PTH administration decreases proximal HCO3 reabsorption and inhibits the brush border Na-H antiporter. We studied the effect of PTH on the renal Na-HCO3 cotransporter and examined whether this effect is mediated through the adenylate cyclase/cyclic AMP system or through the phospholipase A pathway. We studied the effect of PTH [1-34] on the Na-HCO3 cotransporter activity in rabbit renal basolateral membranes incubated with 50 microM ATP by measuring the 22Na uptake in the presence of HCO3 and gluconate. Na-HCO3 cotransporter activity (expressed in nmol/mg protein/3 seconds) was taken as the difference in 22Na uptake in the presence of HCO3 and gluconate. PTH (10(-10) M) completely inhibited Na-HCO3 cotransporter activity from 1.23 +/- 0.14 to -0.58 +/- 0.23, P < 0.001. This effect of PTH to inhibit the Na-HCO3 cotransporter was prevented by the polyclonal antibody against G alpha s indicating that PTH acts through G alpha s protein. Because G alpha s stimulates adenylate cyclase/cyclic AMP system, we examined the effect of PTH in the presence and in the absence of the adenylate cyclase inhibitor, dideoxyadenosine (DDA). DDA alone (10(-4) M) stimulated the Na-HCO3 cotransporter activity. In the presence of DDA, the net inhibitory effect of PTH was the same magnitude as that of control, suggesting the existence of other pathways for the effect of PTH on the cotransporter. Calmodulin inhibition also partially prevented the effect of PTH. To determine whether the inhibitory effect of PTH is mediated at least in part, through phospholipase A, we first examined the effect of PTH on arachidonic acid release and then measured the Na-HCO3 cotransporter activity in presence and in absence of arachidonic acid or eicosatetraynoic acid (ETA), an inhibitor of arachidonic acid metabolism. PTH significantly increased the release of arachidonic acid by isolated proximal tubule cells and arachidonic acid inhibited the Na-HCO3 cotransporter in basolateral membranes. ETA (3 microM) partially prevented the inhibitory effect of PTH. In cultured proximal tubule cells, PTH inhibited the HCO3-dependent 22Na uptake and ethoxyresorufin, an inhibitor of cytochrome P-450, blocked the inhibitory effect of PTH on the cotransporter. These results demonstrate that PTH inhibits the renal Na-HCO3 cotransporter through multiple mechanisms, that are mediated through G proteins, G alpha s and GP, and CaM-KII.