INTRACELLULAR FREE NA-MUSCLE CELLS( IN RESTING AND ACTIVATED A7R5 VASCULAR SMOOTH)

Regulation of intracellular Na+ ([Na+]i) in cultured vascular smooth m uscle cells (A7r5 line) was studied with Na+-sensitive fluorescent dye sodium-binding benzofuran isophthalate. Digital imaging microscopy wa s used to study single-cell fluorescence. Na+ was distributed uniforml y in cytoplasm and nucleus; mean Na+ concentration in resting cells wa s 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 evoke d 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 latte r case, [Na+]cyt fell rapidly when external K+ was later restored. Exp osure to Ca2+-free medium also caused [Na+]cyt to rise; this effect wa s augmented by Na+ pump inhibition and was reversed by 10(-5) M verapa mil or nitrendipine or by restoration of external Ca2+. The implicatio n is that this Na+ entry in absence of external Ca2+ is mediated by Ca 2+ 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 AV P 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 stimulat ed Na+ influx; this could be detected when Na+ pump was inhibited by o uabain. Both AVP and 5-HT activated the Na+ pump, as detected by ouaba in-sensitive decrease in [Na+]cyt when Na+ influx was inhibited. Agoni st-evoked increases in [Na+]cyt were dependent on a rise in cytosolic Ca2+ concentration ([Ca2+]cyt); these [Na+]cyt responses were abolishe d by prolonged exposure to Ca2+-free media, when cytoplasmic Ca2+ was chelated with ,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 increase [Na+]cyt. We conclude that 1) AVP- and 5-HT-evoked increases in [Na+]cyt are agoni st specific and depend on the balance between stimulated Na+ influx an d 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 i n [Ca2+]cyt is necessary but not sufficient to trigger agonist-evoked rise in [Na+]i.