INCREASED INTRACELLULAR NA-MUSCLE CELLS( AUGMENTS MOBILIZATION OF CA2+ FROM SR IN VASCULAR SMOOTH)

The effect of a rise in intracellular Na+ concentration ([Na+](cyt)) o n the amount of Ca2+ in intracellular stores was studied in vascular s mooth muscle cells from the A7r5 line. The relative amount of stored C a2+ was estimated in fura 2-loaded cells by the rise in cytosolic free Ca2+ concentration ([Ca2+](cyt)) evoked by Ca2+ release from the sarc oplasmic reticulum (SR). To improve the detection of released Ca2+, ex trusion of Ca2+ from the cytosol was minimized by using nominally Na+/ Ca2+-free medium containing 0.5 mM La3+ [for vasoconstrictor experimen ts, the medium contained 0.5 mM ethylene glycol-bis(beta-aminoethyl et her)-N,N,N',N'-tetraacetic acid and no La3+]. Ca2+ release was trigger ed by thapsigargin (TG), an SR Ca2+-ATPase inhibitor, and by the vasoc onstrictors arginine vasopressin (AVP) and serotonin (5-HT). Incubatio n 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 12 2 nM). However, ouabain greatly augmented the release of Ca2+ evoked b y 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 mu M verapamil; this im plies that the effect of ouabain was not due to Ca2+ entry through vol tage-gated Ca2+ channels. The response to TG was not augmented when ou abain was applied for 20 min in Na+-free medium (Na+ replaced by equim olar N-methyl-D-glucamine) to prevent [Na+](cyt) from rising. Direct e vidence that ouabain augmented accumulation of Ca2+ into intracellular Ca2+-containing organelles was provided by digital imaging of cells l oaded with chlortetracycline (CTC), a fluorescent indicator sensitive to membrane-bound Ca2+. Ouabain caused the fluorescence of CTC-loaded A7r5 cells to rise; this rise in CTC fluorescence was reversed by TG o r prevented by preincubation of the cells with TG and caffeine. In sum mary, ouabain appears to increase SR Ca2+ by raising [Na+](cyt) and in directly increasing cell Ca2+ via Na+-Ca2+ exchange across the sarcole mma; most of the Ca2+ that enters the cytoplasm is then stored in the SR. This extra stored Ca2+ can be mobilized so that subsequent vasocon strictor-evoked transient increases in [Ca2+](cyt) are amplified.