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The pharmacology of cannabidiol, the non-psychoactive major component of Cannabis sativa, is of growing interest as it becomes more widely prescribed. This study aimed to examine the effects of cannabidiol on a wide range of contractile agents in rat small resistance arteries, in comparison with large arteries, and to explore its mechanism of action. The vascular actions of cannabidiol were also contrasted with effects on the contractions of bronchial, urogenital, cardiac and skeletal muscles. Isolated small or large arteries were incubated with cannabidiol (0.3-3 μM) or vehicle and concentration-contraction response curves were completed to various agents, including endothelin-1, arginine vasopressin, methoxamine, 5-HT, α-methyl 5-HT and U46619. In small arteries, the effects of cannabidiol were tested in the presence of antagonists of CB1 or CB2 receptors, calcitonin gene-related peptide (CGRP), nitric oxide synthase, cyclooxygenase, PPARγ or a combination. The role of L-type voltage-operated calcium channels was also assessed. Cannabidiol 1-3 μM significantly inhibited the contraction of small resistance arteries to all tested agents through a combination of mechanisms that include CGRP and L-type calcium channels. However, large arteries were insensitive to cannabidiol. Cannabidiol (10-100 μM) was largely without effect in bronchi, atria and hemidiaphragm, but 100 μM attenuated maximum contractions in vasa deferentia. Cannabidiol's effects in the clinical range (1-3 μM) appear to be specific to small resistance arteries. This high sensitivity of the resistance arterial circulation to cannabidiol may offer a therapeutic opportunity in peripheral vascular disease that excludes off-target sites such as the heart and non-vascular smooth muscle.

Extracellular signal regulated kinase (ERK) is known to regulate vascular smooth muscle contraction. However, a role for ERK in airway smooth muscle contraction has yet to be demonstrated conclusively, although contractile agents increase ERK activity in airway smooth muscle. Rather than initiating contraction, ERK could regulate airway tone by interfering with relaxation. Therefore, the aim of this study was to determine whether ERK regulates contraction or relaxation of airway smooth muscle. Segments of porcine peripheral bronchioles were mounted in an isolated tissue bath in Krebs–Henseleit buffer and maintained at 37 °C. Cumulative concentration–response curves to histamine, endothelin-1, or the muscarinic agonist carbachol were then carried out in the absence or presence of the MEK inhibitor PD98059. In separate experiments, cumulative concentration response curves to the β-adrenoceptor agonist isoprenaline or the adenylyl cyclase activator forskolin were carried out in the absence or presence of the MEK inhibitors PD98059 or U0126. ERK activity was measured by Western blotting. All three contractile agents increased ERK activity, but the contractile responses were unaffected by PD98059. On the other hand, both PD98059 and U0126 enhanced the relaxations to isoprenaline but not relaxations to the adenylyl cyclase activator forskolin. The enhancement of isoprenaline-induced relaxations with PD98059 was prevented by the K+ channel blocker tetraethylammonium. These data suggest that ERK regulates airway smooth muscle tone by inhibiting β-adrenoceptor-mediated relaxations, rather than an initiation of contraction. The effect on β-adrenoceptor-mediated responses appears to be through a cAMP-independent mechanism, possibly through an interaction with K+ channels.

The chemical compound [4-(4-bromo-2-cyano-phenylcarbamoyl)-benzyl]-phosphonic acid diethyl ester (NO-1886) is a lipoprotein lipase activator having beneficial effects on both diabetes control and the cardiovascular system. Preventing accumulation of lipids in the cell wall, in addition to improving insulin actions on vasculature, may indirectly contribute to the reducing effect of NO-1886 on vascular resistance. However, the direct effect of NO-1886 on vascular resistance, i.e., whether NO-1886 directly modulates the function of vascular endothelium and/or smooth muscle cells has not been investigated. In this study we therefore investigated the direct effect of NO-1886 on vascular contractility using rat aortic rings and cultured smooth muscle cell-line A10. The results show that administration of NO-1886 attenuated aortic contraction induced by phenylephrine and/or a high K(+) environment, in both the presence and absence of aortic endothelium. 1-(5-Chloronaphthalene-1-sulfonyl)homopiperazine hydrochloride (ML-9), a myosin light chain kinase (MLCK) inhibitor, blocked this inhibitory effect of NO-1886, whereas inhibitors of other signaling molecules such as calmodulin, protein kinase C and Rho-kinase had no effect. The vasorelaxant effect of NO-1886 was blocked in the absence of extracellular Ca(2+), or in the presence of the Ca(2+) channel inhibitor, verapamil. NO-1886 attenuated smooth muscle contraction induced by the cumulative addition of CaCl(2). In A10 cells, NO-1886 inhibited the membrane depolarization-induced initial peak of [Ca(2+)](i) in the presence of extracellular Ca(2+). This inhibition did not occur in the absence of extracellular Ca(2+). Taken together these results demonstrate that NO-1886 attenuates smooth muscle contraction and causes vasorelaxation by an extracellular Ca(2+)- and MLCK-dependent mechanism.

In vitro studies have provided evidence that Cl(-) ion currents are important for activation of vascular smooth muscle contraction. The stilbene, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), disrupts Cl(-) metabolism by blocking Cl(-) channels and by inhibiting Cl(-) bicarbonate exchange. The aims of this study were to: (i) characterize the hemodynamic responses produced by DIDS in pentobarbital anesthetized rats, and (ii) examine vasoconstrictor responses to norepinephrine before and after administration of DIDS. DIDS (2.5-50 micromol/kg, 92.5 micromol/kg total dose, i.v.) produced dose-dependent but transient reductions in mean arterial blood pressure and in hindquarter, renal and mesenteric vascular resistances. Prior to the administration of DIDS, norepinephrine (1. 0-5.0 microgram/kg, i.v.) produced dose-dependent increases in mean arterial pressure, renal resistance and mesenteric resistance, but decreases in hindquarter resistance that were inversely related to dose. After administration of DIDS, the peak pressor responses produced by norepinephrine were either slightly diminished (1.0, 2.5 microgram/kg) or unchanged (5.0 microgram/kg). Peak norepinephrine-induced changes in hindquarter and renal vascular resistance were unaffected by DIDS, while increases in mesenteric resistance were augmented. The total norepinephrine-induced increases in mean arterial pressure (mm Hgxs) were markedly reduced by DIDS. These effects of DIDS on norepinephrine-induced responses were similar, but not identical to those of the voltage-sensitive Ca(2+) channel blocker, nifedipine (500 nmol/kg, i.v.). These findings suggest that DIDS may interfere with norepinephrine-induced depolarization of resistance arteries, thereby preventing activation of voltage-sensitive Ca(2+) channels.

Adrenomedullin is a novel peptide known to be one of the most potent vascular smooth muscle relaxing agents in vivo. The aim of this study is to investigate the effect of adrenomedullin in relation to nitric oxide, prostaglandins and endothelium-derived hyperpolarized factor (EDHF). A 0.1 -ml bolus of I nmol human adrenomedullin is a potent inhibitor of the pressor response to exogenous norepinephrine infusion in an ex vivo canine tibia perfusion model for a duration of at least 70 min (P < 0.005). This attenuation of vascular smooth muscle contraction occurs even when nitric oxide production is blocked by N G -monomethyl-L-arginine acetate (L-NMMA) infusion and EDHF is blocked by tetraethylammonium infusion, although the effect is of shorter duration (at least 10 min). Indomethacin as well does not affect the suppression of norepinephrine-induced vascular smooth muscle contraction. Based on these data, human adrenomedullin has both nitric oxide- and EDHF-dependent mechanism as well as a nitric oxide- and EDHF-independent mechanism.

Prostaglandin E2 (PGE2) is one major prostanoid produced under inflammatory situation. Although PGE2 is known to induce vascular contraction, its detailed mechanism remains unknown. In the present study, we investigated the signaling pathway underlying PGE2-induced smooth muscle contraction in rat mesenteric artery. PGE2 (0.3-30 μM) concentration-dependently caused contraction in endothelium-denuded artery. RT-PCR showed that this artery expresses mRNAs for all four prostanoid EP receptors (prostanoid EP1-4). Among selective agonists for PGE2 receptors, only a prostanoid EP3 receptor agonist, ONO-AE-248 (0.3-30 μM) induced contraction. Consistently, pretreatment with a prostanoid EP3 antagonist (L-798106, 1 μM) significantly but not completely inhibited the PGE2-induced contraction. Interestingly, pretreatment with a prostanoid FP antagonist (AL8810, 1 μM) or a TP antagonist (SQ29548, 10 nM) also partially inhibited the PGE2-induced contraction. Since ONO-AE-248 (10 μM) did not influence intracellular Ca2+ concentration in mesenteric artery, we next examined the involvement of Ca2+-independent contractile pathway including PKCs and ROCK in prostanoid EP3-mediated contraction. Pretreatment with bisindolyl-maleimide I (a general PKC inhibitor, 1 μM), Ro-31-8425 (a conventional PKC and PKCe inhibitor, 1 μM), rottlerin (a selective PKCδ inhibitor, 1 μM) and Y-27632 (a ROCK inhibitor, 1 μM) but not Go 6976 (a conventional PKC inhibitor, 1 μM) attenuated 10 μM ONO-AE-248-induced vascular contraction. In western blot analysis, we confirmed that the treatment with ONO-AE-248 (10 μM, 30 min) phosphorylated PKCδ (Thr505) and PKCe (Ser729). These results suggest that PGE2 induces vascular smooth muscle contraction via prostanoid EP3, FP and TP receptors in rat mesenteric artery. Prostanoid EP3-mediated contraction is ascribed to Ca2+-independent contractile pathway including PKCδ, e and ROCK.

Balloon catheter injury results in hyper-reactivity to phenylephrine in contralateral carotids. Decreased nitric oxide (NO) modulation and/or increased intracellular calcium concentration triggers vascular smooth muscle contraction. Therefore, this study explores the participation of NO signaling pathway and calcium mobilization on hyper-reactivity to phenylephrine in contralateral carotids. Concentration-response curves for calcium (CaCl(2)) and phenylephrine were obtained in control and contralateral carotids four days after balloon injury, in the presence and absence of the inhibitors (L-NAME, L-NNA, 1400W, 7-NI, Oxyhemoglobin, ODQ or Tiron). Confocal microscopy using Fluo-3AM or DHE was performed to detect the intracellular levels of calcium and reactive oxygen species, respectively. The modulation of NO on phenylephrine-induced contraction was absent in the contralateral carotid. Phenylephrine-induced intracellular calcium mobilization was not altered in contralateral carotids. However, extracellular calcium mobilization by phenylephrine was reduced in the contralateral carotid compared to control arteries, and this result was confirmed by confocal microscopy. L-NAME increased phenylephrine-induced extracellular calcium mobilization in the contralateral carotid to the control levels. Results obtained with L-NNA, 1400W, 7-NI, OxyHb, ODQ or Tiron showed that this response was mediated by products from endothelial NOS (eNOS) different from NO and without soluble guanylate cyclase activation, but it involved superoxide anions. Furthermore, Tiron or L-NNA reduced the levels of reactive oxygen species in contralateral carotids. Data suggest that balloon catheter injury promoted eNOS uncoupling in contralateral carotids, which generates superoxide rather than NO, and reduces phenylephrine-induced extracellular calcium mobilization, despite the hyper-reactivity to phenylephrine in contralateral carotids.

Protein kinase C (PKC) is involved in the regulation of vascular smooth muscle contraction. However, the role of PKC in erectile function is poorly understood. This study investigated whether PKC mediates agonist-induced contractions in mouse penile tissue (corpora cavernosa). We also compared the effects of PKC activators and inhibitors on contractile responses in mouse corpus cavernosum with those in mouse aorta. Aortic rings and corpus cavernosal strips from C57BL/6J mice was isolated, mounted in the organ bath for isometric tension recording. Our data showed that a PKCα/β selective inhibitor, Gö6976 (10 µM), inhibited phenylephrine and 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619, a thromboxane mimetic)-induced contractions in mouse aorta, reducing the maximum contraction from 123 ± 2% of KCl-induced maximum contraction to 7 ± 2% and 13 ± 1%, respectively. A non-selective PKC inhibitor, chelerythrine (30 µM), also significantly reduced phenylephrine-and U46619-induced maximum contractions in mouse aorta. However, Gö6976 and chelerythrine had no significant effects on phenylephrine-and U46619-induced contractions in corpus cavernosum. Furthermore, a PKC activator, phorbol-12,13-dibutyrate (0.1 µM), significantly increased contractions in aorta (208 ± 14% of KCl-induced maximum contraction) but failed to cause contractions in corpus cavernosum at 1 and 10 µM. Western blot analysis data suggested that protein expression of PKC was similar in aorta and corpus cavernosum. Taken together, our data indicate that PKC does not have a significant role in agonist-induced contractions in mouse corpus cavernosum, whereas it mediates the contractile response to agonists in the aorta.

K(+)-depolarization (KCl)-activated Ca(2+) entry permitting sustained force-maintenance in tonic vascular smooth muscle has long been attributed solely to activation of L-type voltage-operated Ca(2+) channels (VOCs). We used the transient receptor potential channel (TRP) blocker, 2-aminoethoxydiphenyl borate (2-APB), to test the hypothesis that KCl activates additional Ca(2+) entry pathways. 2-APB alone caused a transient weak increase in force, a sustained weak increase in basal [Ca(2+)](i) and myosin light chain phosphorylation, and inhibition of KCl-induced force, [Ca(2+)](i) and myosin light chain phosphorylation. 2-APB did not appear to block VOCs, because 2-APB did not inhibit 30 nM Bay k 8644-induced increases in [Ca(2+)](i). Moreover, although 1 microM nifedipine abolished the increase in [Ca(2+)](i) produced by alpha-adrenergic receptor activation, 2-APB produced an additional reduction in [Ca(2+)](i) below the basal level. These data support the conclusion that membrane depolarization activates 2-APB-sensitive TRPs in addition to VOCs to permit strong force-maintenance in tonic vascular smooth muscle.

In order to elucidate the signal transduction pathway of vascular smooth muscle contraction induced by the activation of receptors for angiotensin II and endothelin-1, we examined whether tyrosine kinases and mitogen-activated protein (MAP) kinases are involved in the development of force of contraction in the rat aorta. Isolated aortic smooth muscles without endothelium were incubated in a modified Krebs-Henseleit solution and stimulated with angiotensin II (100 nM) or endothelin-1 (10 nM). A tyrosine kinase inhibitor genistein (10 microM) reduced the angiotensin II- and endothelin-1-induced aortic contraction, while 10 microM of daidzein (an inactive analogue of genistein) did not. The K(+) depolarization-induced contraction was not attenuated by 10 microM of genistein. Selective inhibitors of MAP kinase/extracellular signal-regulated kinase (Erk) kinase (MEK) such as PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] inhibited the angiotensin II- and endothelin-1-induced vasocontraction. The p44/42 MAP kinases were phosphorylated in cultured aortic smooth muscle cells and in physiologically contracted aortic vessels stimulated with angiotensin II and endothelin-1 for 5 min. The angiotensin II- and endothelin-1-induced phosphorylations of p44/42 MAP kinases were inhibited by PD98059 as well as U0126 in the intact aorta. These results suggest that the activation of genistein-sensitive tyrosine kinases and p44/42 MAP kinases is involved in the angiotensin II- and endothelin-1-induced rat aortic contraction.

We studied the effects of the tyrosine kinase inhibitors genistein and tyrphostin and the tyrosine phosphatase inhibitors sodium orthovanadate and phenylarsine oxide on endotoxin-mediated induction of nitric oxide (NO) synthase in rat aorta and its effects on vascular contractility. Genistein (i.p. 10 mg kg −1 ) inhibited the ex vivo vascular hyporesponsiveness to noradrenaline and the aminoguanidine-sensitive nitrite accumulation induced by endotoxin (i.p. 5 mg kg −1 ) in aortic rings. Low concentrations of genistein (10 −6 M) and tyrphostin (3×10 −6 M) inhibited both endotoxin-induced hyporesponsiveness and nitrite and NO x accumulation in vitro in rat aorta without affecting control nitrite or NO x accumulation or contraction. Higher concentrations of genistein (10 −5 and 5.5×10 −5 M), sodium orthovanadate (10 −4 M) and phenylarsine oxide (10 −6 M) produced an irreversible depression of noradrenaline-induced contractions. In the presence of these drugs, endotoxin did not induce further depression of vascular contractility and did not increase nitrite or NO x production. In conclusion, there is a dissociation between the effects of these drugs on vascular smooth muscle contraction and NO synthase induction, the latter being more sensitive to inhibition by these drugs. Surprisingly, tyrosine phosphatase inhibitors produced similar effects to those of tyrosine kinase inhibitors, suggesting that there is a complex relationship between tyrosine kinases and phosphatases in the signalling pathway of agonist-induced vascular smooth muscle contraction and NO synthase induction.

Stimulation of vascular smooth muscle by bacterial lipopolysaccharide has been shown to produce interleukin-1beta and to induce vasodilation in septic shock. To understand the mechanisms of interleukin-1beta-induced relaxation, we examined the effects of interleukin-1beta on contractility and cyclic GMP contents of vascular smooth muscle. After treatment of the rat aorta with interleukin-1beta (20 ng/ml) for 6 h, the cyclic GMP content increased and the contraction induced by phenylephrine (1 microM) was partially inhibited. An inhibitor of nitric oxide (NO) synthase, N(G)-monomethyl-L-arginine (L-NMMA, 100 microM), prevented the inhibitory effect of interleukin-1beta. After treatment with interleukin-1beta for 24 h, the phenylephrine-induced contraction was inhibited more strongly. Neither L-NMMA (100 microM) nor aminoguanidine (100 microM) reversed the inhibition, whereas methylene blue (10 microM) partially reversed the inhibition. After treatment with interleukin-1beta for 12 or 24 h, the cyclic GMP content increased but to a level lower than that obtained with a 6-h treatment. The effects of sodium nitroprusside (1 microM) to inhibit the phenylephrine-induced contraction and to increase the cyclic GMP content were markedly suppressed by the 24-h interleukin-1beta treatment. In contrast, the 24-h interleukin-1beta treatment did not change the ability of 8-bromo-cGMP to relax the phenylephrine-stimulated aorta. Addition of L-NMMA (1 mM) during the 24 h treatment prevented NO production and preserved the sodium nitroprusside-induced cGMP generation by interleukin-1beta. The 24 h interleukin-1beta treatment increased the threshold concentration of KCl needed to induce contraction without changing the maximum contraction. In the presence of 25.4 mM KCl or the non-selective K+ channel inhibitor, tetraethylammonium, the inhibitory effect of the 24-h interleukin-1beta treatment on phenylephrine-induced contraction was restored. These results suggest that interleukin-1beta inhibits vascular smooth muscle contraction by a time-dependent, dual mechanism. After a 6-h treatment with interleukin-1beta, the NO/cyclic GMP system is activated. After a 24-h interleukin-1beta treatment, in contrast, the NO/cyclic GMP system may be desensitized and the contraction of vascular smooth muscle is inhibited by another mechanism, possibly membrane hyperpolarization.

The effects of three tyrosine kinase inhibitors: genistein, quercetin and psi-tectorigenin, were investigated on contractions evoked in de-endothelised rat aortic rings, either by phenylephrine or 70 mM K+. A dose-dependent inhibition of both contractions by all three compounds was observed, the phenylephrine-mediated contractions being more sensitive to genistein. No differences between genistein or quercetin effects in pre-treatment or post-treatment protocols were found. Ca2+ store refilling, expressed in terms of phenylephrine-induced tension in Ca(2+)-free medium, was dose-dependently blocked by quercetin and genistein. Sodium orthovanadate, an inhibitor of tyrosine phosphatase, contracted the rat aortic rings with an IC50 of 0.66 microM. Its presence during the refilling period after exposure to Ca(2+)-free medium completely prevented the subsequent response to phenylephrine. One can conclude that the use of the above-mentioned protein tyrosine kinase inhibitors in the rat aorta blocks a step involved in Ca2+ entry and Ca2+ store refilling. A definite conclusion regarding the vanadate effects is not possible due to the fact that this compound also affects Ca2+ ATP-ases.

This study investigates the role of primary prostaglandin receptor activation in the modulation of agonist-induced vascular smooth muscle contraction. Prostaglandin F2 alpha induced a concentration-dependent contraction of the rat aorta that was nearly abolished by the thromboxane A2 receptor antagonist, SQ29548. Prostaglandin F2 alpha in the presence of SQ29548 induced leftward shifts of the norepinephrine and KCl concentration-response curves. Nifedipine abolished the leftward shift of the norepinephrine concentration-response curve observed in the presence of prostaglandin F2 alpha and SQ29548. These results suggest that a function of primary prostaglandin receptor activation may be to potentiate agonist-induced contraction. The potentiation is dependent upon the opening of dihydropyridine-sensitive Ca2+ channels.

The effects of (S)-nafenodone, a new antidepressant, were studied on contraction and 45Ca2+ fluxes in rat vascular smooth muscle. In isolated rat aorta (S)-nafenodone, 10(-7) - 10(-4) M, inhibited the contractions induced by 80 mM KCl (IC50 = 1.4 +/- 0.4 x 10(-5) M) and 10(-5) M noradrenaline (IC50 = 1.2 +/- 0.2 x 10(-5) M). (S)-Nafenodone relaxed the contractions induced by both high K+ and noradrenaline, this effect being independent of the presence of functional endothelium. It also inhibited the contractions induced by addition of CaCl2 to Ca(2+)-free high-K+ solution IC50 = 2.5 +/- 0.9 x 10(-6) M) and the phasic contractions induced by noradrenaline in Ca(2+)-free medium, but was a very weak relaxant of the contractions induced by phorbol 12-myristate-13-acetate in Ca2+-free medium. In addition, (S)-nafenodone inhibited the spontaneous mechanical activity in portal vein segments (IC50 = 1.4 +/- 0.8 x 10(-6) M). (S)-Nafenodone inhibited the 45Ca2+ uptake stimulated by high KCl or noradrenaline without altering 45Ca2+ uptake in resting strips and decreased the net 45Ca2+ content in aortic strips non-stimulated as well as stimulated by noradrenaline. In conclusions, (S)-nafenodone inhibited voltage- and agonist-stimulated Ca2+ entry in isolated rat aortas. In addition, it decreased Ca2+ content in both resting and noradrenaline-stimulated muscles, suggesting that it may deplete noradrenaline-sensitive intracellular Ca2+ stores. As a consequence, (S)-nafenodone would reduce the concentration of intracellular free Ca2+ available at the contractile apparatus for vascular smooth muscle contraction.

The effects of the sodium channel activators veratridine and batrachotoxin on isolated rat aorta were investigated. Veratridine caused gradual contraction, independent of the presence of endothelium, with an EC50 of 35 microM. Batrachotoxin (1 microM) also induced contraction. Both effects were completely inhibited by the sodium channel blocker tetrodotoxin (1 microM). The veratridine (60 microM)-induced contraction was inhibited by nifedipine (0.1 microM). In the absence of extracellular Ca2+, veratridine (60 microM) did not cause contraction. Sodium nitroprusside (80 nM), acetylcholine (10 microM) and isoproterenol (1 microM) caused relaxation of rings precontracted with veratridine (60 microM). An inhibitor of endothelium-derived relaxing factor (EDRF) synthase, N omega-nitro-L-arginine methyl ester (L-NAME) (0.65 mM), enhanced the veratridine-induced contraction in rings with an intact endothelium, which suggests that EDRF was being released during the veratridine-induced contraction. These results show that the activation of sodium channels on smooth muscle cells induces a contraction that is probably mediated by Ca2+ influx through voltage-dependent Ca2+ channels.

The inhibitory effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle contraction and cytosolic Ca2+ level ([Ca2+]i) were examined using isolated rabbit aorta loaded with a fluorescent Ca2+ indicator, fura-2. TMB-8 (100 microM) decreased the high K(+)-induced increase in muscle tension, and [Ca2+]i and 45Ca2+ influx to their respective resting levels. TMB-8 (100 microM) almost completely inhibited the increase in [Ca2+]i and 45Ca2+ influx due to norepinephrine although muscle tension was only partially decreased. A higher concentration of TMB-8 (300 microM) inhibited the remaining portion of the contraction without additional decrease in [Ca2+]i. The inhibitory effect of TMB-8 on high K(+)-induced contraction, but not on the norepinephrine-induced contraction, was antagonized by the increase in external Ca2+ concentrations or by the Ca2+ channel activators, CGP 28,392 and by Bay K8644. In Ca(2+)-free solution, norepinephrine-induced transient increases in [Ca2+]i and muscle tension and 100 microM TMB-8 inhibited these changes. The caffeine-induced transient increases in [Ca2+]i and muscle tension were also inhibited by TMB-8 at concentrations higher than those needed to inhibit the norepinephrine-induced transient changes. In permeabilized smooth muscle, TMB-8 (300 microM) did not inhibit the Ca(2+)-induced contraction. These results suggest that TMB-8 inhibits vascular smooth muscle contractility by inhibiting Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.

The inhibitory effects of a hypotensive agent, cadralazine and its metabolite, ISF-2405, on the level of cytosolic Ca2+ ([Ca2+]cyt) and on contractions were examined in isolated vascular smooth muscle. Cadralazine slightly inhibited the transient norepinephrine-induced contraction in rabbit aorta and canine femoral, renal and mesenteric arteries and saphenous vein, and prostaglandin F2 alpha-induced contractions in canine basilar and coronary arteries. In contrast, ISF-2405 inhibited the contractions induced by prostaglandin F2 alpha in canine basilar and coronary arteries and those induced by norepinephrine in canine renal and femoral arteries and rabbit aorta. In aorta, ISF-2405 inhibited the increase in [Ca2+]cyt and muscle tension caused by norepinephrine. A Ca2+ channel blocker, verapamil, inhibited the norepinephrine-stimulated increase in [Ca2+]cyt more potently than it inhibited the increase in muscle tension, and ISF-2405 inhibited the verapamil-resistant part of the contraction. In Ca2(+)-free solution, norepinephrine induced transient increases in [Ca2+]cyt and muscle tension. ISF-2405 inhibited these changes. However, ISF-2405 did not inhibit the transient contraction induced by caffeine in the aorta. These results suggest that cadralazine is metabolized to ISF-2405 and inhibits vascular smooth muscle contraction by inhibiting receptor-mediated Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.

Vasoconstriction in pithed, normotensive rats elicited via stimulation of postsynaptic α - 2 -adrenoceptors by B-HT 920 was antagonized by EDTA and the calcium antagonists nifedipine, D 600 and verapamil, whereas pressor responses to the α 1 -agonist methoxamine were unaffected. This indicates that vasoconstriction in vivo initiated via postsynaptic α - 2 -adrenoceptors requires an influx or extracellular calcium. Thus, the antihypertensive affect of calcium antagonists may be based upon a diminution of vascular tone maintained by postsynaptic α - 2 -adrenoceptors.

Several compounds have been examined for their inhibitory action on a rat aortic ring preparation stimulated by four different agonists, noradrenaline bitartrate (NA), Ba2+, Ca2+ and K+. The compounds tested include phentolamine, papaverine, cinnarizine, cyclandelate, SKF-525A, prenylamine, guancydine, verapamil and compound D-600. Phentolamine inhibited NA reponses at much lower concentrations than those required to antagonize K+, Ca2+ or Ba2+ responses. Guancydine was also more effective against NA but was much less potent than phentolamine in this respect. Papaverine, cinnarizine, cyclandelate, prenylamine and SKF-525A were non selective agents and inhibited responses to all agonists approximately equally at various concentrations. Verapamil and D-600 were highly potent compounds which were more effective against cation-induced responses, especially those to Ca2+, than against those induced by NA.

The effect of catecholamines and angiotensin on 3 H-cyclic AMP levels and on adenyl cyclase activity in the rat aorta and tail artery was investigated in order to study the role of cyclic AMP in vascular smooth muscle contraction. The results are consistent with the concept that vasodilatation is associated with an increase and vasoconstriction with a decrease, in 3 H-cyclic AMP levels in vascular smooth muscle. Vasoactive drugs probably influence the 3 H-cyclic AMP level by changing adenyl cyclase activity. However, the link between alterations in amounts of cyclic AMP and vascular smooth muscle contraction is not yet known.

Since two different types of α-adrenoceptors (α1- and α2-) subserve vasoconstriction in pithed rats and noradrenaline behaves as a non-selective agonist of both receptors (for review see Timmerman and van Zwieten, 1981), we have investigated this interaction between noradrenaline and captopril in more detail to identify the class of α-adrenoceptor involved. This study confirms the findings reported recently (Antonaccio and Kerwin, 1981; Clough et al., 1981) that captopril inhibits postjunctional vasocontrictor responses to noradrenaline in pithed rats. However, captopril interfered with the vasoconstriction elicited by noradrenaline only in those animals in which the vascular postsynaptic α2-adrenoceptor system could be triggered (saline-and prazosin-treated rats). Captopril was without effect after removal of the contribution of post-synaptic α2-adrenoceptor stimulation to the in vivo vascular smooth muscle contraction due to (-)-noradrenaline (rauwolscine-treated rats). These results indicate that captopril selectively impairs the vasoconstriction subserved in vivo by postjunctinal α2-adrenoceptors. The vasoconstrictor process initiated by α1-adrenoceptor activation is not affected by captopril.