The role of protein kinase C in the regulation of gallbladder sodium transport during gallstone formation

Background and aims. Gallbladder Na⁺ and water absorption increases prior to gallstone formation and promotes cholesterol nucleation. Na⁺/H⁺ exchange (NHE) is a major pathway for gallbladder Na⁺ transport. Data suggest that NHE activity is altered during gallstone formation and may be partly due to altered NHE regulation. Studies show that Ca²⁺-dependent second messengers, including protein kinase C (PKC), inhibit basal gallbladder Na⁺ transport. Multiple PKC isoforms have been reported with species and tissue-specific expression. PKC isoform expression and functional role in regulating gallbladder NHE are not known. This dissertation characterized gallbladder PKC isoforms and examined if increased Na⁺ absorption via NHE observed during gallstone formation is in part due to altered Ca²⁺-dependent PKC signaling pathways. Methods. Gallbladders were harvested from prairie dogs fed either nonlithogenic chow or 1.2% cholesterol-enriched diet for varying lengths of time to induce various stages of gallstone formation. PKC regulation of gallbladder NHE activity was examined by measuring unidirectional mucosa-to-serosa Na⁺ flux (J^[Na]_[ms]) and DMA-inhibitable ²²Na uptake, respectively. Gallbladder PKC activity was assayed by histone III-S phosphorylation under maximal stimulation conditions. PKC-[alpha] contributions were determined with Gö 6976, a PKC-[alpha] specific inhibitor. Gallbladder PKC isoform mRNA and protein expression were examined by Northern and Western blot analysis, respectively. Results. PKC activation significantly decreased GB J^[Na]_[ms] and reduced baseline apical ²²Na⁺ uptake. Prairie dog and human GB expresses PKC-[alpha], [beta]II and [delta] isoforms. PKC-[alpha] appeared dominant and mediated ~42% of total PKC activity under basal conditions. PKC-[alpha] regulates basal GB Na⁺ transport via NHE2 stimulation and NHE3 inhibition. PKC-[alpha] blockade significantly reversed (~45%) PKC-induced inhibition of J^[Na]_[ms] and reductions in NHE-mediated ²²Na⁺ uptake in controls but was progressively less effective during the various stages of gallstone formation. PKC-[alpha] contribution to total PKC activity was progressively reduced during gallstone formation. PKC-[alpha] mRNA and protein expression significantly increased (~60%) during gallstone formation. Conclusions. PKC-[alpha]-mediated regulation of gallbladder NHE becomes progressively dysfunctional during gallstone formation, paralleling a progressive loss of PKC-[alpha] phosphorylation activity, despite compensatory increases in mRNA and protein expression. Loss of PKC-[alpha]-mediated regulation of gallbladder NHE may in part account for increased Na⁺ absorption observed during gallstone formation.