Anthony P. Albert

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 μm) activated Icat1 and Icat2 but inositol 1,4,5‐trisphosphate did not evoke either conductance. The protein kinase C (PKC) inhibitor chelerythrine (3 μm) potentiated Ang II‐evoked Icat1 and inhibited Icat2 whereas the PKC activator phorbol‐12,13‐dibutyrate (1 μm) 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.

Diverse properties of store-operated TRPC channels activated by protein kinase C in vascular myocytes

In vascular smooth muscle, store‐operated channels (SOCs) contribute to many physiological functions including vasoconstriction and cell growth and proliferation. In the present work we compared the properties of SOCs in freshly dispersed myocytes from rabbit coronary and mesenteric arteries and portal vein. Cyclopiazonic acid (CPA)‐induced whole‐cell SOC currents were sixfold greater at negative membrane potentials and displayed markedly different rectification properties and reversal potentials in coronary compared to mesenteric artery myocytes. Single channel studies showed that endothelin‐1, CPA and the cell‐permeant Ca2+ chelator BAPTA‐AM activated the same 2.6 pS SOC in coronary artery. In 1.5 mm[Ca2+]o the unitary conductance of SOCs was significantly greater in coronary than in mesenteric artery. Moreover in 0 mm[Ca2+]o the conductance of SOCs in coronary artery was unaltered whereas the conductance of SOCs in mesenteric artery was increased fourfold. In coronary artery SOCs were inhibited by the protein kinase C (PKC) inhibitor chelerythrine and activated by the phorbol ester phorbol 12,13‐dibutyrate (PDBu), the diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and a catalytic subunit of PKC. These data infer an important role for PKC in activation of SOCs in coronary artery similar to mesenteric artery and portal vein. Anti‐TRPC1 and ‐TRPC5 antibodies inhibited SOCs in coronary and mesenteric arteries and portal vein but anti‐TRPC6 blocked SOCs only in coronary artery and anti‐TRPC7 blocked SOCs only in portal vein. Immunoprecipitation showed associations between TRPC1 and TRPC5 in all preparations but between TRPC5 and TRPC6 only in coronary artery and between TRPC5 and TRPC7 only in portal vein. Finally, flufenamic acid increased SOC activity in coronary artery but inhibited SOCs in mesenteric artery and portal vein myocytes. These data provide strong evidence that vascular myocytes express diverse SOC isoforms, which are likely to be composed of different TRPC proteins and have different physiological functions.

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.

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.

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 Ca2+‐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 (Icat) 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 Ca2+ was the charge carrier, illustrating significant Ca2+ permeability. ET‐1‐induced responses were inhibited by the ETA receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) also stimulated Icat, but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG‐ or ET‐1‐induced Icat. Inositol 1,4,5‐trisphosphate (IP3) did not activate Icat, 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 Icat, 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, Gd3+, La3+ and extracellular Ca2+ inhibited Icat with IC50 values of 2.45 μm, 3.8 μm, 7.36 μm and 22 μm, respectively. These results suggest that in rabbit coronary artery myocytes ET‐1 evokes a Ca2+‐permeable non‐selective cation channel with properties similar to TRPC3 and TRPC7, and indicates that these proteins may be important components of this conductance.

Activation of native TRPC1/C5/C6 channels by endothelin‐1 is mediated by both PIP3 and PIP2 in rabbit coronary artery myocytes

We investigate activation mechanisms of native TRPC1/C5/C6 channels (termed TRPC1 channels) by stimulation of endothelin‐1 (ET‐1) receptor subtypes in freshly dispersed rabbit coronary artery myocytes using single channel recording and immunoprecipitation techniques. ET‐1 evoked non‐selective cation channel currents with a unitary conductance of 2.6 pS which were not inhibited by either ETA or ETB receptor antagonists, respectively BQ‐123 and BQ788, when administered separately. However, in the presence of both antagonists, ET‐1‐evoked channel activity was abolished indicating that both ETA and ETB receptor stimulation activate this conductance. Stimulation of both ETA and ETB receptors evoked channel activity which was inhibited by the protein kinase C (PKC) inhibitor chelerythrine and by anti‐TRPC1 antibodies indicating that activation of both receptor subtypes causes TRPC1 channel activation by a PKC‐dependent mechanism. ETA receptor‐mediated TRPC1 channel activity was selectively inhibited by phosphoinositol‐3‐kinase (PI‐3‐kinase) inhibitors wortmannin (50 nm) and PI‐828 and by antibodies raised against phosphoinositol‐3,4,5‐trisphosphate (PIP3), the product of PI‐3‐kinase‐mediated phosphorylation of phosphatidylinositol 4,5‐bisphosphate (PIP2). Moreover, exogenous application of diC8‐PIP3 stimulated PKC‐dependent TRPC1 channel activity. These results indicate that stimulation of ETA receptors evokes PKC‐dependent TRPC1 channel activity through activation of PI‐3‐kinase and generation of PIP3. In contrast, ETB receptor‐mediated TRPC1 channel activity was inhibited by the PI‐phospholipase C (PI‐PLC) inhibitor U73122. 1‐Oleoyl‐2‐acetyl‐sn‐glycerol (OAG), an analogue of diacylglycerol (DAG), which is a product of PI‐PLC, also activated PKC‐dependent TRPC1 channel activity. OAG‐induced TRPC1 channel activity was inhibited by anti‐phosphoinositol‐4,5‐bisphosphate (PIP2) antibodies and high concentrations of wortmannin (20 μm) which depleted tissue PIP2 levels. In addition exogenous application of diC8‐PIP2 activated PKC‐dependent TRPC1 channel activity. These data indicate that stimulation of ETB receptors evokes PKC‐dependent TRPC1 activity through PI‐PLC‐mediated generation of DAG and requires a permissive role of PIP2. In conclusion, we provide the first evidence that stimulation of ETA and ETB receptors activate native PKC‐dependent TRPC1 channels through two distinct phospholipids pathways involving a novel action of PIP3, in addition to PIP2, in rabbit coronary artery myocytes.

Role of phosphoinositol 4,5-bisphosphate and diacylglycerol in regulating native TRPC channel proteins in vascular smooth muscle

Stimulation of receptor-operated (ROCs) and store-operated (SOCs) Ca(2+)-permeable cation channels by vasoconstrictors has many important physiological functions in vascular smooth muscle. The present review indicates that ROCs and SOCs with diverse properties in different blood vessels are likely to be explained by composition of different subunits from the canonical transient receptor potential (TRPC) family of cation channel proteins. In addition we illustrate that activation of native TRPC ROCs and SOCs involves different phospholipase-mediated transduction pathways linked to generation of diacylglycerol (DAG). Moreover we describe recent novel data showing that the endogenous phospholipid phosphoinositol 4,5-bisphosphate (PIP(2)) has profound and contrasting actions on TRPC ROCs and SOCs. Optimal activation of a native TRPC6 ROC by angiotensin II (Ang II) requires both depletion of PIP(2) and generation of DAG which leads to stimulation of TRPC6 via a PKC-independent mechanism. The data also indicate that PIP(2) has a marked constitutive inhibitory action of TRPC6 and DAG and PIP(2) are physiological antagonists on TRPC6 ROCs. In contrast PIP(2) stimulates TRPC1 SOCs and has an obligatory role in activation of these channels by store-depletion which requires PKC-dependent phosphorylation of TRPC1 proteins. Finally, we conclude that interactions between PIP(2) bound to TRPC proteins at rest, generation of DAG and PKC-dependent phosphorylation of TRPC proteins have a fundamental role in activation mechanisms of ROCs and SOCs in vascular smooth muscle.

Potent vasorelaxant activity of the TMEM16A inhibitor T16Ainh‐A01

T16Ainh‐A01 is a recently identified inhibitor of the calcium‐activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16Ainh‐A01 for inhibition of calcium‐activated chloride channels in vascular smooth muscle and consequent effects on vascular tone.

Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5‐bisphosphate in mesenteric artery myocytes

The present work investigates the effect of phosphatidylinositol‐4,5‐bisphosphate (PIP2) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co‐immunoprecipitation methods. Inclusion of 100 μm diC8‐PIP2 in the patch pipette and bathing solutions, respectively, inhibited angiotensin II (Ang II)‐evoked whole‐cell cation currents and TRPC6 channel activity by over 90%. In inside‐out patches diC8‐PIP2 also inhibited TRPC6 activity induced by the diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) with an IC50 of 7.6 μm. Anti‐PIP2 antibodies potentiated Ang II‐ and OAG‐evoked TRPC6 activity by about 2‐fold. Depleters of tissue PIP2 wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP2 scavenger poly‐l‐lysine. Wortmannin reduced Ang II‐evoked TRPC6 activity by over 75% but increased OAG‐induced TRPC6 activity by over 50‐fold. Co‐immunoprecipitation studies demonstrated association between PIP2 and TRPC6 proteins in tissue lysates. Pre‐treatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP2. These results provide for the first time compelling evidence that constitutively produced PIP2 exerts a powerful inhibitory action on native TRPC6 channels.

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 μm) 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.