cAMP/PKA-activated CFTR is required for acidosis-induced release of ATP from skeletal myocytes

Abstract

ATP is an extracellular signaling molecule involved in the regulation of skeletal muscle blood flow. Our previous study showed that depression of pH using lactic acid treatment stimulated the efflux of ATP from skeletal myocytes through a mechanism that involved the cystic fibrosis transmembrane conductance regulator (CFTR). The present study was undertaken to further explore the signal transduction mechanism linking the decrease in pH to the ATP release from muscle. Accumulation of ATP in the medium surrounding the myocytes was measured using a luminescence assay. Incubation of the myocytes in lactic acid (10 mM) for 3 hours increased the extracellular ATP from 0.67±0.08 to 1.15±0.11 nM (n=36; P<0.001). The specific inhibitor of CFTR, CFTRinh-172, inhibited the lactic-acid-induced increase in extracellular ATP. The CFTR expression was significantly increased with 3 hours incubation with lactic acid. Lactic acid treatment also increased the intracellular cAMP from 3.2±0.3 to 7.1±1.0 nM, and the Protein Kinase A (PKA) activity from 30.6±4.5 to 37.0±5.4 pmol/ml, whereas the lactic-acid-induced increase in extracellular ATP was inhibited by the PKA inhibitor, KT5720, but enhanced by the phosphodiesterase inhibitor IBMX, suggesting that the PKA/cAMP pathway was involved in CFTR activation. Furthermore, an in vivo phosphorylation study using 32P-ATP showed that CFTR phosphorylation was increased by forskolin alone or foskolin with dibutyryl-cAMP and IBMX, further supporting a role for the cAMP/PKA pathway in CFTR activation in muscle. Amiloride, an inhibitor of the Na/H exchanger (NHE), prevented the lactic-acid-induced increases in intracellular cAMP and extracellular ATP; inhibitors of the Na/Ca exchanger (NCX), SN-6 and KB-R7943, also inhibited the lactic-acid-induced accumulation of ATP in the medium surrounding the cultured myocytes. Based on these data, we propose that depression of the pH increases the efflux of ATP through a mechanism that involves CFTR activation through the cAMP/PKA pathway, and that NHE and NCX may be involved.