Multiple pathways for entry of calcium and other divalent cations in a vasculr smooth muscle cell line (A7r5)

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The influx of calcium in response to vasopressin receptor stimulation is an important component of excitation-contraction coupling. We have examined the routes by which Ca 2+ and other divalent cations enter vascular smooth muscle cells using a cultured vascular smooth muscle cell line (A7r5). Confluent A7r5 cells were loaded with Fura-2 to permit measurement of intracellular divalent cation concentration (Ca 2+, Ba 2+, Mn 2+). Combinations of excitation wavelengths ( 340 380 , 340 356 , 356 380 and 340 370 ) were used depending on the divalent cation being studied. Emission was measured at 510 nm for all studies. Ca 2+, Ba 2+ and Mn 2+ permeated unstimulated A7r5 cells. Vasopressin increased intracellular Ca 2+ in cells both in the presence and absence of extracellular Ca 2+, although responses in the absence of extracellular Ca 2+ were smaller and had no sustained component. Amlodipine, a voltage-dependent calcium channel blocker, had no effect on Ca 2+ entry, but Ni 2+ did block Ca 2+ influx. Vasopressin-induced elevations of intracellular Ca 2+ in Ca 2+-free physiological saline were abolished by ionomycin and thapsigargin. In the presence of extracellular Ba 2+ vasopressin increased intracellular Ca 2+ transiently and caused a small sustained increase in intracellular Ba 2+ concentration. Ionomycin and thapsigargin increased intracellular Ca 2+ but had no effect on Ba 2+ influx. In contrast vasopressin, ionomycin and thapsigargin had no effect on Mn 2+ influx. Econazole and SKF 96365, imidazoles reported to be blockers of receptor-induced cation entry, increased intracellular Ca 2+ by releasing intracellular Ca 2+ from a different site to that mobilized by vasopressin or thapsigargin in A7r5 cells. Econazole and SKF 96365 partially inhibited passive influx of Ca 2+ and Ba 2+ but did not inhibit passive influx of Mn 2+, or vasopressin-induced influx of Ba 2+. U73122, a putative inhibitor of phospholipase C partially inhibited passive entry of Ca 2+ but not passive entry of Mn 2+ and Ba 2+. U73122 also inhibited vasopressin-induced release of intracellular Ca 2+ and agonist-induced Ca 2+ influx but did not block vasopressin-induced Ba 2+ influx. Divalent cations enter A7r5 cells by a number of routes — ‘passive’ pathway(s) that admit Ca 2+, Ba 2+ and Mn 2+ and receptor-operated pathway(s) that are permeable to Ca 2+, Ba 2+ but not Mn 2+. On the basis of ionic permeabilities and the effect of various blocking agents, there appear to be two distinct passive influx routes. One is permeable to Ca 2+ and Ba 2+ and is blocked by econazole or SKF 96365. The other is permeable to Mn 2+ and is blocked by Ni 2+. There also appear to be two different routes of divalent cation entry involved in responses to receptor activation. One allows both Ba 2+ and Ca 2+ to enter and is blocked by Ni 2+. The other allows only Ca 2+ to enter and is blocked by U73122 and may therefore involve the activation of phospholipase C.