Biomedical subjects

A Mülsch

Publications and source records attributed to A Mülsch.

At least 73 records · Page 4Linked to original sources

PubMed1990

Induction and activity of NO synthase in bone-marrow-derived macrophages are independent of Ca2+.

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2'-amino-5'-methylphenoxy)ethane-NNN'N'-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.

Aluminum↗

PubMed1990

Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin.

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.

Amino Acid Oxidoreductases↗

PubMed1990

cAMP attenuates autacoid release from endothelial cells: relation to internal calcium.

The effects of elevated levels of adenosine 3',5'-cyclic monophosphate (cAMP), in cultured endothelial cells from bovine aorta, on the ATP-induced increases in the intracellular free calcium concentration [( Ca2+]i) and the release of prostaglandin I2 (PGI2) and endothelium-derived relaxant factor (EDRF) were investigated. Endothelial cAMP production was assessed in terms of cAMP release in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine; this release was increased fivefold by isoproterenol (1 microM), 1.6-fold by isoproterenol (0.1 microM), and 1.5-fold by the stable PGI2 analogue iloprost (10 microM). [Ca2+]i, measured with the fluorescent probe indo-1, was increased by ATP (1 microM) from 150 +/- 20 (SE) to 410 +/- 50 nM. Neither isoproterenol nor iloprost changed [Ca2+]i in unstimulated cells, but they significantly reduced [Ca2+]i levels in the presence of ATP. Similar inhibitions of increases in [Ca2+]i as by iloprost and isoproterenol (0.1 microM) were evoked by dibutyryl-cAMP (100 microM). Release of PGI2 was enhanced from 3.9 +/- 0.5 to 34.6 +/- 6 ng.min-1.5 x 10(6) cells-1 by ATP (3 microM); in the presence of isoproterenol, the ATP-stimulated release was reversibly reduced to 18.1 +/- 4.9 ng/min. Release of EDRF was assayed in terms of its stimulatory action on purified soluble guanylate cyclase. EDRF release in the first minute after stimulation with ATP (10 microM) was significantly attenuated by isoproterenol from 32.3 +/- 4.8 to 23.0 +/- 4.6 nmol.min-1.mg-1 (activity of soluble guanylate cyclase).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1990

L-arginine-dependent nitric oxide formation and nitrite release in bone marrow-derived macrophages stimulated with bacterial lipopeptide and lipopolysaccharide.

This study shows that stimulating bone marrow-derived macrophages with either lipopolysaccharide (LPS) or the lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)- cysteinyl-alanyl-glycine (Pam3Cys-Ala-Gly), a synthetic analogue of the N-terminal part of bacterial lipoprotein, leads to the formation of nitric oxide (NO) and nitrite (NO2-), a stable analogue of NO. NO was detected by applying the chemiluminescence method and by measuring the activity of exogenously added soluble guanylate cyclase (GC), which is strongly and selectively activated by NO. Synthesis of NO and NO2- occurs via activation of the L-arginine and NADPH-dependent enzyme(s) present in the cytosol of bone marrow-derived macrophages. No produced by this non-constitutive L-arginine pathway is thought to be responsible for the cytostatic and killing properties of macrophages (Stuehr & Nathan, 1989). Macrophages stimulated either with LPS or Pam3Cys-Ala-Gly exhibited a 6-hr lag time before engaging in nitrite synthesis, a time at which expression of the NO-forming enzyme had already reached its maximum. The regulation of NO and NO2- synthesis during macrophage development seems to differ from that of cytokine synthesis. Whereas cytokine release varies during a culture period up to 20 days, NO synthesis and expression of the NO-forming enzyme remain unaltered. These studies show that, similar to LPS, Pam3Cys-Ala-Gly is a potent activator of 'the oxidative L-arginine pathway' in bone marrow-derived macrophages. Whether both stimuli use the same signal transfer mechanism to induce this pathway and whether NO synthesized by this pathway is involved in the activation of the enzyme guanylate cyclase in macrophages requires clarification.

Animals↗

PubMed1989

Under anaerobic conditions, soluble guanylate cyclase is specifically stimulated by glutathione.

Various thiols exert non-specific effects on the activity of soluble guanylate cyclase under aerobic conditions. We studied the effects of thiols under anaerobic conditions (pO2 less than 6 Torr) on soluble guanylate cyclase, purified from bovine lung. Reduced glutathione stimulated the enzyme concentration-dependently with half-maximal enzyme stimulation at a concentration of about 0.5 mM. The extend of maximal enzyme stimulation (up to 80-fold) was comparable with the activation by NO-containing substances. The activation by glutathione was additive with the effect of sodium nitroprusside. Cysteine and various other thiols increased the enzyme activity 20-fold and 2- to 5-fold, respectively. The stimulatory effect of these thiols was not related to their reducing potency. Activation of soluble guanylate cyclase by glutathione was dose-dependently reduced in the presence of other thiols (cysteine greater than oxidized glutathione greater than S-methyl glutathione). Under aerobic conditions or with Mn-GTP as substrate, the effect of glutathione on soluble guanylate cyclase was suppressed. The results suggest a specific role for glutathione in the regulation of soluble guanylate cyclase activity and a modulation of this effect by redox reactions and other intracellular thiols.

Anaerobiosis↗

PubMed1989

Generation of nitric oxide by human neutrophils.

Human neutrophils were evaluated for their ability to generate nitric oxide. Neutrophils incubated with superoxide dismutase at 37 degrees C produce nitrite anion at a rate of 1.8 nmols/2 x 10(6) cells/30 min, providing indirect evidence of nitric oxide production. Incubation of the neutrophils with concentrations of serum-opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine, or phorbol myristate acetate sufficient to stimulate the respiratory burst and lysosomal enzyme release caused no additional nitrite anion production. Glass wool-adherent neutrophils exhibited a similar dissociation of nitrite anion production from the respiratory burst and lysosomal enzyme release. Direct evidence for nitric oxide production was also obtained using nitric oxide-specific chemiluminescence. These results demonstrate that human neutrophils are capable of generating nitric oxide.

Anions↗

PubMed1989

Endothelium- and sydnonimine-induced responses of native and cultured aortic smooth muscle cells are not impaired by nitroglycerin tolerance.

Tolerance to the cyclic GMP-mediated vasodilator action of nitroglycerin develops with prolonged exposure and may be mediated either by formation of less nitric oxide from nitroglycerin or by desensitization of soluble guanylate cyclase to activation with nitric oxide. In the latter case, smooth muscle cells tolerant to nitroglycerin should show cross-tolerance to nitric oxide released from sydnonimines and endothelial cells (endothelium-derived relaxing factor). Therefore cultured smooth muscle cells from rabbit aorta were pretreated for 1 h with vehicle or high concentrations (0.55 mM) of nitroglycerin or the sydnonimine SIN-1. The formation of cyclic GMP induced by subsequent small doses of nitroglycerin, sydnonimine SIN-1 and endothelium-derived relaxing factor (released from cultured endothelial cells) was compared with the changes in activation of soluble guanylate cyclase, cyclic GMP formation and vasodilation in response to the same stimuli in similarly pretreated segments from rabbit thoracic aortae. Both cultured and native smooth muscle cells remained responsive to stimulation with sydnonimine SIN-1 and endothelium-derived relaxing factor after pretreatment with nitroglycerin, vehicle, or sydnonimine SIN-1, even though they were tolerant to nitroglycerin after pretreatment with nitroglycerin. In contrast, activation of soluble guanylate cyclase by nitroglycerin and sydnonimine SIN-1 was attenuated in homogenates of nitrate-tolerant aortae. The findings suggest that nitroglycerin tolerance in intact cells does not involve desensitization of soluble guanylate cyclase, because in intact cells nitrate tolerance can be overcome by direct activators of soluble guanylate cyclase.

Animals↗

PubMed1989

LY 83583 (6-anilino-5,8-quinolinedione) blocks nitrovasodilator-induced cyclic GMP increases and inhibition of platelet activation.

We studied the effects and the mechanism of action of the cyclic GMP-lowering substance 6-anilino-5,8-quinolinedione (LY 83583) on cyclic GMP-mediated inhibition of platelet function. The activation of washed human platelets by thrombin was counteracted by 8-bromo-cyclic GMP and the direct activators of soluble guanylate cyclase, sodium nitroprusside and endothelium-derived relaxant factor (EDRF = nitric oxide). LY 83583 significantly antagonized the inhibitory effect of sodium nitroprusside and EDRF, but not that of 8-bromo-cyclic GMP, on thrombin-induced aggregation, ATP-release, adhesion to native endothelial cells and increase in concentration of free intracellular calcium ions. In accordance, increases in intracellular cyclic GMP by sodium nitroprusside and EDRF were attenuated by LY 83583. The inhibition of cyclic GMP-mediated effects on platelets by LY 83583 could be related to inhibition of platelet soluble guanylate cyclase, as the activation of the purified enzyme from platelets by sodium nitroprusside was directly inhibited by LY 83583. This effect of LY 83583 was attenuated in the presence of superoxide dismutase. Our findings support the hypothesis that sodium nitroprusside and EDRF inhibit platelet activation by stimulation of soluble guanylate cyclase via nitric oxide. Consequently, inhibition of nitric oxide-induced cyclic GMP formation by LY 83583, which may act by intracellular generation of superoxide anions, facilitates platelet activation.

Adenosine Triphosphate↗

PubMed1989

Nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism.

Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase co-incubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 +/- 0.5-fold (from 31 +/- 9 to 153 +/- 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 +/- 15% (with 2 microM free calcium; EC50 0.3 microM). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 microM) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation NG-nitro-L-arginine (1 mM) and by LY 83583 (1 microM), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells.

Aminoquinolines↗

PubMed1989

Modulation of the vasodilator action of SIN-1 by the endothelium.

We studied the influence of endothelium-derived relaxing factor (EDRF) on sydnonimine (SIN-1)-induced vasodilatation and the accumulation of cyclic GMP in the rabbit femoral artery. The potency of SIN-1 to elicit vasodilatation in norepinephrine-contracted femoral arteries was significantly enhanced in the absence of the endothelium or following impairment of the synthesis of EDRF with gossypol or NG-nitro-L-arginine, whether the application of SIN-1 was intra- or extraluminal. The increase in cyclic GMP in the femoral segments by a combination of SIN-1 and endothelium-derived relaxant factor (released by the endothelium of either the rabbit thoracic aorta or the femoral artery) was significantly less than the sum of the increases in cyclic GMP induced by each agent alone. In contrast, stimulation of purified soluble guanylate cyclase by submaximal concentrations of SIN-1 was additive with the effect of EDRF, released from acetylcholine-stimulated rabbit aortas. This indicates the absence of a direct interaction between the factor and SIN-1 at the level of soluble guanylate cyclase. The interaction seems to be specific for cyclic GMP-mediated responses, since cyclic AMP-induced dilatations elicited by isoproterenol were not affected by the presence of the endothelium. The results indicate that the endothelium can modulate the vascular reactivity to SIN-1. This modulation may be mediated either by EDRF or by another endothelial substance that alters the metabolism or the action of cyclic GMP in vascular smooth muscle.

Animals↗

PubMed1989

Nitrate tolerance in epicardial arteries or in the venous system is not reversed by N-acetylcysteine in vivo, but tolerance-independent interactions exist.

N-acetylcysteine is assumed to reverse nitrate tolerance by replenishing depleted intracellular sulfhydryl groups, but data on interactions of N-acetylcysteine and nitrates in patients with stable angina are controversial and disappointing. Therefore, we studied the effect of N-acetylcysteine on nitrate responsiveness of epicardial arteries and of the venous system (assessed as changes in effective vascular compliance) in dogs (n = 12) during long-term nitroglycerin treatment (1.5 micrograms/kg/min i.v. for 5-6 days). In dogs with nitroglycerin-specific tolerance (shift of venous or epicardial artery dilation to 15-17-fold higher dosages), N-acetylcysteine (100 mg/kg i.v.) had no dilator effect and did not alter the dose-response relations of nitroglycerin. Yet, in nontolerant dogs (n = 17), N-acetylcysteine augmented (1.5-2.0-fold) the dilation of epicardial arteries and the reduction of peripheral vascular resistance induced by 0.5-1.5 micrograms/kg/min nitroglycerin. In vitro, the augmentation of purified guanylate cyclase activity by nitroglycerin (10-100 microM) was potentiated by N-acetylcysteine (0.01-1.0 mM) in saline or in canine plasma, but N-acetylcysteine alone was ineffective. We conclude that 1) N-acetylcysteine does not restore nitroglycerin responsiveness in tolerant epicardial arteries or veins in vivo, 2) a small, tolerance-independent augmentation of nitroglycerin-induced dilation may result from N-acetylcysteine-induced extracellular formation of a stimulant of guanylate cyclase from nitroglycerin.

Acetylcysteine↗

PubMed1989

Physiological targets of superoxide anion and hydrogen peroxide in reperfusion injury.

Current dogma associates reperfusion injury with the introduction of reactive oxygen species (ROS) into the ischemic tissue. The sources of ROS under discussion are xanthine oxidase in the endothelium of small vessels and/or invaded polymorphonuclear leukocytes (PMN). The beneficial effects of both superoxide dismutase and catalase suggest an involvement of superoxide anions and hydrogen peroxide in this pathophysiological process, without describing the targets of their action. In our work we demonstrate that these two ROS effectively interact with two enzymes. Superoxide anions inhibit soluble guanylate cyclase. Its product, cGMP, is considered to antagonize platelet activation and to cause smooth muscle relaxation. Thus O2- can intensify platelet aggregability and small vessel occlusion. Similar effects are elicited by H2O2, which shifts the dose response curve of several agonists towards smaller concentrations by activating cyclooxygenase. This enzyme provides the substrate for thromboxane synthase which generates TxA2, the most potent physiologically occurring platelet aggregating and smooth muscle contacting agonist. These results lead us to the suggestion that the influence of the oxidative burst of PMN in the phenomenon of reperfusion injury should be reconsidered.

Arachidonic Acid↗

PubMed1989

Anti-ischemic actions of molsidomine by venous and large coronary dilatation in combination with antiplatelet effects.

Molsidomine and its vasoactive metabolite SIN-1 elicit anti-ischemic properties by a therapeutically useful pattern of different vasoactive actions combined with a cyclic GMP-mediated inhibition of platelet adhesion and aggregation. Following molsidomine or SIN-1, local venodilatation and an increase in total effective vascular compliance (as an integrated parameter of venodilatation) were observed. This reduction in preload caused a decrease in myocardial oxygen consumption. Molsidomine caused a slowly progressing dilatation (still present and significant after 4 h), while SIN-1 caused an immediate increase in diameter of the large coronary arteries. The cyclic GMP-mediated dilator effects were accompanied by similar increases in platelet cyclic GMP levels (in the effluent of perfused hearts), indicating an increased potential against platelet activation, adhesion, and aggregation. The combined effect on cardiac preload, conductance of the epicardial arteries, and inhibition of platelet activation improves the ratio between myocardial oxygen supply and demand and suppression of ischemic events.

Animals↗

PubMed1989

Failure of the sulfhydryl donor N-acetylcysteine (NAC) to reverse nitrate tolerance in large epicardial arteries and the venous capacitance system of the dog.

NAC has been thought to reverse nitrate tolerance by replenishing depleted intracellular sulfhydryl groups, however data on interactions between N-acetylcysteine and nitrates in patients with stable angina are controversial and disappointing. Therefore, we studied the effect of NAC on nitrate responsiveness of epicardial arteries and of the venous system (assessed as changes in effective vascular compliance) in dogs (n = 12) during long-term nitroglycerine (GTN)-treatment (1.5 micrograms/kg/min for 5 to 6 days). In dogs with GTN-specific tolerance (shift of venous or epicardial artery dilation with 15- to 17-fold higher dosages), NAC (100 mg/kg i.v.) had no dilator effect and did not alter the dose response relations of nitroglycerin. However, in nontolerant dogs (n = 7) NAC augmented (1.5- to 2-fold) the reduction of peripheral vascular resistance induced by 0.5-1.5 microgram/kg/min GTN. In vitro, the augmentation of purified guanylate cyclase activity by GTN (100 microM) was potentiated by NAC (0.01-1.0 mM) in saline or in canine plasma, whereas NAC alone was ineffective. Therefore, NAC does not restore GTN-responsiveness in epicardial arteries or veins in vivo and a small, tolerance-independent augmentation of GTN-induced dilation may result from NAC-induced extracellular formation of a stimulant of guanylate cyclase from GTN.

Acetylcysteine↗

PubMed1989

Clinical tolerance to nitroglycerin is due to impaired biotransformation of nitroglycerin and biological counterregulation, not to desensitization of guanylate cyclase.

UNLABELLED: We studied the effect of nitroglycerin (NTG), endothelium-derived relaxing factor (EDRF), sydnonimine SIN-1, and sodium nitroprusside (SNP) on vascular tone, cyclic GMP content and activity of soluble guanylate cyclase (GC) (in homogenates) of tolerant (1 h 0.55 mM NTG) and non-tolerant (1 h vehicle) de-endothelialized rabbit aortae (RA) as well as on cyclic GMP content of cultured smooth muscle cells (SMC) from RA. Nitrate tolerance significantly attenuated NTG-induced vasodilation of precontracted (1.0 microM norepinephrine) RA, increase in cyclic GMP in RA and SMC, and activation of guanylate cyclase in homogenates as compared to controls. In contrast, vasodilation and cyclic GMP increases to NNP, SIN-1, and EDRF (from cultured bovine aortic endothelial cells) were not affected in RA and SMC, despite desensitization of guanylate cyclase to activation with SNP and SIN-1 in homogenized tolerant RA. CONCLUSION: A desensitization of soluble guanylate cyclase to activation with NO can be demonstrated under non-physiological conditions (disrupted cells) in homogenates from nitrate tolerant RA. However, in intact cells (in situ or in culture) soluble guanylate cyclase is not desensitized to EDRF, SIN-1 or SNP. Therefore reduced generation of NO from NTG because of impaired biotransformation of NTG must be regarded as the basis of nitrate tolerance.

Animals↗

PubMed1988

Desensitization of guanylate cyclase in nitrate tolerance does not impair endothelium-dependent responses.

Tolerance of vascular smooth muscle to nitroglycerin could be induced by an impaired biotransformation of nitroglycerin to nitric oxide, the activator of soluble guanylate cyclase, or by desensitization of guanylate cyclase to activation with nitric oxide. The latter would imply that there would also be tolerance to nitric oxide delivered from sodium nitroprusside or endothelial cells. Therefore, endothelium-denuded segments of rabbit aorta were treated with nitroglycerin to induce tolerance, and were then assessed for mechanical response, cyclic GMP content, and activity of soluble guanylate cyclase after addition of nitrovasodilators. Nitrate tolerance decreased the vasodilation and the increase in cyclic GMP elicited by nitroglycerin, but not that elicited by sodium nitroprusside or endothelium-derived relaxing factor, in norepinephrine-contracted segments. However, soluble guanylate cyclase in the supernatants of homogenates of nitrate-tolerant aortas was desensitized to activation with nitroglycerin and sodium nitroprusside. As the guanylate cyclase was still responsive to activation by nitric oxide in the intact, tolerant smooth muscle, an impaired biotransformation of nitroglycerin rather than desensitization of soluble guanylate cyclase may be the mechanism by which nitrate tolerance develops.

Animals↗

PubMed1988

The primary structure of the 70 kDa subunit of bovine soluble guanylate cyclase.

The primary structure of the 70 kDa subunit of soluble bovine guanylate cyclase, which catalyzes the formation of cyclic GMP from GTP, has been determined. The alignment of six different clones out of two bovine libraries yielded a total of 3.1 kb with a coding region of 1857 bases. The open reading frame encodes a protein of 619 amino acids and a molecular mass of 70.5 kDa. Antibodies raised against a synthetic peptide, which corresponded to the C-terminus of the deduced sequence precipitated guanylate cyclase activity from guanylate cyclase-enriched preparations.

Amino Acid Sequence↗

PubMed1988

Fendiline and calmidazolium enhance the release of endothelium-derived relaxant factor and of prostacyclin from cultured endothelial cells.

We investigated the effects of fendiline, calmidazolium and trifluoperazine, compounds described as calmodulin antagonists, on the release of the endothelial autacoids prostacyclin (PGI2) and endothelium-derived relaxant factor (EDRF). Cultured bovine aortic endothelial cells were grown on microcarrier beads and continuously superfused with Tyrode's solution. Samples collected from the superfusate were assayed for PGI2 concentration (6-keto PGF1 alpha radioimmunoassay) and for EDRF activity (stimulation of soluble guanylate cyclase in vitro). Stimulation of endothelial cells by ATP (3 microM) resulted in a 6.9 +/- 1.4-fold increase of PGI2 concentration in the superfusate (p less than 0.01) and an 8.6 +/- 3.4-fold enhanced guanylate cyclase activity (p less than 0.01). In the presence of calmidazolium (10 microM), the basal values of PGI2 concentration increased 28-fold (p less than 0.01) and the guanylate cyclase activity 10-fold (p less than 0.01). Further enhancement of both was observed after additional administration of ATP. Fendiline (30 microM) did not affect autacoid release by non-stimulated cells. However, the ATP-induced release of PGI2 and EDRF was more than doubled (p less than 0.01) in the presence of this drug compared to ATP-stimulation alone. Trifluoperazine (10 microM) had no enhancing effect on EDRF release, and the ATP-induced release of PGI2 was even significantly attenuated by 84 +/- 12% (p less than 0.01). Calmidazolium and fendiline were also applied to endothelial cells loaded with the fluorescent indicator of free calcium concentration (Ca2i+), indo-1. However, effects of calmidazolium on Ca2i+ could not be quantified since calmidazolium caused some leakage of indo-1 out of the cells. A smaller leakage was observed during the combined application of fendiline and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗