cAMP-activated anion conductance is associated with expression of CFTR in neonatal mouse cardiac myocytes

• Published in: American Journal of Physiology: Cell Physiology 2000-02, Vol.278 (2), p.C436-C450
• Main Authors: Lader, Alan S, Wang, Yihan, Jackson, G. Robert, Jr, Borkan, Steven C, Cantiello, Horacio F
• Format: Article
• Language: English
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Adenosine Triphosphate - metabolism; Animals; Animals, Newborn; Antibodies - pharmacology; Biological Transport - drug effects; Biological Transport - physiology; Calcium Channel Blockers - pharmacology; Cardiotonic Agents - pharmacology; Cells, Cultured; Chlorides - metabolism; Cyclic AMP - pharmacology; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - immunology; Cystic Fibrosis Transmembrane Conductance Regulator - isolation & purification; Electric Conductivity; Female; Gene Expression - physiology; Glyburide - pharmacology; Hypoglycemic Agents - pharmacology; Isoproterenol - pharmacology; Magnesium Chloride - pharmacokinetics; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Fibers, Skeletal - chemistry; Muscle Fibers, Skeletal - physiology; Myocardium - chemistry; Myocardium - cytology; ortho-Aminobenzoates - pharmacology; Osmotic Pressure; Patch-Clamp Techniques; Precipitin Tests; Pregnancy
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.2000.278.2.c436

1  Renal Unit, Massachusetts General Hospital East, Charlestown 02129; 2  Department of Medicine, Harvard Medical School, Boston 02115; and 3  Renal Section, Boston University Medical Center, Boston, Massachusetts 02118 In this study, patch-clamp techniques were applied to cultured neonatal mouse cardiac myocytes (NMCM) to assess the contribution of cAMP stimulation to the anion permeability in this cell model. Addition of either isoproterenol or a cocktail to raise intracellular cAMP increased the whole cell currents of NMCM. The cAMP-dependent conductance was largely anionic, as determined under asymmetrical (low intracellular) Cl conditions and symmetrical Cl in the presence of various counterions, including Na + , Mg 2+ , Cs + , and N -methyl- D -glucamine. Furthermore, the cAMP-stimulated conductance was also permeable to ATP. The cAMP-activated currents were inhibited by diphenylamine-2-carboxylate, glibenclamide, and an anti-cystic fibrosis transmembrane conductance regulator (CFTR) monoclonal antibody. The anti-CFTR monoclonal antibody failed, however, to inhibit an osmotically activated anion conductance, indicating that CFTR is not linked to osmotically stimulated currents in this cell model. Immunodetection studies of both neonatal mouse heart tissue and cultured NMCM revealed that CFTR is expressed in these preparations. The implication of CFTR in the cAMP-stimulated Cl - and ATP-permeable conductance was further verified with NMCM of CFTR knockout mice [ cftr ( / )] in which cAMP stimulation was without effect on the whole cell currents. In addition, stimulation with protein kinase A and ATP induced Cl -permeable single-channel activity in excised, inside-out patches from control, but not cftr ( / ) NMCM. The data in this report indicate that cAMP stimulation of NMCM activates an anion-permeable conductance with functional properties similar to those expected for CFTR, thus suggesting that CFTR may be responsible for the cAMP-activated conductance. CFTR may thus contribute to the permeation and/or regulation of Cl - and ATP-permeable pathways in the developing heart. cystic fibrosis; heart; adenosine 5'-triphosphate channels; chloride channels; adenosine 5'-triphosphate release