Use of La3+ to distinguish activity of the plasmalemmal Ca2+ pump from Na+/Ca2+ exchange in arterial myocytes.
La3+ was tested for its ability to distinguish external Na+ (Nao)-independent Ca2+ efflux via the plasma membrane (PM) Ca2+ pump from Nao-dependent Ca2+ efflux via Na+/Ca2+ exchange. Fura-2 loaded cultured rat aortic myocytes were used with digital imaging to measure the cytosolic free Ca2+ concentration ([Ca2+]cyt) and to monitor La3+ entry. At a La3+ concentration ([La3+]o) of 0.25 mM, but not at lower concentrations, La3+ entered the cells; 0.01 mM verapamil blocked this entry. Transient increases in [Ca2+]cyt were evoked by unloading the sarcoplasmic reticulum with cyclopiazonic acid (CPA)+caffeine (CAF) in Na,Ca-free medium (to inhibit Ca2+ extrusion via Na+/Ca2+ exchange and Ca2+ influx). La3+ (0.03-0.25 mM with verapamil) augmented the Ca2+ transients and slowed Nao-independent [Ca2+]cyt recovery in a dose-dependent manner (IC50 approximately 0.01 mM La3+). This La(3+)-sensitive recovery was apparently mediated by the PM Ca2+ pump. The effects of La3+ were reversible: [Ca2+]cyt returned promptly toward base line when La3+ was washed out in Na,Ca-free medium containing CPA + CAF. Reintroduction of extracellular Na+ during [Ca2+]cyt recovery ([La3+]o = 0.06-0.25 mM) significantly speeded recovery, indicating that the Na+/Ca2+ exchanger was not inhibited by [La3+]o < or = 0.25 mM. The La(3+)-sensitive (Nao-independent) and Nao-dependent [Ca2+]cyt recovery rates were comparable. In Na(+)-loaded cells, < or = 0.25 mM La3+ also did not affect Na+/Ca2+ exchange mediated Ca2+ influx. In medium containing Na+ and Ca2+, 0.125 mM La3+ abolished the serotonin (5-HT) evoked plateau responses that resulted from Ca2+ entry via Ca2+ channels. In Na,Ca-free medium, but not Ca-free medium, however, La3+ converted 5-HT evoked Ca2+ transients into sustained responses. We conclude that low [La3+]o (0.06-0.25 mM) inhibits the PM Ca2+ pump, but spares Na+/Ca2+ exchanger mediated Ca2+ influx and efflux in arterial myocytes.
Shimizu
H
H
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA.
Borin
M L
ML
Blaustein
M P
MP
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003470:Culture Media; D003600:Cytosol / Q000187:drug effects; D007811:Lanthanum / Q000494:pharmacology*; D008297:Male; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*
D003470:Culture Media; D012701:Serotonin; D007811:Lanthanum; D012964:Sodium; D000252:Calcium-Transporting ATPases; D002118:Calcium