cAMP activates an ATP-permeable pathway in neonatal rat cardiac myocytes

ALAN S. LADER, YONG-FU XIAO, CATHERINE R. O'RIORDAN, ADRIANA G. PRAT, G.ROBERT JACKSON, JR. AND HORACIO F. CANTIELLO

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY

Año: 2000 p. 173 - 187

0363-6143

cAMP activates an ATP-permeable pathway in neonatal rat cardiac myocytes. Am J Physiol Cell Physiol 279: C173–C187, 2000.—The molecular mechanisms associated with intracellular ATP release by the heart are largely unknown. In this study the luciferin-luciferase assay and patch-clamp techniques were used to characterize the pathways responsible for ATP release in neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM was significantly increased after cAMP stimulation under physiological conditions. cAMP stimulation also induced an anion-selective electrodiffusional pathway that elicited linear, diphenylamine-2-carboxylate (DPC)-inhibitable Cl2 currents in either symmetrical MgCl2 or NaCl. ATP, adenosine 59-O-(3-thiotriphosphate), and the ATP derivatives ADP and AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP currents were inhibited by DPC and glibenclamide and by a monoclonal antibody raised against the R domain of the cystic fibrosis transmembrane conductance regulator (CFTR). The channel-like nature of the cAMP-induced ATP-permeable pathway was also determined by assessing protein kinase A-activated single channel Cl2 and ATP currents in excised inside-out patches of NRCM. Single channel currents were inhibited by DPC and the anti-CFTR R domain antibody. Thus the data in this report demonstrate the presence of a cAMP-inducible electrodiffusional ATP transport mechanism in NRCM. Based on the pharmacology, patch-clamping data, and luminometry studies, the data are most consistent with the role of a functional CFTR as the anion channel implicated in cAMPactivated ATP transport in NRCM.Am J Physiol Cell Physiol 279: C173–C187, 2000.—The molecular mechanisms associated with intracellular ATP release by the heart are largely unknown. In this study the luciferin-luciferase assay and patch-clamp techniques were used to characterize the pathways responsible for ATP release in neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM was significantly increased after cAMP stimulation under physiological conditions. cAMP stimulation also induced an anion-selective electrodiffusional pathway that elicited linear, diphenylamine-2-carboxylate (DPC)-inhibitable Cl2 currents in either symmetrical MgCl2 or NaCl. ATP, adenosine 59-O-(3-thiotriphosphate), and the ATP derivatives ADP and AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP currents were inhibited by DPC and glibenclamide and by a monoclonal antibody raised against the R domain of the cystic fibrosis transmembrane conductance regulator (CFTR). The channel-like nature of the cAMP-induced ATP-permeable pathway was also determined by assessing protein kinase A-activated single channel Cl2 and ATP currents in excised inside-out patches of NRCM. Single channel currents were inhibited by DPC and the anti-CFTR R domain antibody. Thus the data in this report demonstrate the presence of a cAMP-inducible electrodiffusional ATP transport mechanism in NRCM. Based on the pharmacology, patch-clamping data, and luminometry studies, the data are most consistent with the role of a functional CFTR as the anion channel implicated in cAMPactivated ATP transport in NRCM.