title: Multiple pathways for entry of calcium and other divalent cations in a vascular smooth muscle cell line (A7r5).
abstract: 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 Ca2+ 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 (Ca2+, Ba2+, Mn2+). 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. Ca2+, Ba2+ and Mn2+ permeated unstimulated A7r5 cells. Vasopressin increased intracellular Ca2+ in cells both in the presence and absence of extracellular Ca2+, although responses in the absence of extracellular Ca2+ were smaller and had no sustained component. Amlodipine, a voltage-dependent calcium channel blocker, had no effect on Ca2+ entry, but Ni2+ did block Ca2+ influx. Vasopressin-induced elevations of intracellular Ca2+ in Ca(2+)-free physiological saline were abolished by ionomycin and thapsigargin. In the presence of extracellular Ba2+ vasopressin increased intracellular Ca2+ transiently and caused a small sustained increase in intracellular Ba2+ concentration. Ionomycin and thapsigargin increased intracellular Ca2+ but had no effect on Ba2+ influx. In contrast vasopressin, ionomycin and thapsigargin had no effect on Mn2+ influx. Econazole and SKF 96365, imidazoles reported to be blockers of receptor-induced cation entry, increased intracellular Ca2+ by releasing intracellular Ca2+ from a different site to that mobilized by vasopressin or thapsigargin in A7r5 cells. Econazole and SKF 96365 partially inhibited passive influx of Ca2+ and Ba2+ but did not inhibit passive influx of Mn2+, or vasopressin-induced influx of Ba2+. U73122, a putative inhibitor of phospholipase C partially inhibited passive entry of Ca2+ but not passive entry of Mn2+ and Ba2+. U73122 also inhibited vasopressin-induced release of intracellular Ca2+ and agonist-induced Ca2+ influx but did not block vasopressin-induced Ba2+ influx. Divalent cations enter A7r5 cells by a number of routes - 'passive' pathway(s) that admit Ca2+, Ba2+ and Mn2+ and receptor-operated pathway(s) that are permeable to Ca2+, Ba2+ but not Mn2+. 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 Ca2+ and Ba2+ and is blocked by econazole or SKF 96365. The other is permeable to Mn2+ and is blocked by Ni2+. There also appear to be two different routes of divalent cation entry involved in responses to receptor activation.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Clinical Pharmacology, St Mary's Hospital Medical School, Imperial College of Science Technology and Medicine, London, UK.
authors.forename: A D
authors.initials: AD
authors.lastname: Hughes
authors.forename: M
authors.initials: M
authors.lastname: Schachter
mesh_terms: D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism*; D002460:Cell Line; D004464:Econazole / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D004963:Estrenes; D007093:Imidazoles; D011760:Pyrrolidinones; D014667:Vasopressins; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D001464:Barium; D008345:Manganese; D004464:Econazole; D009532:Nickel; D010738:Type C Phospholipases; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium