Increased intracellular Na+ augments mobilization of Ca2+ from SR in vascular smooth muscle cells

Abstract

The effect of a rise in intracellular Na+ concentration ([Na+]cyt) on the amount of Ca2+ in intracellular stores was studied in vascular smooth muscle cells from the A7r5 line. The relative amount of stored Ca2+ was estimated in fura 2-loaded cells by the rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) evoked by Ca2+ release from the sarcoplasmic reticulum (SR). To improve the detection of released Ca2+, extrusion of Ca2+ from the cytosol was minimized by using nominally Na+/Ca(2+)-free medium containing 0.5 mM La3+ [for vasoconstrictor experiments, the medium contained 0.5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and no La3+]. Ca2+ release was triggered by thapsigargin (TG), an SR Ca(2+)-ATPase inhibitor, and by the vasoconstrictors arginine vasopressin (AVP) and serotonin (5-HT). Incubation with 1-3 mM ouabain for 20 min, which raises [Na+]cyt from 4.4 to 9.0 mM, increased "resting" [Ca2+]cyt only slightly (from 87 to 122 nM). However, ouabain greatly augmented the release of Ca2+ evoked by TG [from 639 nM (control) to 1,021 nM], by AVP (from 993 to 1,597 nM), and by 5-HT (from 559 to 1,486 nM). Ouabain-induced augmentation of TG-evoked Ca2+ release was not affected by 10 microM verapamil; this implies that the effect of ouabain was not due to Ca2+ entry through voltage-gated Ca2+ channels. The response to TG was not augmented when ouabain was applied for 20 min in Na(+)-free medium (Na+ replaced by equimolar N-methyl-D-glucamine) to prevent [Na+]cyt from rising.(ABSTRACT TRUNCATED AT 250 WORDS)