In immortalized human kidney epithelial (IHKE-1) cells derived from proximal tubules, two natriuretic peptide receptors (NPR) were identified. In addition to NPR-A, which is bound by atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and urodilatin (URO), a novel form of NPR-B that might be bound by C-type natriuretic peptide (CNP) was identified using PCR. This novel splice variant of NPR-B (NPR-Bi) was also found in human kidney. Whereas ANP, BNP, and URO increased intracellular cGMP levels in IHKE-1 cells in a concentration-dependent manner, CNP had no effect on cGMP levels. To determine the physiologic responses to these agonists in IHKE-1 cells, the membrane voltage (Vm) was monitored using the slow whole-cell patch-clamp technique. ANP (10 nM), BNP (10 nM), and URO (16 nM) depolarized these cells by 3 to 4 mV (n = 47, 7, and 16, respectively), an effect that could be mimicked by 0.1 mM 8-Br-cGMP (n = 15). The effects of ANP and 8-Br-cGMP were not additive (n = 4). CNP (10 nM) also depolarized these cells, by 3+/-1 mV (n = 28), despite the absence of an increase in cellular cGMP levels, indicating a cGMP-independent mechanism. In the presence of CNP, 8-Br-cGMP further depolarized Vm significantly, by 1.6+/-0.3 mV (n = 5). The depolarizations by ANP were completely abolished in the presence of Ba2+ (1 mM, n = 4) and thus can be related to inhibition of a K+ conductance in the luminal membrane of IHKE-1 cells. The depolarizations attributable to CNP were completely blocked when genistein (10 microM, n = 6), an inhibitor of tyrosine kinases, was present. These findings indicate that natriuretic peptides regulate electrogenic transport processes via cGMP-dependent and -independent pathways that influence the Vm of IHKE-1 cells.