Potassium channels sensitive to combination of charybdotoxin and apamin regulate the tone of diabetic isolated canine coronary arteries.

AIMS: Functional roles of calcium-activated potassium channels on the mechanical activity of epicardial coronary arteries obtained from a canine model of diabetes were investigated. METHODS: Coronary arteries were isolated from healthy, alloxan-diabetic and insulin-treated diabetic dogs. Basal tensions, contractions induced by the prostaglandin (PG) analogue, U46619, and endothelium-dependent relaxations to acetylcholine (ACh) were modified with charybdotoxin (CHTX) + apamin (APA), inhibitors of calcium-activated potassium channels, as well as with N(omega)-nitro-l-arginine (LNA) + indomethacin (INDO) to suppress the synthesis of nitric oxide (NO) and PGs. The relaxing effect of nitroprusside-sodium (SNP), an NO donor, was also determined. RESULTS: In diabetic coronary arteries, CHTX + APA did not change while LNA + INDO elevated the basal tension. PG-induced contractions were enhanced by CHTX + APA and by LNA + INDO in all the three groups of animals. CHTX + APA decreased the maximal relaxations to ACh in a partly insulin-dependent manner. LNA + INDO abolished the endothelium-dependent relaxations to ACh. In diabetic coronary arteries, the sensitivity to SNP-induced relaxation was enhanced, insulin independently, suggesting that NO could be partly responsible for maintaining intact ACh-induced vasorelaxation. CONCLUSION: In diabetic canine coronary artery, the vasomotor responses reflect up-regulation of calcium-activated potassium channels. This endothelial mechanism of the canine epicardial coronary artery may oppose vasoconstrictions in diabetic vascular tissue.

Role of calcium-activated potassium channels in the regulation of basal and agonist-elevated tones in isolated conduit arteries. Short communication.

Functional role of calcium-activated potassium (KCa) channels on the basal and agonist-elevated arterial tones was investigated in isolated rabbit aorta, porcine and canine coronary arteries as well as in human internal mammary artery. The vascular tones enhanced by contractile agents were increased further by preincubation of these conduit blood vessels with selective (charybdotoxin or iberiotoxin) or nonselective (tetraethylammonium) inhibitors of KCa channels. The basal tone (without an agonist) was increased only in the canine coronary artery. The results indicate a feed-back regulatory role of KCa channels counteracting the vasospasm of conduit arteries.

Improvement by phosphoramidon of damaged endothelial function in porcine coronary artery.

BACKGROUND: The bradykinin (BK)-induced endothelium-dependent relaxation is impaired in the presence of elevated potassium concentration enhancing the vasospastic tendency of large coronary arteries. Inhibition of the angiotensin-converting enzyme responsible for bradykinin degradation was found to enhance the endothelium-dependent relaxation by BK. The aim of the present study was to investigate the effect of phosphoramidon, known to inhibit a BK-metabolizing neutral endopeptidase enzyme, on relaxation of porcine-isolated coronary artery in depolarizing solution. METHODS: Endothelium intact porcine coronary artery rings were studied in organ chambers. The rings were isometrically contracted with potassium chloride (30 mmol/L) and the response to BK (1 to 1,000 nmol/L)-induced relaxation was investigated in the presence of nitric oxide synthase inhibitor Nomega-nitro-L-arginine (300 micromol/L) alone and in combination with the cyclooxygenase inhibitor indomethacin (10 micromol/L), and that of the inhibitor of calcium-dependent potassium channels tetraethylammonium (7 mmol/L). Under these conditions, phosphoramidon (10 micromol/L), an inhibitor of a neutral endopeptidase enzyme (EC.3.4.24.11.), which is responsible for the degradation of BK, was used to enhance the endothelium-dependent relaxation. RESULTS: Phosphoramidon potentiated the maximum vasorelaxant effect of BK in Nomega-nitro-L-arginine (control 26.6%+/-10.86% versus phosphoramidon 49.05%+/-4.52%; n = 6, p < 0.05) or in Nomega-nitro-L-arginine + indomethacin-pretreated rings (control 20.7%+/-9.92% versus phosphoramidon 42.0%+/-12.26%; n = 5, p < 0.05) and this increased vasodilation was not modified by tetraethylammonium. CONCLUSIONS: In the present study phosphoramidon potentiated the effect of BK in the absence of nitric oxide and prostaglandins in porcine-isolated coronary artery. This effect did not depend on tetraethylammonium-sensitive potassium channels. Phosphoramidon may be a useful pharmacologic tool for preserving the vasorelaxing capacity of coronary arteries after cardioplegia.

Comparison of the vasorelaxing effect of cromakalim and the new inodilator, levosimendan, in human isolated portal vein.

In the present study the vasorelaxing capacity of cromakalim, an ATP-sensitive potassium-channel (KATP channel) activator, and that of levosimendan, a new positive inotropic and vasodilating drug with calcium sensitizing and potassium-channel-activating properties, were compared in human isolated portal vein. Based on the 50% effective concentrations (EC50), levosimendan was found to be about 16-fold more potent (EC50 = 0.281+/-0.03 microM) as a relaxing agent than cromakalim (EC50 = 4.53+/-0.12 microM) in noradrenaline-precontracted portal venous preparations. Glibenclamide, the known inhibitor of KATP channels, was able to prevent the cromakalim-induced venodilation completely. Glibenclamide (15 microM) decreased the quasi-maximal effect of levosimendan (at 1.27 microm by about 60%) and also the effects of those submicromolar concentrations of the inodilator (at 0.1 microM by 23%, at 0.3 microM by 27% and at 0.7 microM by 19%, on average) which were therapeutically effective in preliminary human studies. These findings indicate that, in the human portal vein, both cromakalim and levosimendan are powerful vasorelaxants and that a considerable part of the relaxing effect induced by levosimendan is of cromakalim type.

Effects of pituitary adenylate cyclase-activating polypeptide on the cyclooxygenase pathway of rat platelets and on platelet aggregation.

Several data suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of local circulation. One possible role of PACAP in the regulation of circulation is that, it may modify the cyclooxygenase pathway of the arachidonate cascade in platelets. Our study was designed to study the effect of PACAP on the cyclooxygenase pathway of rat platelets and on platelet aggregation. PACAP (10(-7) and 10(-6) M) significantly inhibited the cyclooxygenase pathway of platelets, mostly the thromboxane synthesis. Pretreatment with a PACAP receptor antagonist, PACAP(6-38), or with an inhibitor of protein kinase A, H-89, shows that the effects of PACAP on the cyclooxygenase pathway were diminished. In the aggregation studies, PACAP inhibited both the arachidonic acid-induced and the thrombin-induced platelet aggregation. It can be concluded that PACAP inhibits the cyclooxygenase pathway of rat platelets via a specific PACAP receptor-activated, cAMP-dependent pathway, and these effects of PACAP are involved in the inhibition of platelet aggregation.

Vasoactive substances produced by cultured rat brain endothelial cells.

The vasoactive substances synthesized by primary cultures of rat brain endothelial cells were investigated and compared to those from two, immortalized cell lines, RBE4 and GP8. The vasoactivity of endothelium-derived substances was measured on isolated canine coronary artery. Vascular tone was significantly decreased by both primary and GP8, but not by RBE4 cells. Indomethacin pretreatment of primary and GP8 cells turned vasorelaxation into contraction while N(omega)-nitro-L-arginine pretreatment decreased the vasorelaxation induced by primary, but not by GP8 cells. Eicosanoid production was determined after incubation with [14C]arachidonic acid. The predominant vasoactive eicosanoid was prostaglandin E2 in both primary and GP8 cells. RBE4 cells synthetized mainly prostaglandin E2 and thromboxane B2 and significantly less prostaglandin E2 than did either primary or GP8 cells. The capacity of cerebral endothelium to regulate vascular tone by production of dilator and constrictor substances can be preserved under certain circumstances in immortalized cell lines.

Differential efficacy of vasodilators in hypercholesterolaemic rabbits.

The effects of hypercholesterolaemia on the endothelium-dependent and -independent vascular reactivity of the superior mesenteric artery has been examined in anaesthetized rabbits in-vivo. Rabbits were fed with either standard or cholesterol-enriched diet for 24 weeks. Plasma lipids and changes in the endothelin content of plasma and vascular tissue were measured in the superior mesenteric artery and in the thoracic aorta. The functional severity of atherosclerosis was determined by examining vascular responses in the isolated thoracic aorta. The blood flow in the superior mesenteric artery was measured by transit-time flowmetry and drugs were injected through an intra-abdominal aortic catheter. Acetylcholine (5, 10, 20 microg kg(-1)) elicited dose-dependent, mesenteric vasodilation in normocholesterolaemic rabbits. In hypercholesterolaemic animals the response to acetylcholine was completely abolished and even became a vasoconstriction. Endothelin levels in plasma and in the vascular tissue were significantly elevated in hypercholesterolaemic animals compared with controls. Cromakalim at a dose of 3 microg kg(-1), elicited similar mesenteric vasodilation in hypercholesterolaemic and normocholesterolaemic animals. These experiments show that the endothelium-dependent responses of the superior mesenteric artery to acetylcholine are functionally impaired by prolonged hypercholesterolaemia, that this altered vascular reactivity is associated with the elevation of endothelin levels in the circulation and in vascular tissues, and that in hypercholesterolaemia the mesenteric vasodilator effect of the K+-channel opener cromakalim is entirely preserved, suggesting that severe hypercholesterolaemia does not depress the function of ATP-sensitive potassium channels in mesenteric vascular smooth muscle.

Polarographic detection of nitric oxide released from cardiovascular compounds in aqueous solutions.

In order to detect the concentration of nitric oxide, known to be one of the biologically active principles of certain cardiovascular compounds, a highly selective polarographic/amperometric device was used. The nitric oxide-releasing properties of sodium nitroprusside, nitroglycerine, nicorandil, and the molsidomine metabolite, 3-morpholinosydnonimine, were compared in the following cell-free experimental solutions in vitro: in Krebs-Henseleit solution with and without a sulfhydryl donor, L-cysteine, in an acidic, reducing medium, and in Krebs-Henseleit solution with superoxide dismutase enzyme. Sodium nitroprusside released similar concentrations of nitric oxide in Krebs-Henseleit solution and in the acidic, reducing medium. L-Cysteine inhibited the release of nitric oxide at physiological pH. In the presence of nitroglycerine, nitric oxide signals were detected in the acidic, reducing environment and in L-cysteine-rich Krebs-Henseleit solution but not in the absence of the sulfhydryl donor. Amperometric signals could not be detected after adding nicorandil in all the experimental conditions used. 3-Morpholinosydnonimine released nitric oxide only in the presence of the superoxide dismutase enzyme. Our results suggest that the polarographic electrode is able to detect the release of nitric oxide from sodium nitroprusside, nitroglycerine, and 3-morpholinosydnonimine in the absence of biological material. The present observations support the importance of the chemical environment during the detection of nitric oxide from donor compounds in the common in vitro bathing systems.

Endothelium-dependent vasorelaxant and anti-aggregatory effect and mechanism of action of some antifibrinogen RGD (Arg-Gly-Asp-containing) peptides.

Vasorelaxation caused by some antifibrinogen RGD (Arg-Gly-Asp-containing) peptides and their basic mechanism of action was studied on rabbit isolated thoracic aortic rings preconstricted with 0.25 microM phenylephrine. GRGDS (Gly-Arg-Gly-Asp-Ser-OH) and RGDV (Arg-Gly-Asp-Val-OH) caused dose-dependent relaxation. RGDS (Arg-Gly-Asp-Ser-OH) had a biphasic effect (a transient relaxation followed by a contraction) while GRGDS-[SE] (Gly-Arg-Gly-Asp-Ser(SO3)-OH) did not change the isometric tone of precontracted aortic preparations. GRGDS and RGDV exerted no relaxing effect on endothelium-denuded blood vessels suggesting that the vascular action of these peptides was entirely dependent on the presence of functionally intact endothelium. L-NG-Nitro-arginine (30 microM) attenuated the relaxation induced by GRGDS and abolished that induced by RGDV. All of the four RGD congeners inhibited ADP-induced aggregation of human platelets. These findings indicate that the relaxant effect of RGDV is mediated exclusively by the nitric oxide pathway, but GRGDS could cause, besides nitric oxide release, the release of another substance which is different from nitric oxide. Because the rank order of the vasorelaxant potencies of RGD peptides differed from that found for their anti-aggregatory activities, a vascular effector mechanism mediated by an RGD-recognizing structure other than the known glycoprotein IIb/IIIa-like RGD-binding site is suggested.

Nitric oxide activates an iberiotoxin-sensitive potassium channel in human saphenous vein.

Synthetic iberiotoxin (IBTX), an inhibitor of large conductance calcium-activated potassium channel (BKCa), was used to study the possible involvement of a specific hyperpolarizing ion channel in nitric oxide (NO)-induced venodilation. Serotonin (0.125 microM)-induced contraction of isolated human saphenous vein was dose-dependently relaxed with 50-1550 nanoM exogenous NO. 30 min preincubation of venous preparations with 90 nanoM IBTX decreased vasorelaxation induced by NO. In conclusion, a hyperpolarizing potassium channel, BKCa, may be involved in the venodilator effect of endogenous NO.

Effect of selective inhibition of potassium channels on vasorelaxing response to cromakalim, nitroglycerin and nitric oxide of canine coronary arteries.

A comparative study was performed on the sensitivity of in-vitro vasorelaxation by nitroglycerin and cromakalim to block glibenclamide, a blocker of ATP-sensitive potassium channels, and iberiotoxin, a selective inhibitor of large-conductance calcium-activated potassium channels. In isolated canine coronary arteries preconstricted with 25 microM prostaglandin F2 alpha, nitroglycerin (0.005-1.8 microM) and cromakalim (0.15-9.6 microM) produced dose-dependent vasodilations. Glibenclamide (30 microM) had no significant effect on relaxation of the dose-response curve to nitroglycerin and almost completely abolished the relaxation by cromakalim, a known opener of ATP-sensitive potassium channels. Iberiotoxin (90 nM) decreased the maximal response to nitroglycerin and had no effect on the vasodilation induced by cromakalim. The effect of iberiotoxin on the vasorelaxing action of nitric oxide, the active metabolite of nitroglycerin, was also examined. In a low potassium chloride (14.4-20.4 mM) medium, as a contractile stimulus, iberiotoxin inhibited relaxations by exogenous nitric oxide (100-200 nM). Enhancement of potassium concentrations to 35.4-40.4 mM significantly decreased relaxation by nitric oxide and under these conditions the inhibitory action of iberiotoxin disappeared. The present study demonstrated that in canine coronary arteries, the functional role of two potassium channels can be separated by pharmacological means. Nitroglycerin-induced vasorelaxation may be mediated, at least in part, by its enzymatic breakdown product, nitric oxide that activates large-conductance calcium-activated potassium channels.

Synthesis of two peptide scorpion toxins and their use to investigate the aortic tissue regulation.

The 37 amino acid residue polypeptides iberiotoxin and charybdotoxin, which contain three disulfide bridges, were chemically synthesized and characterized. The physiological effectiveness of these peptides was tested on rabbit aorta in vitro.

Cardioprotection: endogenous protective mechanisms promoted by prostacyclin.

Evidence is accumulating that acute stress situations such as ischemia, adrenergic dominance, and ouabain intoxication enhance production of endogenous substances (PgI2, adenosine, NO) which may protect the myocardium from harmful consequences of these stress situations. PgI2 and its stable analogue 7-oxo-PgI2 exert an early direct- and induce a delayed indirect antiischemic, antiarrhythmic, and cytoprotective effect. The direct action is shortlasting; it protects from myocardial ischemia and arrhythmias, at least partly, by its vasodilating, antiaggregatory, and "membrane-stabilizing" effects. The delayed, long-lasting PgI2-induced protection from postocclusion, reperfusion- and ouabain-arrhythmias is dose- (optimal 50 micrograms/kg) and time- (optimal 48 h after treatment) dependent. Its mechanism is probably based on a 7-oxo-PgI2 induced increase in the activity of Na/K-ATP-ase, and further, on a reduced sensitivity to beta-adrenergic agonists and to changes at the cardiac membrane level, resulting in a prolongation of the action potential duration and the effective refractory period.

The use of microcarrier beads in the production of endothelium-derived relaxing factor by freshly harvested endothelial cells.

This study is concerned with the use of freshly harvested bovine endothelial cells attached to microcarrier beads in the production of the endothelium-derived relaxing factor (EDRF). The results are compared to production of EDRF by endothelial cells grown in tissue cultures. We found that freshly harvested cells attach themselves to microcarrier beads within minutes. This results in large surface/area volume ratio and permits superfusion of cells suspension on a filter (pore size of 25-30 microns), resulting in cell free filtrate. When superfusing an endothelium-deprived pulmonary artery strip, the effluent causes relaxation; the response depends on the number of superfused endothelial cells. The number of viable freshly harvested cells attached to microcarrier beads in 5 ml Krebs-Henseleit solution is small (30%), as compared to almost 100% for cultured cells. Despite this difference, percent relaxation induced for the same number of viable cells is identical for both groups. Scanning electromicrographs confirm anchorage of endothelial cells to microcarrier beads. While cultured cells cover the entire surface and are individually attached, freshly harvested cells are anchored as cell aggregates leaving some of the surface free. Attachment of freshly harvested endothelial cells to microcarrier beads offers an alternative for the study of the role of endothelial cells in the production of vasoactive substances.

Pressure-induced contractions in bovine pulmonary artery preparations.

Experiments were conducted in a superfusion bioassay system in which the effluent of a bovine main pulmonary artery (generator) was allowed to superfuse a spirally cut precontracted bovine arterial strip (detector), which had been deprived of endothelium. Effluents of endothelium-intact generator relaxed the precontracted detector by an average of 22 +/- 4.1%. Following removal of endothelium of the generator, the relaxing effect of the effluent was only 1%. A rapid increase of transmural pressure in the generator to about 40 mm Hg for 3 min induced contraction of the detector (8.4 +/- 2%) above the histamine-induced tension. In the absence of endothelium, smaller contractions were obtained from the pressurized generator (4.9 +/- 1.8%). This effect was reversible after pressure release. Effluents of endothelium-deprived generator showed no significant vasoactivity. NG-Mono-methyl-L-arginine (L-NMMA, 3.3 x 10(-5) mol/l), a specific inhibitor of nitric oxide (NO), also depressed endothelium-dependent relaxation and caused a 10.5% increase in contraction of the endothelial effluent. When the endothelium-intact generator was exposed to both pressure and L-NMMA, an additive effect was noted. Based on these observations, it was concluded that transient increase of transmural pressure in bovine main pulmonary arteries depresses the endothelium-dependent relaxation and unmasks a small but significant contractile activity of the endothelial effluent.

A model to study physiological activation of phospholipase A2 and vasorelaxation by lysophosphatidylcholine.

Earlier we demonstrated that micellar solutions of LPC caused endothelium-dependent relaxation of rabbit thoracic aorta and bovine intrapulmonary artery and vein through a cyclic GMP-dependent mechanism. The availability of LPC for vasorelaxation depends on its production by deacylation of PC by PLA2. We assessed the possible activation of PLA2 by commonly used vasorelaxants such as acetylcholine, bradykinin, calcium ionophore A23187 and thrombin and vasoconstrictors like histamine and phenylephrine in the presence of indomethacin in a model system where 14C PC was incorporated into bovine intrapulmonary arterial segments. Taking the ratio of 14C PC:LPC formed by exogenous PLA2 as an index of deacylation, we found that while all the agents relaxed the strips in an endothelium-dependent manner, only thrombin caused relaxation followed by an increase in 14C LPC and a concomittant decrease in 14C PC indicating activation of PLA2. Our data show that PC/PLA2 system can be activated to generate LPC for vascular relaxation under specific physiological conditions. This model system can be used to monitor PLA2 activity and LPC production to compensate flow and pressure induced changes in arteries.

Sympathetic neural mechanisms in cardiac arrhythmias.

In the present experiments the dominant role in the genesis of arrhythmias of stimulation of beta adrenoceptors over that of alpha adrenoceptors has been demonstrated by: (i) infusion of catecholamines in the isolated right ventricular Purkinje fiber preparation of rabbits; and (ii) by subepicardial microinfusion of these substances to anesthetized thoracotomized dogs. Activation of the latent pacemaker areas by local myocardial ischemia was shown. Catecholamine-induced arrhythmias also resulted from premature excitation of the working myocardium as a consequence of an interaction of Purkinje firing and excitation originating from higher pacemaker areas.

Delayed antiischemic effect of prostaglandin I2 and of a new stable prostaglandin I2 analogue, 7-oxo-prostacyclin-Na, in experimental model angina in dogs.

Therapeutic application of prostaglandin I2 (PGI2) is rendered difficult by its instability. The 7-oxo-derivative synthesized by Kovács et al. [8] proved to be stable in aqueous solution. Both drugs were tested in experimental model angina in anesthetized thoracotomized dogs with critical stenosis of the left anterior descending coronary artery, in which drug-induced reduction of ischemic ST-segment elevation evoked by frequency loading was estimated [10]. Both drugs markedly reduced blood pressure (BP) even if given by intravenous infusion for 60 min in a total dose of 20 micrograms/kg for PGI2 and 250 micrograms/kg for 7-oxo-PGI2-Na. BP returned to normal soon after infusion and did not substantially change. After a latency of 90-120 min, a 40-50% reduction of ischemia-induced ST-segment elevation appeared in the epi-, endo-, and intramyocardial ECG. Intracoronary infusion of PGI2 in 1 microgram/kg and 7-oxo-PGI2-Na in 25 micrograms/kg total dose minimized systemic actions, but a delayed protective effect appeared, due probably to some nonvasoactive metabolite of these substances. Although both substances exert a potent platelet-aggregation-inhibiting action, serial ex vivo determination of ADP aggregation at different times has shown a dissociation of antiaggregatory action from the protection, this dissociation reaching its maximum when antiaggregatory action was already over.

On the late antiischaemic action of the stable PgI2 analogue: 7-oxo-PgI2-Na and its possible mode of action.

Earlier we have shown in the dog model mimicking angina on effort a delayed antiischaemic effect of PgI2 and its stable analogue 7-oxo-PgI2-Na, appearing only when the drug induced marked vasodilatation was over [1]. In the present experiments we could show that the protective effect appears at a time when the blood pressure returned to normal and in addition the marked platelet aggregation inhibitory effect has also faded away. In the rat 7-oxo-PgI2 could substantially diminish vasopressine induced T-wave elevation in the ECG if given 2 hours before administration of vasopressin. In addition it could moderate the vasopressin induced metabolic changes appearing as diminution of the myocardial CP and ATP-level and increase of the myocardial lactate content. A similar metabolic protection was found in the heart of rats pretreated with 7-oxo-PgI2 2 hours before taking myocardial samples and exposing them for 1 minute to ischaemia by incubation in Ringer solution. It is concluded that a direct metabolic and hemodynamic effect could be at least partly responsible for the late antiischaemic effect of 7-oxo-PgI2. This effect was also present in the early phase of experimental myocardial infarction in conscious rats if animals were pretreated with 7-oxo-PgI2 2 hours before occlusion. However treatment did not increase survival rate and failed to reduce the incidence and severity of arrhythmias.