calcimycin and linsidomine

The effects of a range of nitric oxide (NO)-related compounds on histamine release from human basophils and rat peritoneal mast cells were studied. Basal and immunologic histamine releases from human basophils were not affected by N(omega)-nitro-L-arginine, N(omega)-nitro-L-arginine methyl ester, aminoguanidine or methylene blue (all inhibitors of NO production), sodium nitroprusside (an NO donor), L-arginine (a substrate for NO synthase) or D-arginine (the inactive enantiomer of L-arginine). In rat peritoneal mast cells, NO donors such as sodium nitroprusside, sodium nitrite and sodium nitrate, and lipopolysaccharide (an inducer of NO synthase) had little effect on basal histamine release, while 3-morpholino-sydnonimine (SIN-1, an NO donor), L-arginine and D-arginine increased this release by up to threefold. None of the inhibitors of NO production had any striking effect on histamine release induced by anti-rat immunoglobulin E (IgE), compound 48/80, sodium fluoride, phospholipase C, 1,2-dioctanoyl-sn-glycerol or ionophore A23187. However, haemoglobin was found to inhibit histamine release by anti-rat IgE or A23187 by ca. 40%. Alone of the NO donors, low concentrations of L-arginine produced a mild inhibition of histamine release induced by anti-IgE, compound 48/80 and A23187, but not other ligands, while sodium nitroprusside dose-dependently inhibited (by a maximum of ca. 30%) histamine release by anti-rat IgE, sodium fluoride or A23187. Stimulation with a variety of secretagogues or treatment with L-arginine, D-arginine, lipopolysaccharide, SIN-1 or sodium nitroprusside had no effect on NO production. Similarly, L-arginine, D-arginine or sodium nitroprusside did not change intracellular cGMP levels. On the basis of these results, it is suggested that NO does not play a significant role in the modulation of histamine release from human basophils or rat peritoneal mast cells. The effects of L-arginine, D-arginine and sodium nitroprusside may involve mechanisms unrelated to NO.

Topics: Animals; Antibodies; Arginine; Basophils; Calcimycin; Dose-Response Relationship, Drug; Guanidines; Hemoglobins; Histamine; Humans; Immunoglobulin E; Male; Mast Cells; Methylene Blue; Molsidomine; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Nitroprusside; p-Methoxy-N-methylphenethylamine; Peritoneal Cavity; Rats; Rats, Sprague-Dawley; Sodium Fluoride; Sodium Nitrite

In some fish blood vessels, the existence of a NO (nitric oxide) system has been reported. We examined the possibility that this NO system acts as an endothelium-derived relaxing factor (EDRF) in carp aorta using the carp aorta alone and in a combined carp-rat aorta donor-detector system. Use of the typical NO stimulating agent in mammal acetylcholine (ACh) only induced constriction of the carp aorta. This response was not modified by denudation or by NO synthesis inhibition with N-nitro-L-arginine methyl ester. Neither the indirect NO stimulating agents bradykinin and histamine nor the direct NO releasers sodium nitroprusside (SNP) and SIN-1 induced vasorelaxation. Both SNP and ACh elevated the cGMP concentration in rat aorta, but not in carp aorta. In the aorta combination set-up, where carp served as a NO donor and rat aorta served as a NO detector, no relaxation of the rat aorta was observed. The calcium ionophore A23187, a known EDRF producer in mammals, induced relaxation of carp aorta through an endothelium- and cyclooxygenase-dependent mechanism. These results indicate that carp aorta does not produce NO as an EDRF nor does it respond to exogenously supplied NO. The major EDRF in carp is apparently a product(s) of cyclooxygenase metabolism.

Topics: Acetylcholine; Animals; Aorta; Calcimycin; Carps; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; In Vitro Techniques; Ionophores; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Phenylephrine; Rats; Rats, Wistar; Vasodilator Agents

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth mu

Topics: Acetylcholine; Animals; Apamin; Calcimycin; Carotid Arteries; Cell Membrane; Cyclic GMP; Glyburide; In Vitro Techniques; Membrane Potentials; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Penicillamine; Peptides; Potassium Channel Blockers; Rabbits

The relaxant effect of two nitric oxide (NO) donors: sodium nitroprusside and 3-morpholino-sydnonimine (SIN-1) on circular smooth muscle strips isolated from guinea pig gastric fundus was studied with the view to elucidating the mechanism, which underlies the NO-induced relaxation of this tissue. Both sodium nitroprusside (10(-9)-10(-5) M) and SIN-1 (10(-9)-10(-4) M) suppressed the spontaneous fundus tone and hyperpolarized the muscle cells by about 5 mV. They antagonized the acetylcholine (10(-6) M)-induced tone and exerted their relaxant effects even when Ca2+ influx into the cells was triggered through the Na+/Ca2+ exchanger. Sodium nitroprusside and SIN-1 antagonized the contraction induced by cyclopiazonic acid (10(-5) M), a specific inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. In the presence of high concentrations of sodium nitroprusside or SIN-1, cyclopiazonic acid (10(-5) M) exerted only a slight if any contractile effect. After the complete relaxation induced by sodium nitroprusside or SIN-1, the K+-channel blockers, tetraethylammonium, apamin and charybdotoxin, as well as the Ca2+ ionophore, A 23187, induced high-amplitude contractions, suggesting that the Ca2+ sensitivity of the contractile myofilaments was not affected. The results suggest that NO, released from NO donors increases the sarcoplasmic reticulum Ca2+ uptake thereby enhancing the vectorial sarcoplasmic reticulum Ca2+ release toward the plasmalemma to elicit membrane hyperpolarization and relaxation in guinea pig gastric fundus.

Topics: Acetylcholine; Animals; Calcimycin; Calcium; Enzyme Inhibitors; Gastric Fundus; Guinea Pigs; Indoles; Ionophores; Male; Membrane Potentials; Molsidomine; Muscle Relaxation; Muscle Tonus; Muscle, Smooth; Nitric Oxide Donors; Nitroprusside; Potassium Channel Blockers; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Thapsigargin; Vasodilator Agents

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N(G)-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall.

Topics: Animals; Basilar Artery; Calcimycin; Chelating Agents; Cyclic GMP; Dogs; Egtazic Acid; Enzyme Inhibitors; Gene Expression; Immunohistochemistry; In Vitro Techniques; Ionophores; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; omega-N-Methylarginine; Recombinant Proteins; Transfection; Uridine Triphosphate; Vasodilation; Vasodilator Agents

Cationic polypeptides are released by activated leukocytes and may play an important role in the regulation of vascular tone. Effects of cationic polypeptides on cerebral vascular tone have not been studied. The present experiments were designed to determine if synthetic cationic polypeptides, poly-L-arginine and poly-L-lysine, affect the function of cerebral arteries. Rings of canine basilar arteries with and without endothelium were suspended for isometric force recording. Poly-L-arginine (10(-8)-10(-7) M) and poly-L-lysine (10(-8)-10(-7).M) caused endothelium-dependent relaxations. A nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (10(-4) M), and a nitric oxide scavenger, oxyhemoglobin (3 x 10(-6) M), inhibited relaxations in response to cationic polypeptides. Negatively charged molecules, heparin (1 U/ml) and dextran sulfate (10 mg/ml), also inhibited relaxations to poly-L-arginine or poly-L-lysine. Higher concentrations of poly-L-arginine (10(-6)-10(-5) M) and poly-L-lysine (10(-6)-10(-5) M) induced endothelium-independent contractions. A protein kinase C inhibitor, staurosporine (10(-8) M), abolished these contractions. Heparin (10 U/ml) and dextran sulfate (100 mg/ml) inhibited the contractile effect of cationic polypeptides but did not affect contractions to phorbol 12,13-dibutyrate. Poly-L-arginine (10(-6) M) and poly-L-lysine (10(-6) M) abolished endothelium-dependent relaxations in response to bradykinin (10(-10)-10(-6) M) or calcium ionophore A23187 (10(-9)-10(-6) M). Heparin (50 U/ml) and dextran sulfate (200 mg/ml) restored endothelium-dependent relaxations to bradykinin (10(-10)-10(-6) M) in arteries exposed to poly-L-arginine (10(-6) M) or poly-L-lysine (10(-6) M). These studies demonstrate that in the lower concentration range (10(-8)-10(-7) M), poly-L-arginine and poly-L-lysine induce endothelium-dependent relaxations by production of nitric oxide via charge-dependent activation of endothelial nitric oxide synthase. In the higher concentration range (10(-6)-10(-5) M), cationic polypeptides cause endothelium-independent contractions as well as impairment of endothelium-dependent relaxations in response to bradykinin and A23187. These contractions and inhibition of endothelium-dependent relaxations are also mediated by a charge-dependent mechanism and may involve activation of protein kinase C.

Topics: Animals; Basilar Artery; Bradykinin; Calcimycin; Dogs; Endothelium, Vascular; Female; In Vitro Techniques; Male; Molsidomine; Nitric Oxide; Peptides; Polylysine; Vasoconstriction; Vasodilation; Vasodilator Agents

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.

Topics: Animals; Calcimycin; Cattle; Cells, Cultured; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Molecular Mimicry; Molsidomine; Nitric Oxide; Nitrogen Oxides; Spin Labels; Spin Trapping; Superoxide Dismutase; Superoxides

The aims of this study were to determine the morphological and functional consequences of balloon angioplasty of the left subclavian artery of Froxfield heritable hyperlipidaemic (FHHL) rabbits and the influence of oral L-arginine therapy on these changes.. Sixteen-week-old FHHL rabbits were subjected to balloon injury of the left subclavian artery under halothane anaesthesia. Control rabbits (n = 7) were given free access to food and normal tap water. L-Arginine-treated rabbits were given L-arginine (5 g.l-1 in the drinking water for 2 days prior to angioplasty and then for either 2 weeks (n = 7) or 4 weeks (n = 7) after surgery. All rabbits were euthanised 28-30 days after surgery and blood and tissue removed for quantification of neointimal size and determination of endothelial function using isolated vessel tension studies. The ability of the endothelium to prevent platelet aggregation was determined by challenging a vessel ring with carbachol when incorporated into a whole blood sample in which platelet aggregation was induced with collagen.. Balloon injury in non-treated rabbits resulted in the development of marked intimal hyperplasia (18.8[3.6]% of the area within the internal elastic lamina) while endothelial function remained intact. Maximum responses to carbachol and calcimycin were, respectively, a 66.6[14.7]% and 46.9[12.9]% relaxation of 5HT-induced tone, compared to 58.0[3.2]% and 39.8[9.4]% in non-injured vessels. Maximum contractile responses to 5HT and KCl were unaffected by injury. L-Arginine therapy alone had no effect on the vasodilator function of the endothelium, but reduced the endothelium-dependent inhibition of platelet aggregation (68.4[7.8] vs 109[10]% of the maximum extent of platelet aggregation in non-treated and 2-week L-arginine-treated non-injured vessels, respectively). L-Arginine significantly reduced the extent of neointimal formation (7.2[3.9]% of the area within the IEL; P < 0.05 vs. non-treated group). However, L-arginine significantly attenuated the relaxant responses to both carbachol (26.5[10.4]% and 31.4[9.4]% for 2- and 4-week L-arginine groups) and calcimycin (38.7[15.4]% and 16.4[10.7]%) in the injured artery (P < 0.05 compared to non-treated controls).. L-Arginine reduces neointimal formation following balloon catheter injury in heritable hypercholesterolaemic rabbits, which is consistent with previous findings in normocholesterolaemic models. However, in the presence of hypercholesterolaemia, L-arginine has a detrimental effect on endothelial function following injury. This may be a consequence of the presence of lipids in the vascular wall on nitric oxide synthase activity.

Topics: Angioplasty, Balloon; Animals; Arginine; Calcimycin; Carbachol; Endothelium, Vascular; Hyperlipidemias; In Vitro Techniques; Male; Molsidomine; Muscle, Smooth, Vascular; Platelet Aggregation; Platelet Aggregation Inhibitors; Potassium Chloride; Rabbits; Serotonin; Tunica Intima; Vasoconstrictor Agents; Vasodilator Agents

Recent reports proposed that nitric oxide was a modulator of cholinergic transmission. Here, we examined the role of NO on cholinergic metabolism in a model of the peripheral cholinergic nervous synapse: synaptosomes from Torpedo electric organ. The presence of NO synthase was immunodetected in the cell bodies, in the nerve ending area of nerve-electroplate tissue and in the electroplates. Exogenous source of NO was provided from SIN1, a donor of NO and O2-., and an end-derivative peroxynitrite (ONOO-). SIN1 increased calcium-dependent acetylcholine (ACh) release induced by KCl depolarization or a calcium ionophore A23187. The formation of ONOO- was continuously followed by a new chemiluminescent assay. The addition of superoxide dismutase, that decreases the formation of ONOO-, did not impair the stimulation of ACh release, suggesting that NO itself was the main stimulating agent. When the endogenous source of NO was blocked by proadifen, an inhibitor of cytochrome P450 activity of NO synthase, both KCl- and A23187-induced ACh release were abolished; nevertheless, the inhibitor Ng-monomethyl-L-arginine did not modify ACh release when applied in a short time duration of action. Both NO synthase inhibitors reduced the synthesis of ACh from the radioactive precursor acetate and its incorporation into synaptic vesicles as did ONOO- chemically synthesized or formed from SIN1. In addition, choline acetyltransferase activity was strongly inhibited by ONOO- and SIN1 but not by the NO donors SNAP and SNP or, by NO synthase inhibitors. Altogether these results indicate that NO and ONOO modulate presynaptic cholinergic metabolism in the micromolar range, NO (up to 100 microM) being a stimulating agent of ACh release and ONOO- being an inhibitor of ACh synthesis and choline acetyltransferase activity.

Topics: Acetylcholine; Animals; Calcimycin; Cell Compartmentation; Choline O-Acetyltransferase; Electric Organ; Enzyme Inhibitors; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Potassium Chloride; Synaptosomes; Torpedo

The aim of the present study was to investigate, whether nitric oxide (NO) modifies prostacyclin synthesis in endothelial cells. Two different NO-donors: SIN-1 (3-morpholino sydnonimine) and GEA 3175 (4-aryl-substituted oxatriazol derivative), and the NO-synthesis inhibitor; L-NAME were used. Endothelial cells were incubated with the tested compounds with or without Ca ionophore A23187 stimulation. SIN-1 (> 33 microM) and GEA 3175 (> 1 microM) increased the endothelial cGMP levels independently of A23187 stimulation. SIN-1 did not influence prostacyclin synthesis. GEA 3175 (> 33 microM) increased prostacyclin synthesis up to 2-fold, when incubated without A23187. GEA 3175 with A23187 induced about 30% inhibition in prostacyclin synthesis. L-NAME decreased unstimulated prostacyclin synthesis and this inhibition was reversed by GEA 3175. Obviously NO is able to modulate prostacyclin synthesis, however, much higher concentrations are needed than those to increase cGMP synthesis.

Topics: Animals; Arginine; Calcimycin; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Epoprostenol; Female; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Wistar; Triazoles

Left lung autotransplantation (LLA) increased the pulmonary vasoconstriction evoked by phenylephrine and attenuated the vasodilatation caused by acetylcholine or bradykinin in conscious dogs. To study the mechanisms responsible for these changes, pulmonary arterial rings were isolated from right (control) and left (LLA) lower lobes of dogs 1-26 mo after LLA and were suspended for isometric tension recording. Compared with control rings from the same anatomic location, contractions to phenylephrine were increased after LLA in rings with or without endothelium. In arterial rings contracted to 50% of their maximal response to phenylephrine, acetylcholine, bradykinin, and calcium ionophore caused endothelium-dependent relaxations that were reduced in LLA compared with control rings. In arterial rings from control and LLA lungs, relaxations to acetylcholine were not altered by inhibition of cyclooxygenase (indomethacin) but were reduced after inhibition of NO synthase [N omega-nitro-L-arginine methyl ester (L-NAME)]. After L-NAME, there was no longer any significant difference in acetylcholine-induced relaxation between arterial rings from control and LLA lungs. Relaxation to SIN-1, a NO donor, was similar in arterial rings (without endothelium) from control and LLA lungs. The results suggest that LLA causes an increased sensitivity of vascular smooth muscle to alpha 1-adrenergic activation and endothelial dysfunction that is mediated by a selective reduction in the activity of endothelium-derived relaxing factor/NO.

Topics: Acetylcholine; Amino Acid Oxidoreductases; Animals; Arginine; Bradykinin; Calcimycin; Dogs; Dose-Response Relationship, Drug; Endothelium, Vascular; In Vitro Techniques; Lung Transplantation; Male; Molsidomine; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phenylephrine; Pulmonary Artery; Pulmonary Circulation; Transplantation, Autologous; Vasoconstriction; Vasodilation; Vasodilator Agents

The effects of Trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a vitamin E analogue, (60-900 microM) and SIN-1 (3-morpholino sydnonimine), a nitric oxide donor, (30-3000 microM) on arachidonic acid metabolism and on cyclic GMP formation in calcium ionophore A23187 (calcimycin)-stimulated human polymorphonuclear leukocytes were investigated. Trolox C elicited a dose dependent decrease in leukotriene B4 levels and increase in prostaglandin E2 levels but did not affect cyclic GMP levels. SIN-1 dose dependently inhibited leukotriene B4 and stimulated prostaglandin E2 and cyclic GMP formation. Dibutyryl cyclic GMP did not affect the formation of leukotriene B4 and prostaglandin E2. Trolox C (180 microM), which itself had no effect on cyclic GMP levels, enhanced the effect of SIN-1 (100 microM) on cyclic GMP levels more than 5-fold. The effects of SIN-1 on arachidonic acid metabolism seem to be independent of cyclic GMP and are probably due to nitric oxide. In this experimental model both Trolox C and SIN-1 have similar actions on the prostaglandin/leukotriene ratio, and Trolox C potentiates the SIN-1-induced increase in cyclic GMP levels.

Topics: Antioxidants; Arachidonic Acid; Calcimycin; Centrifugation, Density Gradient; Chromans; Cyclic GMP; Dibutyryl Cyclic GMP; Dinoprostone; Dose-Response Relationship, Drug; Guanylate Cyclase; Humans; Leukotriene B4; Molsidomine; Neutrophils; Radioimmunoassay; Vasodilator Agents; Vitamin E

The objective of this investigation was to determine the effect of cultured human umbilical vein endothelial cells (HUVEC) on the vascular response to canine coronary arteries in which the endothelium had been either mechanically removed or injured by multiple brief episodes of occlusion and reperfusion in vivo. The endothelium-dependent vasodilator, A23187 (10(-6) mol/l) did not cause any significant relaxation in vessels from which the endothelium had been removed. However, following addition of cultured HUVEC to the tissue bath (75 x 10(3) cells/ml), A23187 produced a significant (p < 0.05) relaxation. This effect was abolished by inhibition of nitric oxide synthase with Nw-nitro-l-arginine methyl ester (L-NAME). Vascular relaxation caused by the nitric oxide donor SIN-1 was significantly (p < 0.05) enhanced when cultured HUVEC were added to vessels mechanically denuded of endothelium. Repetitive ischemia and reperfusion significantly inhibited the relaxant response to A23187. Addition of cultured HUVEC to the tissue bath partially restored the response to A23187. In contrast to the mechanically damaged vessels the relaxant response to SIN-1 was unaffected by cultured HUVEC in reperfusion-injured vessels. These results demonstrate that cultured endothelial cells partially restore endothelium-dependent vasodilation of vessels in which the endothelium is not functional following mechanical- or reperfusion-induced damage. The differential effect of endothelial cells on the response to SIN-1 suggests that mechanical and reperfusion injury alter the coronary vascular response to SIN-1 by different mechanisms.

Topics: Animals; Arginine; Blood Flow Velocity; Calcimycin; Cells, Cultured; Coronary Circulation; Coronary Disease; Coronary Vessels; Dogs; Dose-Response Relationship, Drug; Endothelium, Vascular; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Myocardial Contraction; Myocardial Reperfusion Injury; NG-Nitroarginine Methyl Ester; Nitric Oxide; Umbilical Veins; Vasodilator Agents

Repetitive, brief periods of ischemia and reperfusion ("preconditioning") increase the resistance of myocardial tissue to subsequent prolonged ischemic episodes and limit infarct size. We investigated whether preconditioning also protects against coronary endothelial dysfunction induced by ischemia and reperfusion.. Experiments were performed in four groups of rats (n = 8 in each group): group 1 rats underwent sham surgery, group 2 rats were subjected to 20 minutes of left coronary artery occlusion without reperfusion, group 3 rats underwent 20 minutes of occlusion followed by 1 hour of reperfusion, and group 4 rats (preconditioning group) underwent the same protocol as group 3 rats, preceded by three cycles of 5 minutes of ischemia and 5 minutes of reperfusion. At the end of the experiments, coronary segments (internal diameter, 250 to 300 microns) were removed distal to the occlusion site and mounted in wire myographs for isometric tension recording. Relaxations induced by increasing concentrations of acetylcholine, the calcium ionophore A23187, or the nitric oxide (NO) donor SIN-1 were determined in arteries precontracted by serotonin. Basal NO release was estimated by measuring contractions to NG-nitro L-arginine methyl ester (L-NAME). In addition, we determined the effect of preconditioning on infarct size in two additional groups that were subjected to the same protocols as those of groups 3 and 4. In those animals, area at risk (India ink injection) and infarct size (triphenyltetrazolium stain) were determined by computerized analysis of enlarged sections after video acquisition. Preconditioning markedly limited infarct size (percent of area at risk: controls, 57 +/- 2; preconditioning, 2.2 +/- 0.6; P < .01). Ischemia (without or with reperfusion) or preconditioning did not affect the coronary responses to L-NAME, serotonin, A23187, or SIN-1. Ischemia without reperfusion did not modify the relaxations to acetylcholine (maximal relaxation: sham, 58 +/- 4%; ischemia, 56 +/- 7%; P = NS). In contrast, ischemia followed by reperfusion markedly impaired the response to acetylcholine (26 +/- 6%; P < .01 versus sham). This impaired response was restored by preconditioning (maximal relaxation: 59 +/- 9%; P = NS versus sham; P < .01 versus ischemia/reperfusion).. In addition to protecting myocardial cells, preconditioning also protects coronary endothelial cells against ischemia/reperfusion injury.

Topics: Acetylcholine; Animals; Arginine; Calcimycin; Coronary Vessels; Endothelium, Vascular; Male; Molsidomine; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Wistar; Serotonin; Vasodilator Agents

To explore endothelium-dependent relaxation and the L-arginine (L-ARG)-nitric oxide (NO) pathway during chronic hypoxia, we examined isolated rings from large conduit pulmonary arteries and aorta from rats exposed to either room air (N), 3-week hypoxia (H), or 3-week H followed by 72-h recovery to normoxia (room air). We examined the vasodilatory actions of acetylcholine (ACh), ionophore A23187, and endothelin-3 (ET-3) on extrapulmonary left and right branches of pulmonary arteries and thoracic aorta precontracted by phenylephrine (PE 10(-6) M). Endothelium-dependent relaxation of N rat pulmonary arteries and aorta to ACh and A23187 was abolished in the presence of L-NG nitroarginine methyl ester (L-NAME 10(-4) M) or methylene blue (MB 10(-5) M) but was suppressed only partially by NG-monomethyl-L-arginine (L-NMMA 5 x 10(-4) M). In pulmonary arteries but not in aorta, ET-3 induced endothelium-dependent relaxation that was suppressed by L-NAME, MB, and L-NMMA. Pulmonary arteries from H rats did not relax with ET-3. As compared with those of N rats, they exhibited less relaxation to ACh and A23187, (47 +/- 3 vs. 89 +/- 2 and 53 +/- 2 vs. 85 +/- 4%, p < 0.001, respectively) but exhibited similar relaxation to the nonendothelium-dependent vasodilator linsidomine. In contrast, endothelial-relaxation did not differ between N and H rat aorta.2+ pretreatment with L-ARG.

Topics: Acetylcholine; Animals; Arginine; Calcimycin; Endothelins; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Phenylephrine; Pulmonary Artery; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents

Low output heart failure induces abnormalities of endothelium dependent vasodilation, but the mechanisms responsible for this remain unclear. As blood flow can alter endothelial cell function, in particular nitric oxide (NO) release, the activity of endothelium derived relaxing factor (EDRF) was investigated in a rat model of high output heart failure.. The thoracic aorta upstream of an aorto-caval fistula in rats was submitted to hormonal changes (similar to those in heart failure) and to high blood flow (opposite to that found in low output heart failure). Functional and biochemical arterial properties were studied in aorto-caval fistula rats and in sham operated rats three months after operation. The vascular responses were studied by exposing aortic segments from fistula and sham operated rats to increasing concentrations of agonists. Aortic cyclic guanosine monophosphate (cGMP) concentration was assessed as an index of NO synthase activity. The effect of NO synthase blockade on functional and biochemical arterial properties was also studied.. Plasma atrial natriuretic factor (ANF) was increased in fistula rats compared to sham operated rats. The concentrations of acetylcholine or the calcium ionophore A23187 required to produce 10% and 50% maximum relaxation (EC10 and EC50) were similar in the two groups. Relaxation in response to low concentrations of Sin-1 (an NO donor) was shifted rightwards in fistula rats and EC10 was greater than in the controls. The aortic cGMP concentration was higher in aorto-caval fistula rats than in sham operated rats (p = 0.008). The differences between aorto-caval fistula rats and sham operated rats were probably the result of increased basal EDRF-NO release in the former, since NO synthase blockade abolished the differences in both aortic cGMP and the dose-response curve to Sin-1.. The arterial wall upstream of a chronic aorto-caval fistula has increased cGMP content and hyposensitivity to Sin-1, which may be due to enhanced basal EDRF-NO release. These changes, strikingly different from those found in the low output heart failure, suggest that haemodynamic rather than neuroendocrine factors play a determinant role in the altered vasodilator response in heart failure.

Topics: Acetylcholine; Animals; Atrial Natriuretic Factor; Calcimycin; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Heart Failure; Male; Molsidomine; Nitric Oxide; Rats; Rats, Wistar; Vasodilation

Bovine endothelial cells (ECs, P1) and lipopolysaccharide/gamma-interferon-induced mouse macrophages (MMs) were incubated in the presence of SIN-1 and C 3754 (1 microM to 1 mM), sydnonimine metabolites of the antianginal predrugs molsidomine and pirsidomine, respectively up to 48 h. No change of the endogenous nitric oxide output from MMs and A23187- or adenosine triphosphate-stimulated ECs was found by means of the methemoglobin method. Data indicate that downregulation of the nitric oxide (NO) synthase is not obvious within the intact cells under exogenous NO stress supplied by high concentrations of the spontaneous NO donors. Cytosolic MM NO synthase extracts, however, revealed reduction in the enzymic [3H]arginine turnover to [3H]citrulline by SIN-1, but not by C 3786, the pharmacologically active metabolite of pirsidomine.

Topics: Amino Acid Oxidoreductases; Animals; Aorta; Calcimycin; Cattle; Cells, Cultured; Down-Regulation; Endothelium, Vascular; Interferon-gamma; Lipopolysaccharides; Macrophages; Mice; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Sydnones; Vasodilator Agents

The effects of two nitric oxide (NO) donors were evaluated in a 6-h model of feline myocardial ischemia-reperfusion. After 80 min of a 90-min ischemic period, SIN-1 or C87-3754 or their respective controls (i.e., 0.9% NaCl or C88-3934, a control compound which does not release NO) were given i.v. as a bolus (0.1 mg/kg) and infused at 1 mg/kg/h for the entire 4.5-h reperfusion period. Administration of the active NO donors significantly decreased the necrotic area/area-at-risk ratio from 29 +/- 3% in the vehicle group to 9 +/- 2 and 11 +/- 5% in the SIN-1 and C87-3754 groups, respectively (P less than .001). The inactive NO donor C88-3934 failed to reduce infarct size (31 +/- 3%). Neither NO donor reduced the accumulation of neutrophils in the necrotic area when compared to their respective control groups, but both agents significantly attenuated coronary endothelial dysfunction as shown by a vasorelaxation to acetylcholine of 62 +/- 2 and 64 +/- 3% in the SIN-1- and C87-3754-treated arteries, as compared to only a 27 +/- 3 and 34 +/- 4% vasorelaxation in the vehicle and inactive NO donor groups, respectively (P less than .001). Our studies show that SIN-1 and C87-3754 exert beneficial effects in a 6-h model of myocardial ischemia-reperfusion. Both NO donors decreased myocardial necrosis and decreased the reperfusion-induced endothelial dysfunction without significantly altering the pressure-rate index (i.e., an index of myocardial oxygen demand).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Animals; Calcimycin; Cats; Endothelium, Vascular; Hemodynamics; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Myocardial Reperfusion Injury; Nitric Oxide; Prostaglandin Endoperoxides, Synthetic; Sydnones; Vasodilator Agents

Human epicardial coronary arteries were obtained from the hearts of 15 patients who received cardiac transplantation for cardiomyopathy. Transverse strips of these arteries were mounted in organ baths for isometric tension recording. The arteries were constricted with prostaglandin F2 alpha and then exposed to the endothelium-dependent relaxants substance P (1 nM), bradykinin (1 nM-1 microM), and the Ca2+ ionophore A23187 (1 nM-1 microM). The effects of the endothelium-independent relaxants glyceryl trinitrate (1 nM-10 microM) and SIN-1, the active metabolite of molsidomine (1 nM-10 microM), were also tested. In control strips of human coronary arteries, all vasodilators caused concentration-dependent relaxations. Pretreatment of human coronary arteries with glyceryl trinitrate (0.5 mM for 60 min) shifted the concentration-response curve to glyceryl trinitrate to the right by a factor of approximately 800 (increase in EC50 from 10 nM to 8 microM). In contrast, endothelium-dependent relaxations to substance P, bradykinin, and A23187 and relaxations to SIN-1 were not changed significantly. Previous exposure of human coronary arteries to SIN-1 (0.5 mM for 60 min) did not modify relaxations to any of the agents tested. Our results show that (a) the responsiveness to SIN-1 is not modified in nitrate-tolerant strips of human coronary arteries, (b) prolonged exposure of human coronary arteries to SIN-1 does not cause tolerance, and (c) endothelium-dependent relaxations are not influenced by exposure of human coronary arteries to either glyceryl trinitrate or SIN-1.

Topics: Adult; Analysis of Variance; Bradykinin; Calcimycin; Dinoprost; Drug Tolerance; Humans; In Vitro Techniques; Middle Aged; Molsidomine; Myocardial Contraction; Nitroglycerin; Substance P; Vasodilator Agents