Irén Krassói

Functional Role of Potassium Channels in the Vasodilating Mechanism of Levosimendan in Porcine Isolated Coronary Artery

Levosimendan, a new type of inodilator drugs, is known to activate membrane adenosine 3′,5′-triphosphate-sensitive potassium (KATP) channels in some vascular smooth muscles and causes vasorelaxation. The involvement of potassium channels in the mechanism of the coronary artery relaxing effect of the drug has not been established. In the present study performed in the porcine epicardial coronary artery, the effect of levosimendan (0.009–3.2 μM) was compared to cromakalim (0.0125–5 μM), the known activator of ATP-sensitive potassium (KATP) channels, in the presence of glibenclamide (GLI), an inhibitor of KATP channels and tetraethylammonium (TEA), the non-selective inhibitor of potassium channels. The interaction of levosimendan with the specific calcium-activated potassium channel (KCa) blocker, iberiotoxin (IBTX), and the voltage-sensitive potassium channel (KV) blocker, 4-aminopyridine (4-AP), was also studied. All the experiments were performed in the isometric tension of endothelium denuded porcine isolated epicardial coronary arteries precontracted with 20 mM potassium chloride. 1 μM GLI decreased the maximum of cromakalim-induced relaxation by 60% but did not affect the action of levosimendan. In contrast, 2 mM TEA decreased only the coronary artery relaxing effect of levosimendan. 100 nM IBTX suppressed the maximum effect of levosimendan by only 15% while 0.5 mM 4-AP significantly shifted the concentration-response curve of the inodilator to the right. 5 mM 4-AP caused a maximum of 33% decrease of levosimendan-induced relaxation. These results indicate that, in porcine isolated epicardial coronary artery, the vasorelaxing mechanism of levosimendan involves the activation of voltage-sensitive and, at large concentrations, calcium-activated potassium channels.

C-Type Natriuretic Peptide Hyperpolarizes and Relaxes Human Penile Resistance Arteries

INTRODUCTION In addition to nitric oxide (NO), it is thought that an endothelium-derived hyperpolarizing factor (EDHF) plays an important role in the relaxation of penile arteries. Recently, it has been shown that C-type natriuretic peptide (CNP) shows the characteristics of EDHF in systemic small arteries. AIM To investigate the mechanism involved in CNP-evoked vasodilatation and to address whether CNP is an EDHF in human penile resistance arteries. METHODS Erectile tissue was obtained in connection with transsexual operations. Intracavernous penile resistance arteries were isolated and mounted in microvascular myographs for recording of isometric tension. Membrane potential was recorded by the use of a small glass electrode inserted in the smooth muscle layer. MAIN OUTCOME MEASURE In vitro evidence for hyperpolarization and vasorelaxation induced by CNP. RESULTS Acetylcholine (ACh) and CNP hyperpolarized smooth muscle membrane potential in resting penile resistance arteries. In penile small arteries incubated with inhibitors of NO synthase and cyclooxygenase and contracted with phenylephrine, ACh and CNP evoked concentration-dependent relaxations with maximum of 56 +/- 6% and 71 +/- 6%, respectively. Addition of a combination of blockers of small- and intermediate-conductance calcium-activated K(+) channels, apamin plus charybdotoxin, respectively, and a combination thought to block the smooth muscle response of EDHF-type relaxation, barium plus ouabain, markedly reduced ACh- and CNP-evoked relaxation. Iberiotoxin, a blocker of big-conductance calcium-activated K(+) channels inhibited the vasorelaxant responses evoked by ACh and CNP. A selective natriuretic peptide receptor type C (NPR-C) agonist, C-atrial natriuretic factor(4-23) (cANF(4-23)), induced relaxations with less maximum response compared to CNP. CONCLUSION The present findings suggest that CNP possesses the characteristics of an EDHF in human penile resistance arteries. By activation of natriuretic peptide receptor type B and NPR-C receptors, CNP causes relaxation by activation, respectively, of large-conductance calcium-activated K(+) channels and Na(+)/K(+)-adenosine triphosphatase (ATPase), and barium-sensitive inward rectifier K(+) channels. Modulation of the CNP pathway opens for new treatment modalities of erectile dysfunction.

Suppression of Erythromycin‐induced Early Afterdepolarizations and Torsade de Pointes Ventricular Tachycardia by Mexiletine

Erythromycin is a selective IKr‐blocking, action potential duration (APD)‐prolonging drug, which may induce early afterdepolarizations (EADs) and torsade de pointes ventricular tachycardia. The successful termination of an erythromycin‐induced clinical torsades de pointes by the authors with mexiletine prompted them to investigate in vitro whether erythromycin is able to induce EADs in Purkinje fibers and, if so, whether EADs are suppressible or not by mexiletine. Electrically stimulated canine Purkinje fibers (n=9) were superfused with erythromycin (200 mg/l) and action potentials were recorded by an intracellular microelectrode technique. Erythromycin induced a pronounced prolongation of APD and the appearance of EADs in all Purkinje preparations (9/9). After the addition of mexiletine (10 mM), a marked shortening of APD and the disappearance of EADs (7/9) were observed. Mexiletine, an inhibitor of the tetrodotoxin‐sensitive window Na+‐current, may prevent IKr‐blocking drug‐induced torsade de pointes ventricular tachycardia by abolishing APD prolongation and EADs.

Calcium-dependent vasorelaxant capacity of levosimendan in porcine and human epicardial coronary artery preparations.

In their recent letter, Koppel et al. [1] demonstrated that levosimendan (LEVO), a calcium-sensitizer inodilator agent [2], exerts minimal effects on smooth muscle tone in circular strips of bovine coronary arteries. In order to assess the possible relevance of these findings to other species, including humans, and to clinical settings, it was of interest to us to make similar experiments on epicardial coronary artery preparations obtained from porcine and human donor hearts. In order to validate the role of calcium, the vasorelaxing capacity of therapeutically meaningful concentrations of LEVO [3] was examined at low and high extracellular calcium concentrations ([Ca2+]o).

Improvement by phosphoramidon of damaged endothelial function in porcine coronary artery.

BACKGROUND The bradykinin (BK)-induced endothelium-dependent relaxation is impaired in the presence of elevated potassium concentration enhancing the vasospastic tendency of large coronary arteries. Inhibition of the angiotensin-converting enzyme responsible for bradykinin degradation was found to enhance the endothelium-dependent relaxation by BK. The aim of the present study was to investigate the effect of phosphoramidon, known to inhibit a BK-metabolizing neutral endopeptidase enzyme, on relaxation of porcine-isolated coronary artery in depolarizing solution. METHODS Endothelium intact porcine coronary artery rings were studied in organ chambers. The rings were isometrically contracted with potassium chloride (30 mmol/L) and the response to BK (1 to 1,000 nmol/L)-induced relaxation was investigated in the presence of nitric oxide synthase inhibitor Nomega-nitro-L-arginine (300 micromol/L) alone and in combination with the cyclooxygenase inhibitor indomethacin (10 micromol/L), and that of the inhibitor of calcium-dependent potassium channels tetraethylammonium (7 mmol/L). Under these conditions, phosphoramidon (10 micromol/L), an inhibitor of a neutral endopeptidase enzyme (EC.3.4.24.11.), which is responsible for the degradation of BK, was used to enhance the endothelium-dependent relaxation. RESULTS Phosphoramidon potentiated the maximum vasorelaxant effect of BK in Nomega-nitro-L-arginine (control 26.6%+/-10.86% versus phosphoramidon 49.05%+/-4.52%; n = 6, p < 0.05) or in Nomega-nitro-L-arginine + indomethacin-pretreated rings (control 20.7%+/-9.92% versus phosphoramidon 42.0%+/-12.26%; n = 5, p < 0.05) and this increased vasodilation was not modified by tetraethylammonium. CONCLUSIONS In the present study phosphoramidon potentiated the effect of BK in the absence of nitric oxide and prostaglandins in porcine-isolated coronary artery. This effect did not depend on tetraethylammonium-sensitive potassium channels. Phosphoramidon may be a useful pharmacologic tool for preserving the vasorelaxing capacity of coronary arteries after cardioplegia.

Vasorelaxing effect of levosimendan against 5-hydroxytryptamine-induced contractions in isolated human conduit bypass grafts

Levosimendan is a novel inodilator drug developed for the treatment of heart failure. The possible vasodilating property of the drug in human coronary artery bypass grafts was investigated. Isometric tensions of the left internal thoracic artery (LITA, n = 8) as well as the proximal and distal segments of the radial artery (RA, n = 8 and 8) were measured in isolated organ baths. Concentration‐relaxation curves for levosimendan (0.009‐1.14 μmol L−1) were obtained against 5‐hydroxytryptamine (5‐HT; serotonin, 0.002–9.3 μmol L 1)‐induced contractions. 5‐HT‐induced contraction of LITA was considerably smaller than that of the proximal and distal RAs. Levosimendan relaxed the grafts in the following order of calculated maximum efficacies (Emax): LITA > proximal RA > distal RA (LITA 100.3 ±16.2% of 5‐HT‐induced maximum tension, proximal RA 86.9 ±8.6%, distal RA 59.4 ± 17.5%, P < 0.05 LITA vs distal RA). The potency values of levosimendan, expressed as the negative logarithm of 50% effective concentrations (pD2), were comparable in the three bypass grafts (LITA −6.52 ± 0.44 log mol L−1, proximal RA −6.60 ± 0.49 log mol L−1, distal RA −6.85 ± 0.45 log mol L−1). The results suggest that levosimendan is an effective vasorelaxant of conduit bypass grafts and may serve as a new therapeutic tool, especially in the case of LITA and proximal RA grafts, for relieving perioperative spasm and subsequent graft failure.

Thiorphan enhances bradykinin-induced vascular relaxation in hypoxic/hyperkalaemic porcine coronary artery

Relaxation induced by bradykinin is diminished by hypoxia in epicardial coronary arteries. The bradykinin‐degrading enzyme, neutral endopeptidase (NEP, EC.3.4.24.11), is a potential target for coronary artery vasodilators. In this study, we examined the effect of thiorphan, an inhibitor of NEP, on the tone of porcine isolated coronary artery under hypoxic conditions. Endothelium‐intact porcine isolated coronary artery rings were isometrically contracted with a prostaglandin F2α analogue (U46619, 0.75 μM) and potassium chloride (KCl, 30 mM), and relaxed with bradykinin (1–1000 nM) under normoxic (partial pressure of oxygen, pO2 ˜ 90–100 mmHg) and moderately hypoxic (pO2 ˜ 50–60 mmHg) conditions. Experiments were performed to study the effects of 30 min pre‐treatment with the NEP‐inhibitor, thiorphan (10 μM), both at physiological and at low pO2s. Hypoxia inhibited the bradykinin‐induced relaxation in porcine epicardial coronary arteries. In normoxia, thiorphan significantly enhanced the decrease of coronary tone produced by bradykinin (1–10 nM) when U46619 was used as contractile agent. Under hypoxic conditions, in U46619 contracture, thiorphan did not influence, but in KCl contracture it enhanced the magnitude of relaxations induced by bradykinin. In the absence of bradykinin, thiorphan had no significant effect on the basal, KCl‐ and U46619‐elevated tones and on the hypoxia‐induced decrease of coronary artery tone. Inhibition of NEP‐enzyme activity may effectively improve the relaxing capacity of epicardial coronary arteries under hypoxic/hyperkalemic conditions. This effect could be potentially utilized when the endothelial function and relaxation of the coronary arteries are impaired under clinical conditions.

Low 4-aminopyridine concentration-induced contraction is mediated by neuronal noradrenaline in canine saphenous vein

4-Aminopyridine (4-AP), a known inhibitor of the voltage-dependent potassium channels, is able to increase the basal tone of different types of blood vessel preparations. In order to determine the efficiency of 4-AP in veins and to clarify its possible mechanism of action, the aim of the present study was to determine the basal tone and release of radio-labelled tissue noradrenaline (NA) after administration of low 4-AP concentrations. Experiments were performed in canine saphenous vein in the absence and presence of functional endothelium. 4-AP (0.012-5 microM) enhanced the basal tone of venous rings without and with endothelium (maximum tone at 5 microM 4-AP: 2.20 +/- 1.29 and 1.3 +/- 0.57 mN, respectively). NA stores of the venous tissue were loaded by adding 1 mM NA to the tissue for 10 min and then washed out. After loading the NA-stores of venous tissue, 4-AP-induced contractions were significantly increased both in the absence and presence of endothelium (maximum tone at 5 microM 4-AP after loading with NA: 10.51 +/- 3.64 and 10.52 +/- 4.69 mN, respectively). Following NA loading, chemical denervation of the endothelium denuded venous preparations by 0.5 mM 6-hydroxydopamine (6-OHDA) completely abolished the contractions evoked by 4-AP. After incubation of the saphenous preparations with 3H-NA, 5 microM 4-AP significantly increased tritium-efflux from the tissue. These results provide evidence for the efficiency of 4-AP on the basal tone of isolated canine saphenous vein when applied in low concentrations. Furthermore, it is suggested that this action of 4-AP may considerably depend on the release of NA from the perivascular nerve endings.

Interspecies differences and extracellular calcium dependence in the vasorelaxing effect of cromakalim in isolated human, porcine, and canine coronary arteries.

Coronary arteries isolated from human, porcine, and canine hearts were depolarized with potassium chloride and relaxed by cromakalim (0.0125-10.0 μmol/L) at low (1.5 mmol/L) and high (7.5 mmol/L) extracellular calcium concentration ([Ca2+] o). At low [Ca2+]o, cromakalim (1 μmol/L) relaxed the coronary arteries with the order of porcine > canine > human. Fifty percent effective concentrations of cromakalim revealed the same order: 0.15 μmol/L in porcine, 0.36 μmol/L in canine, and 3.91 μmol/L in human coronary arteries. High [Ca2+]o significantly enhanced the relaxing effect and decreased the potency of cromakalim in porcine and human but not in canine coronary arteries. In human coronary arteries, precontracted with the prostaglandin analogue (U46619), high [Ca2+] o enhanced the effect of 0.1 μmol/L cromakalim more efficiently in the presence than in the absence of endothelium. It appears that the coronary dilating effect of cromakalim largely depends on the species and is modulated by [Ca2+]o, with a partly endothelium dependent manner.