Hormonal regulation of paracellular permeability in isolated rat hepatocyte couplets

Many hormones and drugs exert their effects on cells by increasing cytosolic Ca²⁺ (Ca(i)/²⁺) and activating protein kinase C (PKC). Each of these actions results in cholestasis in the isolated perfused rat liver, but the responsible mechanisms are unclear. We used isolated rat hepatocyte couplets to observe the direct effects of increased Ca(i)/²⁺ and PKC activation on permeability of the hepatocyte tight junction and canalicular volume, two possible determinants of hepatocyte bile secretion. Couplets were stimulated with the Ca²⁺ agonist vasopressin (10^-8 M) in the absence and presence of the Ca²⁺ influx antagonist Ni²⁺ (5 x 10^-3 M) or with the PKC activator phorbol dibutyrate (10^-6 M). Ca(i)/²⁺ was determined by ratio microspectrofluorometry of indo-1, permeability of the couplet tight junctions was assessed by exclusion of horseradish peroxidase from the canalicular space, and changes in canalicular volume over time were measured directly by optical planimetry. Canalicular volume increased by 1.6 ± 2.5%/min (mean ± SD) under basal conditions. In response to vasopressin, there was a rapid 15-fold increase in Ca(i)/²⁺, followed first by an increase in paracellular permeability, then by canalicular collapse (15.9 ± 5.9%/min). Pretreatment with Ni²⁺ markedly decreased the vasopressin- induced increase in Ca(i)/²⁺ and abolished both the increase in paracellular permeability and the canalicular collapse. Phorbol dibutyrate also increased paracellular permeability but resulted in neither increased Ca(i)/²⁺ nor canalicular collapse. The PKC inhibitor H-7 reversed the effects of both vasopressin and phorbol dibutyrate on tight junction permeability. Bile secretory pressure, measured in isolated perfused rat liver preparations, was acutely increased by vasopressin, but the increase was augmented rather than inhibited by Ni²⁺. These results provide evidence that PKC activation increases permeability of the hepatocyte tight junction and that cholestatic agents that increase influx of extracellular Ca²⁺ may modify bile flow by a PKC-induced increase in paracellular permeability.