Hormonal regulation of paracellular permeability in isolated rat hepatocyte couplets.
Many hormones and drugs exert their effects on cells by increasing cytosolic Ca2+ (Cai2+) 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 Cai2+ 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). Cai2+ 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 Cai2+, 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 Cai2+ and abolished both the increase in paracellular permeability and the canalicular collapse. Phorbol dibutyrate also increased paracellular permeability but resulted in neither increased Cai2+ 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+.(ABSTRACT TRUNCATED AT 250 WORDS)
Nathanson
M H
MH
Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510.
Gautam
A
A
Ng
O C
OC
Bruck
R
R
Boyer
J L
JL
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D007211:Indoles; D007365:Intercellular Junctions / Q000187:drug effects / Q000502:physiology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008954:Models, Biological; D009532:Nickel / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors; D014667:Vasopressins / Q000494:pharmacology
D005456:Fluorescent Dyes; D007211:Indoles; D007546:Isoquinolines; D010879:Piperazines; D014667:Vasopressins; D015240:Phorbol 12,13-Dibutyrate; D009532:Nickel; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; C048960:indo-1; D002118:Calcium