Ca efflux via the sarcolemmal Ca ATPase occurs only in the t-tubules of rat ventricular myocytes.
The transverse (t-) tubule network is an important site for Ca influx and release during excitation-contraction coupling in cardiac ventricular myocytes; however, its role in Ca extrusion is less clear. The present study was designed to investigate the relative contributions of Ca extrusion pathways across the t-tubule and surface membranes. Ventricular myocytes were isolated from the hearts of adult male Wistar rats and detubulated using formamide. Intracellular Ca was monitored using fluo-3 and confocal microscopy. Caffeine (20 mmol/L) was used to induce SR Ca release; carboxyeosin (20 μmol/L) and nickel (10 mmol/L) were used to inhibit the sarcolemmal Ca ATPase and Na/Ca exchanger (NCX) respectively. Carboxyeosin decreased the rate constant of decay of the caffeine-induced Ca transient in control cells, but had no effect in detubulated cells, suggesting that Ca extrusion via the Ca ATPase occurs only across the t-tubule membrane. However nickel decreased the rate constant of the caffeine-induced Ca transient in control and detubulated cells, although its effect was greater in control cells, suggesting that Ca extrusion via NCX occurs across the surface and t-tubule membranes. The PKA inhibitor H-89 (10 μmol/L) was used to investigate the role of basal PKA activity in Ca extrusion; H-89 appeared to have no effect on Ca extrusion via the Ca ATPase, but reduced Ca extrusion via NCX at the t-tubules but not the surface membrane. Thus it appears that Ca extrusion via the sarcolemmal Ca ATPase occurs only at the t-tubules, and is not regulated by basal PKA activity, while Ca extrusion via NCX occurs across both the surface and t-tubule membranes, but predominantly across the t-tubule membrane due, in part, to localised stimulation of NCX by PKA at the t-tubules. This may be important in heart disease, in which changes in t-tubule structure and protein phosphorylation occur.
Department of Physiology and Pharmacology, Faculty of Medical and Veterinary Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Anabelle
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Chase
Clive H
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Orchard
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology*; D007545:Isoproterenol / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D012508:Sarcolemma / Q000201:enzymology*; D013449:Sulfonamides / Q000494:pharmacology
D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000252:Calcium-Transporting ATPases; D007545:Isoproterenol; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium