REGULATION OF THE RENAL NA-HCO3 COTRANSPORTER .7. MECHANISM OF THE CHOLINERGIC STIMULATION

Cholinergic agents regulate proximal tubule acidification but the mech anism responsible for this effect is unclear. We examined the effect o f the cholinergic agent carbachol on the activity of the Na-HCO, cotra nsporter in primary cultures of the proximal tubule of the rabbit. The activity of the cotransporter was assayed either as HCO3-dependent Na -22 uptake or as the recovery of intracellular pH in cells perfused co ntinuously with C1-free physiologic solution containing amiloride to b lock the Na-H antiporter. Carbachol caused a dose-dependent stimulatio n of the cotransporter activity with a maximum increase of 90% above c ontrol values at 10(-5) M and half maximal stimulation at 10(-7) M. Th e stimulation was blocked by atropine and pirenzepine indicating an ef fect through the M1 muscarinic receptor. Carbachol increased intracell ular calcium fourfold and the rise in cytosolic calcium was prevented by the intracellular calcium chelator, BAPTA. BAPTA also blocked the e ffect of carbachol on the cotransporter. Because carbachol activates p hospholipase C and protein kinase C, we examined the effect of carbach ol in the presence of the phospholipase C inhibitor, U73122, or the PK C inhibitor, calphostin C, or PKC depletion. The phospholipase C inhib itor prevented both the effect of carbachol on the cotransporter and o n the intracellular Ca. Calphostin C and PKC depletion also prevented the stimulation of the cotransporter. Carbachol increased PKC activity and caused translocation of the PKC to the particulate fraction. We a lso examined the effect of the phosphatase inhibitor, calyculin A or t he calmodulin kinase inhibitor, W-13 on carbachol stimulation. Calycul in A and W13 likewise prevented the carbachol-induced stimulation of t he cotransporter. These results demonstrate that cholinergic stimulati on modulated the activity of the cotransporter through multiple pathwa ys including phospholipase C/PKC and phosphatase systems.