Pascale Gluais

Acetylcholine‐induced endothelium‐dependent contractions in the SHR aorta: the Janus face of prostacyclin

In the spontaneously hypertensive rat (SHR) and aging Wistar–Kyoto rats (WKY), acetylcholine releases an endothelium‐derived contracting factor (EDCF) produced by endothelial cyclooxygenase‐1, which stimulates thromboxane A2 receptors (TP receptors) on vascular smooth muscle. The purpose of the present study was to identify this EDCF by measuring changes in isometric tension and the release of various prostaglandins by acetylcholine. In isolated aortic rings of SHR, U 46619, prostaglandin (PG) H2, PGF2α, PGE2, PGD2, prostacyclin (PGI2) and 8‐isoprostane, all activate TP receptors of the vascular smooth muscle to produce a contraction (U 46619≫8‐isoprostane=PGF2α=PGH2>PGE2=PGD2>PGI2). The contractions produced by PGH2 and PGI2 were fast and transient, mimicking endothelium‐dependent contractions. PGI2 did not relax isolated aortic rings of WKY and SHR. Acetylcholine evoked the endothelium‐dependent release of thromboxane A2, PGF2α, PGE2, PGI2 and most likely PGH2 (PGI2≫PGF2αPGE2>TXA2>8‐isoprostane, PGD2). Dazoxiben abolished the production of thromboxane A2, but did not influence the endothelium‐dependent contractions to acetylcholine. The release of PGI2 was significantly larger in the aorta of SHR than in WKY, and the former was more sensitive to the contractile effect of PGI2 than the latter. The inhibition of PGI‐synthase was associated with an increase in PGH2 spillover and the enhancement of acetylcholine‐induced endothelium‐dependent contractions. Thus, in the aorta of SHR and aging WKY, the endothelium‐dependent contractions elicited by acetylcholine most likely involve the release of PGI2 with a concomitant contribution of PGH2.

Role of SKCa and IKCa in endothelium‐dependent hyperpolarizations of the guinea‐pig isolated carotid artery

1 This study was designed to determine whether the endothelium‐dependent hyperpolarizations evoked by acetylcholine in guinea‐pig carotid artery involve a cytochrome P450 metabolite and whether they are linked to the activation of two distinct populations of endothelial KCa channels, SKCa and IKCa. 2 The membrane potential was recorded in the vascular smooth muscle cells of the guinea‐pig isolated carotid artery. All the experiments were performed in the presence of Nω‐L‐nitro arginine (100 μM) and indomethacin (5 μM). 3 Under control conditions (Ca2+: 2.5 mM), acetylcholine (10 nM to 10 μM) induced a concentration‐ and endothelium‐dependent hyperpolarization of the vascular smooth muscle cells. Two structurally different specific blockers of SKCa, apamin (0.5 μM) or UCL 1684 (10 μM), produced a partial but significant inhibition of the hyperpolarization evoked by acetylcholine whereas charybdotoxin (0.1 μM) and TRAM‐34 (10 μM), a nonpeptidic and specific blocker of IKCa, were ineffective. In contrast, the combinations of apamin plus charybdotoxin, apamin plus TRAM‐34 (10 μM) or UCL 1684 (10 μM) plus TRAM‐34 (10 μM) virtually abolished the acetylcholine‐induced hyperpolarization. 4 In the presence of a combination of apamin and a subeffective dose of TRAM‐34 (5 μM), the residual hyperpolarization produced by acetylcholine was not inhibited further by the addition of either an epoxyeicosatrienoic acid antagonist, 14,15‐EEZE (10 μM) or the specific blocker of BKCa, iberiotoxin (0.1 μM). 5 In presence of 0.5 mM Ca2+, the hyperpolarization in response to acetylcholine (1 μM) was significantly lower than in 2.5 mM Ca2+. The EDHF‐mediated responses became predominantly sensitive to charybdotoxin or TRAM‐34 but resistant to apamin. 6 This investigation shows that the production of a cytochrome P450 metabolite, and the subsequent activation of BKCa, is unlikely to contribute to the EDHF‐mediated responses in the guinea‐pig carotid artery. Furthermore, the EDHF‐mediated response involves the activation of both endothelial IKCa and SKCa channels, the activation of either one being able to produce a true hyperpolarization.

Endothelium-dependent contractions to acetylcholine, ATP and the calcium ionophore A 23187 in aortas from spontaneously hypertensive and normotensive rats

The present study was designed to determine whether or not an increase in endothelial intracellular concentration of calcium ([Ca2+]i) evokes endothelium‐dependent contractions in the aorta from normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR). Acetylcholine, adenosine triphosphate (ATP) and the calcium ionophore, A 23187, produced endothelium‐dependent relaxations in isolated aortic rings of both WKY and SHR. These relaxations in response to the three agonists were significantly smaller in the SHR when compared with the WKY. Endothelium‐dependent contractions to acetylcholine, ATP and A 23187 were observed only in the aorta isolated from the SHR. In the presence of NG‐nitro‐L‐arginine, an NO synthase inhibitor, the endothelium‐dependent contractions in response to acetylcholine, ATP and A 23187 were potentiated significantly in the aorta SHR and were unmasked in that of WKY. However, the contractions were still significantly greater in SHR than in WKY. These contractions were abolished by indomethacin and valeryl salicylate (two cyclo‐oxygenase inhibitors) as well as by S 18886 (a TP‐receptor antagonist), indicating that the endothelium‐dependent contraction produced by the three agonists share the same characteristics. The results of the present study indicate that the release/generation of endothelium‐derived contracting factor, requires an increase in endothelial [Ca2+]i.

Nitric oxide and inactivation of the endothelium-dependent contracting factor released by acetylcholine in spontaneously hypertensive rat.

In the aorta of the spontaneously hypertensive rat (SHR), endothelium-dependent contractions are enhanced by inhibitors of NO synthase and scavengers of NO, but not by methylene blue, an inhibitor of guanylyl cyclase, suggesting that the endothelium-derived contracting factor (EDCF) interacts chemically with NO and is inactivated by the latter. However, in view of the relative lack of specificity of methylene blue this hypothesis was re-examined. Acetylcholine-induced endothelium-dependent contractions of isolated rings of SHR aorta were significantly and similarly potentiated by two NOS inhibitors, by two structurally different NO scavengers, by two inhibitors of guanylate cyclase ODQ and NS2028, but to a lesser extent by methylene blue. The contraction of the isolated rat trachea in response to methacholine and the contraction of the rat aorta in response to both 8-isoprostane and KCl were inhibited significantly by methylene blue. Methylene blue binds to the M3 muscarinic receptor subtype but not to the TP receptor. Therefore, methylene blue is an antagonist of the M3 muscarinic receptor subtype, involved in the release of EDCF, and a non–specific inhibitor of TP receptor-mediated contractions, the receptor involved in the action of EDCF. These inhibitory effects of methylene blue are likely to counteract the effect of the inhibition of soluble guanylate cyclase. These results rule out the hypothesis according to which NO would chemically inactivate EDCF.

Repolarization abnormalities and their arrhythmogenic consequences in porcine tachycardia-induced cardiomyopathy

OBJECTIVES Action potential prolongation related to the alteration of several membrane currents is constantly reported in heart failure (HF) but reports about its role in arrhythmogenesis are sparse. Our aim was to determine, by analogy with long QT syndromes, whether prolonged repolarization is associated with increased dispersion or linked to bradycardia-dependent ventricular arrhythmias in pacing-induced cardiomyopathy. METHODS QT intervals, action potentials and transmural activation-to-recovery intervals (ARIs) along with whole-cell delayed rectifier (I(K)) and transient outward (I(to1)) K+ currents were recorded in left ventricle from pigs with HF and controls. HF was obtained after 14 days of rapid pacing at 250 ms. RESULTS Repolarization was delayed as indexed by corrected QT intervals (13.7% increase, P<0.01) or ARIs (252+/-4 to 340+/-7 ms, P<0.01). ARIs were uniformly prolonged with disappearance of the transmural gradient, spatial dispersion of repolarization decreased by 50% (P<0.05). I(to1) density was reduced in HF from 1.35+/-0.1 to 0.57+/-0.04 pA/pF subepicardially, from 1.05+/-0.19 to 0.55+/-0.08 pA/pF midmyocardially and from 1.04+/-0.1 to 0.48+/-0.04 pA/pF subendocardially. I(K) density was significantly decreased in HF pigs vs. controls: subepicardially from 0.46+/-0.04 to 0.22+/-0.02 pA/pF; midmyocardially from 0.46+/-0.05 to 0.25+/-0.03 pA/pF; and subendocardially from 0.49+/-0.04 to 0.20+/-0.04 pA/pF following depolarization at +50 mV. Electrocardiogram (ECG) monitoring at the time of death did not disclose any polymorphic ventricular tachyarrhythmia. CONCLUSION Despite a profound alteration in K+ currents, repolarization is uniformly prolonged in this model with no proclivity to develop bradycardia-dependent arrhythmias.

Risperidone prolongs cardiac action potential through reduction of K+ currents in rabbit myocytes

Prolongation of QT interval by antipsychotic drugs is an unwanted side effect that may lead to ventricular arrhythmias. The antipsychotic agent risperidone has been shown to cause QT prolongation, especially in case of overdosage. We investigated risperidone effects on action potentials recorded from rabbit Purkinje fibers and ventricular myocardium and on potassium currents recorded from atrial and ventricular rabbit isolated myocytes. The results showed that (1) risperidone (0.1-3 microM) exerted potent lengthening effects on action potential duration in both tissues with higher potency in Purkinje fibers and caused the development of early afterdepolarizations at low stimulation rate; (2) risperidone (0.03-0.3 microM) reduced significantly the current density of the delayed rectifier current and at 30 microM decreased the transient outward and the inward rectifier currents. This study might explain QT prolongation observed in some patients treated with risperidone and gives enlightenment on the risk of cardiac adverse events.

Clarithromycin reduces Isus and Ito currents in human atrial myocytes with minor repercussions on action potential duration.

The macrolide antibacterial agent clarithromycin has been shown to cause QT interval prolongation on the electrocardiogram. In rabbit heart preparations clarithromycin (concentration dependently) lengthened the action potential duration and blocked the delayed rectifier current. The aim of the present study was to investigate the clarithromycin effects: (i) on the Ca2+ L‐type and the main K+ repolarizing currents on human atrial myocytes, using whole‐cell patch clamp recordings and (ii) on action potentials recorded from human atrial and ventricular myocardium using conventional microelectrodes. It has been found that (i) 10–30 μm clarithromycin reduced the sustained current Isus significantly and that a 100 μm concentration was needed to cause a significant reduction in the transient outward current Ito, whereas clarithomycin did not affect the calcium current and (ii) clarithromycin (10–100 μm) prolonged the action potential duration in atrial preparations but did not alter the different parameters of the ventricular action potential. It is concluded that clarithromycin exerts direct cardiac electrophysiological effects that may contribute to pro‐arrythmic potential.

Comparative effects of clarithromycin on action potential and ionic currents from rabbit isolated atrial and ventricular myocytes.

Prolongation of QT interval by several antibacterial drugs is an unwanted side effect that may be associated with development of ventricular arrhythmias. The macrolide antibacterial agent clarithromycin has been shown to cause QT prolongation. To determine the electrophysiologic basis for this arrhythmogenic potential, we investigated clarithromycin effects on (i) action potentials recorded from rabbit Purkinje fibers and atrial and ventricular myocardium using conventional microelectrodes and (ii) potassium and calcium currents recorded from rabbit atrial and ventricular isolated myocytes using whole-cell patch clamp recordings. We found that (i) clarithromycin (3–100 &mgr;M) exerted concentration-dependent lengthening effects on action potential duration in all tissues, with higher efficacy and reverse frequency-dependence in Purkinje fibers. However, clarithromycin did not cause development of early afterdepolarizations, and the parameters other than action potential duration were almost unaffected; (ii) clarithromycin (10–100 &mgr;M) reduced the delayed rectifier current. Significant blockade (approximately 30%) was found at the concentration of 30 &mgr;M. At 100 &mgr;M, it decreased significantly the maximum peak of the calcium current amplitude but failed to alter the transient outward and inwardly rectifier currents. It was concluded that these effects might be an explanation for the QT prolongation observed in some patients treated with clarithromycin.