Abstract
We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTRinh-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTRinh-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na+/H+-exchanger (NHE) with amiloride or the Na+/Ca2+-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway.
Highlights
Adenosine was first proposed as a mediator of exercise hyperaemia more than 50 years ago [1]
We previously reported that lactic acid stimulated ATP release from skeletal muscle through a mechanism that involved the cystic fibrosis transmembrane conductance regulator (CFTR) [17], and that muscle pH was negatively correlated with the extracellular adenosine or ATP concentrations [17,18,19]
We have proposed a model for the CFTR-regulated release of ATP from skeletal muscle, in which a decrease in intracellular pH stimulates hydrogen ion extrusion from the cell by the NHE, resulting in sodium entry to the muscle, which, in turn, leads to sodium extrusion by the Na+/ Ca2+-exchanger (NCX), and allows calcium entry
Summary
Adenosine was first proposed as a mediator of exercise hyperaemia more than 50 years ago [1]. Interstitial adenosine is increased during contractions of both red and white muscles [2,3,4], which is estimated to account for about 40% of the vasodilation [5,6,7,8]. We previously reported that lactic acid stimulated ATP release from skeletal muscle through a mechanism that involved the cystic fibrosis transmembrane conductance regulator (CFTR) [17], and that muscle pH was negatively correlated with the extracellular adenosine or ATP concentrations [17,18,19]. We proposed that the decrease in pH during muscle contractions stimulated ATP release from muscle through a CFTR-dependent mechanism, with that ATP being converted to adenosine in the interstitial space to bring about the muscle vasodilation [17]
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