Vasopressin stimulation of Ca2+ mobilization, two bivalent cation entry pathways and Ca2+ efflux in A7r5 rat smooth muscle cells.
1. Arg8-vasopressin (AVP)-regulated Ca2+ transport were investigated in fura-2-loaded A7r5 cells using both single cell and population measurements. 2. AVP evokes an initial concentration-dependent rise in cytosolic free Ca2+ concentration ([Ca2+ ]i) to a peak which is independent of extracellular Ca2+, and a sustained Ca2+ signal that results from a balance between stimulation of Ca2+ entry and efflux. 3. Depletion of intracellular Ca2+ stores with thapsigargin, ionomycin, or prior treatment with AVP in Ca2(+)-free medium activates 'capacitative' entry of Ca2+, Ba2+ or Mn2+. Capacitative Mn2+ entry is inhibited by refilling stores with Ca2+; neither Sr2+ nor Ba2+ substitute for Ca2+ to give this effect. 4. In cells with empty stores, AVP stimulates further bivalent cation entry, and the effect persists when extracellular Na+ is replaced by N-methyl-D-glucamine or under depolarizing condition (extracellular KCl concentration ([KCl]o), 135 mM). This effect of AVP is not therefore merely a consequence of AVP causing membrane hyperpolarization or stimulation of Na(+)-Ca2+ exchange, but results from opening of a bivalent cation influx pathway. 5. Several lines of evidence indicate that AVP-stimulated bivalent cation entry is not a consequence of more complete emptying of the intracellular stores and consequent further activation of the capacitative pathway. AVP stimulates Ba2+ entry when the intracellular Ca2+ stores have been both emptied by ionomycin and prevented from refilling by thapsigargin. Mn2+ permeates the capacitative pathway, but AVP does not further increase Mn2+ entry, confirming that AVP does not further activate the capacitative pathway and that the two pathways differ in their permeability to Mn2+. When the extracellular [Sr2+] is low, empty stores do not stimulate detectable Sr2+ entry, but addition of AVP causes substantial Sr2+ entry. 6. A decrease in [Ca2+]i occurs when 50 nM AVP is added during a sustained elevation of [Ca2+]i evoked by thapsigargin. Since AVP does not inhibit the capacitative pathway, this result suggests that AVP stimulates Ca2+ extrusion. 7. We conclude that stimulation of Ca2+ mobilization, two modes of bivalent cation entry, and Ca2+ efflux all contribute to the complex concentration-dependent effects of AVP in A7r5 smooth muscle cells.
Byron
K
K
Loyola University Medical Center, Stritch School of Medicine, Cardiovascular Institute, Maywood, Il 60153, USA
Taylor
C W
CW
D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D016257:Fura-2; D015759:Ionomycin / Q000494:pharmacology; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013268:Stimulation, Chemical; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D002120:Calcium Channel Agonists; D002413:Cations, Divalent; D013729:Terpenes; D001127:Arginine Vasopressin; D015759:Ionomycin; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2