Mechanism of the vasodilator effect of 12-O-methylcurine in rat aortic rings

The vasodilator effects of 12-O-methylcurine (OMC), a bisbenzylisoquinoline alkaloid isolated from Chondrodendron platyphyllum (Menispermaceae), and its respective mechanism of action were investigated in rat aorta. In either endothelium-intact or endothelium-denuded aortic rings, OMC induced concentration-dependent relaxation in vessels pre-contracted with 0.1 microM phenylephrine (IC50 = 63.2+/-8.8 microM and 73.9+/-5.3 microM, respectively), 100 microM 5-hydroxytryptamine (IC50=49.6+/-13 microM and 49.9+/-10 microM, respectively) and 50 mM KCl (IC50= 19.9+/-6.8 microM and 21.1+/-4.5 microM, respectively). OMC also inhibited in a concentration-dependent and non-competitive manner the concentration-response curves induced by CaCl2 in high K+ (IC50 = 16.7+/-1.6 microM). In addition, OMC (100 microM) strongly inhibited phenylephrine-induced contractions dependent on calcium influx in the absence and presence of nifedipine (10 microM). In Ca2+-free medium, the transient contractions induced by phenylephrine (0.1 microM) were strongly inhibited by OMC (100 microM), whereas those induced by caffeine (20 mM) were not altered. H-89 (1 microM) and Rp-8-pCPT-cGMPs (3 microM), selective inhibitors of protein kinase A and G, respectively, did not change the relaxant effect of OMC in aortic rings pre-contracted with phenylephrine. Finally, OMC induced a concentration-dependent relaxation (IC50 = 62.8+/-12.5 microM) of the sustained contractions induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate in normal, but not in Ca2+-free, solution. The above results suggest that OMC induces a vasodilator effect in rataortic rings by a mechanism independent of the presence of functional endothelium and dependent on the influx of calcium ions through voltage- and receptor-operated calcium channels. Furthermore, it can also be suggested that the inhibition of calcium influx activated by protein kinase C is involved in the vasodilator effect of OMC.     

Contribution of nitric oxide and K+ channel activation to vasorelaxation of isolated rat aorta induced by procaine

The endothelium-dependent and -independent relaxant effect of procaine was examined in isolated rat aortic rings. Procaine induced relaxation of arteries precontracted with phenylephrine or with 60 mM K+ in a concentration-dependent manner (0.01-3 mM). Procaine (1 mM) inhibited the transient contraction induced by caffeine (10 mM) in Ca2+-free Krebs solution. Removal of the endothelium caused a rightward shift of the concentration-response curve for procaine. N(G)-Nitro-L-arginine (L-NNA, 10-100 microM), N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) and methylene blue (1-10 microM) significantly attenuated the procaine-induced relaxation without affecting the maximal response. L-Arginine (1 mM) partially but significantly antagonized the effect of L-NAME (100 microM). Pretreatment of endothelium-intact aortic rings with procaine (1 mM) or with acetylcholine (10 microM) significantly elevated the tissue contents of cyclic GMP and this increase was inhibited in the presence of 100 microM L-NNA. Tetrapentylammonium ions (1-3 microM) reduced the procaine-induced relaxation in both endothelium-intact and -denuded arteries. Tetrapentylammonium ions (3 microM) did not affect the procaine-induced relaxation of 60 mM K+-contracted arteries. Tetraethylammonium ions (3 mM) inhibited the procaine-induced relaxation. In contrast, iberiotoxin (100 nM), glibenclamide (3 microM), 4-aminopyridine (3 mM) and indomethacin (10 microM) had no effect. These results indicate that the procaine-induced relaxation may be mediated through multiple mechanisms. A substantial portion of the procaine-induced relaxation in rat aorta was caused by nitric oxide but not by other endothelium-derived factors. The activation of tetrapentylammonium- and tetraethylammonium-sensitive K+ channels contributes in part to the procaine-induced vasorelaxation. Besides, procaine may directly inhibit both external Ca2+ entry and internal Ca2+ release in aortic smooth muscle cells.     

Labedipinedilol-C: a third-generation dihydropyridine-type calcium channel antagonist displaying K+ channel opening, NO-dependent and adrenergic antagonist activities

Intravenous and oral labedipinedilol-C showed a dose-dependent long-lasting hypotension and a decrease of heart rate in normotensive and conscious spontaneously hypertensive rats (SHR). In isolated Wistar rat and guinea pig tissues, labedipinedilol-C competitively antagonized (-)isoproterenol-induced cardiac stimulation, tracheal relaxation, and phenylephrine-, CaCl2-, and high-K-induced aorta contractions in a concentration-dependent manner. The estimated pA2 and pKCa values were 8.22+/-0.04 and 7.11+/-0.52, respectively. [H]CGP-12177 binding to ventricle and lung tissues as well as [H]prazosin and [H]nitrendipine binding to brain membranes were inhibited by labedipinedilol-C with Ki values of 2.86, 9.03, 0.39, and 0.05 muM, respectively. The vasorelaxant effects of labedipinedilol-C on phenylephrine (10 microM)-induced contractions were attenuated by removing endothelium, by pretreatment with soluble guanylyl cyclase (sGC) inhibitors ODQ (10 microM) and methylene blue (10 microM), a NOS inhibitor L-NAME (100 microM), a K channel blocker TEA (10 mM), a KATP channel blocker glibenclamide (1 microM), and Ca-dependent K channel blockers apamin (1 microM) and charybdotoxin (0.1 microM). In human umbilical vein endothelial cells (HUVECs), labedipinedilol-C increased NO release, which was significantly inhibited by L-NAME. The Western blot analysis on HUVECs indicated that labedipinedilol-C increased the expression of eNOS. These results indicate that hypotension effects of labedipinedilol-C result from alpha-adrenoceptor and Ca entry-blocking activities and release of NO or NO-related substance from vascular endothelium. The endothelium-independent relaxation of vascular smooth muscle is probably linked to K channel opening and alpha-adrenoceptor-blocking activities.     

Relative contribution of intracellular and extracellular Ca2+ to alpha2-adrenoceptor-mediated contractions of ovine pulmonary artery.

We have examined the mechanism of contractions elicited by guanfacine, a selective agonist for alpha(2A/D)-adrenoceptors and its modulations by cyclic nucleotides in isolated ovine resistance intra-pulmonary artery. Guanfacine (10 nM-30 microM) produced concentration-dependent contraction of the pulmonary artery rings mounted for isometric recording. Yohimbine (0.1 microM), a nonspecific alpha(2)-adrenoceptor antagonist caused a parallel shift to the right (1.2 log unit) in the concentration-response curve of guanfacine without depressing the maxima. Preincubation of the tissues with Ca(2+)-free solution (EGTA 1mM) for 30 min caused a rightward shift (0.8 log unit) of the concentration-response curve of guanfacine with the inhibition of the maxima by 30+/-4.6%. L-type calcium channel blocker, nifedipine (1 microM) slightly inhibited (20%) the maximal contraction elicited with guanfacine (10 microM). On the other hand, brief exposure to cyclopiazonic acid (10 microM), an inhibitor of IP3-sensitive sarcoplasmic reticulum Ca(2+)-ATPase, resulted in marked inhibition of concentration-dependent contractions elicited with guanfacine (10 nM-30 microM), with the maxima being inhibited by 51+/-3.11%. In addition, agents that increase intracellular cAMP and cGMP suppressed guanfacine-induced contractions. The results of the present study suggest that alpha(2)-adrenoceptor-mediated contractions in ovine resistance pulmonary artery is primarily dependent on intracellular Ca(2+) with a small contribution from Ca(2+)-influx through voltage-dependent L-type calcium channels.     

Vasomotor action of insulin on the rabbit normal cavernous smooth muscle

Investigations on the effects of insulin on the normal vasculature have produced conflicting results. This study was aimed at establishing the vasomotor actions of insulin on normal cavernous smooth muscle. Insulin produced dose-dependent (10(-10)-10(-5) M) relaxation of the norepinephrine-precontracted strips of cavernosum, and of Bay K8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-2(trifluoromethylphenyl)pyridine-5-carboxylate]-precontracted strips. Endothelial denudation or indomethacin (10 microM) pre-treatment significantly reduced these insulin-induced relaxations, whereas NG-nitro-L-arginine methyl ester (L-NAME, 5 mM) did not. Moreover, the pre-treatment of the cavernosum strips with a prostacyclin synthesis inhibitor [9,11-diazo-15-deoxy-prostaglandin H2 (U-51605), 10 microM] significantly reduced insulin-induced response, whereas pretreatment with a cyclooxygenase-2 (COX-2) inhibitor (NS-398, 10 microM) did not. In addition, responses to insulin were not inhibited by K+ channel blockers, i.e., tetraethylammonium (TEA, 10 mM) or 4-aminopyridine (4-AP, 10 microM). Moreover, L-type Ca2+ currents were reduced by prostacyclin (2 microM) but not by insulin (10 microM). We conclude that insulin induces the endothelium-dependent relaxation of cavernous smooth muscles and that this relaxation response may emanate from the direct inhibition of L-type Ca2+ channels by prostacyclin.     

Mechanisms of vasorelaxation induced by eicosapentaenoic acid (20:5n-3) in WKY rat aorta

The vasorelaxant activity of eicosapentaenoic acid (EPA, 20:5n-3), the omega-3 polyunsaturated fatty acid, was investigated in isolated Wistar Kyoto (WKY) rat aortae by measuring isometric tension. Eicosapentaenoic acid (1 - 100 microM) relaxed rat aortae contracted with high K(+) (80 mM) or noradrenaline (NA, 1 microM) in a concentration-dependent manner. Contractions induced by Bay K 8644 or increasing concentrations of calcium were unaffected by EPA. The relaxant effect of EPA (3 - 100 microM) was significantly inhibited by indomethacin (10 microM), the cyclo-oxygenase inhibitor, but not by the nitric oxide (NO) synthesis inhibitor, N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 microM). Removal of the endothelium did not alter EPA-induced relaxations. In Ca(2+)-free, EGTA 2 mM solution, EPA (10 - 30 microM significantly inhibited NA-sustained contractions. Incubation with EPA (5, 10 microM) diminished both NA-induced (1 microM) phasic and sustained contractions. The vasorelaxant effects of EPA (> or =30 microM) on NA-induced (1 microM) contractions were significantly inhibited by the K(+) channel blocker, glibenclamide (10 microM), but not tetraethylammonium (1 mM). Moreover, indomethacin and glibenclamide combined significantly inhibited EPA-induced (1 - 100 microM) responses. These results indicate EPA exerts its endothelium-independent vasorelaxant effects in WKY rat aortae through production of prostanoids which activate K(+)(ATP) channels. Inhibition of Ca(2+) mobilization from intracellular pools and influx through the non-L-type, but not the L-type, Ca(2+) channel are also possible mechanisms action of EPA's.     

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.     

Characterisation of the relaxant response to raloxifene in porcine coronary arteries

The present study characterises the vasorelaxant response to raloxifene in isolated rings of porcine coronary artery. Tissues precontracted either with KCl (30 mM) or prostaglandin F(2alpha) (PGF(2alpha); 3 microM) were concentration-dependently relaxed by raloxifene (0.1-10 microM). Relaxation was not inhibited by the estrogen receptor antagonist 7alpha-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17beta-diol (ICI 182,780; 1 microM). Preincubation with raloxifene (1-3 microM) caused an inhibition of the KCl or PGF(2alpha)-induced contraction. The effects of raloxifene were independent of the endothelium. The relaxant response to raloxifene was slow in the onset and could not be reversed after repeated washings. Raloxifene did not affect Ca(2+) release from intracellular stores since it failed to inhibit a transient contraction induced by caffeine (10 mM). Raloxifene-induced relaxation was not influenced by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM; 10-20 microM). Calcium-induced contractions in Ca(2+)-free high K(+) (60 mM) depolarising medium were concentration-dependently inhibited by raloxifene (0.3-3 microM). If arterial rings were incubated with the L-type Ca(2+) channel activator (S)-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester ((S)-(-)-Bay K 8644; 0.1 microM), cumulative concentration-response curves to Ca(2+) were shifted to the left. Raloxifene (0.3-3 microM) inhibited the effect of (S)-(-)-Bay K 8644 in a concentration-dependent manner. 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580; 10 microM), an inhibitor of p38 mitogen-activated protein kinase (MAPK), diminished raloxifene-induced relaxation in endothelium-denuded arterial rings. Western blot analysis demonstrated that raloxifene stimulated p38 MAPK. It is concluded that raloxifene has an inhibitory effect on voltage-gated and receptor-operated L-type Ca(2+) channels in porcine coronary arteries, thus inducing vascular relaxation independent of the endothelium. p38 MAPK is, at least in part, involved in the relaxant response to raloxifene.     

Mechanism of relaxing action of the antiasthmatic drug, azelastine, in isolated porcine tracheal smooth muscle.

Azelastine (1-300 microM) inhibited contractions of isolated porcine trachea induced by high K+, carbachol and endothelin-1 (ET-1) with a decrease in [Ca2+]cyt (as measured by fura-2-fluorescence). Verapamil (0.1-10 microM) also inhibited the high K(+)-induced increases in [Ca2+]cyt and contraction, although it only partially inhibited the responses evoked by carbachol or ET-1. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), carbachol induced a transient increase in [Ca2+]cyt and force by releasing Ca2+ from cellular stores. Azelastine (100 microns) completely inhibited these contransient changes. In the absence of extracellular Ca2+, carbachol and 12-deoxyphorbol 13-isobutyrate (DPB) induced small sustained contractions without increasing [Ca2+]cyt. Azelastine inhibited these contractions. In muscle permeabilized with alpha-toxin, Ca2+ (0.3-3 microM) induced contraction in a concentration-dependent manner. DPB (without GTP) and carbachol or ET-1 (with GTP) enhanced the Ca(2+)-induced contraction. Azelastine partially inhibited the contraction induced by 0.3 microM Ca2+ but not the contraction induced by 3 microM Ca2+, and strongly inhibited the potentiating effects of DPB, carbachol and ET-1. Azelastine had no effect on the content of cyclic AMP or cyclic GMP. These results suggest that azelastine inhibits smooth muscle contraction by (i) decreasing [Ca2+]cyt, by inhibition of Ca2+ channels, (ii) decreasing agonist-induced Ca2+ release, and (iii) direct inhibition of contractile elements.     

Cardiovascular effects induced by reticuline in normotensive rats

The cardiovascular effects of reticuline, isolated in a pure form from the stem of Ocotea duckei Vattimo, were studied in rats by using a combined in vivo and in vitro approach. In normotensive rats, reticuline (5, 10 and 20 mg/kg, i. v., randomly) injections produced an intense hypotension. This hypotensive response was attenuated after either, L-NAME (20 mg/kg, i. v.), a nitric oxide (NO) synthase inhibitor, or atropine (2 mg/kg, i. v.), a muscarinic receptor antagonist. In isolated rat aortic rings with intact endothelium, reticuline (3 x 10 ( - 6), 3 x 10 ( - 5), 3 x 10 ( - 4), 9 x 10 ( - 4) and 1.5 x 10 ( - 3) M) inhibited in a concentration-dependent manner the contractions induced by phenylephrine (1 microM), KCl (80 mM) and KCl (30 mM), [IC (50) = (0.4 +/- 0.1, 2.4 +/- 0.4 and 3 +/- 0.4) x 10 ( - 4) M, respectively). The effect of reticuline on phenylephrine-induced contractions was attenuated by removal of the vascular endothelium [IC (50) = (2.5 +/- 0.7) x 10 ( - 4) M]. Similar results were obtained after pretreatment of the rings with L-NAME 100 microM [IC (50) = (1.3 +/- 0.1) x 10 ( - 4) M], L-NAME 300 microM [IC (50) = (3 +/- 0.3) x 10 ( - 4) M] or atropine 1 microM [IC (50) = (1.2 +/- 0.2) x 10 ( - 4) M]. On the other hand, the effect of reticuline on phenylephrine-induced contractions was not affected by indomethacin 1 microM [IC (50) = (0.7 +/- 0.3) x 10 ( - 4) M]. Reticuline (3 x 10 ( - 6), 3 x 10 ( - 5), 3 x 10 ( - 4), 9 x 10 ( - 4) and 1.5 x 10 ( - 3) M) antagonized CaCl (2)-induced contractions, and also inhibited the intracellular calcium dependent transient contractions induced by norepinephrine (1 microM), but not those induced by caffeine (20 mM). These results suggest that the hypotensive effect of reticuline is probably due to a peripheral vasodilation in consequence of: 1) muscarinic stimulation and NOS activation in the vascular endothelium, 2) voltage-dependent Ca (2+) channel blockade and/or 3) inhibition of Ca (2+) release from norepinephrine-sensitive intracellular stores.     

Mechanisms of hydroxyl radical-induced contraction of rat aorta

The present study was designed to investigate the effects of hydroxyl radicals (*OH), generated via the Fe2+-mediated Fenton reaction, on isolated rat aortic rings with and without endothelium. In the absence of any vasoactive agent, generation of *OH alone elicited an endothelium-independent contraction in rat aortic rings in a concentration-dependent manner. Hydroxyl radical-induced contractions of denuded rat aortic rings appeared, however, to be slightly stronger than those on intact rat aortic rings. The contractile responses to *OH were neither reversible nor reproducible in the same ring; even small concentrations of *OH radicals resulted in tachyphylaxis. Removal of extracellular calcium ions (Ca2+) or buffering intracellular Ca2+ with 10 microM acetyl methyl ester of bis(o-aminophenoxy) ethane-N,N,N',N',-tetraacetic acid (BAPTA-AM) significantly attenuated the contractile actions of *OH radicals. The presence of 1 microM staurosporine, 1 microM bisindolylmaleimide I, 1 microM G?6976 [inhibitor of protein kinase C (PKC)], 2 microM PD-980592 (inhibitor of ERK), 10 microM genistein, and 1 microM wortmannin significantly inhibited the contractions induced by *OH. Proadifen (10 microM), on the other hand, significantly potentiated the hydroxyl radical-induced contractions. Exposure of primary cultured aortic smooth muscle cells to *OH produced significant, rapid rises of intracellular free Ca2+ ([Ca2+]i). Several, specific antagonists of possible endogenously formed vasoconstrictors did not inhibit or attenuate either hydroxyl radical-induced contractions or the elevation of [Ca2+]i. Our new results suggest that hydroxyl radical-triggered contractions on rat aortic rings are Ca2+-dependent. Several intracellular signal transduction systems seem to play some role in hydroxyl radical-induced vasoconstriction of rat aortic rings.     

Effects of hispidulin, a flavone isolated from Inula viscosa, on isolated guinea-pig smooth muscle

1. In small concentrations (10(-7)-3 X 10(-6) M), hispidulin caused concentration-dependent contraction of isolated guinea-pig ileum and only mild relaxation of guinea-pig tracheal rings. 2. Larger concentrations (up to 3 X 10(-4) M) caused concentration-dependent relaxation of the ileum and the trachea. All the effects on the ileum and the trachea are reversible upon removal of the compound. 3. In concentrations from 10(-7) to 3 X 10(-4) M, hispidulin had no effect on the tone of the epinephrine-contracted rings of the guinea-pig main pulmonary artery. 4. Hispidulin caused a shift to the right of the acetylcholine concentration-effect curves on ileum and trachea and significantly inhibited the maximum contractions induced by acetylcholine. 5. In Ca2+-free, depolarizing solution, hispidulin caused both a shift to the right, and an inhibition of the maximum contractions, of the CaCl2 concentration-effect curves on ileum, trachea and pulmonary artery. 6. In Ca2+-free, EGTA-containing solution, hispidulin caused concentration-dependent inhibition of the contractions induced in the pulmonary artery by epinephrine and in the ileum by histamine. 7. These observations suggest that hispidulin may interfere with Ca2+ binding to the Ca2+-receptor protein(s) in the smooth muscle cell and/or with the agonist-induced Ca2+-release from intracellular stores. Less likely, hispidulin may interfere with Ca2+ influx through smooth muscle cell membrane.     

Intracellular calcium mobilization as a target for the spasmolytic action of scopoletin

The coumarin scopoletin was isolated in a pure form from the roots of Brunfelsia hopeana Benth. (Solanaceae). In isolated rat aortic rings, scopoletin (26-520 microM) inhibited to approximately the same extent the contractions induced by a variety of substances, including phenylephrine, potassium chloride, serotonin and PGF(2) (alpha). The effect of the coumarin on phenylephrine-induced contractions was not affected by endothelium removal or NO-synthase blockade by L-NAME (100 microM). Scopoletin (78 - 590 microM) antagonized in a concentration-dependent manner (IC(50) = 300 +/- 20 microM, n = 5), transient contractions in Ca(2+)-free media induced by noradrenaline, but not those induced by caffeine. Also, scopoletin did not interfere with the refilling of noradrenaline-sensitive intracellular calcium stores. It is suggested that the non-specific spasmolytic action of scopoletin can be attributed, at least in part, to its ability to inhibit the intracellular calcium mobilization from the noradrenaline-sensitive stores.     

Vascular smooth muscle contraction induced by Na+ channel activators, veratridine and batrachotoxin.

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.     

Relaxation effect of estradiol on different vasoconstrictor-induced responses in rat thoracal artery

This experiment was designed to compare the relaxant effect of estradiol on the contractions induced by 5-hydroxytryptamine, phenylephrine, and KCl in absence or presence of preincubation with the nitric oxide synthase inhibitor (NOS) N (omega)-nitro-L-arginine methylester (L-NAME). R at thoracic aorta contraction responses to vasoconstrictors were observed in the absence or presence of L-NAME. 17beta-Estradiol was added in increasing cumulative concentrations in the absence or presence of the L-NAME when the contractile response had reached a stable plateau. In the presence of L-NAME, 10(-6) M estradiol on precontracted 5-hydroxytryptamine rings caused significant relaxation in comparison with precontracted phenylephrine, KCl rings. In the presence of L-NAME, 10(-5) M and 10(-6) M estradiol doses on precontracted 5-hydroxytryptamine rings showed no significant difference in relaxation. The 10(-6) M, 10(-5) M, and 10(-4) M estradiol doses on precontracted phenylephrine caused concentration dependent relaxations. The results of this study show that acute vasorelaxation to 17beta-estradiol is largely mediated via NO-independent pathways by inhibiting Ca+2 influx from the extracellular space and Ca+2 released from intracellular stores.     

Vasorelaxation of rat thoracic aorta caused by osthole isolated from Angelica pubescens.

The pharmacological effects of osthole on isolated rat thoracic aorta were examined. Osthole inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta in a concentration-dependent manner (40-200 microM). The tonic contraction elicited by NE was also relaxed by the addition of osthole. This relaxing effect of osthole was not affected by indomethacin (20 microM) and was still observed in endothelium-denuded rat aorta. Methylene blue (50 microM) partially antagonized this relaxing effect of osthole. In high-K+ medium (80 mM), the Ca2+ (0.03-3 mM)-induced vasocontraction was inhibited concentration dependently by osthole (20-100 microM). Addition of osthole (100 microM) at the plateau of the K+ (80 mM)-induced contraction caused relaxation. Methylene blue (50 microM) did not antagonize this relaxation. In Ca(2+)-free medium, the caffeine (10 mM)-induced phasic contraction was also suppressed by osthole in a concentration-dependent manner. Although the cAMP level was not changed by osthole, the cGMP level of rat aorta was increased by osthole in a concentration-dependent manner. The increase in cGMP level caused by osthole was completely blocked by methylene blue. [3H]Inositol monophosphate formation caused by NE was not affected by osthole at a concentration of 200 microM. The 45Ca2+ influx elicited by either NE or high K+ was inhibited by osthole in a concentration-dependent manner. It is concluded that osthole relaxes rat thoracic aorta by virtue of its Ca(2+)-channel blocking properties and by elevating cGMP levels in vascular smooth muscle.     

Differences between the effects of cromakalim and nifedipine on agonist-induced responses in rabbit aorta.

1. The effects of cromakalim on endothelium-denuded rabbit aortic strips were compared with those of the calcium (Ca2+) entry blocking agent, nifedipine. 2. Pre-incubation with cromakalim or nifedipine had no significant effect on the initial phasic component of noradrenaline (NA)-induced responses. 3. Cromakalim (0.3-10 microM), but not nifedipine, inhibited the maintained tonic contractions produced by NA. The effects of cromakalim were antagonized by raising extracellular [K+] or by glibenclamide. 4. Nifedipine inhibited contractions produced by KCl (40 mM) whereas cromakalim had no effect. 5. In Ca2(+)-free physiological salt solution (PSS), cromakalim produced a significant inhibition of both the refilling of and the release of Ca2+ from NA-releasable Ca2+ stores, whereas nifedipine was ineffective. 6. In tissues preloaded with 42K+ cromakalim (0.3-10 microM) produced a concentration-dependent increase in the 42K+ efflux rate coefficient. NA (0.3 microM) also produced an increase in the rate of efflux of 42K+, an effect which was not antagonized by nifedipine (0.3 microM). 7. When microelectrodes were used, cromakalim (1-10 microM) produced a maintained concentration-dependent membrane hyperpolarization. However, low concentrations of cromakalim (less than 1 microM) which relaxed the aorta had no effect on membrane potential. NA had no significant effect on membrane potential. 9. It is concluded that the ability of cromakalim to relax NA-induced contractions in rabbit aorta is not exerted by the indirect closure of nifedipine-sensitive Ca2+ channels. Instead, cromakalim may exert a direct inhibitory action on Ca2+ uptake into and release from Ca2+ stores and additionally inhibit the pathway through which Ca2+ passes from the extracellular fluid to intracellular Ca2+ stores.     

Endothelium-independent vasorelaxant effect of dioclein, a new flavonoid isolated from Dioclea grandiflora, in the rat aorta

We have investigated the endothelium-independent vasorelaxant effect of the new flavonoid dioclein (5,2',5'-trihydroxy-6-7-dimethoxyflavanone) in the rat aorta. In endothelium-denuded vessels, dioclein induced a concentration-dependent relaxation of aortic rings precontracted with noradrenaline (IC50 = 3.5+/-0.89 x 10(-4) M and KCl (IC50 = 5.2+/-1.2 x 10(-4) M). In the absence of extracellular calcium, dioclein reduced the contraction induced by noradrenaline (maximal reduction approximately 33%) but not that induced by caffeine. Dioclein also produced a concentration-dependent inhibition of the sustained contractions induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate in normal (IC50 = 4.0+/-0.2 x 10(-4) M) and Ca2+-free solution (IC50 = 4.0+/-0.3 x 10(-4) M). The results indicate that the endothelium-independent vasorelaxant effect of dioclein may be explained by inhibition of contractions dependent on activation of protein kinase C, voltage-dependent Ca2+ influx and on the release of intracellular Ca2+ stores sensitive to noradrenaline.     

Contractions induced by potassium-free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats.

1. Vascular contractions induced by K(+)-free solution and relaxation responses following the return of K+ to the organ bath were studied in mesenteric arterial rings from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) with particular focus on the role of vascular adrenergic nerve-endings and endothelium. 2. In endothelium-denuded rings the omission of K+ from the incubation medium resulted in gradual contractions, the rate of which was slower in SHR than WKY. Nifedipine (1 microM) inhibited the contractions more effectively in SHR than WKY. 3. Adrenergic denervation in vitro with 6-hydroxydopamine reduced the contractions induced by the K(+)-free medium in endothelium-denuded rings. The remaining contractions after denervation were markedly greater in SHR than WKY. 4. The presence of intact vascular endothelium attenuated the K(+)-free contractions in both strains, the attenuation being smaller in SHR than WKY. NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM) and methylene blue (10 microM), but not indomethacin (10 microM), abolished the attenuating effect of endothelium on the K(+)-free contractions. L-Arginine (1 mM) reversed the effect of L-NAME in WKY but not in SHR. 5. The re-addition of K+ after full K(+)-free contractions dose-dependently relaxed the rings. The rate of this K(+)-induced relaxation was significantly slower in SHR than WKY at all K+ concentrations (0.1-5.9 mM) studied, whether the endothelium or functioning adrenergic nerve-endings were present or not. Ouabain (1 mM) totally inhibited the K+ relaxation in SHR but only partially in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-NAME inhibits Mg(2+)-induced rat aortic relaxation in the absence of endothelium.

1 L-NG-nitro-arginine methyl ester (L-NAME; 100 microM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 microM acetylcholine (ACh) and 2-10 mM Mg2+ in endothelium-intact (+E) rat aortic rings precontracted with 1 microM phenylephrine (PE). In PE-precontracted endothelium-denuded (-E) rat aorta, 3 microM ACh did not, but Mg2+ caused relaxation which was reversed by L-NAME, but not by D-NAME. 2 The concentration response profiles of L-NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 microM ACh were not significantly different. 3 L-NAME (100 microM) also reversed Mg(2+)-relaxation of -E aorta pre-contracted with 20 mM KCl or 10 microM prostaglandin F2alpha (PGF2alpha). L-NG-monomethyl-arginine (L-NMMA; 100 microM) was also effective in reversing the Mg(2+)-relaxation. 4 Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE-pre-contracted -E aorta, which was subsequently reversed by 100 microM L-NAME. 5 Reversal of the Mg(2+)-relaxation by 100 microM L-NAME in PE-precontracted -E aorta persisted following pre-incubation with 1 microM dexamethasone or 300 microM aminoguanidine (to inhibit the inducible form of NOS, iNOS). 6 Pretreatment of either +E or -E aortic rings with 100 microM L-NAME caused elevation of contractile responses to Ca2+ in the presence of 1 microM PE. 7 Our results suggest that L-NAME exerts a direct action on, as yet, unidentified vascular smooth muscle plasma membrane protein(s), thus affecting its reactivity to divalent cations leading to the reversal of relaxation. Such an effect of L-NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.