NO inhibits signal transduction pathway for ATP release from erythrocytes via its action on heterotrimeric G protein Gi

Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri 63104 Submitted 17 February 2004 ; accepted in final form 30 March 2004 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 P O 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 µM, 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 µm, 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 . red blood cell; vascular control; adenine nucleotides; pulmonary circulation Address for reprint requests and other correspondence: J. J. Olearczyk, Dept. of Pharmacological and Physiological Science, St. Louis Univ. School of Medicine, M-208, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: olearcjj{at}slu.edu ).