Intracellular free Na+ in resting and activated A7r5 vascular smooth muscle cells

Abstract

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.

Transient Receptor Potential Channel 6–Mediated, Localized Cytosolic [Na + ] Transients Drive Na + /Ca 2+ Exchanger–Mediated Ca 2+ Entry in Purinergically Stimulated Aorta Smooth Muscle Cells

“…Purinergic stimulation, like vasopressin, 27 increases global [Na ϩ ] i in RASMCs, which we have linked to opening of a Na ϩ -permeable channel. 12 We propose that TRPC6- containing channels mediate this Na ϩ entry.…”

“…28 However, our laboratory and several others have shown that NCX-mediated Ca 2ϩ entry is essential to purinergically and adrenergically stimulated Ca 2ϩ influx in intact blood vessels and cultured aorta smooth muscle cells. 6 27 and were partly attributed to Na ϩ entry via NCX and store-operated channels. 27 We show here that agonist stimulation also causes localized [ …”

“…6 27 and were partly attributed to Na ϩ entry via NCX and store-operated channels. 27 We show here that agonist stimulation also causes localized [ …”

Mutation of the Protein Kinase C Phosphorylation Site on Rat α1 Na+,K+-ATPase Alters Regulation of Intracellular Na+ and pH and Influences Cell Shape and Adhesiveness

“…Osmolality was kept constant by the addition of choline chloride. The solution also contained ouabain (5 ϫ 10 Ϫ3 M) and the Na (16,19). The intracellular Na ϩ (Na ϩ i ) calibration curve was found to be almost linear from about 1 to 20 mM, the physiological intracellular range, and a change of less than 0.5 mM Na …”

“…1A). Values of Na ϩ i are generally lower, when determined with fluorescent probes, which measure free Na ϩ i , than with indirect methods that use, for example, isotopes or an electron probe to measure total Na ϩ i (16,17,19 Fig. 2A).…”