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.     

Different mechanisms involved in the vasorelaxant effect of flavonoids isolated from Satureja obovata

The inhibitory effects of naringenin, eriodictyol, and luteolin (10(-5) and 5 x 10(-5) M), previously isolated from Satureja obovata subsp. obovata var. valentina (Lamiaceae), on rat thoracic aorta were investigated. Flavonoids at the two concentrations assayed (10(-5) and 5 x 10(-5) M) showed different smooth muscle relaxant behaviour in the three phases involved in the noradrenaline (10(-6) M)-induced contractions. The three flavonoids showed an inhibitory effect of the phasic component in order of potency: luteolin > eriodictyol > naringenin. Luteolin and eriodictyol inhibited both tonic-I and tonic-II phases associated to the inhibition of PKC and calcium influx, respectively, whereas naringenin only inhibited the tonic-I phase associated to inhibition of PKC.     

Mechanisms involved in the vasodilator effect of curine in rat resistance arteries

The vasodilator effect of curine was investigated in the rat small mesenteric arteries. In either endothelium-intact or endothelium-denuded mesenteric arteries, curine induced a concentration-dependent relaxation in rings pre-contracted with noradrenline (10 microM; IC 50 = 4.8 +/- 1.3 microM and 4.8 +/- 1.5 microM, respectively) and KCl (80 mM; IC 50 = 6.0 +/- 1.3 microM and 13.0 +/- 5.6 microM, respectively). Curine also inhibited (IC 50 = 4.6 +/- 0.9 microM) the concentration-response curves induced by noradrenaline. Contractions dependent on calcium-influx elicited by KCl (80 mM) and noradrenaline (10 microM) were inhibited by curine (10 microM). Finally, contractions induced by noradrenaline (10 microM), in calcium-free medium, were strongly inhibited by curine (10 microM). The above results suggest that the inhibition of influx of calcium ions through voltage-operated calcium channels and non-selective channels, and mobilization of intracellular calcium stores sensitive to noradrenaline are involved in the vasodilator effect of curine.     

Cardiovascular effects of visnagin on rats

The present article describes the effects of visnagin on systolic blood pressure and heart rate in the anaesthetized rat. Intravenous administration of visnagin (0.3-5 mg kg-1) produced dose-related decreases in blood pressure with no significative changes in heart rate. Under nitric oxide synthase inhibition (L-NAME, 50 mg kg-1) the hypotensive effects of visnagin (5 mg kg-1) were not affected. Visnagin (5 x 10(-6) M-10(-4) M) produced a weak decrease in the rate and amplitude of spontaneous contractions in right atria. Visnagin also caused a weak decrease in peak contractile force and the df/dtmax with no significant changes in the time to peak tension or the time for total contraction in left atria driven at a basal rate of 1 Hz. Visnagin (10(-5) M, 5 x 10(-5) M and 10(-4) M) concentration-dependently decreased pressor response to KCl (IC50 = 5.1 +/- 2.5 x 10(-5) M) and noradrenaline (IC50 = 2.6 +/- 0.9 x 10(-5) M) in rat isolated mesenteric beds. Visnagin (3 x 10(-7) M-10(-4) M) induced a concentration-dependent relaxation of isolated mesenteric arteries contracted by noradrenaline (IC50 = 1.7 +/- 0.8 x 10(-5) M). The relaxant effects in the absence of functional endothelium were not significantly different (IC50 = 1.5 +/- 0.3 x 10(-5) M, P > 0.05) from those observed in segments with intact endothelium. In conclusion, the main mechanism responsible for the acute hypotensive effect of visnagin is the vasorelaxant response induced by this drug in resistance arteries.     

Effects of the novel potassium channel opener, UR-8225, on contractile responses in rat isolated smooth muscle.

1. The effects of UR-8225 [(1,2-dihydro-4-(1,2-dihydro-2-oxo-1-pyridyl)-2,2-dimethyl-1-oxonapht halen-6- carbonitrile)] and levcromakalim were studied on the electrical and contractile responses induced by noradrenaline and KCl and on 86Rb+ efflux in rat aortic rings and on spontaneous mechanical activity in rat portal vein segments. 2. UR-8225 and levcromakalim, 10(-9) M-10(-5) M, relaxed the contractile responses induced by noradrenaline (IC50 = 2.7 +/- 0.4 x 10(-6) M and 6.6 +/- 1.3 x 10(-7) M, respectively) or 30 mM KCl (IC50 = 1.4 +/- 0.2 x 10(-7) M and 9.4 +/- 1.3 x 10(-8) M, respectively) more effectively than those induced by 80 mM KCl. The relaxant effect on noradrenaline-induced contractions was independent of the presence or absence of functional endothelium. 3. The vasorelaxant effect of UR-8225 and levcromakalim can be competitively antagonized by glibenclamide, an ATP-sensitive K+ channel blocker. There were no differences in the calculated pA2 values for glibenclamide to inhibit UR-8225- and levcromakalim-induced relaxations (7.61 +/- 0.08 and 7.69 +/- 0.10, respectively). The slope of the Schild plot yielded values not significantly different from unity (0.95 +/- 0.06 and 0.96 +/- 0.05, respectively). 4. UR-8225 (10(-5) M) hyperpolarized the resting aortic membrane potential from -50.7 +/- 0.7 mV to -66.0 +/- 2.0 mV and stimulated 86Rb+ efflux. 5. UR-8225 and levcromakalim inhibited the contractions induced by Ca2+ in aortae incubated in Ca(2+)-free PSS containing methoxyverapamil in the presence of noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism involved in the spasmolytic effect of a mixture of two triterpenes, cycloartenol and cycloeucalenol, isolated from Herissanthia tiubae in the guinea-pig ileum

The smooth muscle relaxant properties of a mixture of the two triterpenoids cycloeuclalenol and cycloartenol (CC) isolated from Herissanthia tiubae (Malvaceae) were studied in several smooth muscle preparations. CC inhibited contractions induced by carbachol, histamine and KCl in the guinea-pig ileum, but no spasmolytic activity was found in guinea-pig trachea or rat aorta. In guinea-pig ileum, concentration-response curves to carbachol and CaCl (2) in high K(+) were shifted to the right by CC in a concentration-dependent manner with slopes of the Schild plot differing from the unity. IC(50) values were 3.4 +/- 0.8 x 10 (-5) M and 8.44 +/- 1.87 x 10 (-5) M for carbachol and CaCl(2), respectively. The phorbol ester TPA, which activates protein kinase C (PKC), potentiated contractions induced by submaximal concentrations of carbachol. This potentiation was inhibited by CC. Desensitization of PKC by TPA completely abolished the inhibition produced by CC on carbachol-induced contractions. Together, our results indicate that inhibition of PKC is involved in the spasmolytic effect of CC in the guinea-pig ileum.     

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.     

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.     

Effects of hypoxia on the vasodilator activity of nifedipine and evidence of secondary pharmacological properties

The effects of hypoxia on the vasodilator response of endothelium-denuded rat aortic rings to the calcium channel blocker, nifedipine, were examined. Under normoxic conditions, nifedipine (10(-8)-3x10(-6) M) attenuated the contractility of noradrenaline precontracted rings in a concentration-dependent manner, although the sensitivity was less than what occurs with K+ precontracted tissues. Under hypoxic conditions there was no relaxation by nifedipine. When a concentration-response curve to noradrenaline was constructed before and in the presence of a high concentration of nifedipine (10(-5) M), the response to noradrenaline was unaffected in both normoxic and hypoxic conditions. When noradrenaline was replaced by phenylephrine (10(-8)-10(-5) M), the maximum tension was reduced in the presence of nifedipine to 59+/-6% of the pre-nifedipine value. Repetition of the experiment in the presence of cocaine (10(-5) M) revealed the inhibitory effect of nifedipine on noradrenaline-induced contraction, the maximum contraction in the presence of nifedipine falling significantly (P<0.005) to 67+/-6% of the pre-nifedipine response. When propranolol (10(-7) M) was present in the bath, the maximum contraction to noradrenaline was significantly (P<0.05) reduced by nifedipine to 55+/-4% of its previous value. The fact that nifedipine was able to inhibit phenylephrine-induced contractions and relax noradrenaline-precontracted aortic rings confirms its calcium channel blocking activity. The failure to inhibit noradrenaline when added prior to the noradrenaline-induced contractions suggests an opposing effect in addition to calcium channel blockade, which cancels out the attenuation of noradrenaline--but not phenylephrine-induced contractions. When neuronal uptake of noradrenaline was blocked with cocaine or beta-adrenoceptors were blocked with propranolol, the inhibitory effect of nifedipine against noradrenaline-induced contractions was revealed. This suggests that the additional property was due to blockade of neuronal reuptake or antagonism at beta-adrenoceptors. This study also showed that nifedipine is ineffective as a vasodilator in the rat aorta under hypoxic conditions.     

Nantenine blocks muscle contraction and Ca2+ transient induced by noradrenaline and K+ in rat vas deferens

The effect of nantenine, an aporphine alkaloid isolated from Ocotea macrophylla H.B.K., was studied on contractions and Ca(2+) translocation induced by noradrenaline, Ca(2+), or K(+) in the isolated rat vas deferens from reserpinized animals. Concentration-response curves of calcium chloride (CaCl(2)) were performed in the vas deferens, in a Ca(2+)-free nutrient solution, using potassium chloride (KCl, 80 mM) as a depolarizing agent. In these conditions, nantenine (2.35 x 10(-4) and 4.7 x 10(-4) M) significantly reduced the maximum contractions (E(max)) of Ca(2+) (IC(50)=2.6 x 10(-4) M) and noradrenaline (IC(50)=2.9 x 10(-4) M). The contractile responses were totally recovered after the withdrawal of nantenine. In addition, experiments performed to measure simultaneously the contraction and the increase of intracellular Ca(2+) induced by noradrenaline (10(-5) M) or KCl (80 mM) showed that nantenine (2.35 x 10(-4) and 4.7 x 10(-4) M) significantly decreased both effects. The results suggest that a reversible block of Ca(2+) entry could be involved on the non-competitive-like antagonism of nantenine in rat vas deferens.     

Effects of Ro 5-4864 and PK 11195 in rat duodenum and vas deferens.

Ro 5-4864 and PK 11195 inhibit in a concentration-dependent manner carbachol-induced contractions in rat duodenum (IC50: 1.56 +/- 0.07 x 10(-5) M and 1.18 +/- 0.07 x 10(-5) M respectively). The antagonism is non-competitive and is not mediated by peripheral-type benzodiazepine receptors. The Ro 5-4864 effect is modulated by the calcium concentration of the Tyrode-Ringer solution. In the presence of 1 mM NaF/10 microM AlCl3, Ro 5-4864 and PK 11195 do not inhibit carbachol-induced contractions. Moreover, Ro 5-4864 and PK 11195 significantly relax AlF(4-)-induced contractions, with IC50 values of 2.01 +/- 0.12 x 10(-5) M and 1.28 +/- 0.11 x 10(-5) M respectively. This effect is also modulated by the calcium concentration of the medium. Pertussis toxin potentiates the antagonist effects of Ro 5-4864 and PK 11195 on carbachol-induced contractions, but cholera toxin does not affect them. Ro 5-4864 and PK 11195 inhibit 45Ca2+ uptake induced by KCl (120 mM) in rat vas deferens, but do not affect either basal 45Ca efflux or noradrenaline-induced 45Ca2+ efflux. Only high doses of PK 11195 (above 5 x 10(-5) M) are able to produce a slight reduction of the accumulation of inositol phosphates induced by methoxamine in rat vas deferens, while Ro 5-4864 has no significant effect. Finally, Ro 5-4864 and PK 11195 reduce calcium influx, but do not seem to be the only mechanism of the antagonistic effect on carbachol-induced contractions. An alteration of other second messengers, probably cyclic monophosphate nucleotides, may be involved.     

Differences between the vasorelaxant activity of adenosine-receptor agonists on guinea-pig isolated aorta precontracted with noradrenaline or phenylephrine

The relaxant effect of adenosine and 5'-(N-ethylcarboxamido)adenosine (NECA) against alpha-adrenoceptor-mediated contractile tone in guinea-pig isolated aortic rings has been examined to determine if this A2B-receptor-mediated relaxation was dependent upon the contracting agent, and whether the contractions were dependent upon intracellular or extracellular calcium. Relaxation responses were consistently greater for aortic rings pre-contracted with phenylephrine (3x10(-6) M) than for rings pre-contracted with noradrenaline (3x10(-6) M). Maximum inhibition by NECA was significantly greater for phenylephrine-contracted aortae than for noradrenaline-contracted (81.9+/-2.8% compared with 25.0+/-1.5%). These differences persisted in the presence of beta- and alpha2-adrenoceptor blockade and could not, therefore, be attributed to stimulation of these receptors by noradrenaline. The ratio of the contractions obtained before and in the presence of adenosine or NECA was compared with the control ratio obtained before and after vehicle. Experiments were performed both in the presence of normal calcium levels and under calcium-free conditions. In normal-calcium medium, NECA inhibited phenylephrine-induced contractions (test ratio, 76.7+/-3.9%; control ratio, 133.1+/-9.8%) to a greater extent than noradrenaline-induced contractions (108.4+/-4.1 and 123.4+/-4.9%); adenosine similarly inhibited phenylephrine-induced contractions more than those induced by noradrenaline. Under calcium-free conditions, adenosine (36.7+/-11.9 and 110.7+/-26.6%) and NECA (55.2+/-9.1 and 87.1+/-14.9%) were only effective against phenylephrine-induced contractions. This suggests that activation of the A2B-receptor by these agonists inhibited intracellular mobilization of calcium for phenylephrine-induced contractions only. The effects on extracellular calcium influx were examined for phenylephrine- and noradrenaline-induced contractions in normal-calcium medium but in the presence of ryanodine to prevent intracellular calcium mobilization. NECA inhibited phenylephrine-induced contractions (77.3+/-12.4 and 111.4+/-9.3%), presumably by interfering with influx of calcium through receptor-operated calcium channels. In contrast, NECA failed to reduce noradrenaline-induced contractions (121.5+/-10.7 and 122.4+/-11.6%), suggesting that the effect on noradrenaline is predominantly via interaction with intracellular calcium. Adenosine was consistently a more effective relaxant than NECA, possibly because of an additional intracellular component of the response. We conclude that adenosine receptor agonists inhibit phenylephrine-induced contractions of guinea-pig aorta more selectively than noradrenaline-induced contractions. A2B-receptor stimulation might reveal a fundamental difference between the modes of contraction elicited by these two alpha-adrenoceptor agonists.     

HNS-32, a novel azulene-1-carboxamidine derivative, inhibits nifedipine-sensitive and -insensitive contraction of the isolated rabbit aorta

The vasorelaxant profile of a novel azulene-1-carboxamidine derivative, HNS-32 [N1,N1-dimethyl-N2-(2-pyridylmethyl)-5-isopropyl-3,8-dimethyl-azulene-1-carboxamidine, CAS 186086-10-2], was investigated in the isolated rabbit aorta precontracted with high KCl, noradrenaline (NA) or phorbol 12, 13-dibutyrate (PDBu) and compared with those of nifedipine and nitroglycerin. In preparations without endothelium, HNS-32 elicited concentration-dependent, full inhibition of contractions elicited by high KCI (80 mM), NA (3x10(-6) M) or PDBu (10(-6) M). In contrast, nifedipine inhibited only the contraction elicited by membrane depolarization with high KCl. Nitroglycerin also attenuated high-KCl-, NA- and PDBu-elicited contractions effectively, although full suppression was obtained only for NA-elicited contraction. Whilst the relaxant effect of HNS-32 was not affected by the presence of endothelium, the relaxant response to acetylcholine was endothelium dependent. Addition of excess Ca2+ restored both the HNS-32-reduced tension in muscle precontracted with high KCI and the nifedipine-mediated tension decrease. Relaxation elicited by HNS-32 was not affected by the adenylate cyclase inhibitor, 9-(tetrahydro-2'-furyl)adenine (SQ 22,536, 10(-4) M), the soluble guanylate cyclase inhibitor, 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10(-5) M) or a cocktail of K+ channel blockers (glybenclamide 10(-6) M, tetraethylammonium 2x10(-3) M, apamin 10(-7) M, 4-aminopyridine 10(-4) M and Ba2+ 10(-5) M). These findings indicate that HNS-32 inhibits both L-type Ca2+ channel-dependent and -independent vascular contraction. Blockade of Ca2+ entry through L-type Ca2+ channels may be involved in the inhibitory effect of HNS-32 on the contraction due to membrane depolarization with high KCl. On the other hand, HNS-32 seems to inhibit Ca2+ channel-independent contraction via mechanism(s) other than elevation of cyclic nucleotides (cAMP and cGMP) and opening of K+ channels.     

Effects of oxodipine on isolated rabbit aorta and mesenteric resistance vessels.

The inhibitory effects of the dihydropyridine Ca2+ antagonist, oxodipine, on contractions and 45Ca2+ influx stimulated by noradrenaline (NA) and high K+ in rabbit aorta were compared to the same parameters measured in mesenteric resistance arteries. In aortic rings oxodipine, 10(-11)-10(-6) M, inhibited in a concentration-dependent manner the contractions induced by high K+ (IC50 = 9.0 +/- 4.0 x 10(-10) M) or by Ca2+ in high K+ solution (IC50 = 6.2 +/- 2.4 x 10(-9) M), while responses to NA were only slightly affected (IC50 greater than 10(-6) M). In mesenteric resistance vessels oxodipine inhibited the contractions induced by high K+ and NA but was more effective against NA- than high K(+)-induced contractions (IC50 = 5.2 +/- 3.1 x 10(-10) and 1.2 +/- 1.8 x 10(-8) M, respectively). The concentration-inhibition curves for high K(+)-induced contraction and 45Ca2+ influx in aorta were almost superimposable (I50 = 2.2 +/- 2.0 x 10(-9) M), whereas NA-induced contractions were inhibited less than 45Ca2+ influx (I50 = 8.2 +/- 2.6 x 10(-8) M). In mesenteric resistance vessels the curves for contraction and 45Ca2+ influx stimulated by high K+ and NA were also superimposable, but 45Ca2+ influx stimulated by NA was more sensitive to oxodipine than that stimulated by high K+ (I50 = 3.9 +/- 2.0 x 10(-10) and 2.2 +/- 1.2 x 10(-8) M, respectively). It is concluded that the effects of oxodipine can be attributed to its ability to inhibit Ca2+ entry through both potential- and receptor-operated pathways.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effect of calcium and Bay K 8644 on the inhibitory action of estrogens in the rat uterus.

1. The effects of the estrogens estradiol (E2, 10(-7) to 3 x 10(-5) M) and diethylstilbestrol (DES, 10(-7) to 3 x 10(-6) M) on tonic contractions of the rat uterus induced by KCl and CaCl2 have been studied. 2. E2 and DES relaxed, in a dose-dependent way, the tonic contraction induced by KCl (60 mM) (IC50: 5.16 +/- 1.49 x 10(-6) and 4.51 +/- 0.03 x 10(-7) M); the tonic contraction induced by CaCl2 (3 mM) in the rat uterus incubated in depolarizing Krebs (127 mM of K+) have also been relaxed (IC50: 8.6 +/- 0.03 x 10(-7) and 2.56 +/- 0.07 x 10 M) by both drugs. 3. The CaCl2 (0.1 to 10 mM) counteracted the relaxing effect of E2 and DES, respectively, up to 28.13 +/- 10.2% and 34.71 +/- 11.5%, on KCl-induced contractions, and up to 126.36 +/- 19.35% and 95.8 +/- 16.3% on CaCl2-induced contractions. 4. Bay K 8644 (10(-10) to 10(-6) M) reversed the relaxing effect of E2 and DES, respectively, up to 42.49 +/- 2.28% and 43.31 +/- 3.59% on KCl-induced contractions, and up to 21.73 +/- 4.16% and 75.97 +/- 9.63% on CaCl2-induced contractions. 5. Propranolol (10(-6) M) did not modify the relaxing effect of E2 or DES on CaCl2-induced contractions.     

Characterization in rat aorta of the binding sites responsible for blockade of noradrenaline-evoked calcium entry by nisoldipine.

1. The effectiveness of the calcium antagonist, 1,4-dihydropyridine nisoldipine, as an inhibitor of contraction and 45Ca entry evoked by noradrenaline in rat aorta has been investigated and correlated with binding characteristics in intact artery. 2. Contractions evoked by noradrenaline were concentration-dependently depressed by nisoldipine (0.3-300 nM). About 60% of the response was resistant to inhibition, while KCl-induced contractions could be completely blocked. Noradrenaline-induced contractions were also less sensitive to nisoldipine inhibition than were KCl-induced contractions. 3. Preincubation of the aorta with nisoldipine in high KCl depolarizing solution increased the inhibition of the contraction evoked by a short application of noradrenaline or KCl to a similar extent. 4. The inhibition by nisoldipine of 45Ca influx evoked either by KCl depolarizing solution or by noradrenaline correlated well with the inhibition of the contractile responses. However, while KCl-stimulated 45Ca influx was totally abolished by nisoldipine (300 nM), 38% of the noradrenaline-stimulated 45Ca influx was resistant to inhibition by nisoldipine (300 nM). 5. The study of [3H]-(+)-PN 200-10 ([3H]-(+)-isradipine) binding in intact aorta showed the presence of a homogeneous population of specific binding sites. KD values were dependent on the KCl concentration in the bath while Bmax was unaffected. Binding of [3H]-(+)-isradipine was also increased in tissue exposed to noradrenaline; in the presence of 10(-5) M noradrenaline, binding parameters of [3H]-(+)-isradipine were close to the values obtained in aorta bathed in 20 mM KCl solution. 6. Displacement of [3H]-(+)-isradipine specific binding by nisoldipine was determined in segments of mesenteric artery and of aorta. The potency of nisoldipine was dependent on the incubation conditions applied to the vessel, as follows: KCl (100 mM) depolarizing solution greater than noradrenaline (10(-5) M) = KCl (25 mM) solution greater than physiological solution. The Ki value measured in aorta exposed to noradrenaline (10(-5) M) was close to the IC50 value of nisoldipine on the noradrenaline-evoked contraction. 7. The membrane potential value of rat aorta was estimated by the distribution of [3H]-tetraphenylphosphonium bromide ([3H]-TPP+), [3H]-TPP+ uptake concentration-dependently decreased when the KCl concentration in the bath was increased from 5.9 to 130 mM. Noradrenaline also concentration-dependently decreased [3H]-TPP+ uptake; the maximum effect (1-10 microns noradrenaline) was comparable in amplitude to the effect of 25 mM KCl solution.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of calcium channel inhibitors and other procedures affecting calcium translocation on drug-induced rhythmic contractions in the rat vas deferens.

In the rat isolated vas deferens, methoxamine 8.1 microM produced an initial phasic response that declined towards baseline and was followed by rhythmic contractions that continued until wash-out. These responses were predominant in the epididymal half. BaCl2 1 mM produced a similar type of response which was not mediated by noradrenaline release or activation of alpha-adrenoceptors. The barium responses were similar in the epididymal and prostatic halves. Incubation in nominally Ca2+-free solution caused abolition or near abolition of rhythmic contractions produced by barium or methoxamine. The initial phasic response to methoxamine was abolished in Ca2+-free solution, whereas that produced by barium persisted. Rhythmic contractions produced by methoxamine or barium were inhibited by Mg2+ (2.4-20 mM) and by La3+ (1-5 mM). Mg2+ had selectivity for inhibition of the frequency of methoxamine- but not barium-induced rhythmic contractions. Despite their dependence on [Ca2+]o, barium- and methoxamine-induced rhythmic contractions were resistant to inhibition by calcium channel inhibitors. Verapamil, nifedipine and flunarazine inhibited the amplitude of rhythmic contractions more readily than the frequency (methoxamine IC50 for verapamil: amplitude = 29.8 +/- 5.40 microM, n = 6, frequency = 96.7 +/- 31.0 microM, n = 5, for nifedipine: amplitude = 2.42 +/- 0.34 microM, n = 7, frequency = 3.24 +/- 0.75 microM, n = 7, and for flunarizine: amplitude = 15.9 +/- 5.95 microM, n = 7, frequency = 153 +/- 28.6 microM, n = 7). There was no differentiation between inhibition of methoxamine and barium-induced responses. Like Mg2+, methoxyverapamil selectively inhibited the frequency of methoxamine-induced contractions (IC50: amplitude = 16.8 +/- 2.86 microM, n = 5, frequency = 2.07 +/- 0.81 microM, n = 5) but not barium-induced contractions (IC50: amplitude = 13.9 +/- 1.95 microM, n = 5, frequency = 48.5 +/- 8.98 microM, n = 5). Diazoxide (43.3-2167 microM) and nitroprusside (3.36-6712 microM) had only a small effect on barium contractions, but produced a dose-related reduction in the amplitude of methoxamine-induced responses. Diazoxide and nitroprusside caused methoxamine contractions to occur in groups, although they had no effect on their overall frequency. It is concluded that barium- and methoxamine-induced rhythmic contractions in the rat vas deferens are mediated by the entry of [Ca2+]o via membrane calcium channels that have a lower affinity (10-100 X) for calcium channel inhibitors than those mediating the KCl response.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of morphine in rat small mesenteric arteries

We investigated the effect of morphine in phenylephrine (PE)- or KCl-precontracted rat small mesenteric arteries. Morphine (10(-6)-10(-4) M) administration caused concentration-dependent relaxation responses in small mesenteric arteries precontracted by PE or KCl. Removal of endothelium did not significantly alter the relaxation responses to morphine. The relaxant responses to morphine were partially inhibited by pre-treatment of tissues with naloxone (NAL, 10(-5) M) for 20 min. The inhibitory effect of NAL on relaxant responses to morphine in PE- or KCl-precontracted arteries did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of endothelium-intact or endothelium-denuded arterial segments with NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) or cyclooxygenase (COX) inhibitor indomethacin (10(-5) M) or histamine H(1)-receptor blocker diphenhydramine (10(-6) M), for 20 min did not inhibit the relaxation responses to morphine. Small mesenteric arterial segment contractions induced by stepwise addition of calcium to high KCl solution with no calcium were almost completely inhibited by morphine. These findings suggested that morphine-induced relaxation responses in isolated rat small mesenteric arteries were neither dependent on endothelium nor blocked by NOS or COX inhibition but they rather seem to depend on an interaction of morphine with calcium influx pathways.     

Comparison of the calcium antagonistic activity of nifedipine and FR34235 in the canine saphenous vein

The effects of FR34235 (FR) on contractions and 45Ca2+ uptake stimulated by depolarization (80 mM KCl) and activation of postsynaptic alpha 1-adrenoceptors in the canine saphenous vein (CSV) were studied and compared with those of nifedipine. The 45Ca2+ uptake and contractions evoked by KCl were inhibited in a dose-dependent fashion by FR [concentration producing 50% inhibition [(IC50 = 2.0 +/- 0.5 and 1.2 +/- 0.3 X 10(-8) M, respectively)] and nifedipine (IC50 = 6.0 +/- 1.0 and 4.0 +/- 0.9 X 10(-8) M, respectively). FR was a potent inhibitor of the 45Ca2+ uptake and contractions of CSV induced by the selective alpha 1-agonist, SK&F l-89748 (l-[1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine] ) (IC50 = 7.2 +/- 0.7 and 7.0 +/- 1.8 X 10(-8) M, respectively). In contrast, the contractions and 45Ca2+ uptake elicited by SK&F l-89748 were much less sensitive to nifedipine (IC50 = 6.0 +/- 1.4 and 6.6 +/- 0.4 X 10(-6) M, respectively). The results suggest the following: (a) both FR and nifedipine are potent antagonists of the voltage-dependent Ca2+ channels in CSV; and (b) FR is a more effective antagonist of receptor-operated Ca2+ entry in the saphenous vein than nifedipine.     

Effect of somatostatin on resistance and on capacitance rabbit isolated arteries

The effects of somatostatin, a tetradecapeptide isolated from hypothalamus extracts, were studied on the vascular reactivity of aorta and mesenteric arteries isolated from rabbits. We also investigated whether or not Ca(2+) movements were implicated in these effects. Rabbit aorta and mesenteric (fifth branch) arteries were isolated, cleaned off, and mounted in an organ bath containing Godfraind solution or physiological saline solution (PSS), respectively. Somatostatin (10(-8)-10(-4) M) produced a concentration-dependent inhibition of the contractile responses induced by high K(+) (80 mM) or noradrenaline (10(-6) M in aorta or 10(-4) M in mesenteric arteries) in both arteries studied. The inhibitory effect of somatostatin was greater in mesenteric resistance vessels (IC(50) 3.1+/-2.3x10(-5) M, and 5.2+/-4.8x10(-8) M with KCl and noradrenaline, respectively). Contractile responses produced by the addition of Ca(2+) (1-5 mM) to Ca(2+)-free high K(+) solution were also concentration dependently inhibited by somatostatin in aorta. Furthermore, somatostatin decreased noradrenaline-induced contraction attributed to intracellular Ca(2+) release in aorta, and inhibited 45Ca(2+) uptake stimulated by high K(+) or by noradrenaline. However, it did not modify 45Ca(2+) uptake in resting mesenteric resistance arteries. Taken together, these results suggest that somatostatin exerts an inhibitory effect on vascular contractions induced by some stimulating agents in different arteries isolated from rabbits, being more potent in mesenteric arteries.