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The effects of allyl isothiocyanate (AITC), transient receptor potential ankyrin 1 (TRPA1) agonist, on cultured human cardiac fibroblasts were examined by measuring intracellular Ca2+ concentration [Ca2+]i and whole-cell voltage clamp techniques. AITC (200 μM) increased Ca2+ entry in the presence of [Ca2+]i. Ruthenium red (RR) (30 μM), and La3+ (0.5 mM), a general cation channel blocker, inhibited AITC-induced Ca2+ entry. Under the patch pipette filled with Cs+- and EGTA-solution, AITC induced the current of a reversal potential (Er) of approximately +0 mV. When extracellular Na+ ion was changed by NMDG+, the inward current activated by AITC was markedly reduced. La3+ and RR inhibited the AITC-induced current. The conventional RT-PCR analysis, Western blot, and immunocytochemical studies showed TRPA1 mRNA and protein expression. The present study shows the first evidence for functional Ca2+-permeable nonselective cation currents induced by AITC, possibly via TRPA1 in human cardiac fibroblast.

Helianthus annuus L. is an important oilseed crop, which exhibits moderate salt tolerance and can be cultivated in areas affected by salinity. Using patch-clamp electrophysiology, we have characterized Na+ influx and K+ efflux conductances in protoplasts of salt-tolerant H. annuus L. hybrid KBSH-53 under high salinity. This work demonstrates that the plasma membrane of sunflower root cells has a classic set of ionic conductances dominated by K+ outwardly rectifying channels (KORs) and non-selective cation channels (NSCCs). KORs in sunflower show extreme Na+ sensitivity at high extracellular [Ca2+] that can potentially have a positive adaptive effect under salt stress (decreasing K+ loss). Na+ influx currents in sunflower roots demonstrate voltage-independent activation, lack time-dependent component, and are sensitive to Gd3+. Sunflower Na+-permeable NSCCs mediate a much weaker Na+ influx currents on the background of physiological levels of Ca2+ as compared to other species. This suggests that sunflower NSCCs have greater Ca2+ sensitivity. The responses of Na+ influx to Ca2+ correlates well with protection of sunflower growth by external Ca2+ in seedlings treated with NaCl. It can be, thus, hypothesized that NaCl tolerance in sunflower seedling roots is programmed at the ion channel level via their sensitivity to Ca2+ and Na+.

Helianthus annuus L. is an important oilseed crop, which exhibits moderate salt tolerance and can be cultivated in areas affected by salinity. Using patch-clamp electrophysiology, we have characterized Na+ influx and K+ efflux conductances in protoplasts of salt-tolerant H. annuus L. hybrid KBSH-53 under high salinity. This work demonstrates that the plasma membrane of sunflower root cells has a classic set of ionic conductances dominated by K+ outwardly rectifying channels (KORs) and non-selective cation channels (NSCCs). KORs in sunflower show extreme Na+ sensitivity at high extracellular [Ca2+] that can potentially have a positive adaptive effect under salt stress (decreasing K+ loss). Na+ influx currents in sunflower roots demonstrate voltage-independent activation, lack time-dependent component, and are sensitive to Gd3+. Sunflower Na+-permeable NSCCs mediate a much weaker Na+ influx currents on the background of physiological levels of Ca2+ as compared to other species. This suggests that sunflower NSCCs have greater Ca2+ sensitivity. The responses of Na+ influx to Ca2+ correlates well with protection of sunflower growth by external Ca2+ in seedlings treated with NaCl. It can be, thus, hypothesized that NaCl tolerance in sunflower seedling roots is programmed at the ion channel level via their sensitivity to Ca2+ and Na+. [ABSTRACT FROM AUTHOR]

Abstract Muscarinic receptor stimulation induces depolarizing inward currents and catecholamine secretion in adrenal medullary (AM) cells from various mammals. In guinea-pig AM cells muscarine and oxotremorine at concentrations ≤ 1 μM produce activation of nonselective cation channels with a similar potency and efficacy, whereas muscarine at higher concentrations produces not only nonselective cation channel activation, but also TASK1 channel inhibition. In rat AM cells, the muscarinic M 1 receptor is involved in TASK1 channel inhibition in response to muscarinic agonists, and the efficacy of oxotremorine is half that of muscarine. These pharmacological findings might indicate that different muscarinic receptor subtypes are responsible for the regulation of nonselective cation and TASK1 channel activities. The present study aimed to determine the muscarinic receptor subtypes involved in nonselective cation channel activation in guinea-pig and mouse AM cells. The inward current evoked by 1 μM muscarine was completely suppressed by 100 μM quinine, whereas 30 μM muscarine-induced inward currents were comprised of quinine-sensitive and -insensitive components. The electrophysiological and pharmacological properties of the muscarine-induced currents indicated that the quinine-sensitive and insensitive components are due to nonselective cation channel activation and TASK1 channel inhibition, respectively. Muscarine at 30 μM failed to induce any current in AM cells treated with muscarinic toxin 7 or genetically deleted of the M 1 receptor. The K D value of VU0255035 against the muscarinic receptor mediating nonselective cation channel activation was 17.5 nM. These results indicate that the M 1 receptor mediates nonselective cation channel activation as well as TASK1 channel inhibition. [ABSTRACT FROM AUTHOR]

Transient receptor potential canonical 6 (TRPC6) proteins form receptor-operated Ca2+-permeable channels, which have been thought to bring benefit to the treatment of diseases, including cancer. However, selective antagonists for TRPC channels are rare and none of them has been tested against gastric cancer. Compound 14a and analogs were synthesized by chemical elaboration of previously reported TRPC3/6/7 agonist 4o. 14a had very weak agonist activity at TRPC6 expressed in HEK293 cells but exhibited strong inhibition on both 4o-mediated and receptor-operated activation of TRPC6 with an IC50 of about 1 μM. When applied to the culture media, 14a suppressed proliferation of AGS and MKN45 cells with IC50 values of 17.1 ± 0.3 and 18.5 ± 1.0 μM, respectively, and inhibited tube formation and migration of cultured human endothelial cells. This anti-tumor effect on gastric cancer was further verified in xenograft models using nude mice. This study has found a new tool compound which shows excellent therapeutic potential against human gastric cancer most likely through targeting TRPC6 channels. [ABSTRACT FROM AUTHOR]

A thin fluid layer in alveoli is normal and results from a balance of fluid entry and fluid uptake by transepithelial salt and water reabsorption. Conventional wisdom suggests the reabsorption is via epithelial Na+ channels (ENaC), but if all Na_ reabsorption were via ENaC, then amiloride, an ENaC inhibitor, should block alveolar fluid clearance (AFC). However, amiloride blocks only half of AFC. The reason for failure to block is clear from single-channel measurements from alveolar epithelial cells: ENaC channels are observed, but another channel is present at the same frequency that is nonselective for Na+ over K+, has a larger conductance, and has shorter open and closed times. These two channel types are known as highly selective channels (HSC) and nonselective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC number. NSC channels contain α-ENaC since knocking down α-ENaC reduces the number of NSC (knocking down β- or γ-ENaC has no effect on NSC, but the molecular composition of NSC channels remains unclear). We show that NSC channels consist of at least one α-ENaC and one or more acid-sensing ion channel 1a (ASIC1a) proteins. Knocking down either α-ENaC or ASIC1a reduces both NSC and HSC number, and no NSC channels are observable in single-channel patches on lung slices from ASIC1a knockout mice. AFC is reduced in knockout mice, and wet wt-to-dry wt ratio is increased, but the percentage increase in wet wt-to-dry wt ratio is larger than expected based on the reduction in AFC. [ABSTRACT FROM AUTHOR]

Necrosis is considered as a non-specific form of cell death that induces tissue inflammation and is preceded by cell swelling. This increase in cell volume has been ascribed mainly to defective outward pumping of Na+ caused by metabolic depletion and/or to increased Na+ influx via membrane transporters. A specific mechanism of swelling and necrosis driven by the influx of Na+ through nonselective cation channels has been recently proposed (Barros et al., 2001a). We have characterized further the properties of the nonselective cation channel (NSCC) in HTC cells. The NSCC shows a conductance of ~18 pS, is equally permeable to Na+ and K+, impermeant to Ca2+, requires high intracellular Ca2+ as well as low intracellular ATP for activation and is inhibited by flufenamic acid. Hydrogen peroxide induced a significant increase in cell volume that was dependent on external Na+. We propose that the NSCC, which is ubiquitous though largely inactive in healthy cells, becomes activated under severe oxidative stress. The ensuing Na+ influx initiates via positive feedback a series of metabolic and electrolytic disturbances, resulting in cell death by necrosis

The initiation of lens cataract has long been associated with the development of a membrane "leak" in lens fiber cells that depolarizes the lens intracellular potential and elevates intracellular Na+ and Ca2+ concentrations. It has been proposed that the leak observed in cataractous lenses is due to the activation of a nonselective cation (NSC) conductance in the normal electrically tight fiber cells. Studies of the membrane properties of isolated fiber cells using the patch-clamp technique have demonstrated a differentiation-dependent shift in membrane permeability from K+-dominated in epithelial and short fiber cells toward larger contributions from anion and NSC conductances as fiber cells elongate. In this study, the NSC conductances in elongating lens fiber cells are demonstrated to be due to at least two distinct classes: a Gd3+-sensitive, mechanosensitive channel whose blockade is essential for obtaining viable isolated fiber cells, and a second Gd3+-insensitive, La3+-sensitive conductance that appears to be activated by cell shrinkage. This second conductance was eliminated by the replacement of extracellular Na+ with the impermeant cation N-methyl-D-glucamine and was potentiated by both hypertonic stress and isosmotic cell shrinkage evoked by the replacement of extracellular Cl- with the impermeant anion gluconate. This additional cation conductance may play a role in normal lens physiology by mediating regulatory volume increase under osmotic or other physiological challenges. Since the inappropriate activation of NSC channels is implicated in the initiation of lens cataract, they represent potential targets for the development of novel anticataract therapies. [ABSTRACT FROM AUTHOR]

Antenatal maternal long-term hypoxia (LTH) can alter serotonin (5-HT) and calcium (Ca2+) signaling in fetal pulmonary arteries (PAs) and is associated with persistent pulmonary hypertension of the newborn (PPHN). In humans, the antenatal maternal hypoxia can be secondary to smoking, anemia, and chronic obstructive pulmonary disorders. However, the mechanisms of antenatal maternal hypoxia-related PPHN are unresolved. Because both LTH and 5-HT are associated with PPHN, we tested the hypothesis that antenatal maternal LTH can increase 5-HT-mediated PA contraction and associated extracellular Ca2+ influx through L-type Ca2+ channels (CaL), nonselective cation channels (NSCCs), and reverse-mode sodium–calcium exchanger (NCX) in the near-term fetus. We performed wire myography and confocal-Ca2+ imaging approaches on fetal lamb PA (∼140 days of gestation) from normoxic ewes or those acclimatized to high-altitude LTH (3801 m) for ∼110 days. Long-term hypoxia reduced the potency but not the efficacy of 5-HT-induced PA contraction. Ketanserin (100 nmol/L), a 5-HT2A antagonist, shifted 5-HT potency irrespective of LTH, while GR-55562 (1 µmol/L), a 5-HT1B/D inhibitor, antagonized 5-HT-induced contraction in normoxic fetuses only. Various inhibitors for CaL, NSCC, and reverse-mode NCX were used in contraction studies. Contraction was reliant on extracellular Ca2+ regardless of maternal hypoxia, NSCC was more important to contraction than CaL, and reverse-mode NCX had little or no role in contraction. Long-term hypoxia also attenuated the effects of 2-APB and flufenamic acid and reduced Ca2+ responses observed by imaging studies. Overall, LTH reduced 5HT1B/D function and increased NSCC-related Ca2+-dependent contraction in ovine fetuses, which may compromise pulmonary vascular function in the newborn. [ABSTRACT FROM PUBLISHER]

Canonical transient receptor potential (TRPC) channels are Ca2+-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gαs. Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gas [Gαs (Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gas (Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4β was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by Gs/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and β2-adrenergic receptor are involved in the relaxation and contraction of smooth muscles. [ABSTRACT FROM AUTHOR]

1. Coordinated oscillations in diameter occur spontaneously in cerebral vessels and depend on the opening of voltage dependent calcium channels. However, the mechanism that induces the initial depolarisation has remained elusive. We investigated the involvement of canonical transient receptor potential (TRPC) channels, which encode nonselective cation channels passing Na+ and Ca2+ currents, by measuring changes in diameter, intracellular Ca2+ and membrane potential in branches of juvenile rat basilar arteries. 2. Removal of extracellular Ca2+ abolished vasomotion and relaxed arteries, but paradoxically produced depolarisation. 3. Decrease in temperature to 24°C or inhibition of phospholipase C (PLC) abolished vasomotion, hyperpolarised and relaxed arteries and decreased intracellular Ca2+. 4. Reduction in the driving force for Na+ through decrease in extracellular Na+ produced similar effects and prevented the depolarisation elicited by removal of extracellular Ca2+. 5. Nonselective TRP channel blockers, SKF96365 and gadolinium, mimicked the effects of inhibition of the PLC pathway. 6. Depolarisation of vessels in which TRP channels were blocked with SKF96365 reinstated vascular tone and vasomotion. 7. Quantitative polymerase chain reaction revealed TRPC1 as the predominantly expressed TRPC subtype. 8. Incubation with a function blocking TRPC1 antibody delayed the onset of vasomotion. 9. We conclude that nonselective cation channels contribute to vasoconstriction and vasomotion of cerebral vessels by providing an Na+-induced depolarisation that activates voltage dependent calcium channels. Our antibody data suggest the involvement of TRPC1 channels that might provide a target for treatment of therapy-refractory vasospasm. [ABSTRACT FROM AUTHOR]

In articular cartilage inflammation, histamine release from mast cells is a key event. It can enhance cytokine production and matrix synthesis and also promote cell proliferation by stimulating chondrocytes. In this study, the functional impact of Ca2+-activated K+ (Kca) channels in the regulation of intracellular Ca2+ concentration ([Ca2+]i) in chondrocytes in response to histamine was examined using OUMS-27 cells, as a model of chondrocytes derived from human chondrosarcoma. Application of histamine induced a significant [Ca2+]i rise and also membrane hyperpolarization, and both effects were mediated by the stimulation of H1 receptors. The histamine-induced membrane hyperpolarization was attenuated to ∼50% by large-conductance Kca (BK) channel blockers, and further reduced by intermediate (1K) and small conductance Kca (SK) channel blockers. The tonic component of histamine-induced [Ca2+]1 rise strongly depended on the presence of extracellular Ca2+ ([Ca2+]0) and was markedly reduced by La3 or Gd3+ but not by nifedipine. It was significantly attenuated by BK channel blockers, and further blocked by the cocktail of BK, 1K, and SK channel blockers. The Kca blocker cocktail also significantly reduced the store-operated Ca2+ entry (SOCE), which was induced by Ca2+ addition after store-depletion by thapsigargin in [Ca2+]0 free solution. Our results demonstrate that the histamine-induced membrane hyperpolarization in chondrocytes due to Kca channel activation contributes to sustained Ca2+ entry mainly through SOCE channels in OUMS-27 cells. Thus, Kca channels appear to play an important role in the positive feedback mechanism of [Ca2+]i regulation in chondrocytes in the presence of articular cartilage inflammation. [ABSTRACT FROM AUTHOR]

Amiloride-sensitive epithelial sodium channels (ENaC) play an important role in lung sodium transport. Sodium transport is closely regulated to maintain an appropriate fluid layer on the alveolar surface. Both alveolar type I and II cells have several different sodium-permeable channels in their apical membranes that play a role in normal lung physiology and pathophysiology. In many epithelial tissues, ENaC is formed from three subunit proteins: α, β, and γ ENaC. Part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. Thus, lung epithelium has enormous flexibility to alter the magnitude of salt and water transport. In lung, ENaC is regulated by many transmitter and hormonal agents. Regulation depends upon the type of sodium channel but involves controlling the number of apical channels and/or the activity of individual channels. [ABSTRACT FROM AUTHOR]

Tonic physiological activity of RhoAIRho kinase contributes to the maintenance of penile flaccidity through its involvement in the Ca2~ sensitization of erectile tissue smooth muscle. The present study hypothesized that Rho kinase is also involved in the modulation of Ca2~ entry induced by a1- adrenoceptor stimulation of penile arteries. Rat penile arteries were mounted in microvascular myographs for simultaneous measurements of intracellular Ca2~ ([Ca2~]1) and force. The Rho-kinase inhibitor Y-27632 markedly reduced norepinephrine-mediated electrically induced contractions and the increases in both [Ca2~]1 and tension elicited by the ai-adrenoceptor agonist phenylephrine (Phe). In contrast, the protein kinase C (PKC) inhibitor Ro-31-8220 reduced tension without altering the Phe-induced increase in [Ca2~]1. In the presence of nifedipine, Y-27632 still inhibited the non-L-type Ca2~ signal and blunted Phe contraction. Y-27632 did not impair the capacitative Ca2~ entry evoked by store depletion with cyclopiazonic acid but largely reduced the Ba2~ influx stimulated by Phe in fura-2 AM-loaded arteries. The addition of Y-27632 to arteries depolarized with high KCI markedly reduced tension without changing [Ca2~]~. In a-toxin-permeabilized penile arteries stimulated with threshold Ca2~ concentrations, Y-27632 inhibited the sensitization induced by either guanosine 5'-O-(3-thiotriphosphate) (GTP-yS) or Phe in the presence of GTPyS. However, Y-27632 failed to alter contractions induced by a maximal concentration of free Ca2t These results suggest that Rho kinase, besides its contribution to the Ca2~ sensitization of the contractile proteins, is also involved in the regulation of Ca2~ entry through a nonselective cation channel activated by a1-adenoceptor stimulation in rat penile arteries. [ABSTRACT FROM AUTHOR]

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 μmol g−1 h−1 was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5–40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants. [ABSTRACT FROM PUBLISHER]

Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since Ca2+ mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca2+ response and to delineate the Ca2+ pathways activated by hydrogen peroxide (H2O2) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 µM to 1 mM H2O2 elicited concentration-dependent increase in intracellular Ca2+ concentration in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (IP3) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of Ca2+ in external solution. The secondary phase was dependent on extracellular Ca2+ influx. It was unaffected by the voltage-gated Ca2+ channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and La3+, but inhibited concentration dependently by millimolar Ni2+, and potentiated by the Na+/Ca2+ exchange inhibitor KB-R 7943. H+O2 did not alter the rate of Mn2+ quenching of fura 2, suggesting store- and receptor-operated Ca2+ channels were not involved. By contrast, H2O2 elicited a sustained inward current carried by Na+ at -70 mV, and the current was inhibited by Ni2+. These results suggest that H2O2 mobilizes intracellular Ca2+ through multiple pathways, including the IP3-and ryanodine receptor-gated Ca2+ stores, and Ni2+-sensitive cation channels. Activation of these Ca2+ pathways may play important roles in ROS signaling in PASMCs. [ABSTRACT FROM AUTHOR]

The mechanosensitive nonselective cation channel (NSCMS) is a key player in vascular myogenic contraction. The functional channel density and pressure sensitivity of NSCMS in vascular myocytes were compared between pulmonary and systemic arteries (coronary, mesenteric and cerebral arteries) in the rabbit. In cell-attached condition, a negative pressure via patch pipettes commonly activated NSCMS with weak voltage dependence. The threshold pressure for activation was lower, and the density of NSCMS was higher in the pulmonary than the systemic arteries. When the pulmonary arteries were divided into small-diameter (outer diameter, OD < 0.5 mm) and large-diameter (OD > 1.5 mm) arteries, the low threshold and high density of NSCMS were observed only in small-diameter ones. No such difference was observed between the small- and large-diameter coronary arteries. The higher stretch sensitivity and denser functional expression of NSCMS in small pulmonary arteries might suggest an adaptive tuning for the relatively low pulmonary blood pressure in vivo. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Transient receptor potential melastatin- (TRPM) and vanilloid-related (TRPV) channels are nonselective cation channels pertinent to diverse physiological functions. Multiple TRPM and TRPV channel subtypes have been identified and cloned in different tissues. However, their information in vascular tissue is scant. In this study, we sought to identify TRPM and TRPV channel subtypes expressed in rat deendothelialized intralobar pulmonary arteries (PAs) and aorta. With RT-PCR, mRNA of TRPM2, TRPM3, TRPM4, TRPM7, and TRPM8 of TRPM family and TRPV1, TRPV2, TRPV3, and TRPV4 of TRPV family were detected in both PAs and aorta. Quantitative real-time RT-PCR showed that TRPM8 and TRPV4 were the most abundantly expressed TRPM and TRPV subtypes, respectively. Moreover, Western blot analysis verified expression of TRPM2, TRPM8, TRPV1, and TRPV4 proteins in both types of vascular tissue. To examine the functional activities of these channels, we monitored intracellular Ca2+ transients ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs) and aortic smooth muscle cells (ASMCs). The TRPM8 agonist menthol (300 µM) and the TRPV4 agonist 4α-phorbol 12,13-didecanoate (1 µM) evoked significant increases in [Ca2+]i in PASMCs and ASMCs. These Ca2+ responses were abolished in the absence of extracellular Ca2+ or the presence of 300 µM Ni2+ but were unaffected by 1 µM nifedipine, suggesting Ca2+ influx via nonselective cation channels. Hence, for the first time, our results indicate that multiple functional TRPM and TRPV channels are coexpressed in rat intralobar PAs and aorta. These novel Ca2+ entry pathways may play important roles in the regulation of pulmonary and systemic circulation. [ABSTRACT FROM AUTHOR]

Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity. [ABSTRACT FROM AUTHOR]

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca2+-permeable cation channel in mammalian cells. TRPC5 is desensitized rapidly after activation by G protein-coupled receptor. Herein we report our investigation into the desensitization of mTRPC5 and localization of the molecular determinants of this desensitization using mutagenesis. TRPC5 was initially activated by muscarinic stimulation using 100 μM carbachol (CCh) and then decayed rapidly even in the presence of CCh (desensitization). Increased EGTA or omission of MgATP in the pipette solution slowed the rate of this desensitization. The protein kinase C (PKC) inhibitors, 1 μM chelerythrine, 100 nM GF109203X, or PKC peptide inhibitor (19–36), inhibited this desensitization of TRPC5 activated by 100 μM CCh. When TRPC5 current was activated by intracellular GTPγS, PKC inhibitors prevented TRPC5 desensitization and the mutation of TRPC5 T972 to alanine slowed the desensitization process dramatically. We conclude that the desensitization of TRPC5 occurs via PKC phosphorylation and suggest that threonine at residue 972 of mouse TRPC5 might be required for its phosphorylation by PKC. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that TRPC3, a member of the canonical transient receptor potential (TRP) family of channels, mediates agonist-induced depolarization of arterial smooth muscle cells (SMCs). In support of this hypothesis, we observed that suppression of arterial SMC TRPC3 expression with antisense oligodeoxynucleotides significantly decreased the depolarization and constriction of intact cerebral arteries in response to UTP. In contrast, depolarization and contraction of SMCs induced by increased intravascular pressure, i.e., myogenic responses, were not altered by TRPC3 suppression. Interestingly, UTP-evoked responses were not affected by suppression of a related TRP channel, TRPC6, which was previously found to be involved in myogenic depolarization and vasoconstriction. In patch-clamp experiments, UTP activated a whole cell current that was greatly reduced or absent in TRPC3 antisense-treated SMCs. These results indicate that TRPC3 mediates UTP-induced depolarization of arterial SMCs and that TRPC3 and TRPC6 may be differentially regulated by receptor activation and mechanical stimulation, respectively. [ABSTRACT FROM AUTHOR]

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store- operated channels) In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intraceltular Ca2+ store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca2+ store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca2+ store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation. [ABSTRACT FROM AUTHOR]

Proteins related to the Drosophila transient receptor potential (TRP) gene product are key players in Ca2+ homeostasis of mammalian cells. TRP proteins form cation channels, which function as sensors for a variety of stimuli including oxidative stress. The canonical TRP proteins TRPC3 and TRPC4 are likely to play a role in endothelial physiology and pathophysiology. Here, we report that overexpression of TRPC3 or TRPC4 generates a redox-sensitive cation conductance in human embryonic kidney cells (HEK293). Redox activation of these TRPC species appears to be due to oxidant-induced promotion of phospholipase C activity and/or sensitisation of TRPC proteins to regulation by phospholipase C. Our results suggest TRPC species as attractive, novel targets for cardiovascular therapy. [Copyright &y& Elsevier]

Considerable work has led many to conclude that interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. These cells form electrically coupled networks within the pacemaker regions of the GI tract, and ICC are electrically coupled to smooth muscle cells. ICC express unique ion channels that periodically produce inward (pacemaker) currents. Recent work has suggested that the inward current is produced by a calcium (Ca2+)-regulated, nonselective cation conductance. Channels responsible for this conductance oscillate in open probability in response to the periodic drop in intracellular Ca2+ concentration during the slow wave cycle. Pacemaker activity generates slow waves that are propagated actively through ICC networks. Depolarization coordinates the pacemaker activity through the ICC network by activating a dihydropyridine-resistant Ca2+ conductance. Entry of small amounts of Ca2+ into ICC entrains spontaneous pacemaker activity and produces cell-to-cell propagation of slow waves. This review discusses the mechanisms and conductances involved in generation and propagation of electrical slow waves in ICC. [ABSTRACT FROM AUTHOR]

Arachidonic acid (AA; 20:4, n-6) has been implicated in cell damage in the brain under ischemia–reperfusion and other pathological conditions. In our experiments, PC12 cells exposed to >10 μM AA died within 1–2 hr, as assessed by the LDH release assay. Since AA is known to induce Ca2+/cation-permeable conductance in the plasma membrane, we investigated whether Ca2+ influx plays a role in this acute cell death. We found that extracellular Ca2+ was not required for the toxic effect of AA. In fact, the removal of extracellular Ca2+ dramatically accelerated its development: the half-time of the toxic effect of 40 μM AA decreased from 70.1±0.3 min in the presence of 5 mM Ca2+ to 7.4±0.3 min in the Ca-free solution. The extent of cell killing depended only weakly on AA concentration and ion composition, remaining within the 70–95% range. The AA-induced acute death was not affected by inhibitors of AA metabolism (nordihydroguaiaretic acid, indomethacin, proadifen), whereas some antioxidants tested (deferoxamine and ellagic acid), but not all (melatonin), partially suppressed it. Also, it was not affected by changes in the extracellular ionic strength or mimicked by an acetylenic analog of AA 5,8,11,14-eicosatetraynoic acid. We conclude that lethal injuries sustained by cells during short exposures to AA were caused by the fatty acid itself and were not mediated by the AA-induced influx of Ca2+/cations. Moreover, direct physical effects of AA on the plasma membrane (changes in membrane fluidity or detergent-like action) were also excluded. [Copyright &y& Elsevier]

Calcium-activated nonselective cation channels (NSCCa) in brown adipocytes are inhibited by several nucleotides acting on the cytosolic side of the membrane. We used excised inside-out patches from rat brown adipocytes to identify important nucleotide structures for NSC-channel inhibition. We found that 100 mM 5?-AMP inhibited NSC-channel activity more than did ATP or ADP. Adenosine was a weak inhibitor, whereas adenine and ribose-5-phosphate had no effect. The channel activity was effectively blocked by 10 mM AMP, but it was unaffected by 10 mM cAMP, CMP, GMP, IMP, TMP or UMP. Dose-response studies yielded IC50-values of 4 mM for AMP and 32 mM for cAMP. dAMP was as effective as AMP, but all 5?-phosphate group modifications on AMP dramatically lowered the inhibitory effect. 10 mM of the AMP precursor adenylosuccinate weakly inhibited the channel activity. An increase in AMP concentration from 1 to 10 mM shifted the EC50 for Ca2+ activation almost 1 order of magnitude; a Schild plot analysis yielded a KB value of 0.3 mM for AMP. We conclude that AMP is the most efficacious endogenous nucleotide inhibitor of the brown adipocyte nonselective cation channel (NSCCa/AMP) yet identified and that there is functional competition between Ca2+ and AMP. The brown adipocyte NSCCa/AMP thus appears to be functionally different from the NSCCa,PKA in the exocrine pancreas and the NSCCa,cAMP in the endocrine pancreas, but similar to the NSCCa/AMP in the endocrine pancreas. [ABSTRACT FROM AUTHOR]

Previous studies have shown that the mRNA of cyclic-nucleotide-gated nonselective cation (CNG) channels is expressed in rat airway epithelia and that these channels contribute to sodium-mediated short-circuit currents in cultured rat tracheal epithelia. Patch-clamp studies from human A549 cells indicate that these channels contribute to cGMP-stimulated l-cis-diltiazem- and dichlorobenzamil-inhibited whole-cell sodium currents. This study demonstrates that mRNA for primary and secondary subunits of CNG channels, hαCNG1 and hβCNG1 respectively, are expressed in several human airway cell lines, including normal and cystic fibrosis bronchial airway cells, in normal and cystic fibrosis tracheal airway cell lines and nasal polyp tissue from a cystic fibrosis patient. The mRNA of rαCNG1 in rat lung increased in response to increased circulating glucocorticoids and decreased in animals with lowered circulating glucocorticoids after aminoglutethimide treatment. Likewise the mRNA of hαCNG1 increased in the presence of glucocorticoids in cultured alveolar airway cells. The mRNA of αCNG1 in rat lung was also increased in response to a low-salt diet and lowered in animals fed a high-salt diet. Likewise the mRNA of αCNG1 was increased in response to increased aldosterone and decreased in animals given spironolactone. These results suggest that mRNA for αCNG1 increases in response to elevated glucocorticoids or mineralocorticoids. Because αCNG1 is a functional sodium entry channel in both rat and human airway epithelial cells, if channel protein is also elevated this channel could mediate an increase in sodium absorption across lung epithelia in response to circulating hormones. [ABSTRACT FROM AUTHOR]

It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from -10.8 +/- 1.5 pA to -211 +/- 10.2 pA (n = 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly between monovalent cations with a selectivity sequence NH(4) (1.2) > or = Na(+) (1) approximately K(+) (0.9) approximately Li(+) (0.9). In contrast there was no measurable permeability for Ca(2+) or Ba(2+) and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg(2+) and was inhibited by staurosporine (1 microm) and calphostin C (1 microm). Moreover, cytochalasin D (10 microm) and taxol (2 microm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements. [ABSTRACT FROM AUTHOR]

Apoptosis appears to be implicated in the pathogenesis and therapeutic applications of cancer. In this study we investigated the induction of apoptosis by 4-aminopyridine (4-AP), a K[sup +] channel blocker, and its mechanism in HepG2 human hepatoblastoma cells. 4-AP reduced cell viability and induced DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, 4-AP induced a sustained increase in intracellular Ca[sup 2+] concentration, which was completely inhibited by the extracellular Ca[sup 2+] chelation with EGTA. 4-AP also induced Mn[sup 2+] influx, indicating that the 4-AP-induced increased intracellular Ca[sup 2+] levels were due to activation of Ca[sup 2+] influx pathway. 4-AP also depolarized membrane potential that was measured by using di-O-C[sub 5] (3), a voltage-sensitive fluorescent dye. 4-AP-induced Ca[sup 2+] influx was significantly inhibited not by voltage-operative Ca[sup 2+] channel blockers (nifedipine or verapamil), but by flufenamic acid (FA), a known nonselective cation channel blocker. Quantitative analysis of apoptosis by the flow cytometry revealed that treatment with either FA or BAPTA, an intracellular Ca[sup 2+] chelator, significantly inhibited the 4-AP-induced apoptosis. Taken together, these results suggest that the observed 4-AP-induced apoptosis in the HepG2 cells may result from Ca[sup 2+] influx through the activation of voltage-sensitive Ca[sup 2+] -permeable non-selective cation channels. These results further suggest that membrane potential change by modulation of K[sup +] channel activity may be involved in the mechanism of apoptosis in human hepatoma cells.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In mammalian gastrointestinal myocytes, it is known that muscarinic stimulation activates nonselective cation channels through a G-protein and a Cadependent pathway. We recorded inward cationic currents following application of carbachol ( I) to guinea-pig gastric myocytes, which were held at −20 mV using the whole-cell patch-clamp method. I was suppressed by nicardipine or removal of Ca from the bath solution. The peak value of inward current induced by repetitive applications of carbachol (CCh) decreased progressively (run-down phenomenon). This run-down was significantly alleviated by the addition of calmodulin to the pipette solution (0.15 mg/ml) or by using the perforated-patch whole-cell voltage-clamp technique. Moreover, W-7 [ N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a calmodulin antagonist, was a reversible inhibitor of I. However, W-7 had only a weak inhibitory effect on the same cationic current which was induced by guanosine 5′- O-(3-thiotriphosphate) (GTP[ γS] 0.2 mM) in the pipette solution. This GTP[ γS]-induced cationic current was still markedly suppressed by the Ca-free bath solution. W-7 itself had a weak inhibitory effect on voltage-operated Ca channels as well as the effects on I. These data suggest that multiple Ca-dependent pathways are involved in the activation of CCh-gated cation channels in guinea-pig antral myocytes and a Ca/calmodulin-dependent pathway would be one of them. [ABSTRACT FROM AUTHOR]

Ion channels in the basolateral membrane of colonic crypts were investigated with the patch-clamp technique during stimulation of secretion. Intact crypts were isolated from rat distal colon and the cell potential was recorded by addition of nystatin to the pipette solution. The cell resting potential in the base of the crypt was −74±1 mV ( n=90). Addition of 100 μM carbachol to the bath resulted in a transient hyperpolarization by 9 mV, which was probably due to the opening of basolateral K channels. In contrast, application of prostaglandin E (PGE, 1 nM−1 μM) caused a dose-dependent depolarization in the base of the crypt. With 1 μM PGE cells depolarized from -74±1 to −27±2 mV ( n=26). Cell potential recordings in the midcrypt showed only a slight and transient depolarization after application of PGE, whereas cells close to the surface of the crypt had no response. In the base of the crypt the PGE-induced depolarization could be completely inhibited by addition of 50 μM flufenamic acid, a known blocker of nonselective cation channels. After substitution of all monovalent cations by N-methyl- D-glucamine in the bath, PGE had no significant effect on the cell potential. Cell-attached experiments with no nystatin in the patch pipette revealed the activation of ion channels in the basolateral membrane after application of PGE. After excision of the membrane patch, these channels could be identified as nonselective cation channels. Experiments involving substitution of the bath solution showed that the channel is impermeable for Cl and scarcely permeable for Ca ions. The permeability sequence for monovalent cations, as calculated from reversal potentials, is NH>Na=K>Rb=Li≫TRIS=NMDG. Single channels are completely inhibited by flufenamic acid (50 μM), mefenamic acid (200 μM), as well as by 3′, 5-dichlorodiphenylamine-2-carboxylate. In conclusion, PGE activates nonselective cation channels in the basolateral membrane of cells in the base of colonic crypts. It is suggested that this mechanism initiates the secretion of K ions. Na influx through the nonselective cation channel will stimulate the Na/K pump and active uptake of K at the basolateral side. K can leave the cell at the luminal side through K-selective channels. [ABSTRACT FROM AUTHOR]

Nonselective Ca-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca on the cytosolic side, the mean openstate probability P of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3′,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 μmol/l of NPPB or DPC decreased P from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 μmol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reduced P, but in contrast to the abov mentioned substances, it induced fast flickering. Ba (70 mmol/l) and tetraethylammonium (TEA; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 4,4′-dinitro-2,2′-stilbenedisulfonate (DNDS). 10 μmol/l SITS applied to the cytosolic side increased P from 0.5 to 0.7 and with 100 μmol/l SITS the channels remained nearly permanently in its open state ( P≅1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca concentration to 0.1 μmol/l, addition of 100 μmol/l of SITS had no effect. Similarly, reducing bath Ca concentration from 1.3 mmol/l in presence of 100 μmol/l SITS (channels are maximally activated) to 0.1 μmol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca ions. [ABSTRACT FROM AUTHOR]

Transient receptor potential canonical (TRPC) channels constitute a group of receptor-operated calcium-permeable nonselective cation channels of the TRP superfamily. The seven mammalian TRPC members, which can be further divided into four subgroups (TRPC1, TRPC2, TRPC4/5, and TRPC3/6/7) based on their amino acid sequences and functional similarities, contribute to a broad spectrum of cellular functions and physiological roles. Studies have revealed complexity of their regulation involving several components of the phospholipase C pathway, G i and G o proteins, and internal Ca2+ stores. Recent advances in cryogenic electron microscopy have provided several high-resolution structures of TRPC channels. Growing evidence demonstrates the involvement of TRPC channels in diseases, particularly the link between genetic mutations of TRPC6 and familial focal segmental glomerulosclerosis. Because TRPCs were discovered by the molecular identity first, their pharmacology had lagged behind. This is rapidly changing in recent years owning to great efforts from both academia and industry. A number of potent tool compounds from both synthetic and natural products that selective target different subtypes of TRPC channels have been discovered, including some preclinical drug candidates. This review will cover recent advancements in the understanding of TRPC channel regulation, structure, and discovery of novel TRPC small molecular probes over the past few years, with the goal of facilitating drug discovery for the study of TRPCs and therapeutic development. [ABSTRACT FROM AUTHOR]