Calcium dependence of ouabain-induced contraction in aortas from spontaneously hypertensive rats

The calcium sensitivity of ouabain-induced contractions of aortic strips from spontaneously hypertensive rat (SHR) was examined using several drugs which affect Na+ and Ca2+ movements across the cell membrane, and the results were compared with those obtained with age-matched Wistar-Kyoto rat (WKY). The Ca2+ concentration-response curves (10(-3) M ouabain-treated preparations) made with aortic strips from SHR lay to the left of those made with aortic strips from WKY (Ca EC50 values: SHR, 0.51 +/- 0.16 mM, n = 6; WKY, 1.23 +/- 0.41 mM, n = 7; P less than 0.05). Amiloride (a Na+ entry blocker) and nifedipine (a Ca2+ entry blocker) attenuated the sensitivity to Ca2+ of SHR and WKY aortic strips. With 2 x 10(-4) M amiloride, WKY vessels showed a 1.3-fold increase in the Ca EC50 value and SHR a 2.1-fold increase. With 10(-6) M nifedipine. WKY vessels showed a 1.1-fold increase in the Ca EC50 value and SHR a 1.5-fold increase. Addition of monensin (Na ionophore) produced a dose-dependent potentiation in ouabain-treated aorta from WKY, but not in ouabain-treated aorta from SHR. Addition of 1.5 x 10(-5) M A23187 (Ca ionophore) eliminated the difference between the Ca2(+)-induced contractions in aortas from SHR and WKY. These results suggest that enhancement of Ca2+ influx by Na(+)-Ca2+ exchange and/or voltage-dependent Ca2+ channels in vascular smooth muscle cell membranes may be an important factor in the difference between ouabain-induced contractions in aorta from SHR and WKY.     

Alpha 1-adrenoceptor reserve and effects of a Ca2+ entry blocker (Ro 18-3981) on aorta of spontaneously hypertensive rats

The relationship between alpha 1-adrenoceptor reserve and the sensitivity of vasoconstrictor responses to Ca2+ entry blockade was investigated in isolated aortas from age-matched (13-15 weeks) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Noradrenaline (NA) elicited contractile responses with a greater potency (log EC50) in aorta from WKY (-8.7) than in those from SHR (-8.05). The dihydropyridine Ca2+ entry blocker, Ro 18-3981 (10(-6) M), suppressed the maximal NA responses more in aorta of SHR (-54%) than WKY (-14%). The dissociation constant (KA) of NA was similar in aortas of both strains. However, the difference between KA and EC50 values was greater in aorta of WKY (7.2 X) than in those from SHR (1.4 X). Pretreatment of WKY aorta with the irreversible alpha-blocker phenoxybenzamine (10(-9) M) enhanced the inhibitory effect of Ro 18-3981 (10(-6) M) against NA-induced contractions (-14 to -47%). Thus, a smaller alpha 1-adrenoceptor reserve could explain the greater sensitivity of NA-induced contractions in SHR aorta to Ca2+ entry blockade.     

Possible mechanism of the potent vasoconstrictor actions of ryanodine on femoral arteries from spontaneously hypertensive rats.

1. The Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of ryanodine that removes the function of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. The addition of ryanodine to the resting strips caused a concentration-dependent contraction in SHR. This contraction was extremely small in WKY. In the presence of 10(-5) M ryanodine, caffeine (20 mM) failed to cause a further contraction in SHR, but it caused a small contraction in WKY. After washout of the strips with a Krebs solution, the resting tone was greatly elevated in SHR when compared with WKY. 3. The elevated resting tone in SHR strips was abolished by 10(-7) M nifedipine. The ryanodine-induced contraction was also abolished by 10(-7) M nifedipine. Nifedipine itself caused a relaxation from the resting tone of SHR strips, suggesting the maintenance of myogenic tone. 4. In strips preloaded with fura-PE3, the addition of 10(-5) M ryanodine caused a large and moderate elevation of cytosolic Ca2+ level ([Ca2+]i) in SHR and WKY, respectively. After washout, the resting [Ca2+]i was greatly elevated in SHR. The ryanodine-induced elevation of [Ca2+]i was decreased by 5 x 10(-6) M verapamil in SHR. Verapamil itself caused a decrease in resting [Ca2+]i which was significantly greater in SHR than in WKY, and caused a relaxation only in SHR. 5. The resting Ca2+ influx in arteries measured by a 5 min incubation with 45Ca was significantly increased in SHR when compared with WKY. The resting Ca2+ influx was not increased by 10(-5) M ryanodine in both SHR and WKY. The net cellular Ca2+ uptake in arteries measured by a 30 min incubation with 45Ca was decreased by 10(-5) M ryanodine in both strains. 6. The resting Ca2+ influx was decreased by 10(-7) M nifedipine in the SHR artery, but it was unchanged in the WKY artery. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional removal of SR by ryanodine caused a potent contraction in this artery.     

Effects of H-7 (protein kinase inhibitor) and phorbol ester on aortic strips from spontaneously hypertensive rats

We investigated the vascular responsiveness to vasoactive agents and the inhibition by H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine), which inhibits cyclic nucleotide-dependent protein kinases and protein kinase C(PKC) equally potently in helically cut strips of thoracic aortas from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The susceptibility of norepinephrine (NE)- and angiotensin II (Ang II)-induced contractions to H-7 was significantly higher in the aortas from SHR than in those from WKY. H-7 decreased the contractile responses to KCl to a similar extent in both strains without affecting the high K(+)-stimulated Ca2+ influx. H-7 produced a shift to the right of the dose-response curve for the PKC activator, 12-o-tetradecanoylphorbol-13-acetate (TPA) in the case of SHR aortas, while no such shift was noted in tissues from WKY. Functional alterations in the PKC branch of the Ca2+ messenger system in vascular smooth muscle may play an important role in SHR during the sustained contraction.     

Effects of bepridil compared to those of its quaternary analogue on femoral arteries from spontaneously hypertensive rats

The effects of bepridil and its quaternary analogue, methylated bepridil, were studied on femoral arterial strips from spontaneously hypertensive rats (SHR). Methylated bepridil relaxed precontracted strips more rapidly than did bepridil, but bepridil was a more potent vasorelaxant. Both drugs non-competitively inhibited arterial contraction evoked by voltage-dependent or alpha-adrenoceptor-operated Ca2+ influx with bepridil being more potent than methylated bepridil. In the absence of extracellular Ca2+, the contractile responses of Wistar-Kyoto rats (WKY) and SHR arteries to norepinephrine and caffeine were reduced similarly by methylated bepridil and bepridil. It is suggested that the effects of bepridil involve both extracellular and intracellular actions. The altered sensitivity of the contraction mechanism with norepinephrine-releasable Ca2+ to the Ca2+ antagonist may be due to an abnormality in the intracellular vasocontraction process distal to Ca2+ release from storage sites of the SHR blood vessels.     

Effects of Bay K 8644 and nifedipine on femoral arteries of spontaneously hypertensive rats.

Vasoconstrictor effects of Bay K 8644 (an agonist known to increase Ca2+ influx through the voltage-dependent Ca2+ channels) on femoral arteries of 6 week old spontaneously hypertensive rats (SHR) were investigated, and data compared with findings in age-matched normotensive Wistar-Kyoto rats (WKY). The addition of Bay K 8644 (1 X 10(-10)-3 X 10(-7) M) elicited a dose-dependent contraction in SHR femoral artery in the absence of any contractile agent. Maximum contraction induced by this agonist was the same as the maximum induced by either K+-depolarization or alpha-adrenoceptor stimulation. Bay K 8644 was less effective in eliciting a contraction in the WKY femoral artery. Increased sensitivity to K+ was also observed in the SHR femoral artery. In contrast, contractions in response to alpha-adrenoceptor stimulation were the same in the SHR as those in the WKY. The addition of nifedipine, a Ca2+ channel antagonist, to an unstimulated preparation produced a dose-dependent relaxation in femoral arteries from SHR, but not from WKY. When the arteries were contracted with 60 mM K+, nifedipine produced similar relaxations in the SHR as those in the WKY, suggesting that the Ca2+ channels in the SHR femoral arteries are more activated than those in the WKY femoral arteries. Contractile responses to SHR femoral arteries to Bay K 8644 were antagonized competitively by nifedipine. Contractile responses to Ca2+ determined in K+-depolarized strips were also antagonized competitively by nifedipine. However, Schild plot analysis demonstrated a different pA2 value for nifedipine, suggesting that there may be a difference in the state of voltage-dependent Ca2+ channels in SHR femoral artery between the stimulation with Bay K 8644 and K+-depolarization.     

Influence of ouabain on the cholinergic neurotransmission in the canine trachea

The effect of ouabain on the cholinergic neurotransmission of the trachea was investigated using isolated tracheal strips in dogs. Tracheal strips without epithelium were suspended in organ chambers filled with modified Krebs-Henseleit solution. Ouabain (3 X 10(-7)-10(-5) M) concentration-dependently caused a slow sustained tracheal contraction. The contractile response was significantly inhibited by 10(-6) M atropine and was enhanced by 10(-8) M physostigmine. The ouabain-induced tracheal contraction was unaffected by 10(-7) M tetrodotoxin, but was significantly reduced by 10(-3) M hemicholinium-3. In superfusion experiments, ouabain (10(-5) M) produced an increase in the ACh release. Superfusion with Ca(++)-free solution almost eradicated the ACh release and abolished the tracheal contraction induced by ouabain. omega-Conotoxin (5 X 10(-8) M), but not nicardipine (10(-6) M), inhibited significantly the increase in ACh release induced by ouabain. These results suggest that the ouabain-induced tracheal contraction may be mainly due to acceleration of presynaptic ACh release by enhancing the influx of Ca++, and the Ca(+)+ entry may occur through the N-type Ca(+)+ channels in the canine airway presynaptic site.     

Differential effects of tyrosine kinase inhibitors on contraction and relaxation of the aortas of normotensive and hypertensive rats.

The contribution of tyrosine kinase activity to vasoreactivity in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats was investigated on isolated aortic preparations by the use of two tyrosine kinase inhibitors: methyl-2,5-dihydroxycinnamate (30 microM) and genistein (30 microM). The pretreatment of endothelium denuded aorta with methyl-2,5-dihydroxycinnamate reduced the sensitivity of the rings to noradrenaline to a larger extent in SHR than in WKY. The relaxing effects evoked by methyl-2,5-dihydroxycinnamate and genistein on the sustained contraction induced by endothelin-1 were also more pronounced in SHR denuded rings. Furthermore, in presence of methyl-2,5-dihydroxycinnamate, the endothelium-independent contractile responses to equipotent doses of cyclopiazonic acid were more depressed in SHR than in WKY. In WKY and SHR endothelium-intact aortas contracted with either phenylephrine or endothelin-1, carbachol and cyclopiazonic acid evoked endothelium derived relaxing factor (EDRF)/nitric oxide (NO)-dependent relaxations which were reduced by pretreatment of the rings with methyl-2,5-dihydroxycinnamate or genistein. These inhibitory effects were larger in WKY rings and more important on the cyclopiazonic acid response. In addition, sodium orthovanadate (30 microM) potentiated the noradrenaline-mediated contractions of endothelium-denuded SHR rings and reduced the cyclopiazonic acid-induced relaxation of endothelium-intact WKY rings. The present study suggests a regulatory role for tyrosine kinase in the smooth muscle contraction and the endothelium-dependent relaxation in WKY and SHR aortas and demonstrates the existence of a different relationship in the effect of tyrosine kinase inhibitors on vasoreactivity between SHR and WKY. We propose that an increase in the tyrosine kinase activity in SHR could lead to an enhanced reactivity of Ca2+-linked contractile mechanisms. In addition, our results suggest a link between the loss of tyrosine kinase activity and the altered endothelium-dependent relaxation associated with hypertension.     

Comparative effects of bepridil and diltiazem on systemic blood pressure and isolated peripheral arteries in spontaneously hypertensive rats

Bepridil and diltiazem were studied for their effects on blood pressure (BP) and heart rate (HR) of spontaneously hypertensive rats (SHR) and on vascular tone of femoral and mesenteric arterial strips from SHR. The drugs (i.v.) reduced BP and HR more markedly in SHR than in normotensive Wistar Kyoto rats (WKY). The effects of bepridil were less pronounced and less prolonged than those of diltiazem. Bepridil relaxed arterial strips precontracted by KCl or prostaglandin F2 alpha to almost the same extent as diltiazem in both SHR and WKY. Bepridil was almost as potent as diltiazem in inhibiting non-competitively the Ca2+-evoked contractions of arteries depolarized in a Ca2+-free, high K+ buffer. alpha-Adrenoceptor agonist-induced contractions accompanied and not accompanied by Ca2+ influx were inhibited more markedly by bepridil than diltiazem under certain conditions. The inhibitions were more marked in SHR than in WKY. Thus, it was suggested that both drugs acted as Ca2+ influx inhibitor to reduce vascular tone. Bepridil inhibited intracellular vasoconstriction mechanisms linked with alpha-adrenoceptors more potently than did diltiazem in SHR. Taken together, these actions can explain the antihypertensive properties of both drugs in SHR.     

Charybdotoxin-sensitive K+ channels regulate the myogenic tone in the resting state of arteries from spontaneously hypertensive rats.

1. To determine the possible role of Ca(2+)-activated K+ (KCa) channels in the regulation of resting tone of arteries from spontaneously hypertensive rats (SHR), the effects of agents which interact with these channels on tension and 86Rb efflux were compared in endothelium-denuded strips of carotid, femoral and mesenteric arteries from SHR and normotensive Wistar-Kyoto rats (WKY). 2. Strips of carotid, femoral and mesenteric arteries from SHR exhibited a myogenic tone; that is, the resting tone decreased when either the Krebs solution was changed to a 0-Ca2+ solution or 10(-7) M nifedipine was added. 3. The addition of charybdotoxin (ChTX, 10(-9)-10(-7) M), a blocker of large conductance KCa channels, to the resting strips of these arteries produced a concentration-dependent contraction, which was significantly greater in SHR than in WKY. Relatively low concentrations of tetraethylammonium (0.05-5 mM) produced a concentration-dependent contraction which was similar to the ChTX-induced contraction in these strips. 4. The ChTX-induced contractions in SHR were greatly attenuated by 10(-7) M nifedipine and by 3 x 10(-6) M cromakalim, a K+ channel opener. Cromakalim alone abolished the myogenic tone in SHR. 5. The addition of apamin (a blocker of small conductance KCa channels, up to 10(-6) M), or of glibenclamide (a blocker of ATP-sensitive K+ channels, up to 5 x 10(-6) M), to the resting strips failed to produce a contraction. 6. In resting strips of carotid, femoral and mesenteric arteries preloaded with 86Rb, the basal 86Rb efflux rate constants were significantly greater in SHR than in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased Na+-K+-ATPase activity and [3H]ouabain binding sites in various tissues of spontaneously hypertensive rats

Na+-K+-ATPase activity and [3H]ouabain binding were studied in cerebral cortex, kidney and heart isolated from spontaneously hypertensive rats (SHR) in both the prehypertensive (6 week old) and the hypertensive stages (14 week old). Na+-K+-ATPase activity of heart and kidney was found to be decreased by about 38 and 16% in the prehypertensive and hypertensive stages of SHR respectively; that of cerebral cortex decreased by 23.5% only in the hypertensive stages. Similar results were obtained by pretreatment of membranes with either 0.001% Triton X-100 or by increasing the K concentration from 4.7 to 12.7 mM in the Krebs solution. No significant differences in microsomal protein yield were noted between prehypertensive or hypertensive SHR and the age-matched WKY rats. The study of binding of [3H]ouabain to cerebral cortex, kidney and heart showed that the decreased Na+-K+-ATPase in hypertensive SHR was due to a 31.6, 21.8 and 41.3% reduction in the number of high affinity binding sites respectively, while the affinity constants (Kd) of ouabain binding sites on this enzyme in cerebral cortex, kidney and heart of the normotensive WKY rats were 26.5, 455.9 and 74.7 nM respectively and those from the hypertensive SHR were not altered. The plasma K concentration of the SHR in the prehypertensive and hypertensive stages was 4.07 and 4.13 mM, respectively, significantly less than that of the age-matched WKY rats. It appears that the decrease of plasma K and Na+-K+-ATPase activity in heart and kidney in SHR is derived from a genetic defect and may be related to the abnormal Na handling in this genetically hypertensive strain.     

Analysis of ouabain-induced contractions in isolated coronary arteries

Contractile responses to ouabain in helical strips of dog and monkey coronary arteries were investigated. Ouabain (5 X 10(-8) to 5 X 10(-6) M) caused a dose-related contraction in dog and monkey arteries; the response of monkey coronary arteries was significantly greater. In dog coronary arteries, contractile responses to high concentrations of ouabain were potentiated by treatment with propranolol. In the arteries contracted with ouabain, the addition of phentolamine caused a relaxation. Contractile responses of dog coronary arteries to ouabain were markedly suppressed by exposure to Ca2+-free media or by treatment with verapamil. Reduction of external concentration of K+ or lowering the temperature of bathing media did not selectively influence the ouabain-induced contraction. These results suggest that ouabain-induced contractions of dog coronary arteries are associated mainly with an increase in the Ca2+-influx, which does not result from an inhibition of the Na+, K+-activated ATPase nor from an activation of alpha adrenoceptors by noradrenaline released from adrenergic nerves. Ouabain in high concentrations seems to liberate noradrenaline from adrenergic nerves, which preferentially activates beta adrenoceptors in dog coronary artery.     

Strain-specific effects of acidic pH on contractile state of aortas from Wistar and Wistar Kyoto rats

The effects of acidosis were investigated on the resting and precontracted aortas from Wistar and Wistar Kyoto (WKY) rats. Decrease in pH from 7.4 to 6.5, having no effect on the resting tension of Wistar aorta, induced a marked contraction of WKY aorta. Acidic pH markedly relaxed the contraction to 300 nM phenylephrine in Wistar aorta, whereas in WKY aorta, it produced a biphasic response, an initial relaxation followed by potentiation of the contraction. In aortas loaded with fura 2-AM, phenylephrine caused an increase in intracellular Ca2+ ([Ca2+]i) and a contraction in both Wistar and WKY rats. pH 6.5 produced a decrease in [Ca2+]i to a near-basal level and almost abolished the phenylephrine-induced contraction in Wistar rat aorta. However, in WKY aorta, a biphasic response, an initial decline and later a recovery of [Ca2+]i level, was observed. Interestingly, at similar sustained [Ca2+]i, the contractile response to phenylephrine in WKY aorta was potentiated under acidic pH conditions. Acidic pH-induced inhibition of the contraction to phenylephrine was unaffected by iberiotoxin, 4-aminopyridine, and glibenclamide (Ca2+-activated, delayed rectifier and ATP-sensitive K+ channel inhibitors, respectively), in aortas from both Wistar and WKY. Decrease in extracellular pH was associated with a rapid fall in intracellular pH (pHi) and the intracellular acidification profile was not different in both strains. All these results show that acidic pH induces strain-specific inhibitory and excitatory effects on the contractile state of aortas from Wistar and WKY rats, respectively. The sustained and transient relaxant responses to acidic pH in Wistar and WKY aortas, respectively, are due to decrease in [Ca2+]i levels, but this decrease in [Ca2+]i is independent of the activation of K+ channels.     

Ca2+ buffering function of sarcoplasmic reticulum in rat tail arteries: comparison in normotensive and spontaneously hypertensive rats

The superficial buffer barrier function of the sarcoplasmic reticulum (SR) during rest and that during stimulation with Bay k 8644, an agonist of L-type Ca2+ channels, were compared in endothelium-denuded strips of tail arteries from 13-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), by measuring the effects of cyclopiazonic acid (CPA) and thapsigargin that inhibit SR Ca2+-ATPase and the effect of ryanodine that depletes SR Ca2+. The addition of 10 microM CPA induced a transient contraction that was not significantly different between WKY and SHR. The CPA-induced contraction was strongly inhibited by 100 nM nifedipine and was abolished by Ca2+-free solution in both strains. Thapsigargin (100 nM) or ryanodine (10 microM) induced similar, small transient contractions in the two strains. The addition of Bay k 8644 (1-100 nM) almost failed to induce a contraction in both WKY and SHR. When the strips were preincubated with 10 microM CPA, 100 nM thapsigargin or 10 microM ryanodine, Bay k 8644 induced similar concentration-dependent contractions in the two strains. The amount of Ca2+ stored in the SR, as estimated from the 20 mM caffeine-induced contraction, was not significantly different between WKY and SHR. Our results suggest that the SR of rat tail arteries can buffer a large amount of Ca2+ that enters the cell during the rest and the Bay k 8644 stimulation, and these functions are not altered in SHR.     

Effect of high extracellular Ca(2+) levels in spontaneously hypertensive rat aorta.

The release of endothelial relaxing factors has been suggested to be important in modulating the inhibition of the contractile activity caused by the increase in extracellular Ca(2+) concentration in arterial tissue. Since the hypertensive process in spontaneously hypertensive rats (SHR) could be associated with the release of endothelial vasoconstrictor factors (mainly cyclooxygenase-dependent endoperoxides and endothelin-1), we studied the contractile responses to KCl, methoxamine and phenylephrine in different aorta ring preparations (intact, de-endothelized, 10(-5) M indomethacin-treated, 10(-6) M CGS-27830 [meso-1,4-dihydro-5-methoxycarbonyl-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridine carboxylic acid anhydride]-treated, and treated simultaneously with 10(-5) M indomethacin and 10(-6) M CGS-27830) from SHR and normotensive Wistar Kyoto rats (WKY), at various Ca(2+) concentrations (1.25, 2.5, 5 and 10 mM) in the organ bath. In endothelium-intact preparations from WKY rats we observed a decrease in KCl, methoxamine and phenylephrine contractions with high Ca(2+) concentrations (5 and 10 mM), but in the endothelium-intact preparations from SHR, the increase in extracellular Ca(2+) concentration potentiated methoxamine contractions and caused no change in KCl and phenylephrine contractions. When the endothelium was disrupted in preparations from both WKY rats and SHR, we observed a decrease in KCl and methoxamine contractions with high Ca(2+) concentrations. The decrease in phenylephrine contractions caused by high Ca(2+) concentrations was clear in de-endothelized preparations from WKY rats but slight in de-endothelized preparations from SHR. In all indomethacin- and CGS-27830-treated preparations, and also in the preparations from WKY rats and SHR treated with both drugs, we observed a decrease in all the contractile responses with increased Ca(2+) concentration. Besides, there was a clear reduction in the responses of the alpha(1)-adrenoceptor agonists in the WKY and SHR preparations treated with both drugs. The results indicate that, in the hypertensive arteries, endothelium-derived contractile factors can counteract the relaxing effect of high extracellular Ca(2+) concentrations.     

Further study of possible direct vascular actions of indapamide in the conduit and renal arteriolar vessels of spontaneously hypertensive rats

The effect of indapamide on vascular reactivity and its properties as a calcium antagonist were studied in both isolated aorta and perfused renal vasculature of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. Indapamide was given orally to SHR and WKY rats for 2 weeks at a dose of 5 mg/kg per day. During this period indapamide did not lower blood-pressure in SHR and WKY rats although there was an adequate concentration of indapamide in the blood. There were no differences observed in the vascular reactivity towards noradrenaline and high-K+ in both the above mentioned vessels in either indapamide- or vehicle-pretreated SHR and WKY rats. Verapamil (10(-9)-10(-5) M) caused a concentration-dependent relaxation of high-K+-depolarized aortas and a decrease in the renal-arteriolar perfusion pressure elevated by high-K+ in both strains of rat. However, indapamide (10(-7)-10(-4) M) did not affect the K+-induced effect on either vessel type. Preloading of the vessels in vivo with indapamide for 2 weeks did not influence the results. In conclusion, further evidence has been presented to show that indapamide does not have calcium-antagonist properties in conduit (aorta) or resistance (renal) vessels under hypertensive conditions. Preloading of the vessels with indapamide was not a prerequisite for the demonstration of a pharmacological action of indapamide.     

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.     

Functional role of Cl- channels in acidic pH-induced contraction of the aorta of spontaneously hypertensive and Wistar Kyoto rats

pH regulates various cellular functions. Previously, we have described that acidic pH produces depolarization and contraction in isolated aorta from spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats [Br. J. Pharmacol. 118 (1996) 485]. The aim of the present study was to investigate the involvement of Cl- channels in acidic pH-induced contraction. Changing the pH of the bathing solution from 7.4 to 6.5 induced a contraction in both SHR and WKY aorta, which was 127.50+/-13.32% and 79.27+/-0.94% of the 64.8 mM KCl-induced contraction, respectively. The acidic pH-induced contraction was partially inhibited by the voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (1 microM) and nifedipine (0.1 microM). The Cl- channel inhibitors, diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) (0.5 mM), 9-anthracene chloride (0.5 mM), indanyloxyacetic acid (30 microM) and niflumic acid (3 microM) also inhibited the acidic pH-induced contraction and the degree of attenuation was comparable to that of VDCC blockers. DIDS, 9-anthracene chloride and niflumic acid at concentrations used to inhibit the acidic pH-induced contraction also inhibited the 10 microM phenylephrine-induced contraction partially, without affecting the 64.8 mM KCl-induced contraction, whereas both the contractions were inhibited by indanyloxyacetic acid with equal efficacy. Indanyloxyacetic acid but not DIDS, 9-anthracene chloride or niflumic acid inhibited the 24.8 mM KCl-induced contraction. Simultaneous measurement of cytosolic Ca2+ and tension showed that niflumic acid reversed the increase in intracellular Ca2+ level and inhibited the contraction caused by acidic pH. Similarly, acidic pH depolarized the cultured vascular smooth muscle cells from SHR and the depolarization was completely reversible after the administration of niflumic acid. All these results suggest that the activation of Cl- channels is an important mechanism underlying the depolarization and contraction induced by acidic pH in SHR and WKY aortas.     

Protein kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats.

This study investigates the effects of phorbol dibutyrate (PDB) on protein kinase C (PKC) activation, as assessed by the translocation of PKC activity from the cytosolic to the particulate fraction, in aortas and mesenteric arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The basal distribution of PKC activity between the cytosolic and particulate fractions of SHR and WKY aortas, and mesenteric arteries, was not significantly different. PDB induced a concentration-dependent decrease in cytosolic PKC activity in SHR and WKY aortas. PDB (0.01 microM) decreased cytosolic PKC activity to a greater magnitude in SHR aorta as compared to WKY aorta, while 1.0 microM PDB decreased cytosolic PKC activities to similar magnitudes in SHR and WKY aortas, and mesenteric arteries. These results suggest that the increased sensitivity of SHR vessels to contraction by phorbol esters may be due, at least in part, to the greater sensitivity of PKC in these vessels to phorbol ester activation.     

Potentiation by ouabain of the response to vasoconstrictor agents of isolated dog cerebral and mesenteric arteries soaked in Ca2+-free media

In helical strips of dog cerebral arteries soaked in Ca2+-free media and treated with prostaglandin (PG) F2 alpha or K+, the addition of Ca2+ produced a transient contraction, transient relaxation, and slowly developing, persistent contraction. Treatment with 2 X 10(-7) M ouabain did not alter the contractile response to PGF2 alpha in Ca2+-free media, but potentiated the response to Ca2+. Relaxations following the transient contractions were abolished by ouabain. On the other hand, ouabain potentiated the contractile response to serotonin in Ca2+-free media, and also the response to Ca2+. In mesenteric arterial strips soaked in Ca2+-free media, ouabain at 2 X 10(-7) M insufficient to produce contractions increased the contractile response to Ca2+, and the increase in the concentration to 2 X 10(-5) M potentiated the response to PGF2 alpha and serotonin. It may be concluded that ouabain increases the influx of Ca2+ across cell membrane caused by vasoconstrictors and enhances the drug-induced release of Ca2+ from intracellular storage sites.