Norbormide: a calcium entry blocker with selective vasoconstrictor activity in rat peripheral arteries.

Norbormide is a unique vasoactive substance endowed with species- and tissue-specific, endothelium independent, vasoconstrictor activity that is restricted to the peripheral arteries of rat. In rat aorta and in all tested arteries of other species norbormide exhibits vasorelaxant property presumably due to the blockade of calcium channels. A calcium entry blocker effect of norbormide has also been described in isolated, perfused guinea pig hearts. In these preparations norbormide produced coronary vasodilator, as well as negative inotropic and dromotropic effects. In single ventricular myocytes of guinea pigs norbormide reduces L-type calcium current. The mechanism underlying the selective vasoconstrictor effect of norbormide is unknown. In rat caudal artery, a vessel contracted by norbormide, the drug activates phospholipase C (PLC) signal cascade which is the biochemical pathway involved in the contractile effect triggered by most receptor-activating vasoactive agents. Therefore, norbormide-induced contraction of rat peripheral vessels is likely to be due to the activation of a PLC-coupled receptor abundantly or selectively expressed in vascular smooth muscle cells. The identification of this putative receptor could facilitate the development of tissue-selective pharmacological agents.

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 mM ouabain, 0.1 microM phenylephrine, 10 microM serotonin, and 60 mM K+ were relaxed by canrenone (50-250 microM), 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 mM 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.

Signaling mechanisms for the selective vasoconstrictor effect of norbormide on the rat small arteries.

Norbormide (NRB) is a selective vasoconstrictor agent of the rat small vessels. The mechanisms underlying the selective vasoconstrictor effect of NRB are unknown. To investigate whether phospholipase C (PLC) signaling pathway plays a role in NRB-induced vasoconstriction, we performed experiments in NRB-contracted tissues, namely, rat caudal arteries (RCA) and smooth muscle cells derived from rat mesenteric arteries (MVSMCs). An NRB-insensitive vessel, namely rat aorta (RA), served as a control tissue. In RCA and RA we measured either isometric tension or formation of inositol phosphates (IPs), the latter taken as an index of PLC activation. In MVSMCs, we measured intracellular free calcium concentration ([Ca2+]cyt). In the presence of external Ca2+, NRB (2-50 microM) stimulated IPs formation in RCA but not in RA, and increased [Ca2+]cyt in MVSMCs. In the absence of external Ca2+, NRB (50 microM) increased IPs formation in RCA but was unable to increase [Ca2+]cyt in MVSMCs. In RCA, in the presence of external Ca2+, NRB-induced contraction was inhibited by calphostin C (0.2-1 microM), an inhibitor of protein kinase C (PKC), and by SK&F 96365 (30 microM), an inhibitor of the store-operated calcium channels, but was poorly affected by verapamil, an L-type calcium channel blocker. However, verapamil was much more effective when external Ca2+ was substituted by Sr2+. These results suggest that NRB elicits its tissue and species-selective vasoconstrictor effect by stimulating PLC-PKC pathway and increasing Ca2+ influx through both verapamil-sensitive and -insensitive calcium channels. Ca2+ release from sarcoplasmic reticulum seems not involved in NRB vasoconstriction.

Callipeltin A, a cyclic depsipeptide inhibitor of the cardiac sodium-calcium exchanger and positive inotropic agent.

Callipeltin A, a cyclic depsipeptide from the New Caledonian Lithistida sponge Callipelta sp., is a macrocyclic lactone containing four amino acids in the L configuration, Ala, Leu, Thr (2 residues); one (Arg) in the D configuration; two N-methyl amino acids, N-MeAla and N-MeGln; a methoxy tyrosine, a 3, 4-dimethyl-l-glutamine; and a 4-amino-7-guanidino-2,3 dihydroxypentanoic acid (AGDHE), formally derived from L-Arg. In cardiac sarcolemmal vesicles Callipeltin A induces a powerful (IC(50) = 0.85 microM) and selective inhibition of the Na(+)/Ca(2+) exchanger. In electrically driven guinea-pig atria, at concentrations ranging between 0.7 and 2.5 microM, Callipeltin A induces a positive inotropic effect, which at the highest concentrations is accompanied by a rise in resting tension. It is suggested that the positive inotropic effect is linked to the inhibition of the Na(+)/Ca(2+) exchanger and that Callipeltin A may be an useful tool to study the role of the cardiac Na(+)/Ca(2+) exchanger in physiological and pathological conditions.

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.

Alpha1-adrenoceptor-mediated formation of glycerophosphoinositol 4-phosphate in rat heart: possible role in the positive inotropic response.

In the present study, we investigated whether phospholipase A2 (PLA2)/lysophospholipase activity producing glycerophosphoinositols from phosphoinositides was operating in rat heart and could be stimulated by alpha1-adrenergic agonists. PLA2/lysophospholipase activity was found in homogenates from rat right ventricles. The stimulation of PLA2/lysophospholipase activity by noradrenaline (NA) was prevented either by the alpha1-adrenergic antagonist prazosin or arachidonyl trifluoromethyl ketone, a selective inhibitor of the 85-110 kDa, sn-2-arachidonyl-specific cytosolic PLA2. The selective alpha1-adrenergic agonist phenylephrine induced a concentration- and time-dependent increase in glycerophosphoinositol (GroPIns) and glycerophosphoinositol 4-phosphate (GroPIns4P) in rat right ventricle slices prelabelled with D-myo-[3H]inositol. In electrically driven strips of rat right ventricles, prelabelled with D-myo-[3H]inositol, the positive inotropic effect induced by 20 microM NA in the presence of propranolol was accompanied by the formation of GroPIns and GroPIns4P. The concentration of the formed GroPIns4P (1.33+/-0.12 microM, N = 6) was similar to that previously reported to inhibit the Na+/Ca2+ exchanger in cardiac sarcolemmal vesicles (Luciani S, Antolini M, Bova S, Cargnelli G, Cusinato F, Debetto P, Trevisi L and Varotto R, Biochem Biophys Res Commun 206: 674-680, 1995). These findings show that the stimulation of alpha1-adrenoceptors in rat heart is followed by an increase in the formation of GroPIns4P, which may contribute to the positive inotropic effect of alpha1-adrenergic agonists by inhibition of the Na+/Ca2+ exchanger.

Changes of blood pressure and aortic strip contractile responses to ET-1 of heterozygous female transgenic rats, TGR(mRen2)27.

We studied the contractile responses to endothelin-1 (ET-1) of aortic strips from female transgenic rats, TGR(mRen2)27, heterozygous for the Ren-2 mouse gene, during the phases of developing (blood pressure in rats aged 5 weeks; 156 +/- 8 mmHg), steady (blood pressure in rats aged 11 weeks: 206 +/- 27 mmHg), and reversed (blood pressure in rats aged 35 weeks: 151 +/- 17 mmHg) hypertension. These responses were compared with those of aortae from sex- and age-matched, genetically homogeneous, normotensive Sprague-Dawley (SD) rats. Aortic strips from both transgenic and SD rats were deprived of endothelium before isometrically recording developed tension to cumulatively added ET-1. Aortic strips from 5- and 11-week-old female transgenic TGR(mRen2)27 (hfTG) rats responded to ET-1 with higher Emax values and lower EC50 values than those of age-matched SD rats. Conversely, aortic strips from 35-week-old hfTG rats exhibited lower Emax and higher EC50 values than aortic strips from SD rats. Within the hfTG rats, aortic strips from 11-week-old rats showed increased Emax and decreased EC50 of ET-1 as compared with either 5- or 35-week-old hfTG rats. These data are in keeping with the hypothesis that ET-1 contributes to the hypertension of hfTG rats and suggest that an altered vascular responsiveness to the peptide may be implicated in the changes of their systolic blood pressure occurring with ageing in this animal model.

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

1. Cardiac effects on norbormide and verapamil were compared in single ventricular myocytes, right atria, and Langendorff perfused hearts isolated from guinea-pigs. 2. In ventricular myocytes, norbormide 50 microM inhibited the peak calcium current (ICa) by 49.6 +/- 3.9% without altering the shape of the current-voltage relationship; verapamil 1 microM 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. 3. 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. 4. 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. 5. 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.

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 microM) 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 microM) 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 microM) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2(+)-free medium. Norbormide (up to 100 microM) 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-hydroxytryptamine-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.

Inhibition of cardiac sarcolemmal sodium-calcium exchanger by glycerophosphoinositol 4-phosphate and glycerophosphoinositol 4-5-bisphosphate.

The enrichment in phosphatidylinositol 4-phosphate and phosphatidylinositol 4-5-bisphosphate of cardiac sarcolemmal vesicles stimulate Na+/Ca2+ exchange activity. On the contrary the deacylation products of polyphosphoinositides are powerful inhibitors of Na+/Ca2+ exchange: half maximal inhibition of 1.6 and 2.1 microM have been observed for glycerophosphoinositol 4-phosphate and glycerophosphoinositol 4-5-bisphosphate, respectively. The data indicate a bidirectional regulation of Na+/Ca2+ exchanger and suggest that phosphorylated glycerophosphoinositols can be regarded as novel signal transducers of intracellular Ca2+ regulation in heart cell.

Effect of amiloride on inotropic and toxic actions of ouabain in guinea-pig left atria.

The effect of amiloride on the positive inotropic and toxic effects of ouabain in guinea-pig left atria has been studied. In atria driven at 1 Hz, amiloride (0.3 and 0.5 mM) decreased the EC50 but did not affect the maximal tension developed by ouabain. At 0.1 Hz, amiloride did not change either the EC50 or the maximal tension developed by ouabain. Ouabain toxicity (onset of arrhythmias) was not changed by amiloride at either frequency of stimulation. Therefore, amiloride did not antagonize either the positive inotropic or the toxic effect of ouabain. The positive inotropic effect of amiloride has been ascribed to the inhibition of the Na+/Ca2+ exchanger. Since amiloride inhibits also the Na+/H+ exchanger, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an amiloride derivative which selectively inhibits the Na+/H+ exchange, has been tested to evaluate the role of the Na+/H+ exchange in the amiloride-ouabain interaction. EIPA increased the EC50 values of ouabain and decreased the maximal developed tension by the glycoside in atria driven at 0.1 and 1 Hz, but did not antagonize the toxic response (arrhythmias) of atria to ouabain. It is suggested that the inhibition of Ca2+ exit through the Na+/Ca2+ exchange by amiloride and ouabain may explain the observation that the positive inotropic effects of amiloride and ouabain are additive.

Effect of subthreshold ouabain on the tone of guinea-pig aortic strips following repeated noradrenaline stimulation.

1. The effect of ouabain at a concentration (0.8 microM) that does not induce contractile response in guinea-pig aortic strips has been studied on endothelium-denuded strips repeatedly stimulated with 1 microM noradrenaline or 60 mM K+ applied for 5 min every 30 min. 2. The resting tone (i.e. the tone between one noradrenaline stimulation and the following) of the aortic strips exposed to ouabain increased progressively, whereas the control strips (no ouabain) completely relaxed on washout of the agonist. In the aortic strips stimulated by 60 mM K+, the resting tone did not increase. 3. The calcium antagonist, verapamil, did not affect the increase in tone, that was nevertheless strictly dependent on external calcium, since the contracted strips completely relaxed on calcium removal and promptly contracted again on calcium readdition. This finding indicates a mechanism independent of voltage-gated calcium channels. 4. Caffeine-induced contractions, taken as a measure of sarcoplasmic reticulum calcium content, were amplified by the presence of ouabain in aortic strips either stimulated by noradrenaline or unstimulated, with a larger increase in the former. 5. These results suggest that the repeated stimulation of guinea-pig aortic strips by noradrenaline in the presence of ouabain, by raising both intracellular Na+ and Ca2+, decreases the ouabain threshold concentration required for contraction, thus increasing the responsiveness of vascular smooth muscle to the glycoside.

On the mechanism of vasodilating action of berberine: possible role of inositol lipid signaling system.

We have studied the effect of the alkaloid berberine on the contraction of guinea pig aortic strips induced by various stimuli. Berberine (25-200 microM) inhibited the response of the strips to norepinephrine and histamine, but did not decrease the high K(+)-elicited contraction. The antagonism of berberine was not competitive because in the presence of the alkaloid, maximum response to agonists could not be obtained. Analysis of the drug's effect on the time course of norepinephrine-induced contraction showed that berberine reduced both the rate and the relative contribution to developed tension of the initial, rapid phase, whereas the slow, later component was less affected. Berberine inhibited the response of aortic strips incubated in 0 mM Ca++ to norepinephrine, but did not reduce caffeine-induced contraction and also inhibited phospholipase C-activated contractile response, which has been ascribed to production of inositol phosphate-3 in smooth muscle cells. In cultured arterial smooth muscle cells (A7r5 line), the alkaloid did not significantly decrease the production of inositol phosphates activated by Arg8-vasopressin. The pattern of berberine action is difficult to reconcile with an involvement of the contractile machinery and suggests that the drug has no effect on the voltage-operated calcium channels. Although an antagonism at the receptors or an increase of cyclic AMP or cyclic GMP cannot be completely excluded, we suggest that at least one component of the berberine inhibitory effect may be due to its action on some step of the chain of events linking receptors to contractile response.

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

1. The effects of pinacidil (10, 30, 50 microM) 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 microM to 100 microM (8 experiments for each concentration). In control conditions and at the lowest concentrations of pinacidil tested (10 microM) VF could never be induced; in the presence of 30 microM pinacidil VF was induced in 5 out of 8 experiments. Drug concentrations higher than 50 microM 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.

[Positive inotropic effect of amiloride and inhibition of Na+/Ca2+ exchange: dependence on temperature]. / Effetto inotropo positivo dell'amiloride e inibizione dello scambio Na+/Ca2+: dipendenza dalla temperatura.

Na+/Ca2+ exchange plays a fundamental role in the regulation of intracellular Ca2+ levels and thus myocardial contractility. The influence of temperature variations on Na+/Ca2+ exchange activity in bovine heart sarcolemmal vesicles has been studied. Furthermore, the temperature dependence of positive inotropic response induced by amiloride, an inhibitor of Na+/Ca2+ exchange activity, has been investigated in isolated guinea-pig left atria driven at 1 Hz. Our results indicate that cooling from 37 degrees to 20 degrees C inhibits Na+/Ca2+ exchange activity in sarcolemmal vesicles, whereas does not change the extent of Na+/Ca2+ exchange inhibition by amiloride. In addition, the positive inotropic effect induced by amiloride in guinea-pig left atria decreases and eventually disappears when temperature is progressively reduced from 35 degrees to 23 degrees C. A possible relationship between the decrease in Na+/Ca2+ exchange activity induced by cooling and the temperature dependence of positive inotropic effect of amiloride is discussed.