iloprost and mastoparan

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

The release of ATP from erythrocytes involves a signal transduction pathway of which cystic fibrosis transmembrane conductance regulator, PKA, adenylyl cyclase, and the heterotrimeric G proteins G(s) and G(i) are components. In the pulmonary circulation, ATP released from the erythrocyte stimulates nitric oxide (NO) synthesis, thereby regulating vascular resistance. We reported that NO liberated from an NO donor inhibited ATP release from erythrocytes in response to decreased Po(2) or mechanical deformation. Here, we investigated the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of G(i). Washed rabbit erythrocytes were incubated in the presence or absence of the NO donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate; 100 nM, 20 min), followed by treatment with agents that activate specific components of the signal transduction pathway promoting ATP release. Neither ATP release nor cAMP accumulation induced by either forskolin (100 microM, n = 7) or iloprost (100 nM, n = 6) was inhibited by spermine NONOate. These experiments suggest that the inhibitory action of NO is not the result of inactivation of adenylyl cyclase or G(s), respectively. However, spermine NONOate completely inhibited ATP release in response to mastoparan (10 microm, P < 0.05, n = 5), a specific activator of G(i). Spermine (100 nM, 20 min), the polyamine remaining after liberation of NO from spermine NONOate, had no affect on mastoparan-induced ATP release (n = 4). These results support the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of the heterotrimeric G protein G(i).

Topics: Adenosine Triphosphate; Animals; Colforsin; Cyclic AMP; Erythrocytes; Female; GTP-Binding Protein alpha Subunits, Gi-Go; Heterotrimeric GTP-Binding Proteins; Iloprost; Intercellular Signaling Peptides and Proteins; Male; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Oxygen; Partial Pressure; Peptides; Pertussis Toxin; Rabbits; Signal Transduction; Spermine; Wasp Venoms