Currents In Guinea Pig Ventricular Myocytes Research Articles

Crebanine inhibits voltage-dependent Na+ current in guinea-pig ventricular myocytes

AimTo study the effects of crebanine on voltage-gated Na+ channels in cardiac tissues. MethodsSingle ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na+ current was recorded using the whole cell voltage-clamp technique. ResultsCrebanine reversibly inhibited Na+ current with an IC50 value of 0.283 mmol·L−1 (95% confidence range: 0.248–0.318 mmol·L−1). Crebanine at 0.262 mmol·L−1 caused a negative shift (about 12 mV) in the voltage-dependence of steady-state inactivation of Na+ current, and retarded its recovery from inactivation, but did not affect its activation curve. ConclusionIn addition to blocking other voltage-gated ion channels, crebanine blocked Na+ channels in guinea-pig ventricular myocytes. Crebanine acted as an inactivation stabilizer of Na+ channels in cardiac tissues.

Effects of Astragaloside IV on Action Potentials and Ionic Currents in Guinea-Pig Ventricular Myocytes

Astragaloside IV (AS-IV) is one of the main active constituents of Astragalus membranaceus, which has various actions on the cardiovascular system. However, its electrophysiological mechanisms are not clear. In the present study, we investigated the effects of AS-IV on action potentials and membrane currents using the whole-cell patch clamp technique in isolated guinea-pig ventricular myocytes. AS-IV prolonged the action potential duration (APD) at all three tested concentrations. The peak effect was achieved with 1×10(-6) M, at which concentration AS-IV significantly prolonged the APD at 95% repolarization from 313.1±38.9 to 785.3±83.7 ms. AS-IV at 1×10(-6) M also enhanced the inward rectifier K(+) currents (I(K1)) and inhibited the delayed rectifier K(+) currents (I(K)). AS-IV (1×10(-6) M) strongly depressed the peak of voltage-dependent Ca(2+) channel current (I(CaL)) from -607.3±37.5 to -321.1±38.3 pA. However, AS-IV was not found to affect the Na(+) currents. Taken together, AS-IV prolonged APD of guinea-pig ventricular myocytes, which might be explained by its inhibition of I(K). AS-IV also influences Ca(2+) signaling through suppressing ICaL.

Effects of trimebutine maleate on delayed rectifier K+ currents in guinea-pig ventricular myocytes.

The effects of trimebutine maleate, a drug commonly used to regulate motility in the gastrointestinal tract, on the delayed rectifier K+ current (I(K)) were evaluated in guinea-pig ventricular myocytes to determine whether the drug has a proarrhythmic effect through blockade of I(K). Trimebutine decreased I(K) in a concentration-dependent manner. To investigate the effects of trimebutine on two components of I(K) (I(Kr) and I(Ks); rapidly activated and slowly activated components, respectively), we performed the envelope-of-tails test. Trimebutine-sensitive I(K) was determined by digital subtraction of I(K) during exposure to trimebutine from control I(K) for each duration of the test pulse over the range 50 ms-2 s. The ratio of deltaI(K,tail)/deltaI(K) plotted against pulse duration for trimebutine-sensitive I(K) gradually decreased to a steady-state value as the duration of the test pulse was lengthened. This finding suggested a weak inhibitory effect of trimebutine on both I(Kr) and I(Ks). The effects of trimebutine on the inward rectifier K+ current (I(K1)) responsible for the resting potential and final repolarization phase of the action potential were investigated by applying voltage clamp ramps over a broad range of potentials. No significant effects were observed at 10 or 100 microM. We next investigated the effects of the drug on the L-type Ca2+ current (I(Ca)). Significant inhibition of I(Ca) was observed at trimebutine concentrations greater than 10 microM. These results suggested that trimebutine maleate has weak inhibitory effects on I(Kr), I(Ks) and I(Ca) at concentrations much higher than those in clinical use.

Ultraviolet Photoalteration of Late Na+ Current in Guinea-pig Ventricular Myocytes

UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca(2+) imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at -80 mV, dialyzed with K(+)-, Na(+)-free pipette solution, and bathed with K(+)-free Tyrode's solution at 22 degrees C. During experiments that lasted for approximately 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from -80 to -40 mV, but had little effect on background current or on L-type Ca(2+) current. Trials with depolarized holding potential, Ca(2+) channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na(+) current (I(Na)). The amplitude of the late inward current sensitive to 100 microM: TTX was increased by 3.5-fold after 20-30 min of irradiation. UVA modulation of late I(Na) may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac I(Na).

Effects of PFOS and PFOA on L-type Ca 2+ currents in guinea-pig ventricular myocytes

Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are amphiphiles found ubiquitously in the environment, including wildlife and humans, and are known to have toxic effects on physiological functions of various tissues. We investigated the effects of PFOS and PFOA on action potentials and L-type Ca 2+ currents, I CaL, in isolated guinea-pig ventricular myocytes using whole-cell patch-clamp recording. In current-clamp experiments, PFOS significantly decreased the rate of spike, action potential duration, and peak potential at doses over 10 μM. In voltage-clamp experiments, PFOS increased the voltage-activated peak amplitude of I CaL, and shifted the half-activation and inactivation voltages of I CaL to hyperpolarization. PFOA had similar effects PFOS, but showed significantly lower potency. These findings are consistent with previous observations for anionic n-alkyl surfactants, suggesting that PFOS and PFOA may change membrane surface potential, thereby eliciting general effects on calcium channels. These findings provide further insights into the mechanisms of PFOA and PFOS toxicities.

Cardiac Na+–Ca2+ exchanger current induced by tyrphostin tyrosine kinase inhibitors

Tyrosine kinase (TK) inhibitors genistein and tyrphostin A23 (A23) inhibited Ca(2+) currents in guinea-pig ventricular myocytes investigated under standard whole-cell conditions (K(+)-free Tyrode's superfusate; EGTA-buffered (pCa-10.5) Cs(+) dialysate). However, the inhibitors (100 microM) also induced membrane currents that reversed between -40 and 0 mV, and the objective of the present study was to characterize these currents. Genistein-induced current behaved like Cl(-) current, and was unaffected by either the addition of divalent cations (0.5 mM Cd(2+); 3 mM Ni(2+)) that block the Na(+)-Ca(2+) exchanger (NCX), or the removal of external Na(+) and Ca(2+). A23-induced current was independent of Cl(-) driving force, and strongly suppressed by addition of Cd(2+) and Ni(2+), and by removal of either external Na(+) or Ca(2+). These and other results suggested that A23 activated an NCX current driven by submembrane Na(+) and Ca(2+) concentrations higher than those in the bulk cytoplasm. Improved control of intracellular Na(+) and Ca(2+) concentrations was obtained by suppressing cation influx (10 microM verapamil) and raising dialysate Na(+) to 7 mM and dialysate pCa to 7. Under these conditions, stimulation by A23 was described by the Hill equation with EC(50) 68 +/- 4 microM and coefficient 1.1, tyrphostin A25 was as effective as A23, and TK-inactive tyrphostin A1 was ineffective. Phosphotyrosyl phosphatase inhibitor orthovanadate (1 mM) antagonized the action of 100 microM A23. The results suggest that activation of cardiac NCX by A23 is due to inhibition of genistein-insensitive TK.

Effects of AN-132, a novel antiarrhythmic lidocaine analogue, and of lidocaine on membrane ionic currents of guinea-pig ventricular myocytes

We studied the effects of AN-132 (10, 30 and 100 mumol/l), an analogue of lidocaine, on membrane currents and action potentials of single guinea-pig ventricular cells using whole-cell clamp techniques. The effects of lidocaine, an authentic class I antiarrhythmic agent were used for comparative purposes. (1) AN-132 decreased the Na current (INa) in a concentration-dependent manner, with a greater efficacy than seen with lidocaine. The concentration of the half maximal inhibition on INa (Kd) was 31.7 mumol/l for AN-132 and 94.9 mumol/l for lidocaine. (2) AN-132 also decreased the Ca current (ICa), concentration-dependently, while lidocaine had only a minor effect on ICa. The half maximal inhibition on ICa (Kd) was 23.1 mumol/l and 27.4 mumol/l for AN-132 and lidocaine, respectively. (3) AN-132 decreased the IK1, in a concentration-dependent manner; lidocaine was without effect. (4) AN-132 increased the unspecified steady state outward current, at positive potentials and depressed the time- and voltage-dependent outward K current (IK). Lidocaine had no effect on either current. (5) AN-132 shortened the action potential duration (APD), in a concentration-dependent manner, without altering the resting potential. From these findings, we conclude that apart from a potent inhibitory effect on INa, AN-132 had a variety of effects on other currents, properties not shared by lidocaine. Such multiple blocking effects on the membrane currents may relate to the alleged potent antiarrhythmic effect of AN-132.

Block of cardiac delayed‐rectifier and inward‐rectifier K+ currents by nisoldipine

The objective of this study was to determine the concentration-dependent effects of nisoldipine, a dihydropyridine Ca2+ channel blocker, on K+ currents in guinea-pig ventricular myocytes. Myocytes in the conventional whole-cell configuration were bathed in normal Tyrode's solution or K+-free Tyrode's solution for the measurement of the effects of 0.01-100 microM nisoldipine on rapidly activating delayed-rectifier K+ current (I(Kr)), slowly activating delayed-rectifier K+ current (I(Ks)), inwardly rectifying K+ current (I(K1)), and reference L-type Ca2+ current (I(Ca,L)). Nisoldipine inhibited I(Kr) with an IC(50) of 23 microM, and I(Ks) with an IC(50) of 40 microM. The drug also had weak inhibitory effects on inward- and outward-directed I(K1); the IC(50) determined for outward-directed current was 80 microM. Investigation of nisoldipine action on I(Ks) showed that inhibition occurred in the absence of previous pulsing, and with little change in the time courses of activation and deactivation. However, the drug-induced inhibition was significantly weaker at >or =+30 mV than at +10 mV.5 We estimate that nisoldipine is about 30 times less selective for delayed-rectifier K+ channels than for L-type Ca2+ channels in fully polarised guinea-pig ventricular myocytes, and several orders less selective in partially depolarised myocytes.

Voltage- and cation-dependent inactivation of L-type Ca2+ channel currents in guinea-pig ventricular myocytes.

L-type Ca2+ channel currents in native ventricular myocytes inactivate according to voltage- and Ca2+-dependent processes. This study sought to examine the effect of beta-adrenergic stimulation on the contributions of voltage and Ca2+ to Ca2+ current decay. Ventricular myocytes were enzymatically isolated from guinea-pig hearts. Inward whole-cell Cd2+-sensitive L-type Ca2+ channel currents were recorded with the patch clamp technique and comparison was made between inward currents carried by Ca2+ and either Ba2+, Sr2+ or Na+. In control conditions the decay of Ca2+ currents was faster than Ba2+, Sr2+ or Na+ currents at negative voltages while at positive voltages there was no difference. The relationship between voltage and inactivation for Ca2+ currents was bell-shaped, while that for Ba2+, Sr2+, and Na+ currents was sigmoid. Thus depolarisation progressively replaced Ca2+-dependent inactivation in the fast phase of decay of Ca2+ channel currents with rapid voltage-dependent inactivation. In the presence of isoproterenol (isoprenaline) the decay of Ca2+ currents was faster than Ba2+, Sr2+ or Na+ currents at all measured voltages (-40 to +30 mV). The relationship between voltage and inactivation for Ca2+, Ba2+ and Sr2+ currents was bell-shaped, while that for Na+ currents was sigmoid with less inactivation than under control conditions. Therefore the fast phase of decay of Ca2+ channel currents was now almost entirely due to Ca2+. It is concluded that the relative contributions of Ca2+- and voltage-dependent mechanisms of inactivation of L-type Ca2+ channels in native cardiac myocytes are modulated by beta-adrenergic stimulation influencing the amount of rapid voltage-dependent inactivation.

Role of tyrosine kinase activity in -adrenergic inhibition of the -adrenergically regulated L-type Ca2+ current in guinea-pig ventricular myocytes

Role of tyrosine kinase activity in -adrenergic inhibition of the -adrenergically regulated L-type Ca2+ current in guinea-pig ventricular myocytes

Role of tyrosine kinase activity in alpha-adrenergic inhibition of the beta-adrenergically regulated L-type Ca(2+) current in guinea-pig ventricular myocytes.

1. The purpose of this study was to investigate the hypothesis that tyrosine kinase activity contributes to alpha(1)-adrenergic inhibition of beta-adrenergic responses in cardiac myocytes. We addressed this question by studying the pharmacological regulation of the L-type Ca(2+) current in acutely isolated adult guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. 2. The selective alpha(1)-adrenergic receptor agonist methoxamine had no effect on the basal L-type Ca(2+) current. Methoxamine also had no effect on cAMP-dependent stimulation of the Ca(2+) current mediated by H(2) histamine receptor activation. However, methoxamine did inhibit cAMP-dependent stimulation of the Ca(2+) current mediated by beta-adrenergic receptor activation. The ability of methoxamine to inhibit beta-adrenergic regulation of the Ca(2+) current was significantly antagonized by the tyrosine kinase inhibitors genistein and lavendustin A. 3. The inhibitory effect of methoxamine was also mimicked by the phosphotyrosine phosphatase inhibitor pervanadate (PVN). PVN had no effect on basal Ca(2+) current or Ca(2+) current stimulated by histamine, but it did inhibit Ca(2+) current stimulated by beta-adrenergic receptor activation. Furthermore, the ability of PVN to inhibit beta-adrenergic stimulation of the Ca(2+) current was antagonized by lavendustin A. 4. These results are consistent with the conclusion that in guinea-pig ventricular myocytes alpha-adrenergic inhibition of beta-adrenergic responses involves a tyrosine kinase-dependent signalling pathway. The fact that methoxamine and PVN antagonized cAMP-dependent responses mediated by beta-adrenergic, but not H(2) histamine, receptor activation suggests that the inhibitory effect of alpha-adrenergic stimulation and tyrosine kinase activity is at the level of the beta-adrenergic receptor.

External anions and volume-sensitive anion current in guinea-pig ventricular myocytes

The objective of this study was to determine the effects of anion replacement on volume-sensitive anion current in guinea-pig ventricular myocytes. Myocytes in the conventional whole-cell voltage-clamp configuration were superfused and dialysed with Na(+)-, K(+)-, and Ca(2+)-free solution, and exposed to external 75 mM Cl- solution of one-half normal osmolality. Prolonged exposures to hyposmotic solution promoted the development of outwardly-rectifying currents that were inactivated at high positive potentials and reversed in a Cl(-)-dependent manner (50 mV per decade pipette Cl- concentration). Replacement of external Cl- by iodide and aspartate affected the reversal potential (E(rev)) and slope conductance of the volume-sensitive current. Relative permeabilities calculated from changes in E(rev) were 1.49 +/- 0.09, 1.00, and 0.29 +/- 0.04 for iodide, Cl-, and aspartate, respectively; relative slope conductances between E(rev) and E(rev) + 40 mV were 1.21 +/- 0.09, 1.00, and 0.43 +/- 0.07, respectively. Replacement of Cl- also affected the time dependence of the volume-sensitive current; replacement by iodide reversibly enhanced the decay of outward current at positive potentials, whereas replacement by aspartate reduced it. These results are compared with earlier findings on noncardiac time- and voltage-dependent anion current activated by hyposmotic solution.

Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism

OBJECTIVESTo investigate the potential for plant derived estrogens (phytoestrogens) genistein, phloretin, biochanin A and zearalanone to relax rabbit coronary arteries in vitro and to determine the mechanism(s) of such relaxation.BACKGROUNDEpidemiological data suggests a reduction in the incidence of coronary heart disease in humans who have a high intake of phytoestrogens.METHODSIsolated rabbit coronary artery rings were suspended in individual organ baths, precontracted with potassium chloride (30mM), and the relaxing effects and mechanisms of relaxation to genistein, phloretin, biochanin A and zearalanone were determined by measurement of isometric tension.RESULTSGenistein, phloretin and biochanin A induced significant gender-independent relaxation in rings with and without endothelium. Inhibition of nitric oxide and prostaglandin synthesis with L-NAME and indomethacin had no effect on genistein-induced relaxation. Relaxation was unaffected by the specific estrogen receptor antagonist ICI 182,780, the ATP-sensitive potassium channel inhibitor glibenclamide and the potassium channel inhibitor, barium chloride. Calcium concentration-dependent contraction curves in high potassium depolarization medium were significantly shifted to the right and downward after incubation with genistein and zearalanone. An inhibitory effect of genistein (2 μM) on L-type calcium current in guinea-pig ventricular myocytes confirmed a calcium antagonist relaxing mechanism of action. In healthy volunteers, plasma genistein levels of approximately 2 μM are achieved after ingestion of a commercially available soy protein drink (Supro) containing 37 mg genistein.CONCLUSIONSThis study demonstrates that phytoestrogens induce endothelium-independent relaxation of coronary arteries; the mechanism involves calcium antagonism. These mechanisms may contribute to the potential long-term cardiovascular protective effect of these substances.

Effects of JTV-519, a novel anti-ischaemic drug, on the delayed rectifier K+ current in guinea-pig ventricular myocytes.

We studied the effects of a newly synthesized anti-ischaemic agent, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2, 3, 4, 5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) on the delayed rectifier potassium current (IK), using guinea-pig ventricular myocytes and whole-cell voltage-clamp techniques, under blockade of the L-type calcium current (ICa,L) by D600 (1 microM) or nitrendipine (5 microM). The IK in guinea-pig ventricular cells consists of two different components; the rapidly activating, E4031-sensitive component (IKr) and the slowly activating E4031-resistant component (IKs). Under steady-state conditions, JTV-519 (1 and 5 microM) did not change the amplitude of IKs remaining after blockade of IKr with 5 microM E4031. The effect of JTV-519 on IKr was assessed using short (50 ms) pulses which evoked a tail current that was sensitive to E4031 but not to chromanol 293B, a specific blocker of IKs. JTV-519 suppressed the IKr with a half-maximal inhibitory concentration of 1.2 microM. Selective inhibition of IKr by this agent was confirmed by using the "envelope of tails" test. These results suggest that the blockade of IKr may underlie the prolongation of action potential duration in ventricular muscle and QT-intervals alleged to occur in animal as well as human hearts.

Use-dependent facilitation and depression of L-type Ca2+ current in guinea-pig ventricular myocytes: modulation by Ca2+ and isoprenaline.

An increase in stimulation frequency can facilitate or depress cardiac Ca2+ current (ICa). The aim was to examine the Ca2+ dependence of these effects, to determine if facilitation is sustained, and to elucidate the mechanism by which isoprenaline modulates facilitation. We examined the effects of increasing the stimulation frequency for 1 min, from 0.05 to 1 Hz, on ICa recorded from guinea-pig ventricular myocytes, using the whole-cell, voltage-clamp technique. 1 Hz stimulation caused a facilitation of ICa that peaked in 5 s and was followed by depression towards the basal level. Metabolic inhibitors or replacement of extracellular Ca2+ with Ba2+ abolished facilitation without affecting depression, implying that they are independent processes and that facilitation required ATP and Ca2+. Subtraction of the depression observed in either condition, from the response to 1 Hz stimulation recorded under control conditions, revealed that ICa facilitation was well maintained during 1 Hz stimulation. Increased intracellular Ca2+ buffering reduced both phases of the response. Furthermore, varying the extracellular Ca2+ concentration ([Ca2+]o) revealed a Ca(2+)-dependent enhancement of depression and a bell-shaped dependence of facilitation on [Ca2+]o. Facilitation increased with [Ca2+]o up to 1 mM, then declined at higher concentrations due to partial masking by the overlaping depression. Isoprenaline produced concentration-dependent inhibition of facilitation and enhancement of depression when pipettes contained 2 mM EGTA, but not BAPTA. For an equivalent increase in ICa amplitude, the effects of isoprenaline and elevated [Ca2+]o on the response to 1 Hz stimulation were quantitatively the same. Facilitation is sustained during increased activity, but appears transient due to overlapping depression. Both responses are promoted by increased submembrane [Ca2+]. Isoprenaline appears to modulate facilitation and depression as a consequence of increased Ca2+ influx, rather than cAMP-dependent phosphorylation. The apparent block of facilitation by isoprenaline may result from masking by the enhanced depression.

Anoxia depresses sodium-calcium exchange currents in guinea-pig ventricular myocytes.

Cardiac Na-Ca exchange is related to the intracellular calcium overload that occurs during ischemia and reperfusion. However, direct observation of the membrane current through Na-Ca exchange during ischemia has not been performed. The purpose of this study was to clarify the effect of simulated ischemia (substrate-free anoxia) and intracellular acidification on the Na-Ca exchange current. The electrogenic Na-Ca exchange current was recorded from isolated guinea-pig ventricular myocytes by using patch-clamp techniques. Exposure to anoxia significantly decreased both the inward and outward directed Na-Ca exchange currents (from -1.21+/-0. 18 to -0.04+/-0.32 pA/pF at -80 mV; from 6.58+/-1.06 to 3.14+/-1.06 pA/pF at +40 mV). The reversal potential of Na-Ca exchange current shifted to negative direction during anoxia. Subsequent reoxygenation rapidly restored the amplitude of exchange currents and the reversal potential. These anoxia/reoxygenation-induced changes were completely inhibited when the intracellular pH was clamped at 7.3 by using 20 m m HEPES-buffer. Furthermore, the anoxia-induced changes of Na-Ca exchange current were mimicked by the intracellular acidosis induced by a brief exposure to ammonium chloride in normoxic conditions. We conclude that the Cardiac Na-Ca exchange is suppressed by anoxia secondary to intracellular acidosis, and that these changes were reversed by reoxygenation.

P–62 - Inhibition by a dilazep derivative, K-7259, of the Na+ current in guinea-pig ventricular myocytes

P–62 - Inhibition by a dilazep derivative, K-7259, of the Na+ current in guinea-pig ventricular myocytes

P–231 - Inhibitory effect of amiodarone on Na+/Ca2+ exchange current of guinea-pig ventricular myocytes

P–231 - Inhibitory effect of amiodarone on Na+/Ca2+ exchange current of guinea-pig ventricular myocytes

P-585 - Specific carbamate insecticide, 2-s-butylphenyl methylcarbamate, inhibits the L-type Ca2+ current of guinea-pig ventricular myocytes

P-585 - Specific carbamate insecticide, 2-s-butylphenyl methylcarbamate, inhibits the L-type Ca2+ current of guinea-pig ventricular myocytes

Differential effects of endothelin-1 on basal and isoprenaline-enhanced Ca2+ current in guinea-pig ventricular myocytes.

1. We examined the effect of endothelin-1 (ET-1) on basal and isoprenaline-enhanced L-type Ca2+ current (ICa,L) in guinea-pig ventricular myocytes under nystatin-perforated patch configuration. 2. ET-1 at concentrations of 1, 5 and 10 nM had little effect on basal ICa,L. However, ICa,L enhanced by isoprenaline (500 nM) was significantly attenuated by 5 nM ET-1 by more than 50%. This effect was reversed upon washout. ICa,L enhanced by forskolin was also decreased by ET-1. 3. The inhibitory effect of ET-1 against isoprenaline was completely blocked by the ETA receptor antagonist BQ-123 (1 microM). In myocytes incubated with pertussis toxin (PTX, 2 micrograms ml-1) for 5 h, ET-1 did not inhibit isoprenaline-enhanced ICa,L. 4. Although ET-1 has been shown to activate specific protein kinase C (PKC) isoforms, a significant inhibitory effect of ET-1 was maintained in the presence of the PKC inhibitor bisindolylmaleimide (20 nM). The nitric oxide (NO) donor SIN-1 (10 microM) attenuated but failed to prevent the ET-1 effect. 5. In summary, our results demonstrate that ET-1 is devoid of any significant effects on basal ICa,L. However, it exerts a potent inhibitory effect against isoprenaline-enhanced ICa,L. This effect is mediated through ETA receptors coupled to PTX-sensitive G-proteins and occurs in the presence of PKC inhibition and NO generation.