CA-2+ OSCILLATIONS IN THE RABBIT RENAL CORTICAL COLLECTING SYSTEM INDUCED BY NA+ FREE SOLUTIONS

The presence of a Na+-Ca2+ exchange system has been previously demonstrated at the basolateral side of the cortical collecting system. The role of such an exchanger in maintaining low intracellular [Ca2+] ([Ca2+]i) in this nephron segment is now investigated. Cells from the connecting tubule and cortical collecting duct of rabbit kidneys were isolated by immunodissection with mAb R2G9 and subsequently cultured on glass coverslips. [Ca2+]i was measured in single cells using quantitative fluorescence microscopy. Surprisingly, isoosmotic substitution of extracellular Na+ ([Na+]o) for N-methylglucamine generated [Ca2+]i oscillations in individual cells instead of an anticipated sustained increase in [Ca2+]i. The amplitude of these oscillations ranged between 150 to 600 nm (average 308 +/- 19 nM) and occurred at a frequency of 0.63 +/- 0.03 min-1, with a duration of 44 +/- 2 seconds per spike. Oscillations were only observed in response to [Na+]o less than 5 mm and lasted until Na+o was re-introduced. The compound U73122 (10 muM), a new phospholipase C inhibitor, inhibited [Ca2+]i oscillations, which strongly suggests that IP3 generation initiates [Ca2+]i oscillations. [Ca2+]i oscillations were independent of extracellular Ca2+ and could not be inhibited by lanthanum ions, indicative for an intracellular source for the generation of Ca2+ spikes. Addition of thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase activity, induced a considerable intracellular Ca2+ release, after which [Ca2+]i oscillations could no longer be provoked. Caffeine (20 mm) reversibly inhibited the Ca2+ oscillations, which implies that Ca2+-induced Ca2+ release is involved in generating these oscillations. Protein kinase C activation by the phorbol ester, TPA (10(-8) M), interrupted [Ca2+]i oscillations instantaneously, and this effect could be partially reversed by the protein kinase inhibitor, staurosporine (10(-7) M). Prolonged exposure to TPA (up to 96 hr) resulted in down-regulation of PKC activity in cultured cells, but oscillations could still be induced, which strongly suggests that PKC activity is not crucial in generating [Ca2+]i oscillations. In conclusion, in rabbit renal collecting duct cells in primary culture, removal of extracellular Na+ induced oscillations of [Ca2+]i which arise from phospholipase C activation in concert with Ca2+-induced Ca2+ release and with no strict requirement for extracellular Ca2+.