diamide and mastoparan

Low oxygen (O(2)) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O(2) tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O(2) tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 μM) increases erythrocyte deformability (5%) and increases low O(2) tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 μM), Y-27632 restored both deformability and low O(2) tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O(2) tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O(2) tension.

Topics: Adenosine Triphosphate; Amides; Cell Hypoxia; Cross-Linking Reagents; Diamide; Erythrocyte Deformability; Erythrocytes; Humans; Intercellular Signaling Peptides and Proteins; Oxygen; Peptides; Protein Kinase Inhibitors; Pyridines; rho-Associated Kinases; rhoA GTP-Binding Protein; Sulfhydryl Reagents; Wasp Venoms

Exposure of erythrocytes to reduced oxygen (O(2)) tension activates the heterotrimeric G-protein Gi, resulting in the accumulation of cyclic AMP (cAMP) and release of ATP. The mechanism by which exposure of erythrocytes to reduced O(2) tension activates Gi is not known. Here we investigate the hypothesis that, in rabbit erythrocytes, ATP release in response to exposure to reduced O(2) tension is linked to erythrocyte membrane deformability. If this hypothesis is correct, then decreasing the deformability of the erythrocyte membrane should decrease the release of ATP in response to reduced O(2) tension. We report that treating erythrocytes with diamide, a compound that decreases erythrocyte deformability, inhibits low O(2) tension-induced ATP release. Treating erythrocytes with diamide does not, however, interfere with cAMP accumulation or ATP release in response to a direct activator of Gi (mastoparan 7) or in response to receptor-mediated activation of Gs (the prostacyclin analog, iloprost). These results demonstrate that diamide (100 micromol/L) does not directly inhibit the signaling pathways for ATP release from rabbit erythrocytes and support the hypothesis that low O(2) tension-induced ATP release from these cells is linked to membrane deformability.

Topics: Adenosine Triphosphate; Animals; Cell Membrane; Cyclic AMP; Diamide; Erythrocyte Deformability; Erythrocyte Membrane; Erythrocytes; Heterotrimeric GTP-Binding Proteins; Iloprost; Intercellular Signaling Peptides and Proteins; Male; Oxygen; Peptides; Rabbits; Signal Transduction; Wasp Venoms

In parotid acinar cells, the activation of β-adrenergic receptors induces the accumulation of intracellular cAMP, and consequently provokes the exocytotic release of amylase, a digestive enzyme. The cellular redox status plays a pivotal role in regulating various cellular functions. Cellular redox imbalance caused by the oxidation of cellular antioxidants, as a result of oxidative stress, induces significant biological damage. In this study, we examined the effects of diamide, a thiol-oxidizing reagent, on amylase release by rat parotid acinar cells. In cells treated with diamide, the formation of cAMP and the release of amylase induced by the β-agonist isoproterenol (IPR) were partially reduced. The inhibitory effect of diamide on the IPR-induced release of amylase could be abrogated by reduced glutathione or dithiothreitol. Diamide had no effect on the amylase release induced by forskolin, an adenylate cyclase activator, or by mastoparan, a heterotrimeric GTPbinding protein activator. In cells treated with diamide, the binding affinity for [(3)H]DHA, but not the number of binding sites, was reduced. These results suggest that β-adrenergic receptor function is reduced by thiol-oxidation, which inhibits amylase secretion by parotid acinar cells.

Topics: Amylases; Animals; Colforsin; Cyclic AMP; Diamide; Dithiothreitol; Glutathione; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Isoproterenol; Male; Oxidation-Reduction; Parotid Gland; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Sulfhydryl Reagents; Wasp Venoms