AJP - Renal Physiology, Vol 271, Issue 1 21-F29, Copyright © 1996 by American Physiological Society

ARTICLES

Extracellular chloride regulates mesangial cell calcium response to vasopressor peptides

Z. S. Stevanovic, M. W. Salter and C. I. Whiteside
Department of Medicine, Hospital for Sick Children, University of Toronto, Ontario, Canada.

The role of extracellular chloride in the regulation of mesangial cell calcium responsiveness to vasopressor peptides was explored. First, the components of vasopressor-stimulated calcium signaling were defined in rat mesangial cells cultured on coverslips and preloaded with fura 2. By spectrofluorometry, manganese uptake (reflecting divalent cation channel activation) was observed by quenching of fura 2, or intracellular cytosolic calcium concentration was calculated by dual-excitation ratiometric measurement. In cells depolarized with KCl (45 mM), enhanced manganese uptake or increased cytosolic calcium were inhibited with verapamil (10 microM). Pretreatment of mesangial cells with verapamil reduced the sustained calcium level in response to endothelin-1 (0.1 microM) by 65 +/- 6% (means +/- SE, n = 12) and to vasopressin (1 microM) by 62 +/- 12% (n = 8). Perforated cell patch-clamp measurement confirmed that endothelin-1 stimulated a sustained increase in cytosolic calcium or divalent cation entry only in the presence of simultaneous depolarization. In chloride-free buffer (chloride replaced with impermeant anions), sustained calcium response to endothelin-1 was reduced by 72 +/- 8 (n = 8) and by 65 +/- 4% (n = 8) in the presence of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (55 microM). In chloride-free buffer, cytosolic calcium (unstimulated) increased to > 200 nM by 30 min. These data indicate that reduced extracellular chloride increases mesangial cell basal cytosolic calcium and decreases the transient and sustained cytosolic calcium response to vasopressor peptides.

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