Attila Kun

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.

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).

Involvement of Large-Conductance Ca2+-Activated K+ Channels in both Nitric Oxide and Endothelium-Derived Hyperpolarization-Type Relaxation in Human Penile Small Arteries

Large‐conductance Ca2+‐activated K+ channels (BKCa), located on the vascular smooth muscle, play an important role in regulation of vascular tone. In penile corpus cavernosum tissue, opening of BKCa channels leads to relaxation of corporal smooth muscle, which is essential during erection; however, there is little information on the role of BKCa channels located in penile vascular smooth muscle. This study was designed to investigate the involvement of BKCa channels in endothelium‐dependent and endothelium‐independent relaxation of human intracavernous penile arteries. In human intracavernous arteries obtained in connection with transsexual operations, change in isometric force was recorded in microvascular myographs, and endothelium‐dependent [nitric oxide (NO) and endothelium‐derived hyperpolarization (EDH)‐type] and endothelium‐independent (NO‐donor) relaxations were measured in contracted arteries. In penile small arteries contracted with phenylephrine, acetylcholine evoked NO‐ and EDH‐type relaxations, which were sensitive to iberiotoxin (IbTX), a selective blocker of BKCa channels. Iberiotoxin also inhibited relaxations induced by a NO‐donor, sodium nitroprusside. NS11021, a selective opener of BKCa channels, evoked pronounced relaxations that were inhibited in the presence of IbTX. NS13558, a BKCa‐inactive analogue of NS11021, failed to relax human penile small arteries. Our results show that BKCa channels are involved in both NO‐ and EDH‐type relaxation of intracavernous penile arteries obtained from healthy men. The effect of a selective opener of BKCa channels also suggests that direct activation of the channel may be an advantageous approach for treatment of impaired endothelium‐dependent relaxation often associated with erectile dysfunction.

Prolonged Antispasmodic Effect in Isolated Radial Artery Graft and Pronounced Platelet Inhibition Induced by the Inodilator Drug, Levosimendan

Radial artery frequently develops spasm and requires vasodilator therapy during coronary artery bypass graft surgery (CABG). Levosimendan was recently shown to oppose 5-hydroxytryptamine-induced contraction of radial artery (RA) grafts. The aim of the present study was to explore whether levosimendan retains its vasodilatory capacity following in vitro pre-incubation of RA segments with the inodilator. A possible cumulative effect of the drug in human platelets was also studied. Human isolated RA segments were pre-incubated in 0.16 μmol/L levosimendan containing solution or in 0.9% NaCl, Bretschneider, 5% albumin and a 5% human serum protein solution (Biseko) as controls for 45 min. Contractions were induced by three consecutive administrations of 5-hydroxytryptamine (0.31 μM) 45, 90 and 120 min. after exchanging the pre-incubation solutions with Krebs-Henseleit solution, uniformly. Receptor-independent contractions (KCl, 80 mmol/L), endothelium-dependent (acetylcholine, 1 μmol/L) and independent relaxations (papaverine, 100 μmol/L) were also investigated. Washed human platelets were pre-incubated with levosimendan (0.06 μmol/L) for 2 or 15 min. and aggregated with thrombin (0.1 IU/mL). Contractions of RA grafts induced by 5-hydroxytryptamine were significantly smaller 45 min. and 90 min. after the replacement of levosimendan with Krebs-Henseleit solution. Biseko solution also decreased the contraction of the graft at 45 min. Contractions did not change in time following the pre-incubations of radial arteries with 0.9% NaCl, Bretschneider and 5% albumin solutions. The grafts remained intact as assessed by their maximum contractions and endothelium-dependent and endothelium-independent relaxations at the end of the investigations. Platelets revealed larger anti-aggregatory effect to levosimendan following the enhancement of the incubation time. Results indicate that the antispasmodic and anti-aggregatory effects of levosimendan cumulate in the vascular tissue and in platelets. The storage of RA with the inodilator before implantation may help to prevent the intraoperative spasm of the graft and also thrombotic occlusion during CABG surgery.

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.

Potassium channels sensitive to combination of charybdotoxin and apamin regulate the tone of diabetic isolated canine coronary arteries.

Aims:  Functional roles of calcium‐activated potassium channels on the mechanical activity of epicardial coronary arteries obtained from a canine model of diabetes were investigated.

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.

Neurogenic contraction induced by the antiarrhythmic compound, AVE 0118, in rat small mesenteric arteries

The purpose of this study was to investigate the vasoactivity of two inhibitors of potassium ion (K+) channels, a potential antiarrhythmic compound, AVE 0118, and 4‐aminopyridine (4‐AP). Basal and stimulated tones of rat small mesenteric arteries as well as the possible involvement of KV1.5 ion channel in the mechanism of vascular effect induced by the compounds were analysed. The standard organ bath technique for vascular tone and immunohistochemistry for the localization of ion channels in the arterial tissue were performed. Third‐ or fourth‐order branch of arterial segments was mounted in myographs for recording the isometric tension. AVE 0118 (10−5 M) and 4‐AP (10−5 M) modulated neither the basal tone nor the contraction induced by noradrenaline but increased the contraction evoked by electrical field stimulation, sensitive to the block of alpha‐1 adrenergic receptors. KV1.5 ion channel‐specific immunostaining demonstrated the presence of immunoreactive nerves, and Schwann‐cell‐specific (S100) immunostaining confirmed the presence of myelin sheath in rat small mesenteric arteries. The study supports an indirect, sympathetic effect of AVE 0118 similar to that of 4‐AP, which is mediated, at least in part, by blocking neuronal KV1.5 type potassium ion channels in the medio‐adventitial layer of rat small mesenteric artery.