Gabriella Cargnelli

Na/Ca exchange and tension development in vascular smooth muscle: effect of amiloride

1 The potassium‐sparing diuretic, amiloride, has been shown to inhibit the Na/Ca exchange system in various preparations. The effects of this drug have been investigated on the contractions of guinea‐pig aortic strips elicited by reduction of external K, by addition of ouabain and by removal of external Na. 2 Amiloride (5 × 10−6 m‐5 × 10−4 m) inhibited the mechanical responses when it was added before giving the stimulus for contractions, but was not effective in relaxing the contracted strips. The drug shifted to the right the dose‐response curve for Ca in low K solution. 3 The calcium antagonist diltiazem had no effect on the ouabain‐, low K‐ and Na‐free‐induced contractions. 4 Amiloride decreased the rate of relaxation of aortic strips induced by removal of the low K solution. 5 The pattern of amiloride action on ouabain‐, low K‐ and Na‐free‐induced contractions suggests that the drug interferes with Ca influx. The effect of amiloride on the relaxation rate of low K‐ contracted aortic strips is consistent with an interference with Ca efflux. 6 It is suggested that amiloride prevents Ca fluxes through the Na/Ca exchange system of guinea‐pig aortic strips.

Endothelin-1-induced arachidonic acid release by cytosolic phospholipase A2 activation in rat vascular smooth muscle via extracellular signal-regulated kinases pathway.

The present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle.

Effects of pinacidil on guinea-pig isolated perfused heart with particular reference to the proarrhythmic effect.

1 The effects of pinacidil (10, 30, 50 μm) on contractility (+ dP/dtmax), coronary perfusion pressure (cP), and ECG intervals (PR, QRS, QT) have been studied on constant‐flow perfused guinea‐pig hearts, driven at four frequencies (2.5, 3, 3.5, 4 Hz). 2 Pinacidil decreased +dP/dtmax, cP and the QT interval in a dose‐dependent manner, whereas the PR interval was increased. QRS duration was not modified. All these effects were independent of driving frequency. Pinacidil decreased the interval from Q‐wave to T‐wave peak (QTpeak) to a greater extent than the QT interval, thus decreasing the QTpeak/QT ratio. This effect, unlike that on QT interval, was more evident at the highest frequency of stimulation. 3 In 4 out of 20 hearts treated with pinacidil sustained ventricular fibrillation (VF) occurred following a short run of premature ventricular beats (R on T phenomenon). 4 In separate experiments, an attempt to induce VF electrically was made at drug concentrations ranging from 10 μm to 100 μm (8 experiments for each concentration). In control conditions and at the lowest concentration of pinacidil tested (10 μm) VF could never be induced; in the presence of 30 μm pinacidil VF was induced in 5 out of 8 experiments. Drug concentrations higher that 50 μm permitted the induction of VF in every case. 5 Although the concentrations of pinacidil producing ventricular fibrillation are 30–40 times higher than those found in patients under long term treatment with this agent, it is suggested that caution should be used in prescribing this drug, at least in patients suffering from myocardial ischaemia.

Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature.

1 The effects of norbormide on the contractility of endothelium‐deprived rat, guinea‐pig, mouse, and human artery rings, and of freshly isolated smooth muscle cells of rat caudal artery were investigated. In addition, the effect of norbormide on intracellular calcium levels of A7r5 cells was evaluated. 2 In resting rat mesenteric, renal, and caudal arteries, norbormide (0.5–50 μm) induced a concentration‐dependent contractile effect. In rat caudal artery, the contraction was very slowly reversible on washing, completely abolished in the absence of extracellular calcium, and antagonized by high concentrations (10–800 μm) of verapamil. The norbormide effect persisted upon removal of either extracellular Na+ or K+. The contractile effect of norbormide was observed also in single, freshly isolated smooth muscle cells from rat caudal artery. 3 In resting rat and guinea‐pig aortae, guinea‐pig mesenteric artery, mouse caudal artery, and human subcutaneous resistance arteries, norbormide did not induce contraction. When these vessels were contracted by 80 mM KCl, norbormide (10–100 μm) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2+‐free medium. Norbormide (up to 100 μm) was ineffective in phenylephrine‐contracted guinea‐pig and rat aorta. 4 In A7r5 cells, a cell line from rat aorta, norbormide prevented high K+‐but not 5‐hydro‐xytryptamine‐induced intracellular calcium transients. 5 These findings indicate that in vitro, norbormide induces a myogenic contraction, selective for the rat small vessels, by promoting calcium entry in smooth muscle cells, presumably through calcium channels. In rat aorta and arteries from other mammals, norbormide behaves like a calcium channel entry blocker.

Calcium‐antagonist effects of norbormide on isolated perfused heart and cardiac myocytes of guinea‐pig: a comparison with verapamil

Cardiac effects of norbormide and verapamil were compared in single ventricular myocytes, right atria, and Langendorff perfused hearts isolated from guinea‐pigs. In ventricular myocytes, norbormide 50 μm inhibited the peak calcium current (ICa) by 49.6±3.9% without altering the shape of the current‐voltage relationship; verapamil 1 μm inhibited ICa by 83.2±3.3%. Neither norbormide nor verapamil affected ICa at the first beat after a 3 min quiescence period; during repeated depolarizations, both drugs cumulatively blocked ICa (use‐dependence), with time constants of 23.0±7.0 s for norbormide and 91.3±8.4 s for verapamil. In constant‐flow perfused hearts electrically driven at 2.5 Hz or 3.3 Hz, both norbormide and verapamil concentration‐dependently decreased ventricular contractility (dP/dtmax), atrio‐ventricular (AV) conduction velocity and coronary pressure. Intraventricular conduction velocity was slightly decreased by norbormide but not by verapamil. At an equivalent change in AV conduction, norbormide depressed heart contractility less than verapamil. The effects of norbormide on AV conduction, intraventricular conduction, and contractility were frequency‐dependent. Furthermore, the curves correlating the mechanical and electrical effects of norbormide at the two frequencies used were apparently coincident, while those of verapamil were clearly separated. In spontaneously beating right atria, norbormide and verapamil decreased the frequency of sinus node (SA) in a concentration‐dependent way. At an equivalent effect on the AV conduction, norbormide exerted a greater effect on sinus frequency than verapamil. These results indicate that in guinea‐pig heart norbormide has the pharmacological profile of a Ca‐antagonist with strong electrophysiological properties. In comparison with verapamil, norbormide is more selective on SA and AV node tissues and exerts a weaker negative inotropic effect on ventricles. In principle, this pattern of effects may be an advantage in treating supraventricular tachyarrhythmias in patients with heart failure. The effect of norbormide on intraventricular conduction may represent an additional antiarrhythmic mechanism.

An abnormal gene expression of the β‐adrenergic system contributes to the pathogenesis of cardiomyopathy in cirrhotic rats

Decreased cardiac contractility and β‐adrenergic responsiveness have been observed in cirrhotic cardiomyopathy, but their molecular mechanisms remain unclear. To study β‐adrenergic–stimulated contractility and β‐adrenergic gene expression patterns, 20 Wistar Kyoto rats were treated with carbon tetrachloride to induce cirrhosis and 20 rats were used as controls. Left ventricular contractility was recorded in electrically driven isolated hearts perfused at constant flow with isoproterenol (10−10 to 10−6 M). A cardiac gene expression profile was obtained using a microarray for the myocyte adrenergic pathway. The cardiac contractility maximal response to isoproterenol was significantly reduced in cirrhotic rats in comparison to control rats, whereas the half‐maximal effective concentration was not different. In cirrhotic rats, cardiac gene expression analysis showed a significant overexpression of G protein alpha–inhibiting subunit 2 (Gαi2), cyclic nucleotide phosphodiesterase (PDE2a), regulator of G‐protein signaling 2 (RGS2), and down‐expression of adenylate cyclase (Adcy3). These results indicate that overexpression of Gαi2, PDE2a, and RGS2 down‐regulates the β‐adrenergic signaling pathway, thus contributing to the pathogenesis of cirrhotic cardiomyopathy. (HEPATOLOGY 2008;48:1913‐1923.)

Ca2+ entry blocking and contractility promoting actions of norbormide in single rat caudal artery myocytes

Aim of the present study was to investigate the effects of norbormide, a selective vasoconstrictor agent of the rat peripheral vessels, on the whole‐cell voltage‐dependent L‐type Ca2+ current (ICa(L)) of freshly isolated smooth muscle cells from the rat caudal artery, using either the conventional or the amphotericin B‐perforated whole‐cell patch‐clamp method. Norbormide decreased L‐type Ca2+ current in a concentration‐ and voltage‐dependent manner, without modifying the threshold and the maximum of the current‐voltage relationship. Norbormide‐induced ICa(L) inhibition was reversible upon wash‐out. Norbormide both shifted the voltage dependence of the steady‐state inactivation curve to more negative potentials by about 16 mV, without affecting the activation curve, and decreased the slope of inactivation. Norbormide, however, did not modify both the activation and the inactivation kinetics of the ICa(L). Norbormide decreased ICa(L) progressively during repetitive step depolarizations, with inhibition depending on the stimulation frequency (use‐dependent block) as well as on the holding potential. Addition of 50 μM norbormide caused the contraction of all freshly isolated cells and also of those impaled with the perforated method, but not of those impaled with the conventional method (i.e. dialysed). In conclusion, these results prove norbormide to be a vascular L‐type Ca2+ channel inhibitor, which preferentially acts on the inactivated and/or open state of the channel. In rat caudal artery smooth muscle, however, this mechanism does not result in a vasodilating effect since it is overwhelmed by the mechanism underlying norbormide‐induced vasoconstriction.

In Vitro Vascular Reactivity to Endothelin: A Comparison Between Young and Old Normotensive and Hypertensive Rats

In this study we have investigated whether the vascular smooth muscle of a large capacitance artery of spontaneously hypertensive rats (SHR) is hyperresponsive to endothelin-1 and whether the arterial responsiveness to endothelin-1 is affected by aging. Isometric contractions of spirally cut aortic strips from SHR of 11, 22, 33 and 44 weeks of age and from age-matched WKY were measured in parallel. The vessels from SHR did not exhibit a greater responsiveness to endothelin-1 than those from WKY. No difference of responsiveness to the peptide was found among the arteries isolated from WKY of different ages. In contrast, a progressive decrease of responsiveness to endothelin-1 with aging was observed in SHR. This finding seems to be specific for endothelin-1, since the responsiveness to norepinephrine was unchanged. The significant decrease of aortic responsiveness in SHR with aging might be due to chronic hypertension and indicate desensitization to endothelin-1. The latter might be related to chronic in vivo hyperproduction of endothelin, either genetically determined or related to the hypertension-induced endothelial damage.

Effect of long-term ouabain treatment on contractile responses of rat aortae.

Male Sprague-Dawley rats were infused with 50 microg/kg/day of ouabain for 4 weeks to address the question whether prolonged exposure to the drug affects blood pressure, the in vitro contractile responses to agonists and high K+ of their aortae, and the influence of endothelium on these responses. Systolic blood pressure was not affected by ouabain treatment. The responsiveness of endothelium-intact aortae from ouabain-treated rats to endothelin-1 increased, that to phenylephrine decreased, and that to high K+ was unchanged, as compared with control. The responses of endothelium-free aortae to endothelin-1, phenylephrine, and high K+ were lower in ouabain-treated than in control rats. The removal of endothelium increased the response to phenylephrine and decreased that to high K+ in either control or ouabain-treated rat aortae, whereas it did not affect the response to endothelin-1 in control rat aortae and decreased it in ouabain-treated rat aortae. The response to caffeine was unaffected by either ouabain treatment or endothelium removal. Thus rat ouabain long-term treatment induces opposing effects on the responsiveness of their intact aortae to an alpha-adrenergic agonist and endothelin-1. If these effects observed in the ex vivo experiments occur also in vivo on rat microvasculature, they could balance out and contribute to the lack of effect on systolic blood pressure of prolonged ouabain treatment.

Effects of canrenone on aorta and right ventricle of the rat.

Canrenone is a major active metabolite of spironolactone and, in addition to the antimineralocorticoid effect, shares with the parent compound the action as a partial agonist with respect to ouabain on the Na+-K+ ATPase. We have investigated whether canrenone, through its action on Na+-K+ ATPase, reverses rat aorta contractions induced by ouabain and has vasorelaxant properties unrelated to its interaction with ouabain. Contractile responses of endothelium-deprived aorta to 1 m M ouabain, 0.1 &mgr;M phenylephrine, 10 &mgr;M serotonin, and 60 m M K+ were relaxed by canrenone (50–250 &mgr;M), with maximum inhibitions of 85.3%, 55.3%, 56.7%, and 64.2%, respectively. Canrenone shifted to the right the concentration-response curve for Ca2+ in depolarized aorta and did not affect the response to 10 m M caffeine. In rat right ventricular strips driven at 0.1 Hz, canrenone exerted negative inotropic effect. The relaxation of ouabain-induced contraction may be due, at least in part, to an interaction between canrenone and ouabain on the Na+-K+ ATPase. Inhibition of calcium entry through calcium channels either in aorta or ventricles is the most parsimonious hypothesis of mechanism underlying the effect of canrenone on contractile responses of rat aorta to agonists and high K+ and the negative inotropic effect on ventricular strips.