A. P. Albert

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 Ca2+‐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 Ca2+ 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 RÖ‐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 Ca2+ stores by CPA and BAPTA‐AM.

A Ca2+-permeable non-selective cation channel activated by depletion of internal Ca2+ stores in single rabbit portal vein myocytes

In vascular smooth muscle cells many agonists cause the release of Ca2+ ions from internal stores. An important problem concerns the mechanism by which the intracellular stores are refilled subsequent to depletion. In the present study, we describe the properties of a Ca2+‐permeable non‐selective cation channel current that is activated in rabbit portal vein myocytes by depletion of internal Ca2+ stores. Application of cyclopiazonic acid (CPA), which depletes internal Ca2+ stores, activated whole‐cell currents that had a reversal potential (Er) of about +50 mV in 1.5 mm external Ca2+ (Ca2o+). In 0 mm Ca2o+, the currents were larger and Er was ∼0 mV. Application of CPA and caffeine during cell‐attached recording activated single inward channel currents at negative potentials, which had a slope conductance of 2–3 pS and an Er of +20 mV. The slope conductance in 0 and 110 mm Ca2o+ was 7 and 1.5 pS, respectively, and Er values indicated that these non‐selective cation channels are highly permeable to Ca2+ ions. Bath application of the cell‐permeant Ca2+ chelator, BAPTA‐AM, also activated similar currents, indicating that these channels are not activated by Ca2+. Spontaneous channel currents with similar properties to store‐operated channels were observed in some patches. Application of W‐7, an inhibitor of the Ca2+‐binding protein calmodulin, also activated similar Ca2+‐permeable channel currents. In conclusion, it is demonstrated that agents that deplete Ca2+ stores and inhibit calmodulin binding activate Ca2+‐permeable non‐selective cation channel currents in rabbit portal vein myocytes. These channels may have an important role in vascular smooth muscle in providing an influx of Ca2+ to refill depleted internal Ca2+ stores and appear to possess different characteristics to store‐operated channels described in other vascular smooth muscle preparations.

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.

Evidence for myosin light chain kinase mediating noradrenaline-evoked cation current in rabbit portal vein myocytes

1 The role of myosin light chain kinase (MLCK) in the activation of the noradrenaline‐evoked non‐selective cation current (Icat) was examined with the whole‐cell recording technique in single rabbit portal vein smooth muscle cells. 2 Intracellular dialysis with 5 μM MLCK(11–19)amide, a substrate‐specific peptide inhibitor of MLCK, markedly reduced the amplitude and rate of activation of noradrenaline‐evoked Icat. A similar result was obtained when the cells were dialysed with 10 μM AV25, which also inhibits MLCK by an action at the auto‐inhibitory domain of MLCK. 3 Inhibitors of binding of ATP to MLCK, wortmannin and synthetic naphthalenesulphonyl derivatives (ML‐7 and ML‐9), at micromolar concentrations, also reduced the amplitude of noradrenaline‐evoked Icat. 4 ML‐7 and ML‐9 (both at 5 μM) reduced the amplitude of Icat induced by both guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG). 5 MLCK(11–19)amide, AV25 and ML‐9 did not inhibit the noradrenaline‐evoked Ca2+‐activated potassium current at a holding potential of 0 mV. In addition, MLCK(11–19)amide and AV25 did not reduce the non‐selective cation current induced by ATP in rabbit ear artery cells. 6 Intracellular dialysis with 2 μM Ca2+ and 9 μM calmodulin activated Icat, which developed over a period of about 5 min. 7 Intracellular dialysis with the non‐hydrolysable analogue of ATP, 5′‐adenylylimidodiphosphate (AMP‐PNP), reduced the amplitude and rate of activation of noradrenaline‐evoked Icat. 8 The results indicate that MLCK mediates noradrenaline‐activated Icat in rabbit portal vein smooth muscle cells.

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.

Comparison of spontaneous and noradrenaline-evoked non-selective cation channels in rabbit portal vein myocytes

1 The properties of non‐selective cation channels were studied in rabbit portal vein smooth muscle cells in K+‐free conditions with patch pipette techniques. 2 In about 45 % of isolated outside‐out patches spontaneous channel currents with a unitary conductance of 23 pS were observed. The reversal potential was +11 mV which was shifted to more negative values and the unitary conductance was reduced when part of the external Na+ was replaced by Tris. 3 At negative potentials the probability of opening (Po) was low and the open time distributions were described by two exponentials with time constants of about 1 and 7 ms. At positive potentials Po and the longer mean open time were greatly increased. 4 The channel exhibited bursting behaviour and the burst duration distributions were described by two exponentials with time constants of about 3 and 15 ms. At positive potentials the longer burst duration was increased substantially. 5 Application of noradrenaline and the diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) to the external membrane of quiescent patches evoked single‐channel activity with a unitary conductance of about 23 pS and with similar kinetic behaviour to that of the spontaneous channel currents. 6 In conclusion, noradrenaline and OAG activate non‐selective cation channel currents in excised patches of rabbit portal vein myocytes with a unitary conductance and kinetic properties that are similar to those of spontaneous channel currents. These channels have two open states and exhibit bursting behaviour. It is suggested that these channels underlie the whole‐cell currents evoked by noradrenaline and OAG.

Stimulation of β-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 Ca2+ store depletion these SOCs could also be activated by α‐adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)‐dependent mechanism. In the present study we have investigated the effect of β‐adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole‐cell recording the selective β‐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 β‐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.

Agents that increase tyrosine phosphorylation activate a non‐selective cation current in single rabbit portal vein smooth muscle cells

1 The effects of agents that increase tyrosine phosphorylation were studied with whole‐cell recording of membrane currents in single smooth muscle cells from the rabbit portal vein. 2 In K+‐free conditions with the chloride equilibrium potential at about ‐50 mV, intracellular application via the patch pipette of 200 μM sodium orthovanadate (Na3VO4), which inhibits tyrosine phosphatases, activated a ‘noisy’ inward current at a holding potential of ‐50 mV. 3 Intracellular dialysis with 100 μM (pY)EEI, a peptide activator of the cytosolic tyrosine kinase pp60c‐src, and bath application of 5 μM insulin, which activates receptor‐coupled tyrosine kinases, also evoked a ‘noisy’ inward current. The current‐voltage relationships and the reversal potential (about +10 mV) of the Na3VO4‐, pp60c‐src‐ and insulin‐induced currents were similar to those of the noradrenaline‐evoked non‐selective cation current (Icat). 4 The inward currents evoked by noradrenaline, Na3VO4, (pY)EEI and insulin were all greatly potentiated when the bathing calcium concentration was reduced from 1.5 mM to 50 μM. 5 The single channel conductance estimated from spectral density analysis of the whole‐cell current was about 20 pS for noradrenaline, Na3VO4, (pY)EEI and insulin. Moreover for all agents the spectra were described by the sum of two Lorentzians with similar corner frequencies. 6 Noradrenaline‐evoked Icat was inhibited to a similar degree by the tyrosine kinase inhibitors genistein and tyrphostin 23 and their inactive analogues daidzein and tyrphostin A1, respectively. 7 In the presence of Na3VO4, application of noradrenaline evoked a cation current of similar peak amplitude to control Icat although the rate of decay of Icat was enhanced in the presence of Na3VO4. 8 This study shows that stimulation of both cytosolic and receptor‐coupled tyrosine kinases evokes a non‐selective cation current and the conductance is similar to that activated by noradrenaline.

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 α‐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 μm 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 μm Ins(1,4,5)P3 increased open channel probability (NPo) by approximately 3‐fold. Ins(1,4,5)P3 also produced a similar increase in NPo 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 μm. 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 NPo 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.

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 Ca2+‐permeable non‐selective cation conductance (Icat) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of Icat 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 GTPγS 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 GDPβS suggesting a G‐protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to Gαq/Gα11 enhanced channel activity whereas anti‐Gα1−3/Gαo antibodies decreased basal channel activity which suggests that Gαq/Gα11 and Gαι/Gαo 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 (NPo) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13‐dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NPo. These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving Gαq/Gα11‐mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NPo and it was evident that the inhibitory pathway was particularly marked in these cases. Moreover in the latter patches GTPγS and OAG caused marked increases in NPo. Together with inhibitory effects of GDPβS and anti‐Gα1−3/Gαo antibodies the results suggest that there is constitutive Gαi/Gαo 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 Icat and the noradrenaline‐evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist‐evoked Icat in these arterial myocytes.