Vasorelaxant effects of the potassium channel opener SR 47063 on the isolated human saphenous vein and rat aorta.

The vasorelaxant effects of SR 47063 (4-(2-cyanimino-1, 2-dihydropyrid-1-yl)-2,2-dimethyl-6-nitr ochromene), a new K(+)-channel opener structurally related to levcromakalim, were examined in isolated human saphenous vein (HSV) and rat aorta (RA). HSV or RA rings were precontracted with either KCl or noradrenaline and cumulative relaxant concentration-response curves were obtained for SR 47063 (0.1 nM to 1 microM) in the presence or absence of 3 microM glibenclamide. SR 47063 potently relaxed HSV and RA precontracted with 20 mM (but not 60 mM) KCl or 10 microM noradrenaline in a concentration-dependent manner, showing slightly greater activity in the aorta. The potency of the effect of SR 47063 on HSV and RA was 12- and 58-fold greater, respectively, than that reported for the structurally related K(+)-channel opener levcromakalim. The vasorelaxant action of SR 47063 in both blood vessels was strongly inhibited by 3 microM glibenclamide, consistent with a mechanism of action involving ATP-dependent K(+)-channels.

Actions of L-NAME and methylene blue on the hypotensive effects of clonidine and rilmenidine in the anesthetized rat.

The antihypertensive mechanism of alpha2-adrenoceptor agonists, such as clonidine and rilmenidine, is not completely elucidated, although it is probably due to reduction of sympathetic tone mediated by stimulation of central alpha2-adrenoceptors. Because activation of alpha2-adrenoceptors on endothelial cells induces release of endothelium-derived relaxing factor (EDRF), we determined whether nitric oxide (NO) release is involved in the antihypertensive action of clonidine and rilmenidine. In chloralose-anesthetised Wistar rats, systolic and diastolic arterial blood pressures were recorded on a polygraph. Intravenous injection of clonidine or rilmenidine (control group) caused a rapid increase of arterial blood pressure. followed by a long-lasting hypotensive effect. The hypotensive effects, estimated as the area enclosed by the decrease in diastolic pressure during the 20 min after clonidine and rilmenidine injections, were 574+/-60 and 410+/-59 mm Hg/min, respectively. The delta decrease in diastolic arterial blood pressure observed 20 min after intravenous injections of clonidine and rilmenidine was 48+/-5 and 34+/-3 mm Hg, respectively. Clonidine and rilmenidine injected 5-10 min after intravenous pretreatment with L-NAME (2 and 1 mg/kg) or methylene blue (10 mg/kg) induced hypotensive effects that were significantly smaller than that observed for the control group. These results suggest that the antihypertensive effects of clonidine and rilmenidine also may be modulated by the NO-cyclic guanosine monophosphate (cGMP) pathway at the level of the central nervous system and/or at the vascular peripheral circulation.

Studies on the anti-allergic activity of Mikania glomerata.

A fraction (MG1) obtained from the ethanolic extract of Mikania glomerata Sprengel (Compositae), popularly known as 'guaco' and used as 'an' anti-allergic and anti-inflammatory agent, was evaluated for these properties on ovalbumin-induced allergic pleurisy and in models of local inflammation induced by biogenic amines, carrageenan and PAF. Plasma exudation as well as neutrophil and eosinophil infiltration evoked by the intrapleural injection of the antigen were significantly reduced by the fraction. Likewise, PAF-induced pleural neutrophil migration was inhibited by the treatment with MG1. On the other hand, pre-treatment of the animals with MG1 failed to modify the pleurisy induced by histamine, serotonin or carrageenan. These results suggest that MG1 is effective in inhibiting immunologic inflammation but did not affect acute inflammatory response caused by other agents.

Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster).

Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0-6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N-alpha-tosyl-L-Arg methyl esterase (955.38 units/mg) and N-Bz-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A-alpha and B-beta chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein.

Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat.

1. The effects of niflumic acid, an inhibitor of calcium-activated chloride currents, were compared with the actions of the calcium channel blocker nifedipine on noradrenaline- and 5-hydroxytryptamine (5-HT)-induced pressor responses of the rat perfused isolated mesenteric vascular bed. 2. Bolus injections of noradrenaline (1 and 10 nmol) increased the perfusion pressure in a dose-dependent manner. Nifedipine (1 microM) inhibited the increase in pressure produced by 1 nmol noradrenaline by 31 +/- 5%. Niflumic acid (10 and 30 microM) also inhibited the noradrenaline-induced increase in perfusion pressure and 30 microM niflumic acid reduced the pressor response to 1 nmol noradrenaline by 34 +/- 6%. 3. The increases in perfusion elicited by 5-HT (0.3 and 3 nmol) were reduced by niflumic acid (10 and 30 microM) in a concentration-dependent manner and 30 microM niflumic acid inhibited responses to 0.3 and 3 nmol 5-HT by, respectively, 49 +/- 8% and 50 +/- 7%. Nifedipine (1 microM) decreased the pressor response to 3 nmol 5-HT by 44 +/- 9%. 4. In the presence of a combination of 30 microM niflumic acid and 1 microM nifedipine the inhibition of the pressor effects of noradrenaline (10 nmol) and 5-HT (3 nmol) was not significantly greater than with niflumic acid (30 microM) alone. Thus the effects of niflumic acid and nifedipine were not additive. 5. In Ca-free conditions the transient contractions induced by 5-HT (3 nmol) were not reduced by 30 microM niflumic acid, suggesting that this agent does not inhibit calcium release from the intracellular store or the binding of 5-HT to its receptor. 6. Niflumic acid 30 microM did not inhibit the pressor responses induced by KCl (20 and 60 mumol) which were markedly reduced by 1 microM nifedipine. In addition, 1 microM levcromakalim decreased pressor responses produced by 20 mumol KCl. These data suggest that niflumic acid does not block directly calcium channels or activate potassium channels. 7. It is concluded that niflumic acid selectively reduces a component of noradrenaline- and 5-HT-induced pressor responses by inhibiting a mechanism which leads to the opening of voltage-gated calcium channels. Our data suggest that the Ca(2+)-activated chloride conductance may play a pivotal role in the activation of voltage-gated calcium channels in agonist-induced constriction of resistance blood vessels.

Participation of kinins in the inhibitory action of captopril on acute hypertension induced by L-NAME in anesthetized rats.

The aim of the present study was to investigate the role of bradykinin in the inhibitory action of captopril in hypertension induced by L-NAME in anesthetized rats. Male Wistar rats (260-320 g) were anesthetized with chloralose and arterial blood pressure was recorded with a polygraph pressure transducer. The hypertensive effect of L-NAME was studied in rats pretreated with saline, captopril or HOE 140 plus captopril. The effect of captopril was also studied during the sustained pressor effect of L-NAME. The acute pressor effect of L-NAME (10 mg/kg, i.v.) was significantly reduced by i.v. pretreatment with 2 mg/kg captopril (delta increase of 49 +/- 4.9 mmHg reduced to 20 +/- 5.4 mmHg, P = 0.01). The pressor effect of L-NAME (delta increase of 38 +/- 4.8 mmHg) observed in rats pretreated with captopril and HOE 140 (0.1 mg/kg, i.v.) was not significantly different from that induced by L-NAME in rats pretreated with saline (P = 0.09). During the sustained pressor effect induced by L-NAME (delta increase of 49 +/- 4.9 mmHg) captopril induced a significant (P < 0.05) reduction in arterial blood pressure (delta decrease of 22 +/- 3.0 mmHg). The present results demonstrate that the acute pressor effect of L-NAME is reduced by captopril and this inhibitory effect may be partly dependent on the potentiation of the vasodilator actions of bradykinin.

Effects of potassium channel modulators cromakalim, tetraethylammonium and glibenclamide on the contractility of the isolated human ureter.

PURPOSE: Experiments were performed to assess the effect of the potassium channel modulators cromakalim, tetraethylammonium (TEA) and glibenclamide on the contractility of isolated human ureteric rings. MATERIALS AND METHODS: Segments of human distal ureter obtained from kidney donors (leftovers) were cut into rings and suspended in an organ bath filled with modified Tyrode solution for measurement of isometric contractile force. The ureter was stimulated electrically or with KCl, and the contractile activity recorded on a polygraph. RESULTS: The amplitude of the contraction induced by electrical stimulation was not changed by glibenclamide but was enhanced by tetraethylammonium. The resting tension of the ureter was not changed by either potassium channel inhibitor. Cromakalim did not change the resting tension of the human ureter per se but induced a concentration-dependent inhibition of the contractions induced by electrical stimulation. This inhibitory effect of cromakalim was not changed by tetraethylammonium but was inhibited by glibenclamide. A phasic and tonic contractile response in the isolated human ureteric ring was induced by 60 mM. KCl. The phasic contractions were abolished by cromakalim whereas the tonic contractions were unaffected. Following sustained contraction induced by 25 mM. KCl, the cumulative addition of cromakalim to the organ bath produced a concentration-dependent relaxation. However, in rings precontracted with 60 mM. KCl, cromakalim at a concentration as high as 10(-5) M. did not induce relaxation. The cromakalim-induced relaxation of rings precontracted with 25 mM. KCl was significantly inhibited by glibenclamide. CONCLUSION: These results suggest that potassium channels are important in the control of human ureter contractility and that potassium channel openers may be an alternative therapeutic indication in the treatment of human ureteric colic.

Effect of niflumic acid on noradrenaline-induced contractions of the rat aorta.

1. The effects of niflumic acid, an inhibitor of calcium-activated chloride channels, were compared with the actions of the calcium channel antagonist nifedipine on noradrenaline-evoked contractions in isolated preparations of the rat aorta. 2. The cumulative concentration-effect curve to noradrenaline (NA) was depressed by both nifedipine and niflumic acid in a reversible and concentration-dependent manner. The degree of inhibition of the maximal contractile response to NA (1 microM) produced by 10 microM niflumic acid (38%) was similar to the effect of 1 microM nifedipine (39%). 3. Contractions to brief applications (30 s) of 1 microM NA were inhibited by 55% and 62% respectively by 10 microM niflumic acid and 1 microM nifedipine. 4. In the presence of 0.1 microM nifedipine, niflumic acid (10 microM) produced no further inhibition of the NA-evoked contractions. Thus, the actions of niflumic acid and nifedipine were not additive. 5. In Ca-free conditions the transient contraction induced by 1 microM NA was not inhibited by niflumic acid (10 microM) and therefore this agent does not reduce the amount of calcium released from the intracellular store or reduce the sensitivity of the contractile apparatus to calcium. 6. Niflumic acid 10 microM did not inhibit the contractions produced by KCl (up to 120 mM) which were totally blocked by nifedipine. Contractions induced by 25 mM KCl were completely inhibited by 1 microM levcromakalim but were unaffected by niflumic acid. 7. It was concluded that niflumic acid produces selective inhibition of a component of NA-evoked contraction which is probably mediated by voltage-gated calcium channels. These data are consistent with a model in which NA stimulates a calcium-activated chloride conductance which leads to the opening of voltage-gated calcium channels to produce contraction.

Differential vasorelaxant effects of levcromakalim and P1060 in the isolated KCl- and RbCl-precontracted human saphenous vein: possible involvement of intracellular Ca2+ stores.

The influence of rubidium-substituted physiological salt solution (Rb-PSS) on the relaxant effects of K+ channel openers was investigated in the human saphenous vein. In tissues precontracted with 20 mM KCl (in K-PSS) levcromakalim and P1060 produced complete, sustained relaxations. However, in Rb-PSS (containing 20 mM RbCl) these effects were inhibited and, although complete relaxations still occurred, were transient. When caffeine was applied at the beginning of this fade of levcromakalim-induced relaxation in Rb-PSS its contractile effect was potentiated. Similarly, the contraction to noradrenaline was potentiated when applied at the beginning of this fade of levcromakalim-induced relaxation, whereas this response was attenuated in control tissues bathed in 20 mM KCl (in K-PSS). Our results show that the relaxant effects of K+ channel openers in human saphenous vein are inhibited in Rb-PSS, in agreement with previous studies in animal tissue, and suggest that an increased Ca2+ uptake into intracellular stores may be contributory to vasorelaxation.

Inhibitory effects of Rb+ on the responses to levcromakalim and P1060 in the isolated human myometrium.

The mechanoinhibitory effects of two structurally dissimilar K+ channel openers, levcromakalim and P1060, and verapamil were compared in strips of human myometrium bathed in either K-PSS (normal Krebs solution) or Rb-PSS (K+ salts replaced by Rb+ equivalents). In Rb-PSS the effects of levcromakalim and P1060 on amplitude and frequency of spontaneous contractions were inhibited by more than 20- and 138-fold, respectively, whereas those of verapamil were unaltered. These results indicate that K+ channel openers possess Rb-sensitive and Rb-insensitive mechanoinhibitory actions on the human uterus, the former being more important in the effects of P1060 than levcromakalim.

Inhibitory effect of cromakalim in human detrusor muscle is mediated by glibenclamide-sensitive potassium channels.

The effects of cromakalim, a potassium channel activating drug, and glibenclamide, a relatively selective antagonist of ATP-sensitive potassium channels, have been investigated on isolated detrusor muscle from human bladder. Specimens of human bladder were cut into strips and suspended in an organ bath filled with modified Tyrode solution for measurement of isometric contractile force. Concentration-response curves to acetylcholine were constructed before and after pretreatment with cromakalim and cromakalim plus glibenclamide. The concentration-response curves to acetylcholine were displaced to the right, and the maximal response to acetylcholine was significantly inhibited by cromakalim in a concentration-dependent manner. The inhibitory effect of cromakalim on acetylcholine-induced contraction was significantly reduced by glibenclamide. Following sustained contraction induced by 20 mM. KCl, the cumulative addition of cromakalim to the organ bath produced a concentration-dependent relaxation. However, in strips precontracted with 60 mM. KCl, the addition of cromakalim in concentrations as high as 10(-5) M. did not induce relaxation. The relaxation induced by cromakalim in strips precontracted with 20 mM. KCl was significantly inhibited by glibenclamide. These results suggest that the inhibitory effect of cromakalim in human bladder involves activation of glibenclamide-sensitive potassium channels.

Actions of cromakalim in isolated human saphenous vein.

The present study was performed to evaluate the effect of cromakalim on human vein in vitro. Branches of human saphenous vein (leftovers), obtained from patients undergoing heart revascularization surgery, were cut into rings and suspended in an organ chamber filled with Krebs-Ringer solution for the measurement of isometric contractile force. Concentration-response curves to norepinephrine and serotonin were constructed before and after pretreatment with cromakalim. The concentration-response curves to norepinephrine and serotonin were displaced to the right, and the maximal responses to both agonists were significantly inhibited by cromakalim in a concentration-dependent manner. Following sustained contraction induced by prostaglandin F2 alpha or 20 mM KCl, the cumulative addition of cromakalim to the organ chamber produced a concentration-dependent relaxation. However, in veins precontracted with 60 mM KCl the addition of cromakalim in concentrations of up to 10(-5) M did not induce relaxation. The relaxation induced by cromakalim in veins precontracted with prostaglandin F2 alpha was significantly inhibited by glibenclamide. These results indicate that cromakalim has a dilator effect in human vein that may play a helpful role in the treatment of angina. The venodilator effect of cromakalim in human saphenous vein probably involves activation of adenosine triphosphate-regulated potassium channels.

Effect of cooling and warming on the vasodilator response of the isolated dog saphenous artery to acetylcholine.

Experiments were performed to investigate the effect of cooling and warming on the relaxation to acetylcholine in isolated canine saphenous arteries. Rings of saphenous artery were stimulated with norepinephrine and cumulative concentration response curves to acetylcholine were compared at 41 degrees C, 37 degrees C, 30 degrees C, 25 degrees C and 15 degrees C. The data showed that the effect of acetylcholine is depressed by warming and a tendency to increase is observed during cooling, suggesting that muscarinic receptors may be sensitive to temperature changes.

Schistosoma mansoni: new method for recording motor activity in vitro.

A new method for recording the motility of Schistosoma mansoni in vitro is described. Spontaneous activity of the worm shows contraction similar to peristalsis. The worm responded to electrical stimulation with an immediate contraction that was voltage dependent. Oxamniquine produced an increase in the tonus and spontaneous activity of the worm. This method provides a new experimental model for the study of drugs that interfere with Schistosoma mansoni motility.

Effect of disopyramide on isolated aortic ring of the rat.

Disopyramide produces a contraction of the isolated aortic ring of the rat which is graded, develops slowly, and has a time course similar to the tonic phase of the noradrenaline response. This effect is not modified by alpha-adrenoceptor blockade (yohimbine) but is completely abolished by a Ca2+ antagonist (verapamil) or by removal of Ca2+ from the bathing solution. These results indicate that, in the rat aorta, disopyramide has a vasoconstrictor effect dependent on Ca2+ influx across the vascular smooth muscle membrane.

Effect of terbutaline on the human fetal placental circulation.

The effect of terbutaline, a beta2-adrenoceptor stimulator, on the vascular resistance of isolated human placenta was examined. Terbutaline produced no change in basal placental vascular resistance, but when the placental vessels were constricted with angiotensin, terbutaline produced a graded decrease in vascular resistance. The vasodilating effect of a high dose of terbutaline was smaller than the effect 20 microgram of isoprenaline and the effect was significantly blocked by propranolol, a beta-adrenoceptor antagonist. The results suggest that the vasodilating effect of terbutaline on the fetal placental circulation can play a role in the improvement of fetal condition during treatment of premature labour and intrapartum fetal distress.