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, CFTR(inh)-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 CFTR(inh)-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(+)/Ca(2+)-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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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1-Methyl-3-isobutylxanthine / pharmacology
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Adenosine Triphosphate / metabolism*
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Animals
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Benzoates / pharmacology
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Cell Line
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Colforsin / pharmacology
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Cyclic AMP / metabolism*
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Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
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Cyclic AMP-Dependent Protein Kinases / metabolism*
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Cystic Fibrosis Transmembrane Conductance Regulator / antagonists & inhibitors
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Cystic Fibrosis Transmembrane Conductance Regulator / metabolism*
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Glyburide / pharmacology
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Hydrogen-Ion Concentration
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Lactic Acid / pharmacology
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Male
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Muscle Contraction / physiology
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Muscle, Skeletal / cytology
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Muscle, Skeletal / drug effects
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Muscle, Skeletal / metabolism*
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Myoblasts / cytology
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Myoblasts / drug effects
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Myoblasts / metabolism*
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Rats
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Rats, Sprague-Dawley
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Signal Transduction / drug effects
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Sodium-Calcium Exchanger / antagonists & inhibitors
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Sodium-Calcium Exchanger / metabolism
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Sodium-Hydrogen Exchangers / antagonists & inhibitors
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Sodium-Hydrogen Exchangers / metabolism
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Thiazolidines / pharmacology
Substances
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3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone
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Benzoates
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CFTR protein, rat
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Sodium-Calcium Exchanger
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Sodium-Hydrogen Exchangers
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Thiazolidines
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Cystic Fibrosis Transmembrane Conductance Regulator
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Colforsin
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Lactic Acid
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Adenosine Triphosphate
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Cyclic AMP
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Cyclic AMP-Dependent Protein Kinases
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Glyburide
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1-Methyl-3-isobutylxanthine
Grants and funding
This work was supported by the Hong Kong Research Grants Council Direct Allocation grant numbers 10207994-14464-21400-323-01, 10400678-14464-21400-323-01 and 10401287-14464-21400-323-01, the Li Ka Shing Foundation, grant number 2046-14464-21400-N01-01, and the University of Hong Kong, grant number 21372567-14464-21400-N01-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.