sequester Ca2+ was inhibited. This suggests that the caffeinesensitive store may have a thapsigargin-insensitive Ca2+-sequestration mechanism. To complement these fura-2 experiments, chlortetracycline was used to visualize the Ca2+ stores directly. The latter studies revealed spatial differences in the location of the thapsigargin-sensitive and caffeine-sensitive Ca2+ stores (measured as thapsigargin-sensitive and caffeinesensitive chlortetracyclin fluorescence). Thus, these two types of stores appear to be both functionally and spatially distinct.

Regulation of intracellular Na+ ([Na+]i) in cultured vascular smooth muscle cells (A7r5 line) was studied with Na(+)-sensitive fluorescent dye sodium-binding benzofuran isophthalate. Digital imaging microscopy was used to study single-cell fluorescence. Na+ was distributed uniformly in cytoplasm and nucleus; mean Na+ concentration in resting cells was 4.4 +/- 0.3 mM in cytoplasmic areas ([Na+]cyt) and 4.5 +/- 0.4 mM in nuclear areas ([Na+]n). Na+ pump inhibition and cell activation evoked uniform changes in [Na+]cyt and [Na+]n. Inhibition of Na+ pump with 1 mM ouabain or K(+)-free medium caused a rise in [Na+]cyt; in the latter case, [Na+]cyt fell rapidly when external K+ was later restored. Exposure to Ca(2+)-free medium also caused [Na+]cyt to rise; this effect was augmented by Na+ pump inhibition and was reversed by 10(-5) M verapamil or nitrendipine or by restoration of external Ca2+. The implication is that this Na+ entry in absence of external Ca2+ is mediated by Ca2+ channels. Activation by 10(-9) M arginine vasopressin (AVP) and 10(-6) M serotonin (5-HT) caused [Na+]cyt to increase, but response to 5-HT was small (0.6 mM on average) and transient, whereas response to AVP was larger (2.4 mM on average) and was maintained as long as AVP was present (to 20 min). AVP and, to a much smaller extent, 5-HT stimulated Na+ influx; this could be detected when Na+ pump was inhibited by ouabain. Both AVP and 5-HT activated the Na+ pump, as detected by ouabain-sensitive decrease in [Na+]cyt when Na+ influx was inhibited. Agonist-evoked increases in [Na+]cyt were dependent on a rise in cytosolic Ca2+ concentration ([Ca2+]cyt); these [Na+]cyt responses were abolished by prolonged exposure to Ca(2+)-free media, when cytoplasmic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or when Ca2+ mobilization was blocked with thapsigargin. Raising [Ca2+]cyt with 40 mM K+ or with thapsigargin did not increases in [Na+]cyt. We conclude that 1) AVP- and 5-HT-evoked increases in [Na+]cyt are agonist specific and depend on the balance between stimulated Na+ influx and efflux; 2) AVP and 5-HT activate the Na+ pump; this is, at least in part, independent of agonist-induced rise in [Na+]cyt; and 3) a rise in [Ca2+]cyt is necessary but not sufficient to trigger agonist-evoked rise in [Na+]i.

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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)