Rabbit Portal Vein Smooth Muscle Research Articles

Lysophosphatidylcholine Increases Ca2+Current via Activation of Protein Kinase C in Rabbit Portal Vein Smooth Muscle Cells

Lysophosphatidylcholine (LPC), a metabolite of membrane phospholipids by phospholipase A(2), has been considered responsible for the development of abnormal vascular reactivity during atherosclerosis. Ca(2+) influx was shown to be augmented in atherosclerotic artery which might be responsible for abnormal vascular reactivity. However, the mechanism underlying Ca(2+) influx change in atherosclerotic artery remains undetermined. The purpose of the present study was to examine the effects of LPC on L-type Ca(2+) current (I(Ca(L))) activity and to elucidate the mechanism of LPC-induced change of I(Ca(L)) in rabbit portal vein smooth muscle cells using whole cell patch clamp. Extracellular application of LPC increased I(Ca(L)) through whole test potentials, and this effect was readily reversed by washout. Steady state voltage dependency of activation or inactivation properties of I(Ca(L)) was not significantly changed by LPC. Staurosporine (100 nM) or chelerythrine (3 microM), which is a potent inhibitor of PKC, significantly decreased basal I(Ca(L)), and LPC-induced increase of I(Ca(L)) was significantly suppressed in the presence of PKC inhibitors. On the other hand, application of PMA, an activator of PKC, increased basal I(Ca(L)) significantly, and LPC-induced enhancement of I(Ca(L)) was abolished by pretreatment of the cells with PMA. These findings suggest that LPC increased I(Ca(L)) in vascular smooth muscle cells by a pathway that involves PKC, and that LPC-induced increase of I(Ca(L)) might be, at least in part, responsible for increased Ca(2+) influx in atherosclerotic artery.

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 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 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.

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC.

It has been suggested that L-type Ca(2+) channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca(2+) channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca(2+) channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca(2+) channel activity but did not alter the voltage-dependent characteristics of Ca(2+) channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31-8425 or Go-6983, prevented I(Ba) enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I(Ba) under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I(Ba) when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca(2+) channel activity in rabbit portal vein smooth muscle cells through activation of PKC.

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.

The effect of external divalent cations on spontaneous non-selective cation channel currents in rabbit portal vein myocytes.

1. The effects of external divalent cations on spontaneous single non-selective cation channel currents were studied in outside-out patches from rabbit portal vein smooth muscle cells in K+-free conditions. 2. In an external medium containing 1.5 mM Ca2+ (Ca2+o) the majority of spontaneous channel currents had a unitary conductance of 23 pS, reversal potential (Vr) of +10 mV and a low open probability (Po) at negative patch potentials. Some channels opened to a lower conductance state of about 13 pS suggesting that the cation channels have two conductance states. Open time and burst duration distributions could both be described by two exponentials with time constants of about of 1 ms and 7 ms for open times and 3 ms and 16 ms for burst durations. 3. In 0 Ca2+o the majority of spontaneous cation channels had a unitary conductance of 13 pS and Vr was shifted to +4 mV. Moreover the longer open time and longer burst duration time constants were both reduced to approximately half the values in 1.5 mM Ca2+o. 4. Compared to 0 Ca2+o the single channel currents in 3 microM and 100 microM Ca2+o had a 5- to 6-fold increase in Po which was accompanied by increases in both open times and burst durations. In 3 microM and 100 microM Ca2+o the unitary conductance of the single channel currents was between 22 and 26 pS. 5. At positive membrane potentials the single channel currents had an increased Po compared to negative potentials which was associated with increased open times and burst durations but these values were similar in 3 microM, 100 microM and 1.5 mM Ca2+o. 6. In 1.5 mM Sr2+o and 1.5 mM Ba2+o channels opened to the higher conductance state of about 22-25 pS and had a 3- to 7-fold greater Po than in 0 Ca2+o. 7. In conclusion, external divalent cations have marked effects on the unitary conductance and kinetic behaviour of non-selective cation channels in rabbit portal vein smooth muscle cells.

The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel.

The Drosophila transient receptor potential protein (TRP) and its mammalian homologues are thought to be Ca(2+)-permeable cation channels activated by G protein (G(q/11))-coupled receptors and are regarded as an interesting molecular model for the Ca(2+) entry mechanisms associated with stimulated phosphoinositide turnover and store depletion. However, there is little unequivocal evidence linking mammalian TRPs with particular native functions. In this study, we have found that heterologous expression of murine TRP6 in HEK293 cells reproduces almost exactly the essential biophysical and pharmacological properties of alpha(1)-adrenoceptor-activated nonselective cation channels (alpha(1)-AR-NSCC) previously identified in rabbit portal vein smooth muscle. Such properties include activation by diacylglycerol; S-shaped current-voltage relationship; high divalent cation permeability; unitary conductance of 25 to 30 pS and augmentation by flufenamate and Ca(2+); and blockade by Cd(2+), La(3+), Gd(3+), SK&F96365, and amiloride. Reverse transcriptase-polymerase chain reaction and confocal laser scanning microscopy using TRP6-specific primers and antisera revealed that the level of TRP6 mRNA expression was remarkably high in both murine and rabbit portal vein smooth muscles as compared with other TRP subtypes, and the immunoreactivity to TRP6 protein was localized near the sarcolemmal region of single rabbit portal vein myocytes. Furthermore, treatment of primary cultured portal vein myocytes with TRP6 antisense oligonucleotides resulted in marked inhibition of TRP6 protein immunoreactivity as well as selective suppression of alpha(1)-adrenoceptor-activated, store depletion-independent cation current and Ba(2+) influx. These results strongly indicate that TRP6 is the essential component of the alpha(1)-AR-NSCC, which may serve as a store depletion-independent Ca(2+) entry pathway during increased sympathetic activity.

Beta-Adrenergic receptor stimulation of L-type Ca2+ channels in rabbit portal vein myocytes involves both alphas and betagamma G protein subunits.

1. Previous studies have shown that purified G protein alphas and betagamma subunits stimulate vascular L-type Ca2+ channels through protein kinase A and C (PKA and PKC), respectively. The present study tested whether activation of endogenous G proteins via beta-adrenergic receptor binding also stimulates vascular Ca2+ channels through both Galphas and Gbetagamma and the subsequent activation of PKA and PKC. 2. Peak Ba2+ current (IBa) in freshly isolated rabbit portal vein smooth muscle cells was significantly increased by bath application of 0.5 microM isoproterenol (isoprenaline; ISO) when measured using the whole-cell patch clamp method (53 +/- 3 % increase, n = 15). Stimulation of IBa by ISO was partially reversed by a PKA inhibitor, KT 5720, or a PKC inhibitor, calphostin C, and completely blocked when cells were pretreated with both KT 5720 and calphostin C. 3. Dialysis of cells with polyclonal antibody to Galphas significantly reduced but did not completely eliminate ISO-induced stimulation of IBa. The remaining stimulation was abolished by calphostin C. Dialysis of cells with a polyclonal antibody to Gbeta also significantly reduced ISO-induced stimulation and the remaining stimulation was abolished by KT 5720. Dialysis of cells with both antibodies completely prevented the stimulation of IBa by ISO. 4. ISO-induced stimulation of IBa was reversed by ICI-118,551, a specific beta2-adrenoceptor antagonist, but not by CGP 20712A, a specific beta1-adrenoceptor antagonist. In addition, the beta2-adrenoceptor agonist zinterol significantly increased peak IBa while the beta1-adrenoceptor agonist dobutamine and beta3-adrenoceptor agonist BRL 37344A had little effect on peak IBa. 5. These data suggest that beta-adrenergic receptor stimulation of vascular L-type Ca2+ channels involves both alphas and betagamma G-protein subunits, which exert their effects through PKA and PKC, respectively.

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

The properties of non-selective cation channels were studied in rabbit portal vein smooth muscle cells in K+-free conditions with patch pipette techniques. 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. 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. 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. 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. 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.

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

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. In K+-free conditions with the chloride equilibrium potential at about -50 mV, intracellular application via the patch pipette of 200 microM sodium orthovanadate (Na3VO4), which inhibits tyrosine phosphatases, activated a "noisy" inward current at a holding potential of -50 mV. Intracellular dialysis with 100 microM (pY)EEI, a peptide activator of the cytosolic tyrosine kinase pp60c-src, and bath application of 5 microM 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). 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 microM. 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. 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. 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. 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.

Dual modulation of swelling-activated chloride current by NO and NO donors in rabbit portal vein myocytes.

1. The effects of authentic NO and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on swelling-activated chloride currents (Iswell) were investigated in freshly dispersed rabbit portal vein smooth muscle cells. Iswell was recorded with the perforated patch configuration of the whole-cell patch clamp technique. 2. In approximately 50 % of cells NO and SNAP inhibited the amplitude of Iswell by about 45 % in a voltage-independent manner. Iswell was also inhibited by an inhibitor of NO-sensitive guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by KT5823, an inhibitor of cGMP-dependent protein kinase. 3. In other cells both NO and SNAP enhanced Iswell by about 40 % in a voltage-independent manner. A similar increase was produced by application of the cell-permeable cGMP analogue 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP). However, 8-Br-cGMP had no effect on current amplitude in cells pre-treated with KT5823. In contrast 8-Br-cGMP increased the amplitude of Iswell in cells which had been pre-treated with ODQ. 4. SNAP also modulated Iswell recorded in the conventional whole-cell configuration with internal solutions containing 10 mM EGTA to rule out any contribution from Ca2+-activated Cl- currents. 5. These data suggest that the amplitude of Iswell can be enhanced by NO via a cGMP-dependent phosphorylation and inhibited by NO in a cGMP-independent manner.

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

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. Intracellular dialysis with 5 microM 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 microM AV25, which also inhibits MLCK by an action at the auto-inhibitory domain of MLCK. 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. ML-7 and ML-9 (both at 5 microM) reduced the amplitude of Icat induced by both guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and 1-oleoyl-2-acetyl-sn-glycerol (OAG). 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. Intracellular dialysis with 2 microM Ca2+ and 9 microM calmodulin activated Icat, which developed over a period of about 5 min. Intracellular dialysis with the non-hydrolysable analogue of ATP, 5'-adenylylimidodiphosphate (AMP-PNP), reduced the amplitude and rate of activation of noradrenaline-evoked Icat. The results indicate that MLCK mediates noradrenaline-activated Icat in rabbit portal vein smooth muscle cells.

Electrophysiological actions of a novel K + channel opener MCC-134 on rabbit portal vein smooth muscle

The effects of a newly synthesized K + channel opener, 1-[4-(1 H-imidazol-1-yl)benzoyl]- N-methylcyclobutane-carbothioamide (MCC-134) on membrane currents and intracellular Ca 2+ mobilization were investigated in rabbit portal vein smooth muscle cells. Under voltage-clamped conditions, MCC-134 dose-dependently induced K +-selective currents ( I MCC; EC 50 5.3 μM) showing little desensitization but fast deactivating properties on washout of drugs. I MCC was completely blocked by 10 μM glibenclamide, not affected by iberiotoxin (500 nM), charybdotoxin (200 nM) or apamin (500 nM), and inhibited by nonspecific K + channel blockers, tetraethylammonium (1–10 mM), 4-aminopyridine (0.1–1 mM) and Ba 2+ (0.01–0.1 mM). Intracellularly applied nucleotide diphosphates (1 mM) were effective at maintaining I MCC (apparent potency; ADP≦GDP≒IDP<UDP), whereas intracellular ATP exhibited a biphasic, i.e., augmentative and inhibitory effect(s) on I MCC. Single channel activities of about 15 pS (40/140 mM extra-/intracellular K +) fully accountable for macroscopic I MCC were activated by MCC-134. MCC-134, at a concentration as high as 100 μM, suppressed voltage-dependent Ca 2+ and noradrenaline-induced cation currents, and concomitant elevations in the intracellular Ca 2+ concentration ([Ca 2+] i).

Comparison of the effects of divalent cations on the noradrenaline‐evoked cation current in rabbit portal vein smooth muscle cells

1. The facilitatory effects of external Ca2+, Sr2+ and Ba2+ (Ca2+o, Sr2+o and Ba2+o) on the noradrenaline-evoked non-selective cation current (Icat) were compared in rabbit portal vein smooth muscle cells using patch pipette techniques. 2. All divalent cations tested potentiated the amplitude of Icat and the potency sequence was Ca2+o > Sr2+o > Ba2+o. Ca2+o and Sr2+o increased the amplitude of Icat by about eight times whereas Ba2+o produced only a threefold facilitation. 3. The current-voltage relationship of Icat was not changed by Ca2+o, Sr2+o or Ba2+o. 4. From noise analysis the single channel conductance (gamma) was approximately 10 pS in divalent cation-free solution but was about 20 pS with Ca2+o, Sr2+o and Ba2+o. 5. From noise and voltage-jump experiments it was apparent that at least three kinetically resolvable channel states are associated with Icat in divalent cation-free solution. Ca2+o and Sr2+o produced marked changes in the characteristics of the power spectrum and relaxations of Icat in response to voltage steps, consistent with a shift in the equilibrium between the channel states, whereas Ba2+o produced minimal effects. 6. The data show that Ca2+o, Sr2+o and Ba2+o increase the amplitude of Icat, which results in part from an increase in the single channel conductance. In addition the results suggest that Ca2+o and Sr2+o alter the kinetic behaviour of the single channels whereas Ba2+o has little effect on the equilibrium between the channel states.

Modulation of the decay of Ca2+-activated Cl- currents in rabbit portal vein smooth muscle cells by external anions.

1. The effects of external anions on the decay kinetics of Ca2+-activated Cl- currents (ICl(Ca)) were studied in smooth muscle cells isolated from rabbit portal vein using the perforated patch whole-cell voltage clamp technique. 2. In normal NaCl-containing external solution the decay of spontaneous Ca2+-activated Cl- currents (STICs) and Ca2+-activated Cl- 'tail' currents (Itail) was described by a single exponential with a time constant (tau) that was prolonged by external anions which are more permeable than Cl- (Br-, I- and SCN-) and accelerated by less permeant anions. However, intracellular I- did not affect the tau of STICs and Itail. 3. There was a positive correlation between the ability of an external anion to affect the decay tau of ICl(Ca) and its permeability relative to Cl-. 4. The voltage dependence of STIC and Itail decay was not affected by external or internal anions. 5. External permeating anions were not obligatory for activation of ICl(Ca) and STIC tau was not altered in Cl--free external solution. 6. Modulation of tau by mole fractions of SCN- and Cl- ions was fitted by a logistic curve, suggesting competition between SCN- and Cl- ions for a binding site. 7. In conclusion, external anions affect the decay of ICl(Ca) by a mechanism compatible with an interaction with a binding site which modulates Cl- channel kinetics.

Facilitatory effect of Ca2+ on the noradrenaline-evoked cation current in rabbit portal vein smooth muscle cells.

1. The facilitatory effect of external calcium ions (Ca2+o) on the alpha1-adrenoceptor-activated non-selective cation current (Icat) was investigated in rabbit portal vein cells using noise and voltage-jump relaxation analysis of the whole-cell macroscopic current. 2. Micromolar concentrations of Ca2+o potentiated the peak amplitude of Icat at a holding potential (Vh) of -50 mV. The effective [Ca2+]o which produced a 50% potentiation (EC50) was 3 microM. 3. From noise analysis the estimated single channel conductance (gamma) was approximately 23 pS with [Ca2+]o between 3 and 100 microM, whereas in < 10 nM or 1 microM Ca2+o gamma was approximately 10 pS. 4. The spectral density function of Icat at negative potentials could be described by the sum of two Lorentzians in every [Ca2+]o examined. The time constant of the lower frequency Lorentzian component (tau1) was about 11 ms in < 10 nM Ca2+o and was about 45 ms in micromolar concentrations of Ca2+o (1-100 microM). In contrast, the time constant of the higher frequency component (tau2) was similar in < 10 nM Ca2+o and 100 microM Ca2+o (between 1 and 2 ms). 5. The lower frequency Lorentzian component was responsible for about half the total current variance in < 10 nM Ca2+o whereas in micromolar concentrations of Ca2+o it was responsible for most of the measured current variance. 6. In voltage-jump experiments, on stepping the voltage from -50 to +50 mV the instantaneous current was followed by an exponential decline of Icat. Stepping back to -30 mV produced an exponential inward relaxation (Irelax,-30 mV) leading to an increase in the steady-state amplitude of Icat in micromolar concentrations of Ca2+o, but this relaxation was not observed in < 10 nM Ca2+o. The relative amplitude of Irelax,-30 mV increased in an [Ca2+]o-dependent manner (EC50 was 2 microM) although the time constant of this relaxation (taurelax,-30 mV) remained unchanged (about 60 ms between 2 and 100 microM Ca2+o). 7. The data suggest that Ca2+o produces marked changes in the kinetics and single channel conductance of cation channels, which may account for the facilitatory effect of micromolar concentrations of Ca2+o on the peak amplitude of Icat.

Properties of a Cl- current activated by cell swelling in rabbit portal vein vascular smooth muscle cells.

In rabbit portal vein smooth muscle cells, application of a hypotonic external solution caused cell swelling and evoked an outwardly rectifying Cl- current. The hypotonicity-activated current was markedly reduced by the anti-estrogen tamoxifen (10 microM) and was inhibited by DIDS in a voltage-dependent manner [the concentration required to inhibit the current by 50% (IC50) at -50 and +100 mV was 21 and 5 microM DIDS, respectively]. Indanyloxyacetic acid 94 (IAA-94) and niflumic acid also inhibited the hypotonicity-activated current, with 50% inhibition produced at concentrations of approximately 200 and 100 microM, respectively. In isotonic conditions, application of tamoxifen and DIDS to cells decreased the holding current due to the inhibition of a resting conductance that was outwardly rectifying and reversed at the Cl- equilibrium potential. These data show that rabbit portal vein myocytes have a resting Cl- conductance that is enhanced by cell swelling; its possible physiological role is discussed.

The unimportance of being (protein kinase C) epsilon.

The purpose of our study was to determine the mechanism through which phorbol esters and smooth muscle myosin phosphatase inhibitors can induce contraction of smooth muscle in the absence of Ca2+. Protein kinase C-epsilon (PKC-epsilon) was previously implicated in this process based largely on its supposed absence in the ferret portal vein, and a correlation was drawn between the presence of this isoform and the ability of smooth muscle to contract independently of Ca2+ and phosphorylation of the 20 kDa regulatory light chains of myosin (MLC20). We demonstrate here, with two antibodies, one to the NH2 terminus and the other to the COOH terminus of PKC-epsilon, that epsilon is present in both ferret portal vein and rabbit portal vein smooth muscle, neither of which exhibits phorbol ester-induced contraction in the absence of Ca2+. However, in the presence of clamped submaximal Ca2+, phorbol es ter increased MLC20 phosphorylation from 17.7+/-1.7% to 46.4+/-3.6% in ferret portal vein smooth muscle and evoked an increase in force. Prolonged (48 h) incubation of ferret portal vein with phorbol esters completely down-regulated PKC-epsilon, as shown by Western blots, and abolished the phorbol ester-evoked contraction at submaximal Ca2+, but not Ca2+-independent, contractions induced by the phosphatase inhibitor microcystin. Contractions induced by microcystin in Ca2+-free solution were associated with increased phosphorylation of myosin light chain kinase (MLCK). Activation of MLCK by autophosphorylation in the absence of Ca2+ occurs in vitro (1). We conclude that PKC-epsilon is neither necessary nor sufficient for Ca2+-independent regulation of myosin II in smooth muscle, but contractions induced by agents that inhibit smooth muscle myosin phosphatase in the absence of Ca2+ may be mediated by MLCK autophosphorylated or activated by another Ca2+-independent kinase.

Inhibitory effects of genistein on ATP-sensitive K+ channels in rabbit portal vein smooth muscle.

1. Effects on the pinacidil-induced outward current of inhibitors of tyrosine kinases and phosphatases were investigated by use of a patch-clamp method in smooth muscle cells of the rabbit portal vein. 2. A specific tyrosine kinase inhibitor, genistein, inhibited the pinacidil-induced current in a concentration-dependent manner with an IC50 of 5.5 microM. Superfusion of Ca2+-free solution did not affect this inhibitory effect of genistein. At higher concentrations, genistein inhibited the voltage-dependent Ba2+ and K+ currents with IC50 values of > 100 microM and 75 microM respectively. Tyrphostin B46 (30 microM), a tyrosine kinase inhibitor, also inhibited the pinacidil-induced current by 70% of the control. 3. Sodium orthovanadate (100 microM), an inhibitor of tyrosine phosphatase, slightly but significantly enhanced both the pinacidil-induced and delayed rectifier K+ currents. Daidzein (100 microM), an inactive analogue of genistein, did not inhibit these currents. 4. Neither herbimycin A (1 microM), lavendustin A (30 microM), tyrphostin 23 (10 microM), which are also tyrosine kinase inhibitors, nor wortmannin (10 microM), a phosphatidylinositol 3-kinase inhibitor, had an effect on either the pinacidil-induced or delayed rectifier K+ currents. Epidermal growth factor (EGF; 1 microg ml(-1)) did not induce an outward current or enhance the pinacidil-induced current. 5. Pinacidil alone, in the cell-attached configuration, or pinacidil with GDP, in the inside-out configuration, activated a 42 pS channel in the smooth muscle cells of the rabbit portal vein. Genistein (30 microM) reduced the channel's open probability without inducing a change in unitary conductance at any holding potential (-30 to +20 mV). 6. In the inside-out configuration, genistein at 30 microM did not change the mean channel open time, but reduced the burst duration. At 100 microM genistein abolished channel opening. The inhibitory potencies with which 30 and 100 microM genistein acted on the unitary current of the ATP-sensitive K+ channel were similar to those seen in the whole-cell voltage-clamp configuration. 7. Although direct inhibitory actions of genistein on the ATP-sensitive K+ channels are not ruled out, our results suggest that a protein tyrosine kinase may play a role in the regulation of ATP-sensitive K+ channel activity in the rabbit portal vein.

Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B.

Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10(-6) M, 48 h; ; ) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)gammaS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPgammaS-induced translocation of cytosolic RhoA () to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPgammaS, which prevents immunoprecipitation of non-ADP-ribosylated RhoA. Dissociation of cytosolic RhoA-rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.