LOCALIZATION OF THE NA CA EXCHANGE-DEPENDENT CA COMPARTMENT IN CULTURED NEONATAL RAT-HEART CELLS/

It has been previously established, in both adult and cultured neonata l cardiac cells, that there is a discrete Na/Ca exchange-dependent Ca compartment. It has been proposed that a component of junctional sarco plasmic reticulum (JSR) Ca and Ca bound to the apposed inner sarcolemm al leaflet represent together the subcellular locus of the compartment . The present study examines this proposal. The amount of Ca in the to tal compartment is measured isotopically in intact functional cells (u sing the on-line ''scintillation disk'' technique) under a variety of perfusion conditions. Under identical labeling conditions, sarcolemmal membranes are rapidly (within a few hundred milliseconds) isolated fr om another set of intact cells by ''gas dissection,'' and the amount o f Ca bound to the membranes is measured. Probes that specifically decr ease SR Ca content (thapsigargin, caffeine, low-dose ryanodine) decrea se total cell content and sarcolemmal binding proportionally. High-dos e ryanodine (producing closure of SR channels) markedly reduces sarcol emmal binding relative to total content of the compartment. The sarcol emmal sites saturate between 1 and 2 mM, extracellular Ca ([Ca](0)), w hereas the total compartment saturates between 4 and 6 mM [Ca](o). Bel ow 1 mM [Ca](o), sarcolemmal binding is maintained relative to total c ompartment content. Finally, the total compartment increases after rev ersal of the intracellular Na to extracellular Na ([Na](i)/[Na](o)) gr adient with sarcolemmal content-to-total content ratio dependent on th e method used to reverse the [Na](i)/[Na](o) ratio. The results are co nsistent with localization of the Na/Ca exchange-dependent compartment to the subsarcolemmal region (''cleft'') where JSR Ca is in equilibri um with anionic inner sarcolemmal leaflet Ca binding sites.