Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocyte doublets.
Bile-canaliculus contraction in rat hepatocyte doublets is postulated to involve activation of an actin-myosin system. We examined this hypothesis by determining the relationship between canalicular contraction and cystolic free Ca2+ ([Ca2+]i) concentration after extracellular addition of ATP or microdialysis of myosin light chain kinase or its Ca(2+)-independent fragment, which retains catalytic activity. After incubation of doublets with 200 mumol/L ATP in the absence of extracellular Ca2+, [Ca2+]i peaked at 40 sec and 71% of canaliculi contracted within 4 min. Decreasing effects were observed with equimolar ADP, AMP and nonhydrolyzable ATP, but no effect was observed with adenosine. The effect of extracellular ATP on [Ca2+]i and canalicular contraction was dose dependent. Addition of extracellular Ca2+ and ATP resulted in a plateau level of [Ca2+]i. Cytochalasin D, which depolymerizes actin filaments, inhibited ATP-induced canalicular contraction, but not the increase in [Ca2+]i. Microdialysis of myosin light chain kinase and its Ca(2+)-independent fragment (but not the heat-denatured fragment, albumin, trypsin plus soybean inhibitor or buffer) into one hepatocyte of a doublet resulted in canalicular contraction in 86% of doublets. Injection of myosin light chain kinase or its Ca(2+)-independent fragment did not increase [Ca2+]i within 5 min. These results indicate that (a) the basolateral plasma membrane of hepatocytes has a P2Y-class purinoceptor, (b) increased [Ca2+]i after incubation with ATP is initially due to mobilization from internal sites and (c) canalicular contraction is directly related to [Ca2+]i and activation of an actin-myosin system. The physiological role of extracellular ATP in canalicular contraction is uncertain.
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
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Kitamura
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Brauneis
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Gatmaitan
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Arias
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001648:Bile Canaliculi / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D003956:Dialysis; D005110:Extracellular Space / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D009994:Osmolar Concentration; D051381:Rats
D009711:Nucleotides; D015638:Cytochalasin D; D000255:Adenosine Triphosphate; D009219:Myosin-Light-Chain Kinase; D002118:Calcium