cyclic-gmp and Hemolysis

Paroxysmal nocturnal hemoglobinuria (PNH) patients display exaggerated intravascular hemolysis and esophageal disorders. Since excess hemoglobin in the plasma causes reduced nitric oxide (NO) bioavailability and oxidative stress, we hypothesized that esophageal contraction may be impaired by intravascular hemolysis. This study aimed to analyze the alterations of the esophagus contractile mechanisms in a murine model of exaggerated intravascular hemolysis induced by phenylhydrazine (PHZ). For comparative purposes, sickle cell disease (SCD) mice were also studied, a less severe intravascular hemolysis model. Esophagus rings were dissected free and placed in organ baths. Plasma hemoglobin was higher in PHZ compared with SCD mice, as expected. The contractile responses produced by carbachol (CCh), KCl, and electrical-field stimulation (EFS) were superior in PHZ esophagi compared with control but remained unchanged in SCD mice. Preincubation with the NO-independent soluble guanylate cyclase stimulator 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (BAY 41-2272; 1

Topics: Anemia, Sickle Cell; Animals; Cyclic GMP; Esophageal Diseases; Esophagus; Guanylate Cyclase; Hemolysis; Male; Mice; Mice, Inbred C57BL; Models, Animal; Muscle Contraction; Muscle, Smooth; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Nitroprusside; Oxidative Stress; Phenylhydrazines; Pyrazoles; Pyridines; Signal Transduction; Soluble Guanylyl Cyclase

We recently reported that addition of a small amount of hemolysate to the salt solution that perfused isolated rat lungs hypersensitized the vasculature to subsequent additions of ANG II or exposure to hypoxia, and addition of NO gas (. NO) to the perfusate that contained hemolysate caused a strong vasoconstrictor rather than a vasodilator response. In the present study, we demonstrate that CO and the secondary messengers cGMP and cAMP (usually associated with vasodilation) exert similar effects in hemolysate-perfused lungs. Analogs of the cyclic nucleotides cGMP or cAMP (8-bromo-cGMP and dibutyryl-cAMP, respectively) caused profound vasoconstriction in the isolated rat lung perfused with a salt solution that contained hemolysate. The cGMP- or cAMP-analog-induced vasoconstriction was inhibited by chemically dissimilar Ca2+ antagonists, by the protein phosphatase inhibitor okadaic acid, and, to a lesser degree, by protein kinase inhibitor H-7. Antiphosphothreonine immunoblotting demonstrated that lungs perfused with hemolysate exhibit increased phosphorylation of several proteins. These data indicate that, in the presence of hemolysate, pulmonary vasculature responds to nominally vasodilatory stimuli, including analogs of cGMP and cAMP, with vasoconstriction rather than vasodilation. The importance of our finding is the paradoxical nature of the response to (analogs of) cyclic nucleotides because, to our knowledge, cyclic nucleotide-induced vasoconstriction has not been previously reported.

Topics: Animals; Blood Pressure; Calcium; Carbon Monoxide; Cyclic AMP; Cyclic GMP; Erythrocytes; Hemolysis; In Vitro Techniques; Nitric Oxide; Phosphorylation; Proteins; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vasoconstriction

The present study in isolated rat lungs demonstrates that nitric oxide gas (.NO, 70 nM) added to the perfusate containing a small amount of hemolysate [175 microliters of lysed red blood cells (RBC) per 50 ml of Earle's balanced salt solution (EBSS)] triggered profound and sustained vasoconstriction. Vasoconstriction was not observed when .NO was added to lungs perfused with washed intact rat or human RBC or with oxyhemoglobin (Hgb 20 microM). The presence of hemolysate in the perfusate also caused vasoconstriction in response to n-acetylcysteine (50 microM), glutathione (10(-4) M), or ascorbic acid (10(-4) M) and potentiated greatly the vasoconstrictor response to 5 mM KCl. Not only .NO, but also nitroprusside (SNP) or L-arginine and paradoxically three .NO synthesis inhibitors, including N-monomethyl L-arginine, L-NAME, and nitroblue tetrazolium, which have different mechanisms of action, each caused in the presence of hemolysate large vasoconstrictive responses. Hemolysate itself enhanced O2 consumption by slices of lung; no effects of this dose of .NO on lung slice respiration were seen in the absence of hemolysate. Both Hgb and hemolysate lowered perfusate cGMP levels to the same degree suggesting that the vasoconstrictive response was not due to unique effects of hemolysate on guanylyl cyclase. Addition of superoxide dismutase (SOD) and catalase (CAT) to the hemolysate containing perfusate, or addition of a cyclooxygenase or 5-lipoxygenase inhibitor, virtually abolished the .NO induced vasoconstriction. The latter data are consistent with the concept that exposure of the vasculature to hemolysate may result in the formation of peroxynitrite. However, SOD and CAT did not abolish the pulmonary vasoconstriction induced by L-arginine or by NAC. Our data indicate that hemolysate has profound effects on lung vessel tone regulation and on lung tissue mitochondrial function, yet the precise molecular mechanisms responsible for the action of hemolysate are likely to be very complex.

Topics: Animals; Arginine; Catalase; Cyclic GMP; Dialysis; Ethylmaleimide; Hemolysis; In Vitro Techniques; Indoles; Lung; Meclofenamic Acid; Nitric Oxide; Nitroprusside; Oxygen Consumption; Perfusion; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Vasoconstriction

To determine the role of nitric oxide (NO)-dependent mechanisms on erythrocyte properties, we exposed red cells to L-arginine competitive analogues, 8Br-cyclic guanosine monophosphate (8Br-cGMP) and neutrophil-eliminating filters. These treatments significantly decreased hypotonic hemolysis, increased potassium efflux and caused a spiculate change in erythrocyte morphology. These effects were related to a decrease of NO caused by the three types of treatments.

Topics: Adult; Animals; Arginine; Cyclic GMP; Erythrocytes; Female; Hemolysis; Humans; Hypotonic Solutions; Kinetics; Male; Neutrophils; NG-Nitroarginine Methyl Ester; Nitric Oxide; omega-N-Methylarginine; Potassium; Rats; Rats, Wistar

Tetanus and botulinum A neurotoxins inhibited exocytosis evoked by various secretagogues in intact and permeabilized chromaffin cells. The block of exocytosis in intact chromaffin cells due to botulinum A neurotoxin could partially be overcome by enhancing nicotine- and veratridine-induced stimulation, whereas the block due to tetanus toxin persisted under the same conditions. The receptor-mediated restoration of 3H-noradrenaline release was specific for nicotinic stimulation, because exocytosis did not occur during muscarinic stimulation. Depolarization of intact chromaffin cells with increasing concentration of K+ failed to restore exocytosis that had been blocked by either toxin. When chromaffin cells, treated with tetanus or botulinum A neurotoxins, were exposed to the Ca2(+)-ionophore A 23187 or permeabilized by staphylococcal alpha-toxin, Ca2(+)-stimulated exocytosis was also inhibited. The inhibition was unaffected by increasing concentrations of free Ca2+. Activation of proteinkinase C and of G-proteins by phorbolester and GMPPNHP, respectively, increased Ca2(+)-induced exocytosis in control cells as well as in cells treated with tetanus and botulinum A neurotoxins. The block, however, could not be relieved by these manipulations, and it could not be relieved by activating the cGMP or cAMP pathways with analoga of cyclic nucleotides, phosphodiesterases inhibitors, and forskolin either. It is concluded that nicotine and veratridine trigger a mechanism within the sequence of events leading to exocytosis that is located beyond the increase in intracellular Ca2(+)-concentration. This pathway may not be affected by botulinum A neurotoxin. The target of tetanus toxin is probably located even closer to the fusion process, i.e. beyond the step upon which botulinum A neurotoxin acts.

Topics: Adrenal Glands; Animals; Bacterial Toxins; Botulinum Toxins; Calcium; Catecholamines; Cattle; Chromaffin System; Cyclic AMP; Cyclic GMP; Exocytosis; GTP-Binding Proteins; Hemolysin Proteins; Hemolysis; In Vitro Techniques; Neurotoxins; Nicotine; Norepinephrine; Potassium; Protein Kinase C; Signal Transduction; Tetanus Toxin

Para-diazobenzenesulfonic acid (DBSA), a non-penetrating compound, which reacts with proteins of the external surface of intact cells, was used for the preparation of AZO-erythrocytes. When coupling of p-diazobenzenesulfonic acid to human red blood cells was carried out in phosphate-buffered saline (PBS), the cells lysed within one hour, before the completion of the reaction. Elimination of sodium chloride from the reaction mixture allowed the completion of coupling by prolonging the survival of red blood cells in he diazonium salt from one hour to three hours. The survival of human erythrocytes could be further prolonged to seven hours by carrying out coupling in the presence of cyclic 3',5'-guanosine monophosphate (cGMP). Azo-erythrocytes prepared in the absence of cyclic GMP and washed free of the diazonium salt lysed in phosphate-buffered saline, but remained stable for hours in isosmotic phosphate buffers devoid of sodium chloride ions. Under identical conditions, azo-erythrocytes prepared in the presence of cyclic GMP remained stable for two days and were suited for studies on the functional and structural aspects of red blood cell membrane.

Topics: Acetylcholinesterase; Cell Membrane Permeability; Cyclic GMP; Diazonium Compounds; Erythrocyte Membrane; Erythrocytes; Glucosephosphate Dehydrogenase; Glyceraldehyde-3-Phosphate Dehydrogenases; Hemolysis; Humans; Indicators and Reagents; Sulfanilic Acids