radioactive photoactivatable estrogen derivatives demonstrated that these compounds bind directly to CFTR expressed in insect cells. Taken together, the data suggest that estrogens can interact directly with CFTR to alter anion transport.
... Previous studies have suggested various signaling pathways involved in activation of the VDACL anion channels in other cell types: G proteins (Schwiebert et al., , 1992Sun et al., 1992Sun et al., , 1993McGill et al., 1993;Mitchell et al., 1997), Ca 2ϩ Kawahara and Takuwa, 1991;Groschner and Kukovetz, 1992), PKC (Groschner and Kukovetz, 1992;Schwiebert et al., 1992), actin (Schwiebert et al., 1994), NK-1 receptor (Sun et al., 1992(Sun et al., , 1993, adenosine A1 receptor (Schwiebert et al., 1992), and estrogen receptor (Hardy and Valverde, 1994;Li et al., 2000). The exact activation mechanism of the VDACL anion channel in mammary C127i cells is under current investigation. ...
In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.
... nent than that of Ca 2 ϩ -activated Cl Ϫ channels (Nilius et al., 1997b). Because it is inhibited by tamoxifen, this current is also different from the tamoxifen-activated large conductance Cl Ϫ channel current in endothelium (Li et al., 2000). Also, exogenous ATP did not activate a similar current in isotonic solutions, indicating that it was not activated by an autocrine action of ATP released from the cell during the hypotonic challenge. ...
Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.
... Even though the role of this channel at the plasma membrane has not been still elucidated, some recent data have suggested a dual action, on one hand, to participate in the maintenance of normal redox homeostasis and, on the other hand, to modulate apoptotic events (Elinder et al. 2005). The modulation of VDAC at the plasma membrane level is still largely unknown, and only a few data in a variety of cell types have suggested that overall Maxi Cl ( channels may be modulated by different factors such as unsaturated fatty acids (Riquelme & Parra 1999), GTP-binding proteins (Schwiebert et al. 1990, McGill et al. 1993, nucleotides (Schwiebert et al. 1992, Mitchell et al. 1997, phosphorylation (Pahapill & Schlichter 1992, Liberatori et al. 2004, and direct interaction with hormones and anti-hormones (Kajita et al. 1995, Li et al. 2000, Díaz et al. 2001, Valverde et al. 2002. ...
Voltage-dependent anion channel (VDAC) is a porin known by its role in metabolite transport across mitochondria and participation in apoptotic processes. Although traditionally accepted to be located within mitochondrial outer membrane, some data has also reported its presence at the plasma membrane level where it seems to participate in regulation of normal redox homeostasis and apoptosis. Here, exposure of septal SN56 and hippocampal HT22 cells to specific anti-VDAC antibodies prior to amyloid beta (Abeta) peptide was observed to prevent neurotoxicity. In these cell lines, we identified a VDAC form associated with the plasma membrane that seems to be particularly abundant in caveolae. The two membrane-related isoforms of estrogen receptor alpha (mERalpha) (80 and 67 kDa), known in SN56 cells to participate in estrogen-induced neuroprotection against Abeta injury, were also observed to be present in caveolae. Interestingly, we demonstrated for the first time that both VDAC and mERalpha interact at the plasma membrane of these neurons as well as in microsomal fractions of the corresponding murine septal and hippocampal tissues. These proteins were also shown to associate with caveolin-1, thereby corroborating their presence in caveolar microdomains. Taken together, these results suggest that VDAC-mERalpha association at the plasma membrane level may participate in the modulation of Abeta-induced cell death.
In animals, steroid hormones can act using genomic and non-genomic mechanisms. Plant steroid hormones, brassinosteroids, are capable of inducing the expression of some gene ensembles, however their non-genomic pathways for triggering the physiological effects are still unclear. In this paper, we propose the hypothesis on existence of brassinosteroid non-genomic effects in plant cells. This non-genomic pathway could due to modulation of ion channel activities and modification of membrane receptors.
Endothelial cells (EC) form a unique signal-transducing surface in the vascular system. The abundance of ion channels in the plasma membrane of these nonexcitable cells has raised questions about their functional role. This review presents evidence for the involvement of ion channels in endothelial cell functions controlled by intracellular Ca ²⁺ signals, such as the production and release of many vasoactive factors, e.g., nitric oxide and PGI 2 . In addition, ion channels may be involved in the regulation of the traffic of macromolecules by endocytosis, transcytosis, the biosynthetic-secretory pathway, and exocytosis, e.g., tissue factor pathway inhibitor, von Willebrand factor, and tissue plasminogen activator. Ion channels are also involved in controlling intercellular permeability, EC proliferation, and angiogenesis. These functions are supported or triggered via ion channels, which either provide Ca ²⁺ -entry pathways or stabilize the driving force for Ca ²⁺ influx through these pathways. These Ca ²⁺ -entry pathways comprise agonist-activated nonselective Ca ²⁺ -permeable cation channels, cyclic nucleotide-activated nonselective cation channels, and store-operated Ca ²⁺ channels or capacitative Ca ²⁺ entry. At least some of these channels appear to be expressed by genes of the trp family. The driving force for Ca ²⁺ entry is mainly controlled by large-conductance Ca ²⁺ -dependent BK Ca channels ( slo), inwardly rectifying K ⁺ channels (Kir2.1), and at least two types of Cl ⁻ channels, i.e., the Ca ²⁺ -activated Cl ⁻ channel and the housekeeping, volume-regulated anion channel (VRAC). In addition to their essential function in Ca ²⁺ signaling, VRAC channels are multifunctional, operate as a transport pathway for amino acids and organic osmolytes, and are possibly involved in endothelial cell proliferation and angiogenesis. Finally, we have also highlighted the role of ion channels as mechanosensors in EC. Plasmalemmal ion channels may signal rapid changes in hemodynamic forces, such as shear stress and biaxial tensile stress, but also changes in cell shape and cell volume to the cytoskeleton and the intracellular machinery for metabolite traffic and gene expression.
A survey of nearly two hundred reports shows that rapid estrogenic actions can be detected across a range of kinds of estrogens, a range of doses, on a wide range of tissue, cell and ion channel types. Striking is the fact that preparations of estrogenic agents that do not permeate the cell membrane almost always mimic the actions of the estrogenic agents that do permeate the membrane. All kinds of estrogens, ranging from natural ones, through receptor modulators, endocrine disruptors, phytoestrogens, agonists, and antagonists to novel G-1 and STX, have been reported to be effective. For actions on specific types of ion channels, the possibility of opposing actions, in different cases, is the rule, not the exception. With this variety there is no single, specific action mechanism for estrogens per se, although in some cases estrogens can act directly or via some signaling pathways to affect ion channels. We infer that estrogens can bind a large number of substrates/receptors at the membrane surface. As against the variety of subsequent routes of action, this initial step of the estrogen's binding action is the key.
The maxi-anion channels (MACs) with a unitary conductance of 200-500 pS are detected in virtually every part of the whole body and found in cells from mammals to amphibia. The channels are normally silent but can be activated by physiologically/pathophysiologically relevant stimuli, such as osmotic, salt, metabolic, oxidative, and mechanical stresses, receptor activation, serum, heat, and intracellular Ca(2+) rise. In some MACs, protein dephosphorylation is associated with channel activation. Among MACs so far studied, around 60 % (designated here as Maxi-Cl) possess, in common, the following phenotypical biophysical properties: (1) unitary conductance of 300-400 pS, (2) a linear current-voltage relationship, (3) high anion-to-cation selectivity with PCl/Pcation of >8, and (4) inactivation at positive and negative potentials over a certain level (usually ±20 mV). The pore configuration of the Maxi-Cl is asymmetrical with extracellular and intracellular radii of ∼1.42 and ∼1.16 nm, respectively, and a medial constriction down to ∼0.55-0.75 nm. The classical function of MACs is control of membrane potential and fluid movement. Permeability to ATP and glutamate turns MACs to signaling channels in purinergic and glutamatergic signal transduction defining them as a perspective target for drug discovery. The molecular identification is an urgent task that would greatly promote the developments in this field. A possible relationship between these channels and some transporters is discussed.
RESUMENLas membranas celulares contienen un canal de Cl- (Maxi-Cl-) que es modulado por estrógenos y antiestrógenos. El trabajo experimental de mi tesis doctoral ha consistido en el estudio de la modulación de este canal y su identidad molecular. El resultado de mi trabajo ha demostrado que la base molecular del canal Maxi-Cl- es una isoforma de la proteína mitocondrial VDAC y que la activación del canal por los antiestrógenos y su inhibición por los estrógenos implica procesos de defosforilación y fosforilación, respectivamente.ABSTRACTCellular membranes contain a Maxi-Cl- channel that is modulated by oestrogen and antioestrogens. The experimental work of my PhD Thesis has focussed in the study of the molecular identity of the Maxi-Cl- channel and its regulation. My results demonstrated that an isoform of the mitochondrial protein VDAC is the molecular correlate of the Maxi-Cl- channel and that the activation of the channel by antioestrogens and its inhibition by oestrogen requires a dephosphorylation and phosphorylation process, respectively.
The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, that forms the main barrier to materno-fetal exchange. The chloride (Cl(-)) channels in other epithelial tissues contribute to several functions, such as maintenance of the membrane potential, volume regulation, absorption and secretion. Additionally, the contributions of Cl(-) channels to these functions are demonstrated by certain diseases and knock-out animal models. There are multiple lines of evidence for the presence of Cl(-) channels in the hSTB, which could contribute to different placental functions. However, both the mechanism by which these channels are involved in the physiology of the placenta, and their molecular identities are still unclear. Furthermore, a correlation between altered Cl(-) channels functions and pathological pregnancies is beginning to emerge. This review summarizes recent developments on conductive placental chloride transport, and discusses its potential implications for placental physiology.
In epidemiological studies tamoxifen has been associated with a reduction in the incidence of fatal myocardial infarction in women. However, the effects of tamoxifen on coronary artery reactivity are unknown. We hypothesized that tamoxifen would relax precontracted isolated rabbit coronary arteries. Rings of coronary artery from adult male and nonpregnant female New Zealand White rabbits were suspended in organ baths containing Krebs' solution; isometric tension was measured. Tamoxifen (0.1-100 microM) induced significant endothelium-dependent relaxation in precontracted rabbit coronary arteries. This relaxation was inhibited by N(omega)-nitro-L-arginine methyl ester and the estrogen receptor antagonist ICI 182,780. There was no significant effect on calcium concentration-dependent contraction curves. These data suggest that tamoxifen has beneficial effects on coronary reactivity that could, at least in part, account for the reduction in risk of fatal myocardial infarction in women taking tamoxifen.
The understanding of the biological effects of estrogen on the vessel wall has improved dramatically since the discovery of estrogen receptors (ERs). Most, but not all estrogen-mediated effects in blood vessels are thought to be mediated by ERs. Two major ER subclasses have been characterized so far: the ERalpha and the more recently described ERbeta. This review will primarily focus on a new perspective that highlights ERs as essential mediators of the vascular effects of estrogen. In view of the rising research interest in this area, it can be also expected that tissue- and ER subclass-selective agonists and antagonists will be developed over the next few years, thus providing invaluable tools for pharmacological and clinical applications.
Endothelial cells (EC) form a unique signal-transducing surface in the vascular system. The abundance of ion channels in the plasma membrane of these nonexcitable cells has raised questions about their functional role. This review presents evidence for the involvement of ion channels in endothelial cell functions controlled by intracellular Ca(2+) signals, such as the production and release of many vasoactive factors, e.g., nitric oxide and PGI(2). In addition, ion channels may be involved in the regulation of the traffic of macromolecules by endocytosis, transcytosis, the biosynthetic-secretory pathway, and exocytosis, e.g., tissue factor pathway inhibitor, von Willebrand factor, and tissue plasminogen activator. Ion channels are also involved in controlling intercellular permeability, EC proliferation, and angiogenesis. These functions are supported or triggered via ion channels, which either provide Ca(2+)-entry pathways or stabilize the driving force for Ca(2+) influx through these pathways. These Ca(2+)-entry pathways comprise agonist-activated nonselective Ca(2+)-permeable cation channels, cyclic nucleotide-activated nonselective cation channels, and store-operated Ca(2+) channels or capacitative Ca(2+) entry. At least some of these channels appear to be expressed by genes of the trp family. The driving force for Ca(2+) entry is mainly controlled by large-conductance Ca(2+)-dependent BK(Ca) channels (slo), inwardly rectifying K(+) channels (Kir2.1), and at least two types of Cl( -) channels, i.e., the Ca(2+)-activated Cl(-) channel and the housekeeping, volume-regulated anion channel (VRAC). In addition to their essential function in Ca(2+) signaling, VRAC channels are multifunctional, operate as a transport pathway for amino acids and organic osmolytes, and are possibly involved in endothelial cell proliferation and angiogenesis. Finally, we have also highlighted the role of ion channels as mechanosensors in EC. Plasmalemmal ion channels may signal rapid changes in hemodynamic forces, such as shear stress and biaxial tensile stress, but also changes in cell shape and cell volume to the cytoskeleton and the intracellular machinery for metabolite traffic and gene expression.
The antioestrogen 4-hydroxytamoxifen (10 or 2 microM) abolished the generation of action potentials and repolarized the membrane potential in rat pancreatic beta-cells stimulated by 16 mM glucose. This effect was slowly reversible upon withdrawal of the drug. In cells stimulated by tolbutamide (100 microM), application of 4-hydroxytamoxifen again inhibited action-potential generation but failed to repolarize the membrane potential. 4-Hydroxytamoxifen inhibited voltage-sensitive calcium currents and activity of the volume-sensitive anion channel. The drug had no effect on net K(+) conductance of the cell. Insulin release stimulated by either glucose or tolbutamide was inhibited by 4-hydroxytamoxifen. It is concluded that 4-hydroxytamoxifen impairs beta-cell electrical and secretory activity by inhibiting calcium and anion channel currents. This effect could contribute towards hyperglycaemia during therapy with tamoxifen, of which 4-hydroxytamoxifen is the major metabolite. This study also reveals differences between the depolarizing actions of glucose and tolbutamide in the beta-cell.
The identification of alternative estrogen actions has been accumulating steadily over the past two decades. Typically, these novel actions are not directly related to nuclear transcriptional events but related to the interaction of estrogens with sites present at plasma membrane or cytosolic locations. These alternative effects, widely known as non-genomic effects, range from the modulation of plasma membrane ion channel activity to the regulation of different intracellular signalling cascades. In the present study we have investigated the modulation of a large conductance chloride channel (Maxi Cl(-)) by estrogens, non-steroidal triphenylethylene antiestrogens and phenothiazines in NIH3T3 fibroblasts and the dependence on guanosine triphosphate (GTP) of the Maxi Cl(-) activation. Our data identifies the non-steroidal antiestrogens toremifene and tamoxifen, and the phenothiazines chlorpromazine and triflupromazine as activators of Maxi Cl(-) channels. In contrast, 17 beta-estradiol and cAMP, added prior to the exposure to antiestrogens, prevent channel activation. The pure antiestrogen ICI 182780 did not activate the channel nor prevent its activation by non-steroidal antiestrogens. The activation of Maxi Cl(-) channels by toremifene and tamoxifen required the presence of intracellular nucleotides and was inhibited by the stable analog, GDP beta -S, suggesting the participation of a G-protein in the activation process. Little is known about the physiological relevance of Maxi Cl(-) channels. However, that fact that its regulation by estrogens and antiestrogens is shared by different cell types might imply a common role which needs to be identified.
There is a growing interest in the effects of estrogen on the vascular wall, due to the marked gender difference in the incidence of clinically apparent coronary heart disease, when comparing premenopausal women with age-matched males. Estrogen has numerous effects on vascular endothelial and smooth muscle cells, both of which express estrogen receptors (ERs). Although ERs are classically defined as ligand-activated transcription factors, it has become increasingly clear that estrogen-stimulated, ER-dependent cellular responses can be rapid consequences of signal transduction cascades. The cellular localization and molecular form of the ER(s) which mediates rapid signaling are poorly defined. In this review, we describe the mounting evidence for membrane-localized ERs that vary in structure from classical forms. We also discuss ER-catalyzed molecular complex formations and a variety of estrogen-triggered signal transduction cascades, including those involving phosphatidylinositol 3-kinase/Akt, MAP kinase and G-protein-coupled receptors, all of which may induce "protective" profiles in vascular cells.
Steroid hormones have been implicated in the modulation of several transport processes, including conductive chloride transport in epithelial cells. Micromolar concentrations of these hormones have been determined in blood of pregnant women. The purpose of this work was to explore the effects of 17beta-Estradiol, a steroid hormone, on the biophysical properties of the Maxi chloride channel present in apical membranes from human placental syncytiotrophoblast. Apical membrane chloride channels from human term placentas were reconstituted in giant liposomes suitable for electrophysiologic studies by the patch-clamp method. Low micromolar concentrations of 17beta-Estradiol inhibit the Maxi chloride channels in excised patches in a potential-dependent manner. The addition of 1 mM 17beta-Estradiol to the bath solution decreased the total current in the patch from 100% control to 71% at -40 mV holding potential and the current was not affected by 17beta-Estradiol at + 40 mV. However, the presence of the hormone did not affect the single-channel conductance, therefore its effect must be due to modulation of its open probability (Po). Interestingly, 17alpha-Estradiol did not change the total current in the patch. Tamoxifen, an antiestrogen, also showed inhibition, but in a voltage-independent manner. Our results suggest that the Maxi Cl- channel from human term placenta may be regulated by direct interaction of both compounds with the channel. From a functional point of view, the control of these channels by steroid hormones may be of great importance in placental physiology and their regulation may help to unravel their possible role in transplacental transport.
The cell membrane large conductance voltage-dependent chloride channel (Maxi Cl- channel) has been recorded in different cell types following excision of membrane patches or stimulation by antiestrogens under whole-cell recording conditions. However, both its molecular nature and relevance to cell physiology await elucidation. Its electrophysiological properties resemble those of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. This observation has led to the controversial hypothesis that VDAC could be the molecular correlate of the plasma membrane Maxi Cl- channel. We have investigated the cellular localization of VDAC and its relationship with the antiestrogen-activated Maxi Cl- current in C1300 neuroblastoma cells. The presence of a plasma membrane VDAC was demonstrated by immunoblotting of membrane fractions with monoclonal antibodies against the VDAC and by reverse transcription-PCR using primers that hybridize to a VDAC sequence coding for an N-terminal leader peptide required for its plasma membrane sorting. Besides, VDAC colocalized with markers of plasma membrane lipid rafts (cholera toxin beta subunit) but not caveolin-1. Transfection of C1300 cells with an antisense oligonucleotide directed against the specific membrane leader sequence of VDAC markedly reduced both VDAC immunostaining and antiestrogen-activated Maxi Cl- currents, suggesting that VDAC forms the plasma membrane Maxi Cl- channel or a part thereof.
The female sex steroid, estradiol 17beta, mediates its effect through its association with estrogen receptor present in the target cell. So far the major emphasis has been given to the genomic actions of the hormone mediated by the nuclear estrogen receptors. Recent years have seen a shift in the ideas revealing the existence of estradiol binding entities both in the plasma membrane and the endoplasmic reticulum. Though the true identity of this membrane associated receptors is far from being known, a functional role for the same have been implicated both at the genomic as well as the non-genomic level. The major focus of the review is to highlight the existence of membrane associated estrogen receptors and receptor-related proteins and the functional roles played by some of them. The signalling events exerted by this class of membrane associated estrogen receptor could partly explain the physiological significance of estrogen in cardiovascular disease, osteoporosis and breast cancer as well as the molecular mechanism associated with xenoestrogen action.
The Maxi-chloride channel was the first ion channel described by electrophysiological methods in placenta. Because it is difficult to access a complex epithelium such as the placenta for electrophysiological procedures, the studies of ion channels from placental membranes have been performed only very recently. It was only in 1993 that a direct demonstration of a high-conductance chloride channel in apical membranes of intact trophoblastic epithelium was mentioned, and two years later, the description of this channel was reported from purified placental apical membranes reconstituted into artificial lipid membranes suitable for patch-clamp recordings. This brief review comments on the work done with regard to the electrophysiological characterization and regulation of the large-conductance or "Maxi" chloride channel and its contribution to the development of a cellular model for syncytiotrophoblast ion transport.
ATP serves not only as an energy source for all cell types but as an 'extracellular messenger' for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg(2+) and/or H(+) salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP(4-) in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.
We have investigated the effects of steroids on the intracellular calcium ion concentration [Ca2+]i in chicken granulosa cells obtained from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 100 +/- 5 nM. There was an immediate (i.e. less than 5 sec) 4- to 8-fold increase in [Ca2+]i in all of the 76 cells examined after the addition of 10(-7) M estradiol-17 bdta. Estradiol-17 beta was effective between 10(-10)-10(-6) M. Estradiol-17 alpha, estrone, and estriol (10(-8)-10(-6) M) were as effective as estradiol-17 beta, but the progestins, pregnenolone, and progesterone, and the androgens, testosterone, androstenedione, or 5 alpha-dihydrotestosterone were ineffective at concentrations up to 10(-5) M. The prompt estradiol-17 beta-induced [Ca2+]i spike was not affected by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca2+ channel blockers lanthanum (1 mM), cobalt (5 mM), methoxyverapamil (D600; 50 microM), or nifedipine (20 microM). The estrogen-triggered [Ca2+]i surge was also not affected by pretreating the cells with the conventional estrogen receptor antagonist tamoxifen (10(-5) M), or the RNA and protein synthesis inhibitors actinomycin D (1 microgram/ml) and cycloheximide (1 microgram/ml), but was abolished by pretreating the cells with inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) and U-73,122 (2.5 microM). The closely related, but inactive, compound U-73,343 (1 microM) did not affect the estrogen-triggered [Ca2+]i surge. Estradiol-17 beta (10(-7) M), but not progesterone (10(-5) M), also triggered a large [Ca2+]i surge in pig granulosa cells, which, like the [Ca2+]i surge in chicken granulosa cells, was almost immediate, transient, and unaffected by incubation in Ca(2+)-free medium or pretreatment with methoxyverapamil (D600; 50 microM), lanthanum (1 mM), or tamoxifen (10(-5)M). However, granulosa cells from immature rats primed with diethylstilbestrol or PMSG did not respond to estradiol-17 beta, even at concentrations as high as 10(-5) M, although they promptly generated a [Ca2+]i transient upon exposure to LHRH (10(-5) M). These results suggest that estrogens almost instantaneously trigger the release of Ca2+ from intracellular stores which may be mediated through phosphoinositide breakdown. The striking rapidity of this estrogen-induced internal Ca2+ mobilization is consistent with the activation of a cell surface receptor which is different from the conventional slowly acting, gene-stimulating nuclear estrogen receptor.
1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
Tamoxifen is an antiestrogen frequently used in the treatment of breast cancer and is currently being assessed as a prophylactic for those at high risk of developing tumors. We have found that tamoxifen and its derivatives are high-affinity blockers of specific chloride channels. This blockade appears to be independent of the interaction of tamoxifen with the estrogen receptor and therefore reflects an alternative cellular target. One of the clinical side effects of tamoxifen is impaired vision and cataract. Chloride channels in the lens of the eye were shown to be essential for maintaining normal lens hydration and transmittance. These channels were blocked by tamoxifen and, in organ culture, tamoxifen led to lens opacity associated with cataracts at clinically relevant concentrations. These data suggest a molecular mechanism by which tamoxifen can cause cataract formation and have implications for the clinical use of tamoxifen and related antiestrogens.
While alterations in cholesterol and lipoprotein profiles partly account for menopause being a risk factor for coronary heart disease, recent studies have suggested that 17 beta-estradiol may have vascular effects. Our aims were to study the short-term effects of 17 beta-estradiol on vascular function in isolated porcine coronary artery rings. Concomitantly, we sought to determine if physiological concentrations of 17 beta-estradiol could acutely potentiate relaxation
17 alpha- and 17 beta-estradiol at pharmacological (> 1 microM) concentrations produced relaxation in U46619-pre-contracted porcine coronary artery rings. Relaxation evoked by 17 beta-estradiol was not reversed by the estrogen receptor antagonists tamoxifen and ICI 182780. Following 20 min exposure to a physiological concentration of 17 beta-estradiol (1 nM), which on its own had no effect, relaxation elicited by cromakalim, levcromakalim and sodium nitroprusside, but not bradykinin or calcium ionophore A23187, were significantly enhanced. This potentiating action was also insensitive to tamoxifen and ICI 182780. Our data provide evidence for an acute indirect relaxant action of 17 beta-estradiol and suggest that it may be via a tamoxifen- and ICI 182780-insensitive estrogen receptor. While this response was only observed at pharmacological concentrations, the potentiation of cromakalim, levcromakalim and sodium nitroprusside relaxation was evident in the presence of a physiological concentration (1 nM) of 17 beta-estradiol.
These results demonstrate that short-term exposure to 17 beta-estradiol, at concentrations that have no effect on their own, can enhance vasorelaxation. These vascular effects may partly account for some of the acute effects of 17 beta-estradiol on blood flow.
Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue1 and for the inhibition of platelet aggregation2 and platelet adhesion3 attributed to endothelium-derived relaxing factor4. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay5, chemiluminescence1 or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.
Objectives: While alterations in cholesterol and lipoprotein profiles partly account for menopause being a risk factor for coronary heart disease, recent studies have suggested that 17β-estradiol may have vascular effects. Our aims were to study the short-term effects of 17β-estradiol on vascular function in isolated porcine coronary artery rings. Concomitantly, we sought to determine if physiological concentrations of 17β-estradiol could acutely potentiate relaxation. Results: 17α- and 17β-estradiol at pharmacological (>1 μM) concentrations produced relaxation in U46619-pre-contracted porcine coronary artery rings. Relaxation evoked by 17β-estradiol was not reversed by the estrogen receptor antagonists tamoxifen and ICI 182780. Following 20 min exposure to a physiological concentration of 17β-estradiol (1 nM), which on its own had no effect, relaxation elicited by cromakalim, levcromakalim and sodium nitroprusside, but not bradykinin or calcium ionophore A23187, were significantly enhanced. This potentiating action was also insensitive to tamoxifen and ICI 182780. Our data provide evidence for an acute indirect relaxant action of 17β-estradiol and suggest that it may be via a tamoxifen- and ICI 182780-insensitive estrogen receptor. While this response was only observed at pharmacological concentrations, the potentiation of cromakalim, levcromakalim and sodium nitroprusside relaxation was evident in the presence of a physiological concentration (1 nM) of 17β-estradiol. Conclusions: These results demonstrate that short-term exposure to 17β-estradiol, at concentrations that have no effect on their own, can enhance vasorelaxation. These vascular effects may partly account for some of the acute effects of 17β-estradiol on blood flow.
Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified
GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth
properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning
and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning
electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences
in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of
Weibel-Palade bodies identified the cells from both sources as endothelial.
Bovine pulmonary microvessel endothelial cells grown on a flexible substrate contract upon the addition of angiotensin II, thrombin, bradykinin, and U44069, a stable analogue of thromboxane A2. All these agents promote inflammation and increase paracellular permeability in vivo or in vitro. The contractile response is mediated by intracellular and extracellular free calcium: the response is inhibited by TMB-8, an intracellular Ca2+ chelator, and EGTA. Contraction is inhibited by trifluoroperazine, a Ca2+-calmodulin antagonist, and by ML-7, an inhibitor of myosin light-chain kinase. Preincubation with PMA, a protein kinase C activator, prevents contraction by angiotensin II. The inactive analogue 4--phorbol 12,13-didecanoate does not inhibit contraction. In contrast cAMP, carbacyclin (a stable PGI2 analogue), and isoproterenol, agonists known to stabilize the microvascular barrier against inflammatory agents, relax pulmonary microvessel EC. This direct evidence of the contractile potential of microvessel endothelial cells lends support to the theory that endothelial contraction leads to increased junctional permeability.
Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial.
OESTROGENS are more readily accumulated and retained in responsive cells
than in cells that are not their targets1. Cytoplasmic
macromolecules2 which specifically interact with oestradiol
and other steroid hormones seem to mediate transfer of the agonist to
the nuclear chromatin, where the complex is believed to promote
expression of the phenotypic effects3-6. It is generally
assumed that the hormone diffuses passively to ``cytoplasmic'' receptors
which determine the cellular specificity of response7. But
some experiments indicate that steroid hormones interact with components
of biological membranes and may enter their respective target cells by a
membrane-mediated process8-12 which is saturable and
temperature-dependent13-17. We have investigated
steroid-binding components associated with the plasma membranes of cells
isolated from endometrium, liver and intestinal mucosa. Endometrial and
liver cells show substantial binding to oestrogen immobilised by
covalent linkage to an inert support, while intestinal cells have no
such binding sites. The relative quantity of these steroid receptors at
the outer surfaces of cells from diverse tissues corresponds well with
the capacity of a given cell to accumulate and retain oestrogen.
Macrophage production of growth factors for fibroblasts, in particular platelet-derived growth factor B [PDGF(B)] and transforming growth factor-beta (TGF-beta), is thought to be central to the pathogenesis of pulmonary fibrosis. In a search for anti-inflammatory agents that might prevent this process, we asked whether colchicine might modulate the abundance of PDGF(B) and TGF-beta mRNA, as well as the mRNA of early growth response gene 2 (EGR2), in human macrophages. Colchicine caused a dose- and time-dependent increase in PDGF(B), but not TGF-beta or EGR2, mRNA in human macrophages derived from culture of peripheral blood monocytes. Similarly, colchicine caused an increase in PDGF(B) mRNA in human alveolar macrophages obtained from normal volunteers. Colchicine also caused an increase in PDGF(B) protein production by macrophages, as determined by enzyme-linked immunosorbent assay. Interferon-gamma further increased the PDGF(B) mRNA abundance in human alveolar but not monocyte-derived macrophages. The effect of coincubation with dibutyryl-cAMP (dBcAMP) was assessed in an attempt to prevent the colchicine-induced increase in PDGF(B) mRNA. dBcAMP alone resulted in no increase in PDGF(B) mRNA or alteration in TGF-beta mRNA but resulted in a reduction in EGR2 mRNA. When added with colchicine, dBcAMP completely abrogated the colchicine-induced increase in PDGF(B) mRNA but had little effect on TGF-beta mRNA. These data, showing that colchicine increased macrophage PDGF(B) mRNA in human macrophages and that this was prevented by coincubation with dBcAMP, lead us to speculate that colchicine may not be helpful in preventing the contribution of macrophage PDGF(B) gene activation to the pathogenesis of lung fibrosis. However, this effect of colchicine may be prevented by increasing intracellular cAMP in macrophages.
The development of tamoxifen therapy to treat selected patients, with all stages of breast cancer, has provided the clinical community with an efficacious and safe drug for long-term therapy. Issues of safety are under constant review, but justified concerns about high doses of tamoxifen acting as a promoter of liver cancer in rats or as a promoter of endometrial cancer in women have not, as yet, proved to be of clinical relevance. The situation will continue to be reviewed during the development of the prevention studies in Europe and the United States because an improvement in women's health is the ultimate goal of these programs. The hallmark for the successful development of tamoxifen has been the close cooperation between the laboratory and the clinic. The clinical strategy of long-term tamoxifen therapy is a direct application of a laboratory concept. Furthermore, potential problems in the clinic have been identified in the laboratory, and the clinical community has responded quickly to evaluate the real risks to the patient population. This close cooperation will continue. Issues of drug resistance, new antiestrogen development, and the application of the knowledge about steroid receptors to develop targeted gene therapies are being addressed so that additional treatment approaches for breast cancer will be in place by the turn of the century.
Expression of P-glycoprotein, the product of the MDR1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). P-glycoprotein (relative molecular mass 170,000) is an ATP-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. It is a member of the ABC (ATP-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major histocompatibility locus, and the product of the cystic fibrosis gene (the cystic fibrosis transmembrane regulator, CFTR). CFTR is located in the apical membranes of many secretory epithelia and is associated with a cyclic AMP-regulated chloride channel. At least two other chloride channels are present in epithelial cells, regulated by cell volume and by intracellular Ca2+, respectively. Because of the structural and sequence similarities between P-glycoprotein and CFTR, and because P-glycoprotein is abundant in many secretory epithelia, we examined whether P-glycoprotein might be associated with one or other of these channels. We report here that expression of P-glycoprotein generates volume-regulated, ATP-dependent, chloride-selective channels, with properties similar to channels characterized previously in epithelial cells.
Single-channel currents of an anionic channel in the plasma membrane of cultured bovine aortic endothelial cells have been recorded with the patch-clamp technique. The channel is selective for chloride over cations, and has an average single channel conductance of 382 picosiemens in symmetric 140 millimoles of chloride. In addition to the main conductance state it shows well-defined subconductance states of about 50, 100, 150 and 200 picosiemens. The channel is very active at membrane potentials close to 0 mV, but steps to either positive or negative membrane potentials above +/- 20 millivolt lead to a rapid inactivation of the channel. Changes in the concentrations of free calcium or adenosine tri-phosphate on the cytosolic surface do not influence channel activity. The chloride channel rarely opens at resting membrane potential, but it may help repolarize endothelial cells following depolarizing stimuli.
Single, large-conductance chloride-selective channels were studied in the membrane of pig aortic endothelial cells. These channels were usually inactive in cell-attached recordings and activated spontaneously upon formation of inside-out patches or amphotericin B-perforated vesicles. Channel activity was voltage dependent, with a maximum open probability within the range of -20 mV to + 20 mV. Addition of 1 mM Zn2+ to either the cytoplasmic or extracellular side blocked channel activity reversibly. Extracellular 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) blocked the channels; the concentration necessary for half-maximum blockade was 100 mumol/l. The frequency of observing channels in cell-attached patches increased from less than 5% to 27% when cells were treated for several minutes with 1 mumol/l bradykinin and to 80% in the presence of the calcium ionophore A23187 (1 mumol/l). Both agents increase the cytoplasmic Ca2+ concentration, thereby stimulating nitric oxide (NO) synthesis and cGMP formation in endothelial cells. Sodium nitroprusside (100 mumol/l), which spontaneously releases NO, did not increase Cl- channel activity in intact cells. Polymyxin B (100 mumol/l), an inhibitor of protein kinase C, clearly enhanced Cl- channel activity in intact cells, resulting in the observation of Cl- channels in 70% of cell-attached patches. Our results demonstrate the existence of a large-conductance (LC-type) Cl- channel in vascular endothelium which is subject to a complex cellular regulation, possibly involving inhibition via phosphorylation by protein kinase C, and activation by a Ca2(+)-dependent process which is different from the NO/cGMP pathway.
The human multidrug resistance P-glycoprotein is an active transporter that pumps cytotoxic drugs out of cells. Expression of P-glycoprotein is also associated with a volume-activated chloride channel. Here we address the relationship between these two functions. Drug transport requires ATP hydrolysis while, in contrast, ATP binding is sufficient to enable activation of the chloride channel. The chloride channel and drug transport activities of P-glycoprotein appear to reflect two distinct functional states of the protein that can be interconverted by changes in tonicity. Transportable drugs prevent channel activation but have no effect on channel activity once it has been preactivated by hypotonicity. The transport and channel functions of P-glycoprotein have been separated by directed mutations in the nucleotide-binding domains of the protein. These data provide further evidence that P-glycoprotein is bifunctional with both transport and channel activities. Implications for the design of chemotherapeutic drugs and for the function of the related cystic fibrosis gene product, CFTR, are discussed.
We examined the role of ovarian steroids in regulating Ca2+ channels in rat uterine smooth muscle. Ca2+ currents (ICa) in myometrial cells from nonpregnant adult rats and immature rats injected with either estrogen or progesterone or estrogen plus progesterone were measured with the whole cell patch-clamp method. ICa was more prominent in cells from diestrous rats than in cells from estrous rats. In cells from immature rats the ICa density was significantly greater in cells from progesterone-injected rats than in cells from estrogen-injected or noninjected rats. ICa in cells from rats injected simultaneously with progesterone and the progesterone antagonist RU-486 was not significantly greater than those from noninjected rats. These increases in ICa density are not the result of changes in ICa activation kinetics or voltage dependence, since both are unaffected by steroid injection. The kinetics and voltage dependence of the ICa current in cells from immature and nonpregnant adult rats are similar, suggesting that they represent a single population of Ca2+ channels.
An 'anti-oestrogen' such as tamoxifen may protect prophylactically against breast cancer. At the Royal Marsden Hospital, the blind randomised feasibility study of tamoxifen 20 mg per day versus placebo in 200 healthy women has been extended into a pilot trial. A total of 435 women with a family history of breast cancer have been accrued. Compliance, acute toxicity, clotting factors, lipids and bone mass were assessed. The pilot trial has confirmed the findings of the feasibility study. Compliance was high and the frequency of side-effects was similar in both groups, except for a significant increase in hot flushes in the tamoxifen-treated women (33 vs. 17%). Bone mass and clotting factors were not affected. Tamoxifen significantly reduced serum cholesterol, low-density lipoprotein cholesterol (LDLC) and apolipoprotein B levels in post-menopausal women. In premenopausal women, the effects on lipids and lipoproteins was smaller with a significant fall in total serum cholesterol and LDLC only. The trial has approval to accrue up to 1000 women.
A Cl- channel with large single-unit conductance and characteristic voltage-dependent inactivation was studied on cultured human fibroblasts. The channel was activated only after excision and lasting depolarization of the membrane patch. In inside-out configuration and in symmetrical 135 mM NaCl, the conductance was 300 pS. The channel was usually open at the membrane potentials between -20 to +20 mV, while more negative or positive voltages closed the channel. The time course of this apparent inactivation process was dependent on increasing potential. Recovery from inactivation was made possible by returning the membrane potential to 0 mV. The channel was selective to Cl- over Na+ with a PCl/PNa of 6. The order of permeability among anions was: I greater than Br = Cl greater than isethionate greater than F greater than glutamate. The channel was blocked by internal application of a derivative of the diphenylamine-2-carboxilate (Blocker 144) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.
In the presence of porcine aortic endothelial cytosol, soluble guanylyl cyclase purified from bovine lung was activated by L-arginine up to 2.5-fold, with an EC50 of about 6 microM. This activation was dependent on NADPH and Ca2+. The EC50 for Ca2+ was about 60 nM. No effect of L-arginine on guanylyl cyclase was observed when the cytosolic proteins were heat-denaturated. The effect of L-arginine was inhibited by NG-monomethyl-L-arginine and hemoglobin. These results indicate that endothelial cells contain a cytosolic enzyme which is directly or indirectly regulated by Ca2+ and converts L-arginine into a compound which in stimulating soluble guanylyl cyclase behaves similar to endothelium-derived relaxing factor.
The biological response to anti-estrogens is very variable and depends on the animal species considered, the target organ, the parameter studied, and the experimental conditions. Anti-estrogens can bind specifically, (1) to the estrogen receptor, (2) to the typical anti-estrogen specific binding site, and (3) to low density lipoproteins in the plasma. Using a monoclonal antibody against the estrogen receptor, different immunological characteristics of the anti-estrogen-receptor complex can be observed. This difference could explain some of the different biological effects. Studies using different human mammary cancer cell lines (hormone-dependent) show that anti-estrogens are active in decreasing cell proliferation. Also, anti-estrogens can block proteins specifically produced by these cells. Some of these proteins could act as growth or inhibitory factors. Estrogen sulfates are the main precursors of estradiol in breast tissues and this conversion is significantly decreased by anti-estrogens. It is accepted that the main pathway of action of anti-estrogens is through the estrogen receptor, but recent information suggests the possibility that this is not the only step in the mechanism of action of anti-estrogens.
Using the patch-clamp technique single-channel parameters and kinetic properties of an anionic channel are studied in cell-attached and excised membrane patches from peritoneal macrophages of mouse and cultured chicken myotubes. The channel has a unit conductance of about 340 pS with a Q10 of 1.3. In addition a subconductance state of about 210 pS is frequently adopted. The selectivity ratio of PCl/PNa is about 5. In excised membrane patches the activation of the channel appears to be independent of Ca either in the cytoplasmic or the extracellular medium. The channel induced current fluctuations appear in a burst like pattern. At least three non-conducting channel states could be distinguished kinetically. The mean lifetime of one of these states exhibits a strikingly steep voltage dependence which could be correlated to the mean shut interval between consecutive bursts. A similar steep voltage dependence was found for the mean lifetimes of bursts. The burst kinetic shows an about bell-shaped dependence on voltage. The results suggest that the burst kinetic and the kinetic within bursts are regulated by independent voltage sensitive mechanisms. The burst kinetic was analyzed by ensemble averages of voltage-jump current relaxations performed on the single channel level. A model of two voltage-sensitive gates is proposed for a description of the burst kinetic.
The present review will deal with the following topics: the characteristics of the relaxation of isolated arteries by ACh and the evidence for its mediation by EDRF, agents and conditions that interfere with the relaxation by ACh, the role of calcium in the release of EDRF, other agents that relax arteries in an endothelium-dependent manner, the role of endothelial cells in the relaxation of veins and peripheral resistance vessels, proposed roles for endothelial cells in facilitating contractions of blood vessels, and speculation about the nature of EDRF and the mechanism by which it produces relaxation.
Women rarely suffer cardiovascular dysfunction before menopause, but by the age of 65 a woman becomes as vulnerable to cardiovascular mortality as a man. It has been proposed that estrogens protect against cardiovascular disease; however, the physiological basis of estrogen protection is unknown. In the present study the mechanism of estrogen-induced relaxation of coronary arteries was investigated at the tissue, cellular, and molecular levels. Tissue studies demonstrate that 17 beta-estradiol relaxes porcine coronary arteries by an endothelium-independent mechanism involving K+ efflux, and subsequent studies employing the patch-clamp technique confirmed that estrogen stimulates K+ channel gating in coronary smooth muscle. Perforated-patch recordings from metabolically intact coronary myocytes revealed that 17 beta-estradiol more than doubles steady state outward currents in these cells at positive voltages. Studies of on-cell patches demonstrated a potent stimulatory effect of 17 beta-estradiol on the gating of the large-conductance, Ca(2+)- and voltage-activated K+ (BKCa) channels, while 17 alpha-estradiol had no effect. Furthermore, blocking BKCa channels in intact arteries inhibited estrogen-induced relaxation. The effect of 17 beta-estradiol on BKCa channels was blocked by inhibiting cGMP-dependent protein kinase (PKG) activity and was mimicked by exogenous cGMP or by stimulating PKG activity. Therefore, we propose that 17 beta-estradiol relaxes coronary arteries by opening BKCa channels via cGMP-dependent phosphorylation. This novel mechanism could account for the hypotensive effect of estrogens and help explain, at least in part, why postmenopausal estrogen therapy lowers the risk of cardiovascular disease.
Epithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n = 10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl- permeability ratio of 0.17 +/- 0.04) and had a unitary conductance of approximately 380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5'-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5' guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both Gi alpha-2 and Gi alpha-3 were present in significant amounts, whereas Gi alpha-1 and Go alpha were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.
Chloride channels play important functions in different aspects of cell physiology including volume regulation, transepithelial ion transport and stabilization of membrane potential. In recent years the molecular identity of the chloride channels defective in cystic fibrosis and myotonia congenita has been elucidated, highlighting the importance of anion-selective channels in cell and tissue function. Concurrently, several proteins have been identified as chloride channels along with proteins that possess channel regulatory behavior. Novel interactions with more potent pharmacological compounds have been reported with different chloride channels. This burgeoning field of interest is reviewed.
Antiestrogens antagonize many genomic effects of estrogen through binding to the nuclear estrogen receptor. We report here that NIH3T3 fibroblasts grown in the presence of colchicine acquire the activation of a large conductance chloride channel upon exposure to extracellular but not intracellular antiestrogens. This effect can be prevented by extracellular 17 beta-estradiol, but not intracellular 17 beta-estradiol or extracellular 17 alpha-estradiol. This is the first demonstration of a regulatory role for antiestrogens and estrogens in the regulation of ionic channels occurring through an interaction of these compounds with a plasma membrane binding site distinct from the classical estrogen receptor and subsequent activation of intracellular second messenger pathway (or pathways).
1. The effects of estrogens estradiol (E2, 10(-6)-10(-4) M) and diethylstilbestrol (DES, 10(-6)-10(-4) M) and the antiestrogens nafoxidine (N, 10(-6)-10(-4) M), tamoxifen (T, 10(-6)-6 x 10(-4) M), tamoxifen ethyl bromide (TEB, 10(-4) M) and ICI 164,384 (ICI, 10(-5) M) on tonic contractions induced by oxytocin (2 x 10(-8) M) or vanadate (3 x 10(-4) M) in rat uterus incubated in calcium-free EDTA treated solution have been assayed. 2. E2 and DES relaxed the tonic contraction induced by oxytocin in a dose dependent way (EC50: 1.11 +/- 0.01 x 10(-4) M and 1.5 +/- 0.07 x 10(-5) M). The vanadate-induced contraction only was relaxed with DES (57.62 +/- 2.38% at 10(-3) M). 3. The effect of DES on oxytocin contraction was unmodified by the protein synthesis inhibitor cycloheximide (10 micrograms/ml) and by the cyclooxygenase inhibitor indomethacin (3 x 10(-6) M), but enhanced by the intracellular calcium release inhibitor TMB-8 (10(-5) M). The antiestrogen tamoxifen (3 x 10(-5) M) promotes the relaxing effect of DES. 4. The antiestrogens N, and T, but not ICI, relaxed the oxytocin-induced contraction (EC50: 4.51 +/- 0.43 x 10(-5) M and 2.27 +/- 0.05 x 10(-4) M). TEB (10(-4) M) produces a relaxation of 74.5 +/- 2.11%. The vanadate contraction is also relaxed by T (EC50: 6.03 +/- 0.04 x 10(-4) M). 5. The effect of T on oxytocin contraction was unmodified with cycloheximide or TMB-8 but decreased with indomethacin.
Using immunocytochemical techniques, cells containing estrogen and progestin receptors have been observed in many discrete regions of the guinea-pig forebrain, including the mediobasal hypothalamus and preoptic area. While most reaction product is located within cell nuclei, we have reported abundant reaction product in perikaryal cytoplasm and neuronal processes as well. Ultrastructural analysis has revealed the presence of estrogen and progestin receptors in atypical subcellular sites within the hypothalamus, including dendrites and axon terminals. In order to determine if microtubule-dependent intracellular transport is involved in intraneuronal transport of steroid hormone receptors, ovariectomized guinea-pigs were injected intracerebroventricularly with the microtubule inhibitor, colchicine, and brain sections at the level of the hypothalamus were immunostained for estrogen receptors. This treatment resulted in the appearance of estrogen receptor immunoreactivity in the paraventricular and mediodorsal thalamic region, areas typically devoid of estrogen receptor-immunoreactive cells in guinea-pigs. In a second study on progestin receptors, we observed the colchicine-induced accumulation of progestin receptor immunoreactivity in the paraventricular thalamic, mediodorsal thalamic and lateral dorsal thalamic areas as well as in the medial amygdala, all areas typically devoid of progestin receptor immunoreactivity. While estradiol injection induced progestin receptor immunoreactivity in the hypothalamus and preoptic area as described previously, it had no effect on the colchicine-induced accumulation in the thalamus and amygdala. These results provide evidence that in some neurons, progestin receptors and estrogen receptors are transported intracellularly, apparently at a rapid enough rate that they do not ordinarily accumulate within the perikaryon.
Estrogen is an important vasoprotective molecule that causes the rapid dilation of blood vessels by activating endothelial nitric oxide synthase (eNOS) through an unknown mechanism. In studies of intact ovine endothelial cells, 17beta-estradiol (E2) caused acute (five-minute) activation of eNOS that was unaffected by actinomycin D but was fully inhibited by concomitant acute treatment with specific estrogen receptor (ER) antagonists. Overexpression of the known transcription factor ERalpha led to marked enhancement of the acute response to E2, and this was blocked by ER antagonists, was specific to E2, and required the ERalpha hormone-binding domain. In addition, the acute response of eNOS to E2 was reconstituted in COS-7 cells cotransfected with wild-type ERalpha and eNOS, but not by transfection with eNOS alone. Furthermore, the inhibition of tyrosine kinases or mitogen-activated protein (MAP) kinase kinase prevented the activation of eNOS by E2, and E2 caused rapid ER-dependent activation of MAP kinase. These findings demonstrate that the short-term effects of estrogen central to cardiovascular physiology are mediated by ERalpha functioning in a novel, nongenomic manner to activate eNOS via MAP kinase-dependent mechanisms.
