CFTR-associated ATP Release in Neonatal And Adult Skeletal Myoblasts

Abstract

ATP release from contracting skeletal muscle cells stimulates extracellular adenosine formation, which contributes to vasodilation. We previously showed that 1) lactic acid (which is formed by contracting muscle) lowers intracellular pH and stimulates ATP release, and 2) cystic fibrosis transmembrane conductance regulator (CFTR) is activated by low pH and it is involved in acidosis-induced ATP release. Patients with Cystic Fibrosis commonly have reduced exercise tolerance. ATP is a large diameter molecule, and CFTR chloride channel is too narrow for passage of ATP: we investigated whether connexins and/or pannexins were involved in CFTR-associated ATP release from rat L6 skeletal myoblasts cell line (a model for neonatal muscle) and human skeletal muscle myoblasts (HSMM; a model for adult muscle). We measured protein expression with western blot, ATP release by the luciferase assay and large-diameter channel opening by Lucifer Yellow (LY) uptake. LY uptake data suggested that both connexins and pannexins function as membrane channels in L6 myoblasts, whereas HSMM expressed Pannexin 1 strongly. Lactic acid increased LY uptake and ATP release in the 2 cell types, confirming that both models are capable of acidosis-induced ATP release, yet CFTR-stimulated ATP release was mediated by pannexins but not connexins in both neonatal and adult skeletal muscle cells, although connexins could mediate non-CFTR-associated ATP release. Further investigation is required to determine the mechanism by which CFTR regulates the opening of pannexin channel.