Protein kinase C-α regulation of gallbladder Na+ transport becomes progressively more dysfunctional during gallstone formation

Gallbladder Na+ absorption and biliary Ca2+ are both increased during gallstone formation and may promote cholesterol nucleation. Na+/H+exchange (NHE) is a major pathway for gallbladder Na+ transport. Ca2+-dependent second messengers, including protein kinase C (PKC), inhibit basal gallbladder Na+ transport. Multiple PKC isoforms with species- and tissue-specific expression have been reported. In this study we sought to characterize Ca2+-dependent PKC isoforms in gallbladder and to examine their roles in Na+ transport during gallstone formation. Gallbladders were harvested from prairie dogs fed either nonlithogenic chow or 1.2% cholesterol-enriched diet for varying periods to induce various stages of gallstone formation. PKC was activated with the use of phorboldibutyrate, and we assessed gallbladder NHE regulation by measuring unidirectional Na+ flux and dimethylamiloride-inhibitable 22Na+ uptake. We measured gallbladder PKC activity with the use of histone III-S phosphorylation and used Gö 6976 to determine PKC-α contributions. Gallbladder PKC isoform messenger RNA and protein expression were examined with the use of Northern- and Western-blot analysis, respectively. Prairie dog and human gallbladder expresses PKC-α, βII, and δ isoforms. The PKC activation significantly decreased gallbladder JNams and reduced baseline 22Na+ uptake by inhibiting NHE. PKC-α mediated roughly 42% of total PKC activity under basal conditions. PKC-α regulates basal gallbladder Na+ transport by way of stimulation of NHE isoform NHE-2 and inhibition of isoform NHE-3. PKC-α blockade reversed PKC-induced inhibition of JNams and 22Na+ uptake by about 45% in controls but was progressively less effective during gallstone formation. PKC-α contribution to total PKC activity is progressively reduced, whereas expression of PKC-α mRNA, and protein increases significantly during gallstone formation. We conclude that PKC-α regulation of gallbladder NHE becomes progressively more dysfunctional and may in part account for the increased Na+ absorption observed during gallstone formation.