Involvement of Ca2+/Calmodulin-Dependent Protein Kinase II in Heparin-Stimulated Release of Hepatic Lipase Activity from Rat Hepatocytes

Abstract

Hepatic lipase (HTGL; hepatic triacylglyceride lipase, EC 3.1.1.3), is synthesized primarily by hepatocytes, hydrolyzes triacylglyceride-rich lipoproteins, such as high and intermediate density lipoprotein, while on the luminal endothelial cell surface. [1][2][3] The enzyme, which is found in plasma membranes, microsomes, and cytosol in hepatocytes, is thought to play important roles in the lipid metabolism including catabolic action for the clearance of circulatory triacylglycerides and the production of free fatty acids that are available for local uptake.1,4-6) It is suggested that HTGL is found in the endothelium by electrostatic interactions with cell surface proteoglycans. It has been shown that the intravenous injection of heparin leads to the appearance of large amounts of HTGL activity in the blood-stream.1,2) Inclusion of heparin in the medium decreases HTGL binding to the surface of cultured cells, and causes release of HTGL into the medium. 1,3,4,6) Heparin releases HTGL by direct interaction with the enzyme or by competing for the binding sites on the cell surface. The mechanism of this action is thought to be due to an interaction with the negative charge of each molecule. 4)Previously, we showed that the release of lipoprotein lipase (LPL) activity from tumor cells is stimulated via a process involving the activation of various protein kinases by dextran sulfate, which is a type of heparinoid. 7,8) However, the involvement of the protein kinases in the heparin-stimulated release of HTGL is still unknown.In this report, we show that the release of HTGL produced by heparin from cultured rat hepatocytes is, in part, associated with an increase in the activities of tyrosine kinase (TK) and Ca 2+ /calmodulin-dependent protein kinase II (CaMK-II). Quin2/AM and collagenase were purchased from Wako Pure Chemical Industries (Osaka, Japan). KN-93, KN-92, W-7 and W-5 were from Seikagaku Industries (Tokyo, Japan). Biochanin A and poly (glutamate : tyrosine, 4 : 1) were purchased from Sigma (MO, U.S.A.). ST-638 was provided by Dr. Tadayoshi Shiraishi (Kanegafuchi Chemical Industry, Osaka). Williams' medium E was from Gibco (N.Y., U.S.A.). All other chemicals used were of analytical grade. MATERIALS AND METHODS MaterialsPreparation and Incubation of Hepatocytes Male Wistar rats, weighing 200-250 g, were fed a commercial laboratory chow ad libitum and fasted for 24 h before the experiments. Hepatocytes were isolated by in vitro collagenase perfusion and low speed centrifugation with modifications.9-11) Cell viability was determined by trypan blue exclusion and ranged from 85 to 95%. The hepatocytes were cultured for 24 h in monolayers in a plastic dish (1ϫ10 5 cells/cm 2 ) in Williams' medium E containing 10% fetal calf serum, 10 nM insulin, 10 nM dexamethasone and 5 KIU/ml aprotinin under 5% CO 2 atmosphere. After removal of the medium by aspiration, monolayers of hepatocytes in the dish were further incubated for 0-90 min in Williams' medium E containing 2% bovine serum albumin with or without the addition of ...

Prazosin Stimulates the Release of Hepatic Triacylglycerole Lipase Caused from Primary-culture Rat Hepatocytes

“…Activation of CaMK-II induced by heparin was also reported to cause the stimulatory release of HTGL activity. 26) However, prazosin-stimulated release of HTGL activity was not suppressed by protein kinase C inhibitors (data not shown).…”

Involvement of Leukotriene Production in Release of Hepatic Lipase Activity Produced by Heparin from Rat Hepatocytes

“…24) Therefore, to determine whether TK is involved in the stimulatory effect on PLA 2 activity by heparin, the hepatocytes were incubated with heparin in the presence of two TK inhibitors, biochanin A and ST-638. 25,26) The heparin-stimulated increase in PLA 2 activity was markedly suppressed by both inhibitors (Fig.…”

“…24) However, the action mechanisms of the heparin-stimulated release of HTGL activity are still unclear. Here, we further investigated the stimulatory effects of heparin on the release of HTGL activity.…”

Rapid Increase in the Leukotriene B4 Concentration of Cultured Rat Hepatocytes by Heparin

“…16) Then, to determine whether the membrane TK activity is involved in the heparin-stimulated increase in LTB 4 in hepatocytes, the hepatocytes were incubated with heparin in the presence or absence of TK inhibitors, such as ST-638 and biochanin A (Table 1). 18,19) When the increase in the membrane TK activity from hepatocytes incubated in the presence of heparin was largely suppressed by ST-638 and biochanin A, the heparin-stimulated increase in LTB 4 was also markedly reduced in the presence of these TK inhibitors.…”

“…Determination of Tyrosine Kinase (TK) Activity ---The preparation of TK activity, containing zero phosphotyrosine phosphatase activity, was partially purified from the membrane fraction of the hepatocytes by the method reported previously. 15,16) TK activity was determined by the method of Braun et al using poly (glutamate : tyrosine, 4 : 1) and [γ-32 P]ATP as substrates.…”