Pharmacological profile of phosphatidylinositol 3-kinases and related phosphatidylinositols mediating endothelin(A) receptor-operated native TRPC channels in rabbit coronary artery myocytes.

BACKGROUND AND PURPOSE: Endothelin(A) (ET(A) ) receptor-operated canonical transient receptor potential (TRPC) channels mediate Ca²âº influx pathways, which are important in coronary artery function. Biochemical pathways linking ET(A) receptor stimulation to TRPC channel opening are unknown. We investigated the involvement of phosphatidylinositol 3-kinases (PI3K) in ET(A) receptor activation of native heteromeric TRPC1/C5/C6 and TRPC3/C7 channels in rabbit coronary artery vascular smooth muscle cells (VSMCs). EXPERIMENTAL APPROACH: A pharmacological profile of PI3K was created by studying the effect of pan-PI3K, pan-Class I PI3K and Class I PI3K isoform-selective inhibitors on ET(A) receptor-evoked single TRPC1/C5/C6 and TRPC3/C7 channel activities in cell-attached patches from rabbit freshly isolated coronary artery VSMCs. The action of phosphatidylinositol 3-phosphate- [PI(3)P], 4-phosphate- [PI(4)P] and 5-phosphate- [PI(5)P] containing molecules involved in PI3K-mediated reactions were studied in inside-out patches. Expression of PI3K family members in coronary artery tissue lysates were analysed using quantitative PCR. KEY RESULTS: ET(A) receptor-operated TRPC1/C5/C6 and TRPC3/C7 channel activities were inhibited by wortmannin. However, ZSTK474 and AS252424 reduced ET(A) receptor-evoked TRPC1/C5/C6 channel activity but potentiated TRPC3/C7 channel activity. All the PI(3)P-, PI(4)P- and PI(5)P-containing molecules tested induced TRPC1/C5/C6 channel activation, whereas only PI(3)P stimulated TRPC3/C7 channels. CONCLUSIONS AND IMPLICATIONS: ET(A) receptor-operated native TRPC1/C5/C6 and TRPC3/C7 channel activities are likely to be mediated by Class I PI3Kγ and Class II/III PI3K isoforms, respectively. ET(A) receptor-evoked and constitutively active PI3Kγ-mediated pathways inhibit TRPC3/C7 channel activation. PI3K-mediated pathways are novel regulators of native TRPC channels in VSMCs, and these signalling cascades are potential pharmacological targets for coronary artery disease.

TRPC6 channels stimulated by angiotensin II are inhibited by TRPC1/C5 channel activity through a Ca2+- and PKC-dependent mechanism in native vascular myocytes.

The present work investigated interactions between TRPC1/C5 and TRPC6 cation channel activities evoked by angiotensin II (Ang II) in native rabbit mesenteric artery vascular smooth muscle cells (VSMCs). In low intracellular Ca(2+) buffering conditions (0.1 mm BAPTA), 1 nm and 10 nm Ang II activated both 2 pS TRPC1/C5 channels and 15-45 pS TRPC6 channels in the same outside-out patches. However, increasing Ang II to 100 nm abolished TRPC6 activity but further increased TRPC1/C5 channel activity. Comparison of individual patches revealed an inverse relationship between TRPC1/C5 and TRPC6 channel activity suggesting that TRPC1/C5 inhibits TRPC6 channel activity. Inclusion of anti-TRPC1 and anti-TRPC5 antibodies, raised against intracellular epitopes, in the patch pipette solution blocked TRPC1/C5 channel currents but potentiated by about six-fold TRPC6 channel activity evoked by 1-100 nm Ang II in outside-out patches. Bath application of T1E3, an anti-TRPC1 antibody raised against an extracellular epitope, also increased Ang II-evoked TRPC6 channel activity. With high intracellular Ca(2+) buffering conditions (10 mm BAPTA), 10 nm Ang II-induced TRPC6 channel activity was increased by about five-fold compared to channel activity with low Ca(2+) buffering. In addition, increasing intracellular Ca(2+) levels ([Ca(2+)](i)) at the cytosolic surface inhibited 10 nm Ang II-evoked TRPC6 channel activity in inside-out patches. Moreover, in zero external Ca(2+) (0 [Ca(2+)](o)) 100 nm Ang II induced TRPC6 channel activity in outside-out patches. Pre-treatment with the PKC inhibitor, chelerythrine, markedly increased TRPC6 channel activity evoked by 1-100 nm Ang II and blocked the inhibitory action of [Ca(2+)](i) on TRPC6 channel activity. Co-immunoprecipitation studies shows that Ang II increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca(2+)](o) and the anti-TRPC1 antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5-mediated Ca(2+) influx and stimulation of PKC, which phosphorylates TRPC6 subunits. These conclusions represent a novel interaction between two distinct vasoconstrictor-activated TRPC channels expressed in the same native VSMCs.

Multiple activation mechanisms of store-operated TRPC channels in smooth muscle cells.

Store-operated channels (SOCs) are plasma membrane Ca2+-permeable cation channels which are activated by agents that deplete intracellular Ca2+ stores. In smooth muscle SOCs are involved in contraction, gene expression, cell growth and proliferation. Single channel recording has demonstrated that SOCs with different biophysical properties are expressed in smooth muscle indicating diverse molecular identities. Moreover it is apparent that several gating mechanisms including calmodulin, protein kinase C and lysophospholipids are involved in SOC activation. Evidence is accumulating that TRPC proteins are important components of SOCs in smooth muscle. More recently Orai and STIM proteins have been proposed to underlie the well-described calcium-release-activated current (ICRAC) in non-excitable cells but at present there is little information on the role of Orai and STIM proteins in smooth muscle. In addition it is likely that different TRPC subunits coassemble as heterotetrameric structures to form smooth muscle SOCs. In this brief review we summarize the diverse properties and gating mechanisms of SOCs in smooth muscle. We propose that the heterogeneity of the properties of these conductances in smooth muscle results from the formation of heterotetrameric TRPC structures in different smooth muscle preparations.

Endothelin-1 activates a Ca2+-permeable cation channel with TRPC3 and TRPC7 properties in rabbit coronary artery myocytes.

In the present work we used patch pipette techniques to study the properties of a novel Ca(2+)-permeable cation channel activated by the potent coronary vasoconstrictor endothelin-1 (ET-1) in freshly dispersed rabbit coronary artery myocytes. With cell-attached recording bath application of 10 nm ET-1 evoked cation channel currents (I(cat)) with subconductance states of about 18, 34 and 51 and 68 pS, and a reversal potential of 0 mV. ET-1 evoked channel activity when extracellular Ca(2+) was the charge carrier, illustrating significant Ca(2+) permeability. ET-1-induced responses were inhibited by the ET(A) receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also stimulated I(cat), but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG- or ET-1-induced I(cat). Inositol 1,4,5-trisphosphate (IP(3)) did not activate I(cat), but greatly potentiated the response to OAG and this effect was blocked by heparin. Bath application of anti-TRPC3 and anti-TRPC7 antibodies to inside-out patches markedly inhibited ET-1-evoked I(cat), but antibodies to TRPC1, C4, C5 and C6 had no effect. Immunocytochemical studies demonstrated preferential TRPC7 expression in the plasmalemma, whereas TRPC3 was distributed throughout the myocyte, and moreover co-localization of TRPC3 and TRPC7 signals was observed at, or close to, the plasma membrane. Flufenamic acid, Gd(3+), La(3+) and extracellular Ca(2+) inhibited I(cat) with IC(50) values of 2.45 microm, 3.8 microm, 7.36 microm and 22 microm, respectively. These results suggest that in rabbit coronary artery myocytes ET-1 evokes a Ca(2+)-permeable non-selective cation channel with properties similar to TRPC3 and TRPC7, and indicates that these proteins may be important components of this conductance.

Angiotensin II activates two cation conductances with distinct TRPC1 and TRPC6 channel properties in rabbit mesenteric artery myocytes.

Angiotensin II (Ang II) is a potent vasoconstrictor with an important role in controlling blood pressure; however, there is little information on cellular mechanisms underlying Ang II-evoked vasoconstrictor responses. The aim of the present study is to investigate the effect of Ang II on cation conductances in freshly dispersed rabbit mesenteric artery myocytes at the single-channel level using patch-clamp techniques. In cell-attached patches, bath application of low concentrations of Ang II (1 nM) activated cation channel currents (Icat1) with conductances states of about 15, 30 and 45 pS. At relatively high concentrations, Ang II (100 nM) inhibited Icat1 but evoked another cation channel (Icat2) with a conductance of approximately 2 pS. Ang II-evoked Icat1 and Icat2 were inhibited by the AT1 receptor antagonist losartan and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol (DAG) lipase inhibitor RHC80267 initially induced Icat1 which was subsequently inhibited to reveal Icat2. The DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (1 microM) activated Icat1 and Icat2 but inositol 1,4,5-trisphosphate did not evoke either conductance. The protein kinase C (PKC) inhibitor chelerythrine (3 microM) potentiated Ang II-evoked Icat1 and inhibited Icat2 whereas the PKC activator phorbol-12,13-dibutyrate (1 microM) reduced Ang II-induced Icat1 but activated Icat2. Moreover in cell-attached patches pretreated with chelerythrine, application of 100 nM Ang II activated Icat1. These data indicate that PKC inhibits Icat1 but stimulates Icat2. Agents that deplete intracellular Ca2+ stores also activated cation channel currents with similar properties to Icat2. Bath application of anti-TRPC6 and anti-TRPC1 antibodies to inside-out patches inhibited Icat1 and Icat2, respectively. Also flufenamic acid and zero external Ca2+ concentration, respectively, potentiated and reduced Ang II-evoked Icat1. Immunocytochemical studies showed TRPC6 and TRPC1 expression with TRPC6 preferentially distributed in the plasma membrane and TRPC1 expression located throughout the myocyte. These results indicate that Ang II activates two distinct cation conductances in mesenteric artery myocytes by stimulation of AT1 receptors linked to PLC. Icat1 is activated by DAG via a PKC-independent mechanism whereas Icat2 involves DAG acting via a PKC-dependent pathway. Higher concentrations of Ang II inhibit Icat1 by activating an inhibitory effect of PKC. It is proposed that TRPC6 and TRPC1 channel proteins are important components of Ang II-induced Icat1 and Icat2, respectively.

TRPC3 properties of a native constitutively active Ca2+-permeable cation channel in rabbit ear artery myocytes.

Previously we have described a constitutively active, Ca2+-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside-out patches with IC50 values of 6.8 microm, 25 nm, 1.5 microm and 0.124 mm, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.

Signal transduction pathways and gating mechanisms of native TRP-like cation channels in vascular myocytes.

Activation of Ca2+-permeable non-selective cation channels produces an increase in excitability of vascular smooth muscle cells which has an important role in vasoconstriction. These channels are activated by various physiological stimuli including vasoconstrictor agents such as noradrenaline, depletion of internal Ca2+ stores and cell stretching. In addition cation channels have been shown to be constitutively active and these channels are thought to contribute to resting membrane conductance and basal Ca2+ influx in vascular myocytes. Recent evidence has suggested that transient receptor potential (TRP) proteins represent strong candidates for these channels in the vasculature. This review discusses proposed signal transduction pathways and gating mechanisms which link physiological stimuli to opening of cation channels in vascular myocytes. It is apparent that G-protein-coupled pathways linked to stimulation of phospholipase activity have a profound effect on regulating channel activity and that generation of diacylglycerol (DAG) is a central event in these signalling cascades with this triglyceride having a pivotal role in gating cation channels via both PKC-independent and -dependent mechanisms. Moreover phosphorylation processes produced by stimulation of protein kinases have been proposed to have an important role in regulating cation channel activity.

Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes.

Previously we have described a constitutively active Ca2+-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel activity through activation of PKC. This study indicates that constitutive cation channel activity in ear artery myocytes is mediated by DAG which is generated by PC-PLD via phosphatidic acid which represents a novel activation pathway of cation channels in vascular myocytes.

Facilitatory effect of Ins(1,4,5)P3 on store-operated Ca2+-permeable cation channels in rabbit portal vein myocytes.

In rabbit portal vein smooth muscle cells, store-operated Ca2+-permeable cation channels (SOCs) display multi-modal gating mechanisms. SOCs are activated by depletion of intracellular Ca2+ stores but also may be stimulated in a store-independent manner by noradrenaline acting on alpha-adrenoceptors and by diacylglycerol (DAG) via protein kinase C (PKC). In the present study we have investigated whether inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modulates SOC activity in freshly dispersed rabbit portal vein myocytes with patch pipette recording techniques. Inclusion of 1 mum Ins(1,4,5)P3 in the patch pipette solution increased whole-cell currents evoked by the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) by about 3-fold at -80 mV. In the cell-attached configuration the cell-permeable Ca2+ chelator BAPTA-AM stimulated SOC activity and after excision of an isolated inside-out patch bath application of 1 mum Ins(1,4,5)P3 increased open channel probability (NP(o)) by approximately 3-fold. Ins(1,4,5)P3 also produced a similar increase in NP(o) of SOCs stimulated by the phorbol ester, phorbol 12,13-dibutyrate (PDBu) in inside-out patches and these channel currents had a unitary conductance of about 2 pS. The equilibrium constant of Ins(1,4,5)P3 on increasing PDBu-evoked SOC activity was about 0.4 mum. The facilitatory effect of Ins(1,4,5)P3 was also manifest as markedly increasing the rate of activation of SOCs. The synergistic effect of Ins(1,4,5)P3 was mimicked by the metabolically stable analogue 3-fluoro-Ins(1,4,5)P3 and Ins(1,4)P2, a metabolite of Ins(1,4,5)P3, but was not inhibited by the classical Ins(1,4,5)P3 receptor antagonist heparin. Finally Ins(1,4,5)P3 also increased NP(o) of SOCs activated by a PKC catalytic subunit. It is concluded that Ins(1,4,5)P3 facilitates SOC opening via a heparin-insensitive mechanism at, or close to, the channel protein.

Stimulation of beta-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes.

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca(2+) store depletion these SOCs could also be activated by alpha-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of beta-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective beta-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl-sn-glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of beta-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.

Inhibitory regulation of constitutive transient receptor potential-like cation channels in rabbit ear artery myocytes.

In the present study we have investigated an inhibitory pathway regulating a constitutively active Ca(2+)-permeable non-selective cation conductance (I(cat)) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of I(cat) was recorded in cell-attached and inside-out patches with similar unitary conductance values. In inside-out patches with relatively high constitutive activity the G-protein activator GTPgammaS inhibited channel activity which was reversed by the protein kinase C (PKC) inhibitor chelerythrine indicating a G-protein pathway inhibits channel activity via PKC. Spontaneous channel activity was also suppressed by the G-protein inhibitor GDPbetaS suggesting a G-protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to G(alphaq)/G(alpha11) enhanced channel activity whereas anti-G(alpha1-3)/G(alphao) antibodies decreased basal channel activity which suggests that G(alphaq)/G(alpha11) and G(alphaiota)/G(alphao) proteins initiate, respectively, the inhibitory and excitatory cascades. The phospholipase C (PLC) inhibitor U73122 increased spontaneous activity which implies a role for PLC in the inhibitory pathway. Bath application of the diacylycerol (DAG) analogue 1-oeoyl-2-acetyl-sn-glycerol (OAG) decreased the probability of channel opening (NP(o)) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NP(o). These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving G(alphaq)/G(alpha11)-mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NP(o) and it was evident that the inhibitory pathway was particularly marked in these cases. Moreover in the latter patches GTPgammaS and OAG caused marked increases in NP(o). Together with inhibitory effects of GDPbetaS and anti-G(alpha1-3)/G(alphao) antibodies the results suggest that there is constitutive G(alphai)/G(alphao) protein activity leading to channel opening via a DAG-mediated but PKC-independent mechanism. Finally, with whole-cell recording it is shown that noradrenaline increases I(cat) and the noradrenaline-evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist-evoked I(cat) in these arterial myocytes.

Direct effect of Ca2+-calmodulin on cGMP-activated Ca2+-dependent Cl-channels in rat mesenteric artery myocytes.

Recently a novel cGMP-activated Ca2+-dependent Cl- channel has been described in rat mesenteric artery smooth muscle cells. In the present work we have investigated the actions of calmodulin (CaM) on single channel cGMP-activated Ca2+-dependent Cl- current (ICl(cGMP,Ca) in inside-out patches. When 1 microm CaM was applied to the intracellular surface of inside-out patches bathed with 10 microm cGMP and 100 nm [Ca2+]i there was approximately a 10-fold increase in channel open probability (NPo). This effect of CaM was not observed with lower [Ca2+]i and 100 nm [Ca2+]i with 1 microm CaM did not activate Cl- channels in the absence of cGMP. The unitary conductance, reversal potential and mean open time of the single-channel currents were similar in the absence or presence of CaM. With 10 microm cGMP and 100 nm [Ca2+]i the relationship between NPo and CaM concentration was well fitted by the Hill equation yielding an equilibrium constant for CaM of about 1.9 nm and a Hill coefficient of 1.7. With 1 microm CaM (+10 microm cGMP) the relationship between [Ca2+]i and NPo was also fitted by the Hill equation which yielded an apparent equilibrium constant of 74 nm [Ca2+]i and a Hill coefficient of 4.8. When [Ca2+]i was increased from 300 nm to 1 microm there was a decrease in NPo. The potentiating effect of CaM was markedly reduced by the selective CaM binding peptide Trp (5 nm) but not by the Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor autocamtide II related inhibitory peptide (AIP). It is concluded that CaM potentiates the activity of single channel ICl(cGMP,Ca) by increasing the probability of channel opening via a CaMKII-independent mechanism.

Single cGMP-activated Ca(+)-dependent Cl(-) channels in rat mesenteric artery smooth muscle cells.

The present study describes the single channel properties of a novel cGMP-activated Ca(2+)-dependent Cl(-) channel in rat mesenteric artery smooth muscle cells. Single channel currents were recorded in cell-attached patches in the presence of 8 Br cGMP in response to the addition of caffeine or noradrenaline and in both outside-out and inside-out patches when the internal patch surface was bathed in cGMP and Ca(2+). The channels were permeable to Cl(-) ions with an anion permeability sequence of SCN(-) (1.7) > Cl(-) (1.0) > I(-) (0.6). Single channel mean open probability (NP(o)) was independent of voltage and the channels displayed three conductance levels of 15, 35 and 55 pS. cGMP was required for channel activation and the single channel NP(o) increased sharply with raised [Ca(2+)](i), maximal activation occurring at a [Ca(2+)](i) of about 100 nM. The relationship between NP(o) and cGMP concentration was voltage independent and could be fitted by the Hill equation giving a K(d) of about 3 microM and a Hill coefficient (n(H)) of 3. cGMP- and Ca(2+)-dependent channel currents were inhibited by 10 microM ZnCl(2) but niflumic acid, an inhibitor of Ca(2+)-activated Cl(-) channels, had no effect. Inhibition of cGMP-dependent protein kinase activity by the cGMP-dependent protein kinase inhibitor KT5823 or replacement of ATP by AMP-PNP reduced NP(o), while activation of cGMP-dependent protein kinase by guanosine 3', 5'-cyclic monophosphate, beta-phenyl-1, N(2)-etheno-8-bromo-sodium salt (8 Br PET cGMP) produced a significant increase in single channel NP(o). It is likely that these single channel currents underlie the noradrenaline-activated inward current important for vasomotion in these resistance arteries.

Synergism between inositol phosphates and diacylglycerol on native TRPC6-like channels in rabbit portal vein myocytes.

In rabbit portal vein myocytes noradrenaline activates a non-selective cation current (Icat) which involves a transient receptor potential protein (TRPC6). Previously we have shown that the diaylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulates Icat via a protein kinase C (PKC)-independent mechanism, and in the present study we have investigated the interaction between inositol phosphates (InsPs) and OAG on Icat. With whole-cell recording of Icat from freshly isolated rabbit portal vein myocytes the amplitude and rate of activation of noradrenaline-evoked Icat were much greater than those of OAG-induced Icat. Inclusion of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the pipette solution did not evoke Icat but greatly potentiated the amplitude and rate of activation of OAG-induced Icat. With isolated outside-out patches Ins(1,4,5)P3 markedly increased the rate of activation and the open probability of OAG-evoked channel activity, with no change in unitary conductance, channel mean open times or burst durations. The effects of Ins(1,4,5)P3 were mimicked by Ins(2,4,5)P3, 3-F-Ins(1,4,5)P3 and Ins(1,4)P2 but not by Ins(1,3,4,5)P4 and the potentiating effects of InsPs were not inhibited by heparin. Therefore it is concluded that both DAG and InsPs are necessary for full activation of Icat by noradrenaline and the effect of InsPs is via a heparin-insensitive mechanism and represents a novel action of InsPs.

Store-operated Ca2+-permeable non-selective cation channels in smooth muscle cells.

Over twenty years ago it was shown that depletion of the intracellular Ca2+ store in smooth muscle triggered a Ca2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca2+ ions. Two activation mechanisms have been identified which involve Ca2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of alpha-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.

Properties of a constitutively active Ca2+-permeable non-selective cation channel in rabbit ear artery myocytes.

In smooth muscle, non-selective cation conductances contribute to agonist-evoked depolarisation and contraction, and in the present study using patch-pipette techniques we describe the properties of a constitutively active cation channel. With whole-cell recording in K+-free conditions, there was a spontaneous current with a reversal potential (Er) that was altered by replacement of external Na+ by an impermeant cation, but not when external Cl- was replaced by an impermeant anion. The tonic cation inward current could be carried by Ca2+ ions and was greatly enhanced when the external Ca2+ concentration was reduced. In outside-out patches there was spontaneous cation channel activity that could be resolved into three conductance states of about 15, 25 and 40 pS, all with the same Er as the whole-cell current. Kinetic analysis revealed that there were two open times of about 1 and 5 ms and that the currents displayed bursting kinetics with burst durations of approximately 5 ms and 25 ms. Removal of external Ca2+ ions increased the probability of channel opening (Po) sixfold, which was associated with an increase in the longer burst duration. Bath application of the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol increased Po, but phorbol 12,13-dibutyrate, which stimulates protein kinase C (PKC), reduced channel activity. In contrast, the PKC inhibitor chelerythrine increased the activity of channel currents. It is concluded that in rabbit ear artery myocytes there is a constitutively active Ca2+-permeable cation channel that is regulated by external Ca2+ ions and suppressed by tonic PKC activity. It is proposed that this mechanism may contribute to the resting membrane conductance and basal Ca2+ influx in this particular arterial preparation.

Multiple conductance states of single Ca2+-activated Cl- channels in rabbit pulmonary artery smooth muscle cells.

Ca2+-activated Cl- channels contribute to agonist-evoked contraction and spontaneous activity in some smooth muscle preparations. Patch pipette techniques were used to study the properties of single Ca2+-activated Cl- channels in freshly dispersed rabbit pulmonary artery myocytes. In the cell-attached recording mode, two conductance states of 3.5 and 1.8 pS were recorded either spontaneously or in response to increasing [Ca2+]i. With inside-out patches, the 3.5 pS channel current predominated at 50 nM [Ca2+]i, but at 500 nM [Ca2+]i most channels opened to the 1.8 pS level and an additional 1.2 pS channel conductance was resolved. At 1 microM [Ca2+]i all of the Cl- channels opened either to the 1.8 pS or 1.2 pS level. In 0 [Ca2+]i, no channel activity was observed at -100 mV to +100 mV, but with 10-250 nM [Ca2+]i the total single channel open probability (NP(o)) increased with depolarisation. This voltage dependence was not seen at higher values of [Ca2+]i. The plot of NPo vs. [Ca2+]i yielded Ca2+ affinity constants of 8 and 250 nM and Hill slopes of 1.3 and 2.3 at +100 and -100 mV, respectively. The distribution of open times was fitted by two exponentials of about 5 and 30 ms, which were neither voltage nor Ca2+ dependent. Replacement of external Cl- by I- shifted the reversal potential by about -30 mV and lengthened the longer of the two mean open times without significant effects on other kinetic parameters. Based on these data, a model for the activation of Ca2+-activated Cl- channels is proposed.

Activation of store-operated channels by noradrenaline via protein kinase C in rabbit portal vein myocytes.

In the present study we have investigated the role of diacylglycerol (DAG) and protein kinase C (PKC) in mediating activation of Ca(2+)-permeable store-operated channels (SOCs) by noradrenaline in rabbit portal vein smooth muscle cells. With cell-attached recording, bath application of noradrenaline, 1-oleoyl-acetyl-sn-glycerol (OAG) and phorbol 12,13-dibutyrate (PDBu) evoked single channel currents. The biophysical properties of these channel currents were similar to those of the channel currents activated by depletion of internal Ca(2+) stores with cyclopiazonic acid (CPA). The activation of SOCs in cell-attached recording by noradrenaline, OAG, PDBu, CPA and the acetoxymethyl ester form of BAPTA (BAPTA-AM) was markedly inhibited by the PKC inhibitors chelerythrine and RO-31-8220. In isolated outside-out patches CPA did not evoke SOCs but noradrenaline stimulated SOC activity, which was reduced by about 90 % by PKC inhibitors. The addition of the serine/threonine phosphatase inhibitors calyculin A and microcystin also stimulated SOCs in isolated outside-out patches. It is concluded that in rabbit portal vein myocytes, noradrenaline activates SOCs via DAG and PKC, possibly by a store-independent mechanism. In addition in this cell type it appears that PKC and phosphorylation may play an important role in stimulating SOC activity in response to depletion of internal Ca(2+) stores by CPA and BAPTA-AM.

Modulation of volume-sensitive chloride current by noradrenaline in rabbit portal vein myocytes.

The effect of noradrenaline on the volume-sensitive chloride current (I(Cl(swell))) was studied with conventional whole-cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of I(Cl(swell)) in some cells and a decrease in others. In the presence of the beta-adrenoceptor antagonist propranolol, noradrenaline increased I(Cl(swell)) and in the presence of the alpha(1)-adrenoceptor antagonist prazosin, noradrenaline reduced I(Cl(swell).) The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of I(Cl(swell)) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu) increased the amplitude of I(Cl(swell)) by approximately 60 and 100 %, respectively, in a voltage-independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin-C decreased the amplitude of I(Cl(swell)) in a concentration-dependent but voltage-independent manner. Bath application of 8-Br-cAMP decreased I(Cl(swell)) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of I(Cl(swell)) by approximately 80-90 %. In the presence of propranolol, chelerythrine prevented the increase of I(Cl(swell)) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances I(Cl(swell)) by acting on alpha(1)-adrenoceptors and reduces I(Cl(swell)) by stimulating beta-adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

Dual effect of blocking agents on Ca2+-activated Cl(-) currents in rabbit pulmonary artery smooth muscle cells.

The effects of the Cl- channel antagonists, niflumic acid (NFA), dichloro-diphenylamine 2-carboxylic acid (DCDPC) and diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) on Ca2+-activated Cl- current (I(Cl(Ca))) evoked by adding fixed intracellular calcium concentrations ([Ca2+]i) to the pipette solution were studied in rabbit pulmonary artery myocytes. With 250 and 500 nM [Ca2+]i bath application of NFA (100 microM) increased inward current at negative potentials, but inhibited outward current at positive potentials. On wash out of NFA, I(Cl(Ca)) was greatly enhanced at all potentials. When external Na+ ions were replaced by N-methyl-D-glucamine (NMDG+) NFA still enhanced I(Cl(Ca)) at negative potentials but the increase of I(Cl(Ca)) on wash out was blocked. When the mean reversal potential (E(r)) of I(Cl(Ca)) was shifted to negative potentials by replacing external Cl- with SCN-, NFA increased inward current but blocked outward current suggesting that the effect of NFA is dependent on current flow. Inclusion of NFA in the pipette solution had no effect on I(Cl(Ca)). Voltage jump experiments indicated that I(Cl(Ca)) displayed characteristic outward current relaxations at +70 mV and inward current relaxations at -80 mV that were abolished by NFA. DCDPC (100 microM) produced similar effects to NFA but 1 mM DIDS produced inhibition of I(Cl(Ca)) at both positive and negative potentials and there was no increase in current on wash out of DIDS. These results suggest that NFA and DCDPC, but not DIDS, simultaneously enhance and block I(Cl(Ca)) by binding to an external site, probably close to the mouth of the chloride channel.