The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels.
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Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance-induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance-mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance-related vascular disease.
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Transient receptor potential canonical (TRPC) proteins assemble to form ion channels that enable influx of calcium and sodium ions into cells. There are 6 TRPC proteins in humans but more TRPC channels may arise through heteromerization among TRPCs and other types of TRP protein. They are widely expressed and have multiple functions throughout the peripheral and central systems of the body. This review summarizes current knowledge of the characteristics of TRPC channels and discusses principles by which the channels operate. Modulators of the channels include lipids, redox factors, and agonists at G-protein and tyrosine kinase receptors. The channels enable coupling between these factors and the calcium ion, which is a master intracellular regulator of multiple cell functions. In the context of this information the review gives specific consideration to TRPC channels in vascular cells, which include endothelial cells, vascular smooth muscle cells, perivascular adipocytes, and cells of the hematopoietic lineage. It is discussed that the channels may have most significance as drivers of change when there is strain or insult in physiology or disease. The TRPC proteins constitute a substantial and important group of calcium-permeable channels. They remain enigmatic but there is increasing understanding of their properties and recognition of their importance in the vasculature as well as in other systems such as the myocardium.  
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Reactive oxygen species such as H2O2 elevates the cytosolic Ca(2+) concentration ([Ca(2+)]c) and causes cell death via poly(ADPR) polymerase (PARP) activation, which also represents the primary mechanism by which H2O2 activate the TRPM2 channel as a Ca(2+)-permeable channel present in the plasma membrane or an intracellular Ca(2+)-release channel. The present study aimed to define the contribution and mechanisms of the TRPM2 channels in macrophage cells in mediating Ca(2+) signalling and cell death during initial response to H2O2, using mouse peritoneal macrophage, RAW264.7 and differentiated THP-1 cells. H2O2 evoked robust increases in the [Ca(2+)]c and such Ca(2+) responses were significantly greater at body temperature than room temperature. H2O2-induced Ca(2+) responses were strongly inhibited by pretreatment with PJ-34, a PARP inhibitor, and largely prevented by removal of extracellular Ca(2+). Furthermore, H2O2-induced increases in the [Ca(2+)]c were completely abolished in macrophage cells isolated from trpm2-/- mice. H2O2 reduced macrophage cell viability in a duration- and concentration-dependent manner. H2O2-induced cell death was significantly attenuated by pretreatment with PJ-34 and TRPM2 channel deficiency, but remained significant and persistent. Taken together, these results show that the TRPM2 channel in macrophage cells functions as a cell surface Ca(2+)-permeable channel that mediates Ca(2+) influx and constitutes the principal Ca(2+) signalling mechanism, but has a limited, albeit significant, role in cell death during early exposure to H2O2.
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Transient receptor potential canonical (TRPC) channels are the canonical (C) subset of the TRP proteins, which are widely expressed in mammalian cells. They are thought to be primarily involved in determining calcium and sodium entry and have wide-ranging functions that include regulation of cell proliferation, motility and contraction. The channels are modulated by a multiplicity of factors, putatively existing as integrators in the plasma membrane. This review considers the sensitivities of TRPC channels to lipids that include diacylglycerols, phosphatidylinositol bisphosphate, lysophospholipids, oxidized phospholipids, arachidonic acid and its metabolites, sphingosine-1-phosphate, cholesterol and some steroidal derivatives and other lipid factors such as gangliosides. Promiscuous and selective lipid sensing have been detected. There appear to be close working relationships with lipids of the phospholipase C and A(2) enzyme systems, which may enable integration with receptor signalling and membrane stretch. There are differences in the properties of each TRPC channel that are further complicated by TRPC heteromultimerization. The lipids modulate activity of the channels or insertion in the plasma membrane. Lipid microenvironments and intermediate sensing proteins have been described that include caveolae, G protein signalling, SEC14-like and spectrin-type domains 1 (SESTD1) and podocin. The data suggest that lipid sensing is an important aspect of TRPC channel biology enabling integration with other signalling systems.
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Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01-10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.
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Orai1 was discovered in T cells as a calcium-selective channel that is activated by store depletion. Recent studies suggest that it is expressed and functionally important also in blood vessels, not only because haematopoietic cells can incorporate in the vascular wall but also because Orai1 is expressed and functional in vascular smooth muscle cells and endothelial cells. This article summarises the arising observations in this new area of vascular research and debates underlying issues and challenges for future investigations. The primary focus is on vascular smooth muscle cells and endothelial cells. Specific topics include Orai1 expression; Orai1 roles in store-operated calcium entry and ionic currents of store-depleted cells; blockade of Orai1-related signals by Synta 66 and other pharmacology; activation or regulation of Orai1-related signals by physiological substances and compartments; stromal interaction molecules and the relationship of Orai1 to other ion channels, transporters and pumps; transient receptor potential canonical channels and their contribution to store-operated calcium entry; roles of Orai1 in vascular tone, remodelling, thrombus formation and inflammation; and Orai2 and Orai3. Overall, the observations suggest the existence of an additional, previously unrecognised, calcium channel of the vascular wall that is functionally important particularly in remodelling but probably also in certain vasoconstrictor contexts.
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Orai1 was discovered in T cells as a calcium-selective channel that is activated by store depletion. Recent studies suggest that it is expressed and functionally important also in blood vessels, not only because haematopoietic cells can incorporate in the vascular wall but also because Orai1 is expressed and functional in vascular smooth muscle cells and endothelial cells. This article summarises the arising observations in this new area of vascular research and debates underlying issues and challenges for future investigations. The primary focus is on vascular smooth muscle cells and endothelial cells. Specific topics include Orai1 expression; Orai1 roles in store-operated calcium entry and ionic currents of store-depleted cells; blockade of Orai1-related signals by Synta 66 and other pharmacology; activation or regulation of Orai1-related signals by physiological substances and compartments; stromal interaction molecules and the relationship of Orai1 to other ion channels, transporters and pumps; transient receptor potential canonical channels and their contribution to store-operated calcium entry; roles of Orai1 in vascular tone, remodelling, thrombus formation and inflammation; and Orai2 and Orai3. Overall, the observations suggest the existence of an additional, previously unrecognised, calcium channel of the vascular wall that is functionally important particularly in remodelling but probably also in certain vasoconstrictor contexts.© 2012 The Author(s).
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Calcium entry through Orai1 channels drives vascular smooth muscle cell migration and neointimal hyperplasia. The channels are activated by the important growth factor platelet-derived growth factor (PDGF). Channel activation is suggested to depend on store depletion, which redistributes and clusters stromal interaction molecule 1 (STIM1), which then coclusters and activates Orai1.
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Calcium entry is pivotal in the heart and blood vessels, but its significance and mechanisms in adipose tissue are largely unknown. An important factor produced by adipocytes is adiponectin, which confers myocardial protection, insulin-sensitization, and antiatherosclerotic effects.
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We recently demonstrated that reducing IGF-1 receptor (IGF-1R) numbers in the endothelium enhances nitric oxide (NO) bioavailability and endothelial cell insulin sensitivity. In the present report, we aimed to examine the effect of increasing IGF-1R on endothelial cell function and repair. To examine the effect of increasing IGF-1R in the endothelium, we generated mice overexpressing human IGF-1R in the endothelium (human IGF-1R endothelium-overexpressing mice[hIGFREO]) under direction of the Tie2 promoter enhancer. hIGFREO aorta had reduced basal NO bioavailability (percent constriction to N(G)-monomethyl-l-arginine [mean (SEM) wild type 106% (30%); hIGFREO 48% (10%)]; P<0.05). Endothelial cells from hIGFREO had reduced insulin-stimulated endothelial NO synthase activation (mean [SEM] wild type 170% [25%], hIGFREO 58% [3%]; P = 0.04) and insulin-stimulated NO release (mean [SEM] wild type 4,500 AU [1,000], hIGFREO 1,500 AU [700]; P<0.05). hIGFREO mice had enhanced endothelium regeneration after denuding arterial injury (mean [SEM] percent recovered area, wild type 57% [2%], hIGFREO 47% [5%]; P<0.05) and enhanced endothelial cell migration in vitro. The IGF-1R, although reducing NO bioavailability, enhances in situ endothelium regeneration. Manipulating IGF-1R in the endothelium may be a useful strategy to treat disorders of vascular growth and repair.
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Rationale: Calcium entry is pivotal in the heart and blood vessels, but its significance and mechanisms in adipose tissue are largely unknown. An important factor produced by adipocytes is adiponectin, which confers myocardial protection, insulin-sensitization, and antiatherosclerotic effects. Objective: To investigate the relevance of calcium channels to adipocytes and the production of adiponectin. Methods and Results: Microarray analysis led to identification of transient receptor potential canonical (TRPC)1 and TRPC5 as channel subunits that are induced when adipocytesmature. Both subunits were found in perivascular fat of patients with atherosclerosis. Intracellular calcium and patch-clamp measurements showed that adipocytes exhibit constitutively active calcium-permeable nonselective cationic channels that depend on TRPC1 and TRPC5. The activity could be enhanced by lanthanum or rosiglitazone, known stimulators of TRPC5 and TRPC5-containing channels. Screening identified lipid modulators of the channels that are relevant to adipose biology. Dietaryω-3 fatty acids (eg, α-linolenic acid) were inhibitory at concentrations that are achieved by ingestion. The adipocyte TRPC1/TRPC5-containing channel was functionally negative for the generation of adiponectin because channel blockade by antibodies, knock-down of TRPC1-TRPC5 in vitro, or conditional disruption of calcium permeability in TRPC5-incorporating channels in vivo increased the generation of adiponectin. The previously recognized capability of α-linolenic acid to stimulate the generation of adiponectin was lost when calcium permeability in the channels was disrupted. Conclusions: The data suggest that TRPC1 and TRPC5 contribute a constitutively active heteromultimeric channel of adipocytes that negatively regulates adiponectin and through which ω-3 fatty acids enhance the anti-inflammatory adipokine, adiponectin. © 2012 American Heart Association, Inc.
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Calcium entry through Orai1 channels drives vascular smooth muscle cell migration and neointimal hyperplasia. The channels are activated by the important growth factor platelet-derived growth factor (PDGF). Channel activation is suggested to depend on store depletion, which redistributes and clusters stromal interaction molecule 1 (STIM1), which then coclusters and activates Orai1. To determine the relevance of STIM1 and Orai1 redistribution in PDGF responses. Vascular smooth muscle cells were cultured from human saphenous vein. STIM1 and Orai1 were tagged with green and red fluorescent proteins to track them in live cells. Under basal conditions, the proteins were mobile but mostly independent of each other. Inhibition of sarco-endoplasmic reticulum calcium ATPase led to store depletion and dramatic redistribution of STIM1 and Orai1 into coclusters. PDGF did not evoke redistribution, even though it caused calcium release and Orai1-mediated calcium entry in the same time period. After chemical blockade of Orai1-mediated calcium entry, however, PDGF caused redistribution. Similarly, mutagenic disruption of calcium flux through Orai1 caused PDGF to evoke redistribution, showing that calcium flux through the wild-type channels had been filling the stores. Acidification of the extracellular medium to pH 6.4 caused inhibition of Orai1-mediated calcium entry and conferred capability for PDGF to evoke complete redistribution and coclustering. The data suggest that PDGF has a nonclustering mechanism by which to activate Orai1 channels and maintain calcium stores replete. Redistribution and clustering become important, however, when the endoplasmic reticulum stress signal of store depletion arises, for example when acidosis inhibits Orai1 channels.
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Acidic pH is an important parameter regulating ion channel activity and its biological function. This study investigated inhibition of the hTRPM2 channels by extracellular acidic pH and compared the sensitivity of human (h) and mouse (m) TRPM2 channel to such an inhibition. The initial inhibition of hTRPM2 channel currents was substantially reversible, but the reversibility progressively diminished as the exposure to acidic pH was prolonged and it was essentially lost in the steady state, suggesting that extracellular acidic pH induces initial reversible inhibition and subsequent irreversible inactivation. Like the hTRPM2 channel, the mTRPM2 channel was sensitive to inhibition by pH 4.0-5.5, but the kinetics was significantly slower. Moreover, in contrast to the complete inhibition of the hTRPM2 channel, the mTRPM2 channel was insensitive to pH 6.0. Replacement of residue Gln(992) in the outer pore with the equivalent residue His(995) in the hTRPM2 channel resulted in a mutant mTRPM2 channel with the pH sensitivity and kinetics of inhibition of the wild-type hTRPM2 channel. Conversely, the reciprocal mutation H995Q in the hTRPM2 channel dramatically slowed down the kinetics of inhibition. Swapping other residues in the pore region failed to produce such opposing effects. Taken together, our results suggest a crucial role of residue His(995)/Gln(992) in the outer pore of TRPM2 channels in determining species-dependent effects of extracellular acidic pH.
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The aim was to advance the understanding of Orai proteins and identify a specific inhibitor of the associated calcium entry mechanism in vascular smooth muscle cells (VSMCs).
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In mice, haploinsufficiency of the IGF-1 receptor (IGF-1R(+/-)), at a whole-body level, increases resistance to inflammation and oxidative stress, but the underlying mechanisms are unclear. We hypothesized that by forming insulin-resistant heterodimers composed of one IGF-1Rαβ and one insulin receptor (IR), IRαβ complex in endothelial cells (ECs), IGF-1R reduces free IR, which reduces EC insulin sensitivity and generation of the antioxidant/anti-inflammatory signaling radical nitric oxide (NO).
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The Transient Receptor Potential Canonical 5 (TRPC5) protein forms calcium-permeable cationic channels that are stimulated by G protein-coupled receptor agonists. The signaling pathways of such agonist effects are poorly understood. Here we investigated the potential for involvement of lysophosphatidylcholine (LPC) and arachidonic acid generated by group 6 (GVI) phospholipase A2 (PLA2) enzymes, focusing on stimulation of TRPC5 by sphingosine-1-phosphate (S1P) which acts via a pertussis toxin-sensitive (Gi/o protein) pathway without Ca2+-release. Experiments were on HEK 293 cells containing conditional expression of human TRPC5. Channel activity was recorded using an intracellular calcium indicator or whole-cell patch-clamp and PLA2 activity was detected using 3H-arachidonic acid. S1P stimulated PLA2 and TRPC5 activities. Both effects were suppressed by the GVI PLA2 inhibitor bromoenol lactone. Knock-down of GVI PLA2 by RNA interference suppressed channel activity evoked by S1P whereas activity evoked by the direct channel stimulator LPC was unaffected. Arachidonic acid did not stimulate the channels. Prior exposure of channels to LPC but not arachidonic acid suppressed channel activity evoked by S1P but not gadolinium, a putative direct stimulator of the channels. The data suggest roles of LPC and GVI PLA2 in S1P-evoked TRPC5 activity.
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Transient Receptor Potential Canonical 1 (TRPC1) is a widely-expressed mammalian cationic channel with functional effects that include stimulation of cardiovascular remodelling. The initial aim of this study was to investigate variation in TRPC1-encoding gene transcripts.
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Acidic pH is an important parameter regulating ion channel activity and its biological function. This study investigated inhibition of the hTRPM2 channels by extracellular acidic pH and compared the sensitivity of human (h) and mouse (m) TRPM2 channel to such an inhibition. The initial inhibition of hTRPM2 channel currents was substantially reversible, but the reversibility progressively diminished as the exposure to acidic pH was prolonged and it was essentially lost in the steady state, suggesting that extracellular acidic pH induces initial reversible inhibition and subsequent irreversible inactivation. Like the hTRPM2 channel, the mTRPM2 channel was sensitive to inhibition by pH 4.0-5.5, but the kinetics was significantly slower. Moreover, in contrast to the complete inhibition of the hTRPM2 channel, the mTRPM2 channel was insensitive to pH 6.0. Replacement of residue Gln in the outer pore with the equivalent residue Hisin the hTRPM2 channel resulted in a mutant mTRPM2 channel with the pH sensitivity and kinetics of inhibition of the wild-type hTRPM2 channel. Conversely, the reciprocal mutation H995Q in the hTRPM2 channel dramatically slowed down the kinetics of inhibition. Swapping other residues in the pore region failed to produce such opposing effects. Taken together, our results suggest a crucial role of residue His /Gln in the outer pore of TRPM2 channels in determining species-dependent effects of extracellular acidic pH. © 2011 Springer-Verlag.
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Voltage-gated K+ channels (K(V) channels) are encoded by the KCNx gene family and have such a wide range of properties that it is necessary to identify the precise expression profile that is instrumental in governing the electrical phenotype of a cell and its response to extrinsic factors. Real-time quantitative RT-PCR methodology has been developed and validated for specific RNA species in vascular smooth muscle cells. We have shown that most of the KCNA gene family, encoding the major K(V)alpha1 subunits, was markedly up-regulated in the resistance artery compared to the thoracic aorta, in line with reported patch-clamp recordings. Thus quantitative real-time RT-PCR data can be translated into physiological response.
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Blood vessels are essential for animal life, allowing flow of oxygen and nutrients to tissues and removal of waste products. Consequently, inappropriate remodelling of blood vessels, resulting in occlusion, can lead to disabling or catastrophic events: heart attacks, strokes and claudication. An important cell type of remodelling is the VSMC (vascular smooth-muscle cell), a fascinating cell that contributes significantly to occlusive vascular diseases by virtue of its ability to 'modulate' to a cell that no longer contracts and arranges radially in the medial layer of the vessel wall but migrates, invades, proliferates and adopts phenotypes of other cells. An intriguing aspect of modulation is switching to different ion transport systems. Initial events include loss of the Ca(V)1.2 (L-type voltage-gated calcium) channel and gain of the K(Ca)3.1 (IKCa) potassium channel, which putatively occur to enable membrane hyperpolarization that increases rather than decreases a type of calcium entry coupled with cell cycle activity, cell proliferation and cell migration. This type of calcium entry is related to store- and receptor-operated calcium entry phenomena, which, in VSMCs, are contributed to by TRPC [TRP (transient receptor potential) canonical] channel subunits. Instead of being voltage-gated, these channels are chemically gated - importantly, by key phospholipid factors of vascular development and disease. This brief review focuses on the hypothesis that the transition to a modulated cell may require a switch from predominantly voltage- to predominantly lipid-sensing ion channels.
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K(V)1.5, a voltage-gated potassium channel, has functional importance in regulating blood vessel tone and cardiac action potentials and is a target for numerous therapeutic drug development programs. Despite the importance of K(V)1.5, there is little knowledge of the mechanisms controlling expression of its underlying gene, Kcna5. We identified a 5' flanking region of the murine Kcna5 gene that drives expression of a luciferase reporter gene in primary smooth muscle cells and a smooth muscle cell line. The promoter contained CACCC nucleotide motifs, which we have shown to bind the Sp1 transcription factor in the aorta under physiological conditions in vivo. Inhibition of Sp1-Kcna5 promoter interactions using mithramycin A, a dominant-negative Sp1 mutant, or disruption of the CACCC boxes by mutagenesis inhibited promoter activity. Conversely, expression of exogenous Sp1 augmented promoter activity. Sp1 has known sensitivity to oxidative stress and, consistent with this property, Kcna5 promoter activity was suppressed by hydrogen peroxide-induced oxidative stress. Our results show that Kcna5 promoter activity in vascular smooth muscle is critically dependent on Sp1 regulation via CACCC box motifs and identify mechanisms that potentially influence the expression of K(V)1.5 channel expression in physiological or pathological conditions.
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Canonical transient receptor potential 5 TRPC5 (also TrpC5, trp-5 or trp5) is one of the seven mammalian TRPC proteins. Its known functional property is that of a mixed cationic plasma membrane channel with calcium permeability. It is active alone or as a heteromultimeric assembly with TRPC1; TRPC4 and TRPC3 may also be involved. Multiple activators of TRPC5 are emerging, including various G protein-coupled receptor agonists, lysophospholipids, lanthanide ions and, in some contexts, calcium store depletion. Intracellular calcium has complex impact on TRPC5, including a permissive role for other activators, as well as inhibition at high concentrations. Protein kinase C is inhibitory and mediates desensitisation following receptor activation. Tonic TRPC5 activity is detected and may reflect the presence of constitutive activation signals. The channel has voltage dependence but the biological significance of this is unknown; it is partially due to intracellular magnesium blockade at aspartic acid residue 633. Protein partners include calmodulin, CaBP1, enkurin, Na(+)-H+ exchange regulatory factor (NHERF) and stathmin. TRPC5 is included in local vesicular trafficking regulated by growth factors through phosphatidylinositol (PI)-3-kinase, Rac1 and PIP-5-kinase. Inhibition of myosin light chain kinase suppresses TRPC5, possibly via an effect on trafficking. Biological roles of TRPC5 are emerging but more reports on this aspect are needed. One proposed role is as a mediator of calcium entry and excitation in smooth muscle, another as an inhibitor of neuronal growth cone extension. The latter is intriguing in view of the original cloning of the human TRPC5 gene from a region of the X chromosome linked to mental retardation. TRPC5 is a broadly expressed calcium channel with capability to act as an integrator of extracellular and intracellular signals at the level of calcium entry.
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Agonist-induced Ca2+ entry via store-operated Ca2+ (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(-/-) mice was severely decreased (by 70%). Further, agonist- and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(-/-) mice. Deletion of TRPC1 also eliminated sustained Ca2+-dependent potassium channel activity, which depends on Ca2+ entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca2+ signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(-/-) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion.
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The most widely studied stimuli for ion channel activation are changes in membrane voltage and binding of a chemical ligand in a pocket of the channel protein. While modulation by redox potential has also been appreciated our study shows previously unrecognised channel activation via electron donation from the extracellular redox protein thioredoxin (TRX).(1) The ion channel type involved is a member of the Transient Receptor Potential (TRP) family. Activation by TRX led us to consider the relevance of TRP channels to the inflammatory condition of rheumatoid arthritis, where functions of ion channels are relatively unknown and TRX concentrations are high. TRP channel activation was found to be inhibitory for secretion of matrix metalloproteinases, suggesting activation by TRX may have a protective role against disease. Here we expand on our original article and discuss the potential wider implications of the findings in terms of concepts for channel activation and relevance to other ion channel types and systems.
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Smooth muscle cells in arterioles have pivotal roles in the determination of blood pressure and distribution of local blood flow. The cells exhibit calcium entry in response to passive store depletion, but the mechanisms and relevance of this phenomenon are poorly understood. Previously, a role for canonical transient receptor potential 1 (TRPC1) was indicated, but heterologous expression studies showed TRPC1 to have poor function in isolation, suggesting a requirement for additional proteins. Here we test the hypothesis that TRPC5 is such an additional protein, because TRPC5 forms heteromultimeric channels with TRPC1, and RNA encoding TRPC5 is present in arterioles. Recordings were from arteriolar fragments freshly isolated from rabbit pial membrane. Ionic current in response to store depletion has properties like that of the TRPC1/TRPC5 heteromultimer, and so the effect of the E3-targeted, externally acting, anti-TRPC5 blocking antibody (T5E3) was explored. T5E3 suppressed calcium entry in store-depleted arterioles but had no effect in the absence of store depletion. T5E3 preadsorbed to its antigenic peptide did not inhibit calcium entry. TRPC6 is commonly detected in smooth muscle and is present in the arterioles, but T5E3 had no effect on TRPC6. The data suggest that calcium entry occurring in response to passive store depletion in smooth muscle cells of arterioles involves TRPC5 as well as TRPC1.
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TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine ( LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.
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Loss of the smooth muscle contractile phenotype is critical in atherosclerosis and in restenosis after angioplasty, but its early signals are incompletely understood. In this study, we have explored the role of transient receptor potential canonical ( TRPC) proteins, which have been suggested to mediate store-operated Ca2+ entry (SOCE). Contractility of rat cerebral arteries in organ culture is preserved for several days, whereas SOCE is increased. In correlation with this increase is that nifedipine-insensitive whole cell current, activated by depletion of intracellular Ca2+ stores, was increased by 50% in cells isolated from arteries cultured for 3 days. TRPC1 and TRPC6 mRNA were more than fivefold increased in cells isolated after organ culture, whereas TRPC3 was decreased. Immunofluorescent staining and/or Western blotting of arteries and isolated cells showed upregulation of TRPC1 and TRPC6 proteins during organ culture. In intact arteries, TRPC4 expression correlated with the amount of endothelium present. Ca2+ addition after store depletion caused a contraction in cultured, but not in freshly dissected, arteries. A polyclonal TRPC1 antibody directed against an extracellular epitope inhibited this contraction by similar to 50%. To investigate the basis of the TRPC upregulation and assess its possible clinical significance, segments of human internal mammary artery were organ cultured for 24 h and then exposed to balloon dilatation in vitro, followed by further culturing for up to 48 h. After dilatation, TRPC1 and TRPC6 mRNA were progressively increased compared with undilated control segments. The results of this study indicate that vascular injury enhances plasticity in TRPC expression, that TRPC expression correlates with cellular Ca2+ handling, and that TRPC1 is a subunit of upregulated store-operated Ca2+ channels.
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Here we describe a strategy for generating ion-channel inhibitors. It takes advantage of antibody specificity combined with a pattern recognition approach that targets the third extracellular region (E3) of a channel. To test the concept, we first focused on TRPC5, a member of the transient receptor potential (TRP) calcium channel family, the study of which has been hindered by poor pharmacological tools. Extracellular application of E3-targeted anti-TRPC5 antibody led to a specific TRPC5 inhibitor, enabling TRPC5 to be distinguished from its closest family members, and TRPC5 function to be explored in a relatively intractable physiological system. E3 targeting was further applied to voltage-gated sodium channels, leading to discovery of a subtype-specific inhibitor of Na(V)1.5. These examples illustrate the potential power of E3 targeting as a systematic method for producing gene-type specific ion-channel inhibitors for use in routine assays on cells or tissues from a range of species and having therapeutic potential.
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1. The influx of Ca2+, Mg2+ and Na+ and the efflux of K+ have central importance for the function and survival of vascular smooth muscle cells, but progress in understanding the influx/efflux pathways has been restricted by a lack of identification of the genes underlying many of the non-voltage-gated cationic channels. 2. The present review highlights evidence suggesting the genes are mammalian homologues of the Transient Receptor Potential (TRP) gene of the fruit-fly Drosophila. The weight of evidence supports roles for TRPC1, TRPP2/1 and TRPC6, but recent studies point also to TRPC3, TRPC4/5, TRPV2, TRPM4 and TRPM7. 3. Activity of these TRP channels is suggested to modulate contraction and sense changes in intracellular Ca2+ storage, G-protein-coupled receptor activation and osmotic stress. Roles in relation to myogenic tone, actions of vasoconstrictors substances, Mg2+ homeostasis and the vascular injury response are suggested. 4. Knowledge that TRP channels are relevant to vascular smooth muscle cells in both their contractile and proliferative phenotypes should pave the way for a better understanding of vascular biology and provide the basis for the discovery of a new set of therapeutic agents targeted to vascular disease.
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Transient receptor potential canonical 1 (TRPC1) is a transmembrane protein expressed in a range of vertebrate cells including smooth muscle, endothelium, neurones and salivary gland cells. It functions as an element of a mixed cationic Ca(2+-)permeable channel, probably commonly as part of a heterotetrameric assembly involving other related proteins such as TRPC5. Wide-ranging biological roles of TRPC1 are suggested, including regulation of smooth muscle and stem cell proliferation, endothelin-evoked arterial contraction, salivary gland secretion, endothelial permeability, glutamatergic neurotransmission, growth cone turning, neuroprotection, neuronal differentiation, lipid raft integrity and the nuclear factor of activated T-cell transcription factor. The mechanisms by which TRPC1 serves these functions are starting to emerge. At one level, it is apparent that TRPC1 is subcellularly compartmentalised, at least in part in cholesterol-rich caveolae closely associated with sub-plasmalemmal endoplasmic reticulum. At another level, TRPC1 is embedded in a protein complex that can include inositol trisphosphate receptor, homer, calmodulin, caveolin-1, FKBP25, I-mfa, MxA, GluR1 alpha, bFGFR-1, G(q/11) protein, phospholipase C-beta/gamma, protein kinase C-alpha and RhoA. It is also apparent that TRPC1 responds to general stimuli-not only depletion of intracellular Ca2+ stores, but also receptor activation, and membrane stretch. We are at the early stages of understanding of how these various signals and components integrate to form a functional channel, and this article provides a brief overview of current progress.
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This study focused on the hypothesis that KCNA genes (which encode K(V)alpha1 voltage-gated K(+) channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance - the small mesenteric artery. Real-time PCR methodology was developed to measure cell type-specific in situ gene expression. Profiles were determined for arterial myocyte expression of RNA species encoding K(V)alpha1 subunits as well as K(V)beta1, K(V)alpha2.1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Ca)beta1. The seven major KCNA genes were expressed and more readily detected in endothelium-denuded mesenteric resistance artery compared with thoracic aorta; quantification revealed dramatic differential expression of one to two orders of magnitude. There was also four times more RNA encoding K(V)alpha2.1 but less or similar amounts encoding K(V)beta1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Cabeta)1. Patch-clamp recordings from freshly isolated smooth muscle cells revealed dominant K(V)alpha1 K(+) current and current density twice as large in mesenteric cells. Therefore, we suggest the increased RNA production of the resistance artery impacts on physiological function, although there is quantitatively less K(+) current than might be expected. The mechanism conferring up-regulated expression of KCNA genes may be common to all the gene family and play a functional role in the physiological control of blood pressure.
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TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC(50) 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca(2+) at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.
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We determined whether voltage gated K+ (KV) channels are expressed and functional in human detrusor smooth muscle
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This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca2+ channels activated by depletion of intracellular Ca(2+) stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca2+ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca2+ by inhibition of SR Ca2+-ATPase (SERCA) led to sustained elevation of [Ca2+]i that depended on extracellular Ca2+ and slightly enhanced Mn2+ entry. Enhanced background Ca2+ influx did not explain the raised [Ca2+]i in response to SERCA inhibitors because it had marked gadolinium (Gd3+) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca2+ depletion activated SOCs. Strikingly, SOC-mediated Ca2+ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca2+]i rise being greater than that evoked by 20 mM [K+]o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca2+ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca2+ channels allowing Ca2+ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.
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The primary objectives of this study were to reveal cell-specific expression patterns and functions of voltage-gated K(+) channel (K(V)alpha1) subunits in precapillary arterioles of the murine cerebral circulation. K(V)alpha1 were detected using peptide-specific antibodies in immunofluorescence and Western blotting assays. K(V)1.2 was localized almost exclusively to endothelial cells, whereas K(V)1.5 was discretely localized to the nerves and nerve terminals that innervate the arterioles. K(V)1.5 also localized specifically to arteriolar nerves in human pial membrane. K(V)1.5 was notable for its absence from smooth muscle cells. K(V)1.3, K(V)1.4, and K(V)1.6 were localized to endothelial and smooth muscle cells, although K(V)1.4 had a low expression level. K(V)1.1 was not expressed. Therefore, we show that different cell types of pial arterioles have distinct physiological expression profiles of K(V)alpha1, conferring the possibility of differential modulation by extracellular and second messengers. Furthermore, we show recombinant agitoxin-2 and margatoxin are potent vasoconstrictors, suggesting that K(V)alpha1 subunits have a major function in determining arteriolar resistance to blood flow.
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The primary objectives of this study were to reveal cell-specific expression patterns and functions of voltage-gated K channel (Kαl) subunits in precapillary arterioles of the murine cerebral circulation. Kαl were detected using peptide-specific antibodies in immunofluorescence and Western blotting assays. Kl.2 was localized almost exclusively to endothelial cells, whereas Kl.5 was discretely localized to the nerves and nerve terminals that innervate the arterioles. Kl.5 also localized specifically to arteriolar nerves in human pial membrane. Kl.5 was notable for its absence from smooth muscle cells. K1.3, Kl.4, and Kl.6 were localized to endothelial and smooth muscle cells, although K1.4 had a low expression level. Kl.1 was not expressed. Therefore, we show that different cell types of pial arterioles have distinct physiological expression profiles of Kαl, conferring the possibility of differential modulation by extracellular and second messengers. Furthermore, we show recombinant agitoxin-2 and margatoxin arepotent vasoconstrictors, suggesting that Kαl subunits have a major function in determining arteriolar resistance to blood flow.
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The subunits of the pancreatic ATP-sensitive potassium channel Kir6.2 and the sulphonylurea receptor (SUR1) contain endoplasmic reticulum (ER) retention signals (RKR), which prevent their plasma membrane expression when expressed individually. When co-expressed, however, these signals are masked and the complex traffics to the plasma membrane. To investigate this further, we have expressed epitope-tagged chimaeras between Kir6.2 and Kir2.1 (which traffics to the membrane independently of SUR1) in Xenopus oocytes alone and together with SUR1. By staining sections of the oocytes, we show that, in addition to the ER retention signal present in the distal C-terminus, the M2 transmembrane and the proximal C-terminal regions also contribute to the inability of Kir6.2 to traffic to the membrane in the absence of SUR1. Furthermore, by staining the whole oocytes for the hexa-histidine tag attached to the N-terminus of SUR1, we provide direct experimental evidence that the N-terminus of SUR1 is extracellular.
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Electrogenicity of the Na(+)/K(+) pump has the capability to generate a large negative membrane potential independently of ion-channel current. The high background membrane resistance of arterioles may make them susceptible to such an effect. Pump current was detected by patch-clamp recording from smooth muscle cells in fragments of arterioles (diameter 24-58 microm) isolated from pial membrane of rabbit cerebral cortex. The current was 20 pA at -60 mV, and the extrapolated zero current potential was -160 mV. Two methods of estimating the effect of pump electrogenicity on resting potential indicated an average contribution of -35 mV. In 20% of the recordings, block of inward rectifier K(+) channels by 10-100 microM Ba(2+) led to a small depolarization, but hyperpolarization was a more common response. Ba(2+) also inhibited depolarization evoked by 20 mM K(+). In arterioles within intact pial membrane, Ba(2+) failed to evoke constriction but inhibited K(+)-induced constriction. The data suggest that cerebral arterioles are vulnerable to the hyperpolarizing effect of the Na(+)/K(+) pump, excessive effects of which are prevented by depolarizing inward rectifier K(+) current
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1. Arteriolar segments were isolated from pial membrane and studied within 10 h. Current-clamp and voltage-clamp measurements were made by patch-clamp recording from smooth muscle cells within arterioles. [Ca2+]i was measured from the smooth muscle cell layer by digital imaging of emission from fura-PE3 which was loaded into arterioles by pre-incubation with the acetoxymethyl ester derivative. The external diameter of arterioles was measured using a video-dimension analyser. 2. Endothelin-1 (ET1) was a potent constrictor of isolated arterioles and induced a sustained depolarization up to -27 mV and reduced membrane resistance (EC50 140-170 pm). At a constant holding potential of -60 mV ET-1 induced a transient followed by a sustained inward current. ET1 inhibited L-type voltage-dependent Ca2+ current. 3. ET1 induced a transient followed by sustained elevation of [Ca2+]i. The sustained effect was dependent on extracellular Ca2+. It occurred at a constant holding potential of -60 mV and was not inhibited by the Ca2+ antagonists nicardipine (1 microM) or D600 (10 microM). Thapsigargin (1 microM) completely depleted Ca2+ from caffeine- and ET1-sensitive sarcoplasmic reticulum but did not inhibit the ET1-induced sustained elevation of [Ca2+]i. ET1 effects on [Ca2+]i were prevented by the ETA receptor antagonist BQ123 (cyclo-D-Asp-Pro-D-Val-Leu-D-Trp). 4. The data suggest that ETA receptors are negatively coupled to L-type Ca2+ channels and positively coupled to receptor-operated Ca2+-permeable channels. Inhibition of L-type Ca2+ channel activity may suppress autoregulation, and Ca2+ influx through receptor-operated channels may have a major functional role in the potent long-lasting constrictor effect of endothelin-1 in the cerebral microcirculation.
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1. The types of K+ channel which determine the membrane potential of arcuate artery smooth muscle cells were investigated by patch-clamp recording from isolated cells and lumenal diameter measurements from intact pressurized renal arcuate arteries. 2. Single cells had a mean resting potential of -38 mV and were depolarized by 130 mM K+ but not by the Cl- channel blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS). 3. Iberiotoxin did not affect the resting potential but inhibited spontaneous transient hyperpolarizations. Iberiotoxin or 1 mM tetraethylammonium (TEA+) constricted intact arteries. 3,4-Diaminopyridine (3,4-DAP)-sensitive delayed rectifier K+ (KV) channel current was elicited by depolarization but 3,4-DAP did not affect the resting potential or induce constriction in the intact artery. 4. A voltage-independent K+ current was inhibited by>= 0.1 mM barium (Ba2+) and unaffected by iberiotoxin, glibenclamide, apamin, 3,4-DAP and ouabain. In six out of ten cells, 1 mM Ba2+ depolarized the resting potential, while in the other cells the potential was resistant to all of the K+ channel blockers and ouabain. Ba2+ (0.1-1 mM) constricted the intact artery, but 10 microM Ba2+, 1 microM glibenclamide or 100 nM apamin had no effect. 5. The data suggest that resting potential is determined by background K+ channels, one type being Ba2+ sensitive and voltage independent, and another type being poorly defined due to its resistance to any inhibitor. Large conductance Ca2+-activated K+ (BKCa) and KV channels do not determine the resting potential but have separate functions to underlie transient Ca2+-induced hyperpolarizations and to protect against depolarization past about -30 mV.
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To investigate the mechanism of K(+)-induced vasodilation in a small artery from the kidney, with a particular emphasis on the role of inward rectifier K+ channels.
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The aim of this project was to develop a method to enable routine application of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. Vessels were identified under a microscope and the majority were small arterioles with a mean external diameter, in Ca2+-containing (1.5 mM) solution, of 29 microm and variable lengths of 100 microm or more. Arterioles excluded trypan blue, constricted in response to 60 mM K+ and dilated in response to levcromakalim. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged -72 mV. Short arteriolar segments could be voltage-clamped. Injection of depolarising current or bath application of 10 mM Ba2+ induced constriction of the entire arteriolar segment. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K+ channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Notably, avoidance of single-cell isolation means that enzymatic treatment is minimised and cells can be studied within their normal environment of the blood vessel wall.
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Lemildipine is a 1,4-dihydropyridine calcium channel blocker which is under phase III development by Banyu (Merck and Co), in Japan, for its potential to treat hypertension and cerebrovascular ischemia. In one study, involving five patients with essential hypertension accompanied by cerebrovascular disorder, lemildipine, administered orally at doses of 5 to 20 mg/day, significantly lowered blood pressure and increased cerebral blood flow [256721]. Another study in 31 patients with essential hypertension demonstrated that lemildipine has significant pressure lowering effects without affecting serum lipids [256722]. Worldwide rights to market the drug have been assigned to Kowa in Japan.
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Ion channels play key roles in determining smooth muscle tone by setting the membrane potential and allowing Ca2+ influx. Perhaps not surprisingly, therefore, they also provide targets for neurotransmitters and other messengers that act on smooth muscle. Application of patch-clamp and molecular biology techniques and the use of selective pharmacology has started to provide a wealth of information on the ion channel systems of smooth muscle cells, revealing complexity and functional significance. Reviewed are the actions of messengers (e.g., noradrenaline, acetylcholine, endothelin, angiotensin II, neuropeptide Y, 5-hydroxytryptamine, histamine, adenosine, calcitonin gene-related peptide, substance P, prostacyclin, nitric oxide and oxygen) on specific types of ion channel in smooth muscle, the L-type calcium channel, and the large conductance Ca(2+)-activated, ATP-sensitive, delayed rectifier and apamin-sensitive K+ channels.
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1. Single smooth muscle cells were isolated from the basilar artery of the guinea-pig and, within 10 h, inward currents through voltage-gated Ca2+ channels were recorded using the amphotericin or conventional whole-cell voltage-clamp techniques. 2. In amphotericin whole-cell recordings, bath application of 2,4-dinitrophenol (DNP, an uncoupler of mitochondrial ATP production) induced an initial stimulation (14% increase in 5 of 11 cells) and then pronounced inhibition (50% decrease in 9 of 11 cells within 9.5 min) of voltage-dependent Ca2+ current (I(Ca)) elicited by depolarizing to +10 mV in 1.5 mM extracellular Ca2+ solution. By contrast, inhibition of glycolysis by replacing glucose in the bath with 2-deoxy-D-glucose had no effect. 3. Na+ current through Ca2+ channels (I[(Ca)(Na)]) recorded in the absence of extracellular divalent cations also responded to DNP, again with stimulation followed by inhibition of current. The stimulation of I[(Ca)(Na)] was associated with a leftward shift of the Ca2+ channel activation curve which averaged -9 mV. A combination of 2-deoxy-D-glucose, mannoheptulose and 3-0-methyl-glucose had only minor effects on I[(Ca)(Na)], whereas rotenone had an effect similar to that of DNP in six of eight cells. 4. The amplitude of I[(Ca)(Na)] in conventional whole-cell recordings was not different from that in amphotericin whole-cell recordings, even without ATP in the recording pipette and with metabolic poisons in the bath solution. Furthermore, attempts to dephosphorylate the Ca2+ channels in ATP-free conditions did not prevent I[(Ca)(Na)], and a high concentration of Mg-ATP with or without a phosphorylation-supporting medium in the recording pipette did not increase its amplitude. 5. In the absence of ATP, Mg2+ inhibited whole-cell I[Ca)(Na)] with a K(d) of about 100 mu M at -10 mV and induced a leftward shift of the Ca2+ channel activation curve. When ATP and a phosphorylation-supporting medium were in the recording pipette the blocking effect of free Mg2+ was reduced but the shift in the Ca2+ channel activation curve was unaffected. 6. From these data it is suggested that inhibition of mitochondrial, but not glycolytic, ATP production has stimulatory and inhibitory effects on voltage-gated Ca2+ channels of basilar artery smooth muscle cells. Effects of intracellular Mg2+ on the Ca2+ channels were modulated by ATP and mimicked the effects of metabolic poisoning by DNP. A hypothesis is discussed in which the intracellular free Mg2+ concentration may be a key factor coupling ATP production to Ca2+ channels.
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1. Whole-cell voltage-clamp recordings were made from smooth muscle cells isolated from guinea-pig seminal vesicle. 2. When the recording pipette solution contained 130 mM KCl and a low concentration of EGTA (0.2 mM), a dominant outward current was elicited by depolarization to positive of -30 mV from a holding potential of -50 mV. The current was non-inactivating, stimulated by intracellular Ca2+ and blocked by bath-applied 1 mM tetraethylammonium but not 1 mM 3,4 diaminopyridine. 3. If 10 mM EGTA was added to the KCl pipette solution and the holding potential was -50 mV, or more negative, the major current elicited by depolarization to positive of -30 mV was an A-type K(+)-current. This current inactivated rapidly (within 100 ms) and was blocked by bath-applied 1 mM 3,4-diaminopyridine but not 10 mM tetraethylammonium. 4. An inward voltage-gated Ca channel current was observed on depolarization to positive of -30 mV with 1.5 mM Ca2+ or 10 mM Ba2+ in the bath solution and when Ca+ replaced K+ in the pipette. The Ba(2+)-current was shown to be abolished by bath-applied 100 microM Cd2+ and inhibited by 90% by 1 microM nifedipine, and thus appeared to be carried by L-type Ca channels. 5. High concentrations of glibenclamide (10-500 microM) inhibited A-type K(+)-current, Ba(2+)-current and contraction of the whole tissue induced by noradrenaline or electrical field stimulation. 6. From these data we suggest that seminal vesicle smooth muscle cells express Ca2+ -dependent K channels, A-type K channels and L-type Ca channels which are inhibited by tetraethylammonium,3,4-diaminopyridine and nifedipine, respectively. In addition, an unexpected relaxant effect of high concentrations of glibenclamide may be explained by inhibition of the Ca channels.
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