CHARACTERIZATION OF CA2-PERMEABILITY OF RENAL A6 CELLS UPON DIFFERENTOSMOTIC CONDITIONS( MEMBRANE)

The nature of the calcium-transporting mechanisms involved in A6 cell calcium homeostasis under iso-and hypo-osmotic conditions was investig ated using fura-2 (AM) as a cell calcium indicator. Under steady-state conditions, intracellular calcium (Ca-i(2+)) was increased by Bay K86 44 or by gramicidin, an ionophore which depolarises A6 cell membranes. The Ca-i(2+) increase following calcium addition (to calcium-depleted cells) or membrane depolarisation was blocked by nifedipine but not b y verapamil or omega-conotoxin, indicating that the membrane calcium p ermeability may be mediated by voltage-dependent and dihydropyridine-s ensitive calcium channels. Ca-i(2+) could also be increased by a hypo- osmotic shock having a linear relationship with the osmolarity change. This osmotically induced Ca-i(2+) increase had an extracellular origi n since it was absent when cells were suspended in a calcium-free medi um and it was not affected by thapsigargin or TMB-8 application. In ad dition, it was inhibited by the calcium channel inhibitor, nifedipine. Furthermore, under hypo-osmotic conditions, an additional Ca-i(2+) in crease, sensitive to nifedipine, was measured when cells were depolari sed by gramicidin or K-gluconate addition. It is proposed that the hyp o-osmotically induced cell calcium increase implies the activation of voltage-dependent and nifedipine-sensitive calcium channels, presentin g the same pharmacological characteristics as those involved in cell c alcium homeostasis under iso-osmotic conditions. The initial Ca-i(2+) increase was transient and stabilised to a value nevertheless higher t han the iso-osmotic level; this secondary and incomplete regulatory ph ase did not occur in the presence of thapsigargin or TMB-8, thus provi ding evidence of intracellular calcium storage.