Effects of amiloride in guinea-pig and rat left atrial contraction as affected by frequency of stimulation and [Ca2+]0-[Na+]0 ratio: role of Na+/Ca2+ exchange.

1. The effect of amiloride (0.5 mM) on guinea-pig and rat left atria driven at various rates of stimulation and different [Ca2+]0-[Na+]0 ratios has been studied. 2. Amiloride elicited a positive inotropic response in guinea-pig left atria driven at 0.1 Hz, 0.5 Hz and 1 Hz when [Ca2+]0 was 3.6 mM, 1.8 mM and 0.9 mM respectively but not when [Ca2+]0 was 2.7 mM at 0.1 Hz, 0.9 mM at 0.5 Hz and 0.45 mM at 1 Hz. 3. A positive inotropic response was obtained in guinea-pig left atria driven at 0.1 Hz and 1 Hz when [Ca2+]0-[Na+]0(2) was increased respectively from 8 x 10(-5) to 16 x 10(-5) and from 2 x 10(-5) to 8 x 10(-5). The positive inotropic effect was evident only when the ratio was increased by increasing [Ca2+]0 and not by decreasing [Na+]0. 4. In the presence of amiloride, the force of contraction of guinea-pig left atria decreased instead of increasing, when the rate of stimulation was lowered from 1 Hz to 0.01 Hz. Amiloride inhibited the post-rest potentiation. 5. In rat left atria amiloride was devoid of any effect in all the above-mentioned experimental conditions. 6. It is suggested that the pattern of cardiac actions of amiloride can be explained by the inhibition of the Na+/Ca2+ exchange system.

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 X 10(-6) M-5 X 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.

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.

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.

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.

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.

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.

The influence of acidosis on the myocardial uptake and electrocardiographic effects of disopyramide.

The time course for the ECG effects and myocardial uptake of disopyramide was studied in isolated perfused guinea pig hearts under different pH conditions. At pH 7.46 the drug depressed the overall AV conduction time (PR) by 16.64%, the His-ventriculum conduction time (HV interval) by 30.46% and delayed the ventricular repolarization (QT interval) by 8.08%, on average. The maximum intraventricular pressure (Pmax) was also depressed by 35.6%. The maximum effect on the QT interval (constant rate: 0.609 min-1) was reached faster than the maximum effect on the PR and HV intervals (constant rates: 0.399 and 0.400 min-1, respectively), while the myocardium uptake process was complete before any ECG parameter reached a steady state (uptake constant: 1.58 min-1). Under conditions of extracellular acidosis (pH 6.92), the disopyramide disposition parameters (uptake rate constant and myocardial concentration) were not modified. However, the drug exerted significantly smaller effects on the HV and QT intervals and on myocardial contractility. These results are in contrast with those obtained previously with lidocaine and quinidine, and indicate that the influence of acidosis on class 1 antiarrhythmic agents may also depend on the characteristics of the individual drug.

Effects of SAS 1310, a new calcium antagonist, on isolated myocardial and smooth muscle preparations.

Some features of the effects of the diltiazem derivative SAS 1310 on in vitro myocardial and smooth muscle preparations were compared to those of the effects of diltiazem. Left atria, aortic strips and taenia coli of guinea-pigs were used. SAS 1310 induced a negative inotropic response of the left atria driven at 1 Hz similar to the response to diltiazem (IC50 values: SAS 1310 1.34 microM, diltiazem 0.8 microM). The inotropic effect of diltiazem (5 microM) was clearly rate-dependent whereas the reduction of left atria contractility induced by SAS 1310 (5 microM) was not modified by changes of the stimulation rate (the range of frequencies used was 0.5-2 Hz, with stepwise changes of 0.5 Hz). Diltiazem (0.1-0.5 microM) was more effective than SAS 1310 (0.1-5 microM) in inhibiting the contractile response to calcium of taenia coli depolarized by high K+ as well as in relaxing the aortic strips contracted by high K+ (IC50 SAS 1310 12.3 microM, diltiazem 0.41 microM). The response of aortic strips to norepinephrine (50 microM) in Ca2+-free medium was inhibited by SAS 1310 (50 microM) and was not affected by diltiazem (2 microM). The drug concentrations used were equiactive in inhibiting the high K+-induced contraction of the aortic strips. The different effects of diltiazem and its derivative on left atria contraction at different force-frequency ratios and on aortic strip contraction induced by norepinephrine in a Ca2+-free medium suggest that the actions of the two drugs differ qualitatively.

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.

Plasma levels and urinary excretion of verapamil, norverapamil, N-dealkylverapamil (D617), N-dealkylnorverapamil (D620) following oral administration of a slow-release preparation.

The kinetics of verapamil and of its N-dealkylated metabolites (norverapamil, D617, D620) were studied in six cardiac patients with normal cardiac indexes after 120 mg oral administration of the drug both as conventional preparation and as slow-release preparation. Following a dose of the slow-release preparation, the drug concentration curves were smoother and the mean bioavailability was lower in comparison with the conventional preparation. A patient taking inducing agents (phenobarbital and phenytoin) exhibited a strikingly low bioavailability. Following administration of the conventional preparation, the mean plasma half-lives of verapamil, norverapamil, D617 and D620 were 4.4, 6.6, 8.5, and 15.8 h respectively and the drug concentrations showed a triexponential decay. Urinary excretion data indicate that a saturation phenomenon may occur at level of renal tubular transport and that a competition may be suspected between D620 and the other compounds. It is concluded that various mechanisms, i.e. changes in hepatic and renal clearances, occurrence of a deep compartment, and the properties of the pharmaceutical preparation may affect verapamil kinetics during long-term treatment.

Competitive inhibition of phosphodiesterase activity by amrinone: its implication in the cardiac effect of the drug.

The effects of amrinone on partially purified bovine heart PDE and on resting tension of isolated guinea-pig left atria were investigated. Amrinone strongly inhibited the high affinity Mg2+ dependent PDE activity present in the 105000xg supernatant fraction of heart homogenate (IC50 = 60 microM), but only weakly blocked the Ca2+-CaM-dependent PDE (IC50 = 2 mM). The inhibition of high affinity PDE was competitive in nature. In guinea-pig left atria driven at 1 Hz, amrinone induced a concentration-dependent positive inotropic effect (EC50 = 200 microM). This action was not followed by a rise of the diastolic tension, even when high amrinone concentrations (1 mM) and/or high external Ca2+ concentrations (up to 12.8 mM) were used. It is suggested that the competitive inhibition of PDE by amrinone can only partially account for its positive inotropic effect. Moreover, because of the lack of effect on diastolic tension, amrinone differs from methylxanthines and digitalis glycosides in regard to its influence on intracellular Ca2+ movements.

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.

[Effects of aprindine on the electromechanical coupling of the myocardium and smooth muscle]. / Effetti della aprindina sull'accoppiamento elettromeccanico del miocardio e del muscolo liscio.

The effect of aprindine on myocardial and smooth muscle preparations activity were studied. Aprindine exerts a marked inhibition of the rate and contractility of guinea pig isolated atria and counteracts the inotropic effect induced by calcium. On the isolated guinea-pig left atrium electrically driven, apridine was found to exert negative inotropic effect both at low and high rates of stimulation, without depressing the "post-stimulation" potentiation. In spontaneously active guinea-pig taenia coli aprindine induces stimulation and regularization of the mechanical and electrical activity; on the other hand, aprindine is able to inhibit the contraction induced by acetylcholine, and this inhibition is counteracted by increasing concentration of calcium. Aprindine exerts no influence on the electrical membrane resistence of the guinea-pig taenia coli.

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 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.

[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.

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.