BACKGROUND: Gallstone formation is characterized by increased biliary calcium (Ca2+) level and altered gallbladder absorption. Recent studies suggest that luminal Ca2+ regulates gallbladder ion transport via intracellular calcium ([Ca2+]ic). Ca2+-calmodulin and protein kinase C (PKC) are two major systems through which [Ca2+]ic carries out second-messenger functions in many cell types. We have previously shown that Ca2+-calmodulin regulates basal gallbladder ion transport in prairie dog. The present study tests the hypothesis that PKC is also essential in regulation of gallbladder ion transport in this model. METHODS: The role of PKC in regulation of gallbladder ion transport was determined by studying the effects of phorbol esters, synthetic analogues of diacylglycerol, which directly activates PKC. Gallbladders were mounted in Ussing chambers, and standard electrophysiologic parameters were recorded after exposing tissues to either 10(-5) mol/L of 4-alpha-phorbol 12,13-didecanoate (PDD), 4-beta-phorbol 12-myristate 13-acetate, 4-beta-phorbol 12,13-dibutyrate (PDB), or 10(-4) mol/L serotonin. Unidirectional Na+, Cl-, and H2O fluxes were measured before and after treatment with only inactive PDD and most active PDB. RESULTS: Mucosal and serosal exposure of tissues to either 4-beta-phorbol 12-myristate 13-acetate or PDB resulted in a decrease in short-circuit current and transepithelial potential difference without any change in tissue resistance. Serotonin induced similar changes in gallbladder electrical properties. PDB caused an inhibition of mucosal to serosal fluxes of Na+, Cl-, and H2O, with a decrease in net Na+ absorption, an increase in net Cl- secretion, and a conversion of net H2O absorption to net H2O secretion. Serosal-to-mucosal fluxes of Na+, Cl-, and H2O did not change. Inactive PDD had no effect on either electrophysiologic parameters or ion and water fluxes. Pretreatment of tissues with PKC antagonist 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine blocked the phorbol ester-induced inhibition of ion transport. CONCLUSION: PKC regulates gallbladder ion transport in the prairie dog by inhibiting Na+ absorption and stimulating Cl- secretion.