OSMO-MECHANICALLY SENSITIVE PHOSPHATIDYLINOSITOL SIGNALING REGULATES A CA2-CELLS( INFLUX CHANNEL IN RENAL EPITHELIAL)

Regulation of dihydropyridine (nifedipine)-sensitive calcium influx wa s studied in rabbit culture proximal tubule cells using the fura 2 flu orescence ratio technique. ''Osmo-mechanically induced'' swelling of c ells by exposure to hypotonic medium (220 mosmol/kgH(2)O) caused a rap id rise in intracellular calcium that was predominantly due to influx of calcium via both dihydropyridine-sensitive (nifedipine-sensitive) a nd -insensitive calcium influx pathways. The dihydropyridine-sensitive pathway was regulated, in part, by the phosphatidylinositol signaling pathway. Inhibition of phospholipase C by treat-ment with 2-nitro-4-c arboxyphenyl-N,N-diphenylcarbamate (NCDC), inhibition of protein kinas e C (PKC) by staurosporine, or long-term (24 h) treatment with phorbol 12-myristate 13-acetate (PMA) to downregulate PKC abolished most of t he osmo-induced, dihydropyridine-sensitive calcium influx signal. Shor t-term (seconds) PMA treatment to activate PKC produced a marked stimu lation of both dihydropyridine-sensitive and -insensitive calcium infl ux in isotonic (2- to 3-fold stimulation) and hypotonic (5-fold stimul ation) conditions. In contrast, elevation of adenosine 3',5'-cyclic mo nophosphate (cAMP) by treatment with forskolin or inhibition of protei n kinase A (PKA) by treatment with the cAMP analog, Rp-8-CPT-cAMPS (th e Rp diastereoisomer of adenosine 3',5'-cyclic monophosphothionate), h ad little or no influence on calcium influx, including dihydropyridine -sensitive calcium influx. It is concluded that osmo-mechanical stress activates a dihyropyridine-sensitive calcium influx pathway that is p redominantly regulated via the phosphatidylinositol signaling pathway and PKC and not through the cAMP/PKA signaling pathway.