VASOPRESSIN-INDEPENDENT REGULATION OF COLLECTING DUCT WATER PERMEABILITY

Studies were conducted in microdissected rat terminal inner medullary collecting duct (IMCD) segments to determine whether agents that activ ate the phosphoinositide signaling pathway regulate osmotic water perm eability independent of the action of vasopressin. Water was withheld from the rats for 24 h before the experiments, a procedure that yields a stably high osmotic water permeability in isolated perfused termina l IMCDs in the absence of vasopressin. In the present studies, the vas opressin-independent osmotic water permeability was sustained at simil ar to 300 mu m/s for at least 90 min. We used the cholinergic agent ca rbachol (10-100 mu M) to activate the phosphoinositide pathway. This a gent has previously been reported to increase the hydrolysis of inosit ol phospholipids in IMCD cells without altering adenosine 3',5'-cyclic monophosphate production. In preliminary studies, we demonstrated (us ing fura 2) that carbachol transiently increases intracellular calcium and that this response was blocked by atropine, confirming that musca rinic receptors are coupled to activation of the phosphoinositide sign aling pathway in these cells. In the absence of vasopressin, both carb achol (10 mu M) and phorbol myristate acetate (1 nM) inhibited osmotic water permeability by 40-50% within 10 min. These effects were partia lly blocked by calphostin C, an inhibitor of protein kinase C. These r esults demonstrate that activation of phosphatidylinositol hydrolysis and/or activation of protein kinase C markedly inhibits osmotic water permeability in isolated perfused IMCD segments, even in the absence o f prior stimulation by vasopressin.