Depolarized Arteries Research Articles

Vascular dysfunction in obese diabetic db/db mice involves the interplay between aldosterone/mineralocorticoid receptor and Rho kinase signaling

Activation of aldosterone/mineralocorticoid receptors (MR) has been implicated in vascular dysfunction of diabetes. Underlying mechanisms are elusive. Therefore, we investigated the role of Rho kinase (ROCK) in aldosterone/MR signaling and vascular dysfunction in a model of diabetes. Diabetic obese mice (db/db) and control counterparts (db/+) were treated with MR antagonist (MRA, potassium canrenoate, 30 mg/kg/day, 4 weeks) or ROCK inhibitor, fasudil (30 mg/kg/day, 3 weeks). Plasma aldosterone was increased in db/db versus db/+. This was associated with enhanced vascular MR signaling. Norepinephrine (NE)-induced contraction was increased in arteries from db/db mice. These responses were attenuated in mice treated with canrenoate or fasudil. Db/db mice displayed hypertrophic remodeling and increased arterial stiffness, improved by MR blockade. Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Vascular hypercontractility in db/db mice was associated with increased myosin light chain phosphorylation and reduced expression of PKG-1α. Vascular RhoA/ROCK signaling and expression of pro-inflammatory and pro-fibrotic markers were exaggerated in db/db mice, effects that were attenuated by MRA. Fasudil, but not MRA, improved vascular insulin sensitivity in db/db mice, evidenced by normalization of Irs1 phosphorylation. Our data identify novel pathways involving MR-RhoA/ROCK-PKG-1 that underlie vascular dysfunction and injury in diabetic mice.

Discovery and Development of Calcium Channel Blockers.

In the mid 1960s, experimental work on molecules under screening as coronary dilators allowed the discovery of the mechanism of calcium entry blockade by drugs later named calcium channel blockers. This paper summarizes scientific research on these small molecules interacting directly with L-type voltage-operated calcium channels. It also reports on experimental approaches translated into understanding of their therapeutic actions. The importance of calcium in muscle contraction was discovered by Sidney Ringer who reported this fact in 1883. Interest in the intracellular role of calcium arose 60 years later out of Kamada (Japan) and Heibrunn (USA) experiments in the early 1940s. Studies on pharmacology of calcium function were initiated in the mid 1960s and their therapeutic applications globally occurred in the the 1980s. The first part of this report deals with basic pharmacology in the cardiovascular system particularly in isolated arteries. In the section entitled from calcium antagonists to calcium channel blockers, it is recalled that drugs of a series of diphenylpiperazines screened in vivo on coronary bed precontracted by angiotensin were initially named calcium antagonists on the basis of their effect in depolarized arteries contracted by calcium. Studies on arteries contracted by catecholamines showed that the vasorelaxation resulted from blockade of calcium entry. Radiochemical and electrophysiological studies performed with dihydropyridines allowed their cellular targets to be identified with L-type voltage-operated calcium channels. The modulated receptor theory helped the understanding of their variation in affinity dependent on arterial cell membrane potential and promoted the terminology calcium channel blocker (CCB) of which the various chemical families are introduced in the paper. In the section entitled tissue selectivity of CCBs, it is shown that characteristics of the drug, properties of the tissue, and of the stimuli are important factors of their action. The high sensitivity of hypertensive animals is explained by the partial depolarization of their arteries. It is noted that they are arteriolar dilators and that they cannot be simply considered as vasodilators. The second part of this report provides key information about clinical usefulness of CCBs. A section is devoted to the controversy on their safety closed by the Allhat trial (2002). Sections are dedicated to their effect in cardiac ischemia, in cardiac arrhythmias, in atherosclerosis, in hypertension, and its complications. CCBs appear as the most commonly used for the treatment of cardiovascular diseases. As far as hypertension is concerned, globally the prevalence in adults aged 25 years and over was around 40% in 2008. Usefulness of CCBs is discussed on the basis of large clinical trials. At therapeutic dosage, they reduce the elevated blood pressure of hypertensive patients but don't change blood pressure of normotensive subjects, as was observed in animals. Those active on both L- and T-type channels are efficient in nephropathy. Alteration of cognitive function is a complication of hypertension recognized nowadays as eventually leading to dementia. This question is discussed together with the efficacy of CCBs in cognitive pathology. In the section entitled beyond the cardiovascular system, CCBs actions in migraine, neuropathic pain, and subarachnoid hemorrhage are reported. The final conclusions refer to long-term effects discovered in experimental animals that have not yet been clearly reported as being important in human pharmacotherapy.

Retina derived relaxation is mediated by Kir channels and the inhibition of Ca2+ sensitization in isolated bovine retinal arteries

Retinal relaxing factor (RRF) has recently been identified as a novel paracrine regulator of retinal circulation acting differently from well known mediators of the endothelium and the retina. Herein, we aimed to characterize the relaxing mechanism of the retina, i.e. RRF, by evaluating the role of Ca+2-dependent and -independent signaling mechanisms as well as inward rectifier K+ (Kir) channels. Retinal relaxation was determined by placing a piece of retinal tissue just on top of the precontracted bovine retinal arteries mounted in a wire myograph. The retina produced a complete relaxation response, which display a biphasic character, in depolarized arteries contracted by L-type Ca2+ channel agonist, Bay k 8644. Blockade of L-type Ca2+ channel by nifedipine, inhibition of sarcoplasmic reticulum Ca2+-ATPase by cyclopiazonic acid or removal of extracellular Ca2+ did not influence the prominent relaxation to the retina. Originally, retinal relaxation was found to be unaffected from the inhibition of myosin light chain kinase by ML7, whereas, completely abolished in the presence of myosin light chain phosphatase (MLCP) inhibitor, Calyculin A. Moreover, the inhibition of Rho kinase by its putative inhibitor, Y-27632 displayed comparable relaxant effects to RRF in retinal arteries precontracted either by prostaglandin F2α or K+, and augmented the moderate response to the retina in K+ precontracted arteries. In addition, retinal relaxation was significantly inhibited and lost its biphasic character in the presence of Kir channel blocker, Ba2+. Our results suggested that inhibition of Ca2+ sensitization through the activation of MLCP, possibly via interfering with Rho kinase, and the opening of Kir channels are likely to be involved in the inhibitory influence of RRF on the retinal arteries.

Contractile responses to rat urotensin II in resting and depolarized basilar arteries

The effects of human urotensin II (hUII) on the vascular tone of different animal species has been studied extensively. However, little has been reported on the vasoactive effects of rat urotensin (rUII) in murine models. The aim of the present study was to investigate the effects of rUII on vasoreactivity in rat basilar arteries. Basilar arteries from adult male Wistar rats (300-350 g) were isolated, cut in rings, and mounted on a small vessel myograph to measure isometric tension. rUII concentrations were studied in both resting and depolarized state. To remove endothelial nitric oxide effects from the rUII response, we treated selected arterial rings with Nω-nitro-L-arginine methyl ester (L-NAME). 10 μM rUII produced a potent vasoconstrictor response in rat basilar arteries with intact endothelium, while isometric forces remained unaffected in arterial rings treated with lower rUII concentrations. Although L-NAME did not have a significant effect on 10 μM rUII-evoked contraction, it slightly increased arterial ring contraction elicited by 1 μM rUII. In depolarized arteries, dose-dependent rUII increased depolarization-induced contractions. This effect was suppressed by L-NAME. Our results show that the rat basilar artery has a vasoconstrictor response to rUII. The most potent vasoconstrictor effect was produced by lower doses of rUII (0.1 and 1 μM) in depolarized arteries with intact endothelium. This effect could facilitate arterial vasospasm in vascular pathophysiological processes such as subarachnoid hemorrhage and hypertension, when sustained depolarization and L-type Ca(2+) channel activation are present.

Abstract 373: Mineralocorticoid Receptor and Rhoa/rho Kinase Contribute to Vascular Dysfunction in Obese Db/db Mice

Increasing evidence indicates that adipose tissue modulates vascular function. Our previous studies demonstrated that adipocytes secrete aldosterone. We hypothesized that adipocyte-releasing factors induce vascular dysfunction through mineralocorticoid receptor (MR)-dependent mechanisms. This study explored the small G protein RhoA/Rho kinase as a pathway linking aldosterone/MR and the paracrine effects of adipose tissue in the vasculature, since the activation of this pathway regulates vascular contraction. Diabetic obese mice (db/db) and their control counterparts (db/+) were treated with MR antagonist (MRA) (K canrenoate, 30mg/Kg/day, S.C., 4 weeks) or RhoA/Rho kinase inhibitor, fasudil (30 mg/Kg/day, S.C., 3 weeks). Blood pressure levels were similar between groups. Fasudil, but not MRA, reduced plasma glucose levels in db/db mice (db/+:12.3±6; db/db: 30.1±3; MRA-treated db/db: 32.5±2; fasudil treated db/db: 17.1±2; mmoL/mL). Arteries from db/db mice displayed reduced relaxation to 10 - 5 M acetycholine (Ach; db/+:79.4±3.9% vs db/db: 14.3±3.1%). Arteries from MRA-treated db/db mice exposed to fat conditioned medium (FCM) had improved responses to Ach (MRA-treated db/db: 28.3±1.9% vs MRA-treated db/db +FCM: 42.8±4.3%). Increased norepinephrine (NE)-induced contraction was observed in db/db mice in endothelium-denuded arteries (10 -5 M NE; db/+: 1.5 ± 0.1 vs db/db: 2.2 ± 0.1; mN/mm). NE responses were reduced in MRA-treated db/db mice (1.7±0.1 mN/mm) or fasudil (1.6±0.2 mN/mm). Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Fasudil treatment reduced calcium-induced contraction in both groups with greater effect in db/db mice (5 mmol/L CaCl 2 ; db/+: 73 % vs db/db: 57 %). MR blockade did not affect vascular RhoA/Rho kinase activity (RhoA membrane to cytosol translocation) in db/db mice. Our data suggest that in db/db mice: MR and RhoA/Rho kinase signaling contribute to adipose modulation of vascular/contraction. While MR plays a role in vascular relaxation, RhoA/Rho kinase signalling is important in contractile dysfunction, adipocyte secretion, and glucose metabolism - processes that may contribute to cardiovascular injury in obesity-associated diabetes.

Calcitonin Gene-Related Peptide Selectively Relaxes Contractile Responses to Endothelin-1 in Rat Mesenteric Resistance Arteries

We tested the hypothesis that endothelin-1 (ET-1) modulates sensory-motor nervous arterial relaxation by prejunctional and postjunctional mechanisms. Isolated rat mesenteric resistance arteries were investigated with immunohistochemistry, wire-myography, and pharmacological tools. ET(A)- and ET(B)-receptors could be visualized on the endothelium and smooth muscle and on periarterial fibers containing calcitonin gene-related peptide (CGRP). Arterial contractile responses to ET-1 (0.25-16 nM) were not modified by blockade of ET(B)-receptors, NO-synthase, and cyclooxygenase or desensitization of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) with capsaicin. ET-1 reversed relaxing responses to CGRP in depolarized arteries. This effect was inhibited by ET(A)-antagonists. It was not selective because ET-1 also reversed relaxing responses to Na-nitroprusside (SNP) and because phenylephrine (PHE; 0.25-16 microM) similarly reversed relaxing responses to CGRP or SNP. Conversely, contractile responses to ET-1 were, compared with PHE, hypersensitive to the relaxing effects of the TRPV1-agonist capsaicin and to exogenous CGRP, but not to acetylcholine, forskolin, pinacidil, or SNP. In conclusion, ET-1 does not stimulate sensory-motor nervous arterial relaxation, but ET(A)-mediated arterial contractions are selectively sensitive to relaxation by the sensory neurotransmitter CGRP. This does not involve NO, cAMP, or ATP-sensitive K(+) channels.

Effect of nitroglycerine in popliteal preparations from patients with peripheral occlusive arteriopathy precontracted with KCl or 5-hydroxytryptamine.

1. At the present time, there are no studies in isolated arteries from patients suffering from peripheral occlusive arteriopathy (POA). In the present study, we attempt to characterize the effect of nitroglycerine (GTN) in isolated popliteal preparations obtained after leg amputation in 60-90-year-old men and women suffering from POA. 2. After surgical operation, arterial samples were stored in a refrigerator at 4 degrees C and, after 12-36 h, they were cut into rings and mounted in organ baths containing Krebs'-Henseleit solution at 37 degrees C and gassed constantly with 95% CO2 and 5% O2. Because noradrenaline elicited very poor contractile responses in these preparations, in the present study we evaluated the concentration-dependent contractions induced by KCl (15-90 mmol/L) and 5-hydroxytryptamine (5-HT; 10-7 to 10-4 mol/L) in arteriopathic popliteal rings and, when the corresponding maximum contractile effect had been obtained, we also evaluated the concentration-dependent relaxing effect produced by GTN (10-10 to 10-5 mol/L) in all precontracted preparations. As a reference, similar experiments were performed in popliteal preparations obtained following surgery on non-arteriopathic vascular tissue where it was necessary to resect a certain percentage of healthy vessel. 3. The responses to KCl and 5-HT were greater in healthy vessel than in arteriopathic rings. The relaxing effect of GTN was greater in preparations precontracted with 5-HT than in those preparations precontracted with KCl. In addition, preparations precontracted with KCl relaxed even less when they were obtained from patients with POA. 4. The present data indicate that GTN is a vasodilator with little effect on depolarized arteries. The results also indicate that the effect of this drug is even less in depolarized arteries from patients with POA.

Regulation of vascular tone by endothelium-derived hyperpolarizing factor.

1. Endothelium-derived hyperpolarizing factor (EDHF) mediates the nitric oxide (NO)-independent component of the relaxation in rat mesenteric arteries. The relationship between hyperpolarization and vascular tone was studied by simultaneous recording of membrane potential with intracellular microelectrodes and tension in ring segments of rat mesenteric arteries. 2. By depolarizing arteries with high potassium solutions, it was determined that the threshold for contraction is approximately -46 mV. Maximum contraction was attained when the arteries were depolarized to -20 mV. Thus, 1 mV depolarization resulted in an approximate 4% increase in tone. This relationship was not altered in spontaneously hypertensive rats. 3. Noradrenaline (0.3 mumol/L) caused contraction and depolarized arteries by 13 mV. Acetylcholine caused endothelium-dependent relaxation and hyperpolarization up to 14 mV. In the presence of N omega-nitro-L-arginine, the EDHF-mediated relaxation was correlated to hyperpolarization. A hyperpolarization of 1 mV corresponded to a 4.3% decrease of the induced tone. 4. At concentrations (10 mumol/L) causing total relaxation, the maximum hyperpolarization induced by NO was only 7.6 mV. 5. A maximum relaxation of 88% was observed with pinacidil (3 mumol/L), despite a 25 mV hyperpolarization. Relaxations to NO and pinacidil were not correlated with hyperpolarization. At similar levels of hyperpolarization, NO and pinacidil elicited more relaxation than EDHF. 6. These studies show that vascular tone is very sensitive to membrane potential change in the range between -46 and -20 mV in the rat mesenteric artery. The relaxation response to EDHF, unlike that to NO and pinacidil, can be accounted for solely by its effect on the membrane potential.

Comparative Relaxant Effects of the NO Donors Sodium Nitroprusside, DEA/NO and SPER/NO in Rabbit Carotid Arteries

1. Sodium nitroprusside (SNP, 10 −9–3×10 −4 M), diethylamine/NO complex (DEA/NO, 10 −9–10 −4 M) and spermine/NO complex (SPER/NO, 10 −8–3×10 −4 M) induced concentration-dependent relaxation of isolated rabbit carotid arteries precontracted with KCl (50 mM) or with histamine (3×10 −6 M). 2. In KCl-precontracted arteries the order of potency was SNP=DEA/NO>SPER/NO, and in histamine-precontracted arteries the order of potency was SNP>DEA/NO>SPER/NO. Relaxations to the three NO donors were significantly higher in histamine-precontracted arteries than in KCl-precontracted arteries. 3. The guanylyl cyclase inhibitor methylene blue (10 −5 M) significantly inhibited relaxations to the three NO donors in histamine-precontracted arteries and, to a lesser extent, in KCl-precontracted arteries. 4. In conclusion, the NO donors SNP, DEA/NO and SPER/NO induce quantitatively different relaxation of rabbit carotid artery. Both, lower relaxant effects in depolarized arteries and inhibition of relaxation by methylene blue indicate the mediation of cGMP formation in the relaxant effects of the three NO donors.

Analysis of relaxation and repolarization mechanisms of nicorandil in rat mesenteric artery.

1. The mechanisms by which nicorandil causes relaxation of rat isolated small mesenteric arteries mounted on a Mulvany myograph was investigated by use of a combination of putatively mechanism-specific antagonists. 2. In arteries precontracted by the thromboxane-mimetic, U46619, the EC50 for cromakalim and levcromakalim-induced relaxation curves were raised by 36 and 17 fold by glibenclamide (3 microM) while the EC50 for nicorandil-induced relaxation was unaffected by either glibenclamide or methylene blue (10 microM). A combination of these antagonists raised the EC50 for nicorandil by 8 fold. 3. In U46619-contracted arteries, nifedipine (100 nM) did not affect the cromakalim relaxation curve but it raised the EC50 for nicorandil by 5 fold. The combination of methylene blue, glibenclamide and nifedipine further inhibited the maximum relaxation to nicorandil. 4. In separate experiments, membrane potential (Em) and force responses were measured simultaneously. In arteries depolarized and contracted by U46619 both nicorandil and cromakalim repolarized (delta Em, 35 mV) and relaxed (100%) the vessels. Glibenclamide (3 microM) did not alter the relaxation-concentration curve to nicorandil but reduced the maximum repolarization to delta 10.8 mV. In contrast to Em and relaxation-response curves to cromakalim were shifted to the right by glibenclamide by 30-100 fold. 5. In unstimulated arteries, nicorandil (but not cromakalim) -induced hyperpolarization was significantly antagonized by methylene blue (10 microM) (6.6 fold rightward shift) or nifedipine (100 nM) (2.6 fold). In depolarized arteries (U46619), nifedipine but not methylene blue inhibited the nicorandil-induced hyperpolarization. 6. In arteries precontracted to 50% tissue maximum by either KCl or U46619, nifedipine (100 nM) relaxed the artery but failed to repolarize the Em. Presumably voltage-operated calcium channels (VOCC) were blocked preventing contraction but the artery remained depolarized, presumably through non VOCC mechanisms. 7. These data suggest that nicorandil may relax small arteries through 3 parallel pathways, (i) NO-donor mediated stimulation of guanylate cyclase and increase in cyclic GMP, (ii) K+ATP channel opening, and (iii) nifedipine-sensitive VOCC inhibition. Em data suggest that nicorandil-induced repolarization is caused principally through opening K+ATP channels. Blockade of this hyperpolarization by glibenclamide is not sufficient to alter the relaxation, indicating dissociation of nicorandil-induced changes in membrane potential and relaxation. 8. These results highlight the 'chameleon' actions of nicorandil where there is no apparent association of Em repolarization with relaxation, in contrast to the parallel responses for cromakalim.

Age, endothelium and aortic reactivity to calcium in wistar rat

The influence of age from (9 to 72 weeks) was investigated on contractions elicited by calcium in Wistar rat aortic rings with and without endothelium. The arteries were either depolarized by KCl or exposed to Bay K 8644 and concentration-effect curves to cumulative additions of CaCl 2 were constructed. Whether the endothelium was present or not, the potency of CaCl 2 but not its maximal effect decreased with age. The same decrease in potency was obtained with Bay K 8644, in the presence of which the maximal contractions were increased at 72 weeks. In K + depolarized arteries, nitrendipine (10 −10 and 10 −9 M) markedly decreased maximal contraction elicited by CaCl 2 and it moderately displaced to the right the concentration-effect curves. The endothelium did not modify the responses to CaCl 2 of depolarized arteries, in the absence of nitrendipine. However it markedly enhanced the depressor effect of nitrendipine in these preparations, and it impaired contractions elicited by calcium in the presence of Bay K 8644. These effects of the endothelium were both less at 72 weeks than at younger ages. These experiments show a possible impairment of the inhibitory effect of the endothelium in 72 week old rats in the presence of both agonist and antagonist dihydropyridines.

CAS 936, a Novel Sydnonimine with Direct Vasodilating and Nitric Oxide-Donating Properties

Organic nitrates and sydnonimines exert their vasorelaxant activity by a common mechanism of action, i.e., release of nitric oxide (NO) and stimulation of the soluble guanylate cyclase of vascular smooth muscle cells. We wished to investigate the vasodilating activity of the novel sydnonimine CAS 936 in guinea pig isolated pulmonary arteries without endothelium. CAS 936 had no effect on contractions induced by norepinephrine (NE) or by the PGF2 alpha-analogue U46 619, but induced a longlasting relaxation of potassium depolarized arteries and of A23 187-contracted vessels. This effect was concentration-dependent (IC50 approximately 16 microM). Oxyhemoglobin and methylene blue had no inhibitory effect on CAS 936, whereas they inhibited the relaxations induced by SIN-1, a sydnonimine which acts by releasing NO. These results suggest that the vasodilating activity of CAS 936 is not related to NO. On the other hand, in vivo metabolites of CAS 936 inhibited NE- and U46 619-induced contractions. Oxyhemoglobin inhibited this effect. Therefore, we conclude that the CAS 936 molecule possesses a vasodilating activity of its own, whereas the metabolites may function as NO donors. The primary target of the intrinsic vasodilating activity of CAS 936 is very likely the vascular smooth muscle cell membrane. To determine which mechanism of action (NO unrelated or NO related) contributes mainly to the in vivo effects of CAS 936, studies of the metabolic fate of CAS 936 may be crucial.

Effects of a chronic treatment by nisoldipine, a calcium antagonistic dihydropyridine, on arteries of spontaneously hypertensive rats.

Earlier studies have shown that relaxation in response to several agents is impaired in arteries from spontaneously hypertensive rats (SHR). We had previously reported that SHR aortas present a delayed relaxation when first exposed for 35 minutes to a 100 mM KCl solution and then transferred into physiological solution. The first phase of relaxation appeared similar in SHR and Wistar-Kyoto rat arteries, but the second phase was markedly slowed down in SHR arteries, giving rise to a postcontraction tone. In this study, we found that this postcontraction tone could be demonstrated not only in the aorta but also in the mesenteric artery, was independent of the presence of endothelium, increased with the age of SHR, and disappeared progressively when arterial segments were submitted to successive cycles of KCl depolarization followed by reimmersion in physiological solution. Chronic treatment of SHR with nisoldipine at doses that blocked the development of hypertension and attenuated the concomitant hypertrophy of heart and aorta, or in vitro pretreatment of SHR arteries with nisoldipine, decreased the contractile force developed by arteries in response to KCl depolarization and normalized the subsequent relaxation. [3H] (+)-PN200-110 binding studies on heart and brain homogenates indicated an increase in apparent Kd in nisoldipine-fed rats without significant change in Bmax. Binding data were compatible with the view that occupation of dihydropyridine receptors by nisoldipine after chronic oral administration was responsible for the modifications observed ex vivo in the mechanical activity of arteries. We conclude that the postcontraction tone of SHR arteries was mainly due to an abnormally prolonged activation of calcium channels after transfer of depolarized arteries into the physiological solution and that a labile or slowly releasable factor was probably involved in this phenomenon. We suggest that the antihypertensive action of nisoldipine might be related to the mechanisms involved in the suppression of the postcontraction tone as observed in vitro and that this mode of action could be more important than the vasodilating effect of this drug.

Effects of ryanodine on tension development in rat aorta and mesenteric resistance vessels.

1. The effect of ryanodine on contractile responses dependent either on intracellular Ca2+ release or on extracellular Ca2+ influx were studied in aorta and mesenteric resistance vessels of the rat. 2. In aorta, in the presence of extracellular Ca2+, pretreatment with ryanodine (10(-5)M) did not modify contractile responses to noradrenaline (NA) (10(-6)M) whereas in the absence of Ca2+, pretreatment with ryanodine reduced to about 25% the contractile response to NA (10(-6)M) and totally abolished the transient contraction elicited by caffeine (5 x 10(-2)M). 3. In mesenteric resistance vessels, ryanodine (10(-5)M) had no effects on NA (10(-5)M)-induced tension in the presence of extracellular Ca2+ but totally abolished contractile responses to caffeine (10(-2)M) in the absence of Ca2+. 4. In K+ -depolarized mesenteric resistance vessels, pretreatment with ryanodine (10(-5)M) significantly enhanced contractile responses to Ca2+ concentrations higher than 10(-4)M and 10(-3)M for arteries depolarized with 30 mM and 40 mM K+ respectively. Concentrations of either diltiazem (6 x 10(-7)M) or nifedipine (10(-8)M) that abolished contractile responses to Ca2+ in depolarized arteries (K+, 40 mM) did not totally inhibit the enhancement of Ca2+ -induced contractions obtained in the presence of ryanodine. 5. Ryanodine did not modify the Ca2+ concentration-effect relationships in mesenteric resistance vessels exposed to NA or arginine vasopressin. 6. These data are consistent with the hypothesis that ryanodine induces a release of Ca2+ from intracellular stores, resulting in a subsequent reduction of the amplitude of contractions dependent upon intracellular Ca2+ liberation. Furthermore, the ability of sarcoplasmic reticulum to buffer rises in cytoplasmic Ca2+ may be reduced in the presence of ryanodine, thereby accounting for the potentiation of contractile responses to Ca2+ in K+-depolarized mesenteric resistance vessels.

Selective modulation by membrane potential of the interaction of some calcium entry blockers with calcium channels in rat mesenteric artery.

1. The effects of flunarizine, (+)-PN 200-110 and nifedipine on [3H]-(+)-PN 200-110 specific binding were investigated in intact rat mesenteric arteries bathed in physiological solution or in KCl-depolarizing solution, and in a membrane fraction from rat mesenteric arteries. 2. Unlabelled dihydropyridines, (+)-PN 200-110 and nifedipine, inhibited [3H]-(+)-PN 200-110 specific binding concentration-dependently in polarized as well as in depolarized intact arteries. The Ki value of (+)-PN 200-110 was decreased in arteries bathed in KCl-depolarizing solution compared to arteries bathed in physiological solution, while the Ki value of nifedipine was not significantly changed. Ki values measured in depolarized arteries were close to the IC50 values (concentrations inhibiting by 50% the KCl-contraction of rat mesenteric artery). 3. Flunarizine (10(-6) M) was unable to displace the specific binding of [3H]-(+)-PN 200-110 in intact arteries bathed in physiological solution. At 10(-7) M-10(-6) M, it inhibited the binding in depolarized arteries, suggesting that prolonged depolarization is required for the interaction of flunarizine with the dihydropyridine receptor. 4. In a membrane fraction isolated from rat mesenteric arteries, (+)-PN 200-110, nifedipine and flunarizine were all able to displace completely the specific binding of [3H]-(+)-PN 200-110. Displacement curves were parallel and Hill coefficients were close to unity. Ki values were close to the values obtained in depolarized intact arteries. 5. These results revealed a good correlation between the data obtained from binding tests and from pharmacological studies for dihydropyridine calcium entry blocking drugs, taking into account the time-dependence associated with their action on KCl-contraction compared to their binding properties. There was an important discrepancy between the concentrations of flunarizine active in binding studies and those active in pharmacological studies, which could be accounted for by the existence of multiple binding sites for calcium entry blockers in calcium channels.

Cerebral antivasoconstrictive effects of flunarizine.

Flunarizine is a selective Ca++-antagonist with weaker H1-histaminergic antagonistic properties. Flunarizine reduces vasospasm caused by an exaggerated Ca++-influx in depolarized arteries and by vasoactive substances released from aggregating platelets. This Ca++-antagonist also antagonizes the mutual amplification of the vasoconstrictor action of the platelet products. Flunarizine is particularly effective in the cerebral blood vessels, also when they are hyperreactive because of hypoxia. The compound does not interfere with normal arteriolar autoregulation or heart function. Thus flunarizine may be particularly effective in preventing vasospasm without lowering blood pressure or inducing a steal phenomenon.

Selectivity of Phenytoin and Dihydropyridine Calcium Channel Blockers for Relaxation of the Basilar Artery

We addressed the questions of whether or not phenytoin is a direct vasodilator and if it is selective for brain blood vessels, by studying the relaxant effects of phenytoin on isolated segments of canine basilar, femoral, and brachial arteries. Two dihydropyridine calcium channel blockers, nifedipine and PY 108-068, were also studied for comparison with phenytoin and to test for cerebral selectivity. Blood vessels were contracted with K+, prostaglandin F2 alpha, or serotonin. Phenytoin relaxed the basilar artery with low potency (pD2, 4.71 +/- 0.14) and moderate selectivity. Phenytoin also antagonized Bay K 8644 contractions of basilar artery in a noncompetitive manner. Basilar arteries contracted with 60 mM K+ were the most sensitive to nifedipine (pD2, 8.72 +/- 0.18), followed by the mesenteric (pD2, 8.24 +/- 0.07), femoral (pD2, 8.04 +/- 0.18), and brachial (pD2, 7.66 +/- 0.23) arteries. A similar pattern was observed in potassium-depolarized arteries relaxed by PY 108-068. The calcium dependence of contraction was studied using intact muscles depolarized in 60 mM K+ as well as chemically skinned basilar artery. Mean pD2 values for Ca2+-induced contractions of intact, depolarized arteries were not different (basilar, 4.15 +/- 0.13; mesenteric, 4.04 +/- 0.07; femoral, 4.24 +/- 0.11). The mean Ca2+ EC50 of chemically skinned basilar arteries was 8.7 X 10(-7) M, which is similar to the Ca2+ sensitivity of other skinned smooth muscles. The beneficial effect of phenytoin in treating cerebral ischemia may be due in part to relaxation of vascular smooth muscle. The dihydropyridines were potent smooth muscle relaxants with selectivity for the basilar artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of calcium entry blockers on Ca 2+-induced contraction of depolarized and noradrenaline-exposed rat resistance vessels

The effects of four calcium entry blockers (CEBs), diltiazem (D), verapamil (V), nifedipine (NF) and nicardipine (NC), were investigated on Ca 2+ concentration-effect curves of rat depolarized (K +, 40 mM) or noradrenaline (NA, 3 μM)-exposed mesenteric resistance vessels. Under control conditions, NA-exposed vessels were more sensitive to Ca 2+ (pD 2: 4.12±0.11) than depolarized vessels (pD 2: 3.16±0.02, P < 0.01) whereas the maximal active wall tensions were not significantly different (2.86 ± 0.11 mN/mm and 2.11±0.34 mN/mm respectively). In depolarized vessels, D,V, NF and NC induced a concentration-dependent shift to the right and a depression of the maximal effect of the Ca 2+ curves, which suggested a non-competitive antagonism. The IC 50 (concentration of CEB producing a 50% inhibition of the maximal contractile response from control curve) values were: D: 3·10 −7, V: 1.3·10 −7, NF: 4.5·10 −9, NC: 3·10 −9 M. In NA-exposed vessels, the CEBs produced a concentration-dependent shift to the right of the Ca 2+ curves before depressing their maximal effect. This suggested that the antagonism was different from that observed in depolarized arteries. In this case, the IC 50 values were: D: 4.5·10 −7, V: 2·10 −7, NF: 9·10 −9, NC: 7·10 −9 M. Although the gating mechanisms activated in this study were differently affected by CEBs, since there were marked qualitative differences in their antagonistic effects on Ca 2+ concentration-effect curves, depolarization and NA promoted the entry of Ca 2+ into smooth muscle cells of rat resistance vessels by mechanisms with the same sensitivity to CEBs as expressed by IC 50 values.