title: Chloride is required for receptor-mediated divalent cation entry in mesangial cells.
abstract: Agonists which stimulate the inositol 1,4,5 trisphosphate ([1,4,5]-IP3)-dependent mobilization of Ca2+ from intracellular stores also stimulate entry of divalent cations across the cell membrane. Under appropriate experimental conditions, divalent cation entry across the cell membrane can be monitored as the rate at which the intracellular fluorescence of divalent cation indicators is quenched by the addition of Mn2+ to the extracellular medium. We report that addition of vasopressin to fura-2-loaded glomerular mesangial cells in culture markedly accelerated the rate at which Mn2+ quenched fura-2 fluorescence at its Ca(2+)-insensitive wavelength in the presence of extracellular NaCl, but that this quench response was attenuated when Cl- was removed from the extracellular medium by equimolar substitution with impermeant anions (gluconate, methanesulfonate, acetate, lactate). Similarly, loss of agonist-induced quench also occurred when Cl- was substituted with gluconate in K(+)-containing media. Addition of the Cl- channel inhibitor, 5-nitro-2-(3-phenylpropylaminobenzoic acid) (NPPB), also inhibited Mn(2+)-induced quench of fura-2 fluorescence following vasopressin addition. In contrast, in the presence of gramicidin to provide an alternate conductance pathway to accompany divalent cation entry, agonist-dependent Mn2+ quench occurred even in the absence of extracellular Cl-, indicating that the requirement for Cl- was not the result of cotransport on a common transporter nor the result of Cl- serving as a necessary cofactor for divalent cation entry. A similar dependence on extracellular Cl- was observed for other Ca(2+)-mobilizing agonists such as endothelin, as well as the intracellular Ca2+ ATPase inhibitor, thapsigargin. Extracellular Cl- dependence for agonist-induced divalent cation entry was also reflected in a corresponding extracellular Cl- dependence for agonist-induced mesangial cell contraction. It has been previously shown by ourselves (Kremer et al., 1992a, Am. J. Physiol., 262:F668-F678) and others that agonist-stimulated calcium mobilization in mesangial cells is accompanied by inhibition of K+ conductance and increased Cl- conductance. Accordingly, we conclude that the current findings suggest that activation of Cl- conductance provides regulated charge compensation for receptor-mediated divalent cation entry in response to Ca(2+)-mobilizing vasoconstrictor agonists in mesangial cells.
authors.affiliation: MRC Group in Membrane Biology, Hospital for Sick Children, Toronto, Canada.
authors.forename: S G
authors.initials: SG
authors.lastname: Kremer
authors.forename: W
authors.initials: W
authors.lastname: Zeng
authors.forename: R
authors.initials: R
authors.lastname: Hurst
authors.forename: T
authors.initials: T
authors.lastname: Ning
authors.forename: C
authors.initials: C
authors.lastname: Whiteside
authors.forename: K L
authors.initials: KL
authors.lastname: Skorecki
mesh_terms: D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002413:Cations, Divalent / Q000493:pharmacokinetics*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000494:pharmacology*; D016232:Endothelins / Q000494:pharmacology; D005453:Fluorescence; D005920:Glomerular Mesangium / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D005942:Gluconates / Q000494:pharmacology; D006096:Gramicidin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D015221:Potassium Channels / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000502:physiology*; D012965:Sodium Chloride / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D018118:Chloride Channels; D002712:Chlorides; D016232:Endothelins; D005942:Gluconates; D009579:Nitrobenzoates; D015221:Potassium Channels; D011956:Receptors, Cell Surface; D013729:Terpenes; D014667:Vasopressins; D006096:Gramicidin; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D008345:Manganese; D012965:Sodium Chloride; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases