title: Membrane hyperpolarization is not required for sustained muscarinic agonist-induced increases in intracellular Ca2+ in arteriolar endothelial cells.
abstract: OBJECTIVE
  Hyperpolarization modulates Ca2+ influx during agonist stimulation in many endothelial cells, but the effects of hyperpolarization on Ca2+ influx in freshly isolated arteriolar endothelial cells are unknown. Therefore, the purpose of the present study was to characterize agonist-induced Ca2+ transients in freshly isolated arteriolar endothelial cells and to test the hypothesis that membrane hyperpolarization augments agonist-induced Ca2+ influx into these cells.
  
  
  METHODS
  Arterioles were removed from hamster cremaster muscles and arteriolar endothelial cells were enzymatically isolated. Endothelial cells were loaded with Fura 2-AM and the Fura 2 ratio measured photometrically as an index of intracellular Ca2+. The cells were then stimulated with the muscarinic, cholinergic agonist, methacholine, and the resulting Ca2+ transients were measured.
  
  
  RESULTS
  Methacholine (1 microM) increased the endothelial cell Fura 2 ratio from a baseline of 0.81 +/- 0.02 to an initial peak of 1.17 +/- 0.05 (n = 17) followed by a sustained plateau of 1.12 +/- 0.07. The plateau phase of the Ca2+ transient was inhibited by removal of extracellular Ca2+ (n = 12, p < .05), or the nonselective cation channel blockers Gd3+ (30 microM; n = 7, p < .05) or La3+ (50 microM; n = 7, p < .05) without significant effect on the baseline or peak (p > .05). The initial peak of methacholine-induced Ca2+ transients was inhibited by the IP3-receptor antagonist xestospongin D (10 microM, n = 5, p < .05). The methacholine-induced Ca2+ transients were accompanied by endothelial cell hyperpolarization of approximately 14-18 mV, as assessed by experiments using the potentiometric dye, di-8-ANEPPS as well as by patch-clamp experiments. However, inhibition of hyperpolarization by blockade of Ca2+-activated K+ channels with charybdotoxin (100 nM) and apamin (100 nM) (n = 5), or exposure of endothelial cells to 80 or 145 mM KCl (both n = 7) had no effect on the plateau phase of methacholine-induced Ca2+ transients (p > .05).
  
  
  CONCLUSIONS
  Freshly isolated arteriolar endothelial cells display agonist-induced Ca2+ transients. For the muscarinic agonist, methacholine, these Ca2+ transients result from release of Ca2+ from intracellular stores through IP3 receptors, followed by sustained influx of extracellular Ca2+. While these changes in intracellular Ca2+ are associated with endothelial cell hyperpolarization, the methacholine-induced, sustained increase in intracellular Ca2+ appears to be independent from this change in membrane potential. These data suggest that arteriolar endothelial cells may possess a novel Ca2+ influx pathway, or that the relationship between intracellular Ca2+ and Ca2+ influx is more complex than that observed in other endothelial cells.
authors.affiliation: Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan 49008-5410, USA.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Cohen
authors.forename: William F
authors.initials: WF
authors.lastname: Jackson
mesh_terms: D000818:Animals; D001160:Arterioles / Q000166:cytology; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology*; D008647:Mesocricetus; D016210:Methacholine Chloride / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology*; D018482:Muscle, Skeletal / Q000098:blood supply
chemical_list: D018721:Muscarinic Agonists; D016210:Methacholine Chloride; D002118:Calcium