EXTRACELLULAR CHLORIDE REGULATES MESANGIAL CELL CALCIUM RESPONSE TO VASOPRESSOR PEPTIDES

The role of extracellular chloride in the regulation of mesangial cell calcium responsiveness to vasopressor peptides was explored. First, t he 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 catio n channel activa tion) was observed by quenching of fura 2, or intrace llular cytosolic calcium concentration was calculated by dual-excitati on ratiometric measurement. In cells depolarized with KCI (45 mM), enh anced manganese uptake or increased cytosolic calcium were inhibited w ith verapamil (10 mu M). Pretreatment of mesangial cells with verapami l reduced the sustained calcium level in response to endothelin-l (0.1 mu M) by 65 +/- 6% (means +/- SE, n = 12) and to vasopressin (1 mu M) by 62 +/- 12% (n = 8). Perforated cell patch-clamp measurement con fi rmed that endothelin-l stimulated a sustained increase in cytosolic ca lcium or divalent cation entry only in the presence of simultaneous de polarization. In chloride-free buffer (chloride replaced with impermea nt 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 chlor ide channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (55 mu M). In chloride-free buffer, cytosolic calcium (unstimulated) incr eased to >200 nM by 30 min. These data indicate that reduced extracell ular chloride increases mesangial cell basal cytosolic calcium and dec reases the transient and sustained cytosolic calcium response to vasop ressor peptides.