Endogenously synthesized liver triglyceride (TG) is secreted into the blood in the form of TG-rich very low density lipoprotein (VLDL) containing lipids and lipoproteins such as phospholipids, cholesterol ester, and apolipoprotein B (apoB). It is known that microsomal TG transfer protein (MTP) in the liver catalyzes the assembly of TG, the conversion of apoB into VLDL, and the secretion of VLDL into the blood.1-3) Recently, it was reported that calcium antagonists inhibit the secretion of VLDL from rat hepatocytes into the blood. [4][5][6] From these results, [4][5][6] it could be speculated that MTP activity might be stimulated by the increase of intracellular Ca 2ϩ . A few studies have reported that the calcium ionophore (A23187) and thapsigargin, an inhibitor of Ca 2ϩ -ATPase in the endoplasmic reticulum (ER), inhibit VLDL secretion by hepatocytes. 7,8) At present, despite many studies on MTP activity, the role of Ca 2ϩ on MTP activity in hepatocytes is poorly understood, and the inhibition of MTP activity is associated with the suppression of hypertriglyceridemia and atherosclerosis. In recent, in another experiment, we found that MTP activity was relatively higher under high Ca 2ϩ level (10.1Ϯ1.8 mM/protein-mg) than under low Ca 2ϩ level (6.2Ϯ1.9 mM/protein-mg) in liver tissues, and its activity was about 2-fold higher than under low Ca 2ϩ level, and had a relative association with hyperlipoproteinemia. It is inferred that MTP activity may have a correlation with Ca 2ϩ level in the liver. Accordingly, we investigated the effects of Ca 2ϩ on MTP activity in hepatocytes, our data demonstrate that MTP activity may be activated by Ca 2ϩ and calmodulin, and may inhibit hypertriglyceridemia by decreasing the level of Ca 2ϩ in hepatocytes. MATERIALS AND METHODS MaterialsHanks' balanced salts (HBSS) buffer (without calcium chloride, magnesium sulfate, and sodium bicarbonate) for collagenase perfusion and Williams' medium E (with L-glutamine but without sodium bicarbonate) for hepatocytes culture were purchased from Sigma (St. Louis, MO, U.S.A.). MTP assay kits were obtained from Calbiochem ® (an affiliate of Merck KGaA, Darmstadt, Germany). Lipid assay kits were obtained from Nissui Pharmaceutical (Tokyo, Japan). Collagenase (type IV) and other chemical reagents were obtained from Sigma (St. Louis, MO, U.S.A.).Preparation of Hepatocytes and Cell Culture Freshly isolated hepatocytes were prepared from adult male Sprague-Dawley rats weighing between 200 and 250 g. The hepatocytes were isolated by the collagenase-liver perfusion method of Berry and Friend. 9) Hepatocytes viability was assessed by trypan blue exclusion. Cell viability assessed by trypan blue exclusion averaged 95%. Hepatocytes with 95% viability were washed three times with washing buffer [Williams' medium E, 23.8 M NaHCO 3 , 0.5 mM dexamethasone, and 15 mM HEPES, pH 7.4 supplemented with 1000 U/l penicillin, 1 mg/l streptomycin, 1 mM insulin]. Hepatocytes (10 6 cells/well) suspended in culture medium (washing buffer supplemented with 10% fetal bovine ...

In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its IC50 value was 175 μg/ml. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated [Ca2+]i mobilization and thromboxane A2 (TXA2) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated [Ca2+]i level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor (IP3R) phosphorylation. These results suggest that the inhibition of [Ca2+]i mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CE-WIB801C suppressed TXA2 production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS). These results suggest that the inhibition of TXA2 production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent Ca2+-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

We have investigated possible roles of intra-glucose supply on microsomal triglyceride (TG) transfer protein (MTP) in the secretion of TG-rich very low-density lipoprotein (VLDL) from the liver. Due to the activation of MTP, TG and apolipoprotein B (apoB) in the liver are assembled into VLDL and then the VLDL is transferred into the blood stream. High MTP activity can increase the release of VLDL into the blood stream, and this would lead high levels of TG and apoB in the blood. High MTP activity was found when the liver (or hepatocytes) contained a high level of total Ca 2؉ as a response of glucose administration. However, the MTP activity was reduced in response to the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7, K i ‫52؍‬ m mM), the intracellular Ca 2؉ chelator BAPTA-AM, and the extracellular Ca 2؉ chelator EDTA. These suggested that there might be a very close relationship between high MTP activity and high Ca 2؉ level in the liver by glucose administration. Glucose-derived hyperglycemic condition resulted from those elevations of TG and total cholesterol in the liver. This hyperglycemic phenomenon may be associated with the increase of TG and apoB levels in blood. The possibility for the regulation of VLDL formation in the liver and, further, those related circulatory diseases due to the excess of VLDL in the blood stream by controlling MTP activity in association with Ca 2؉ was investigated.

In this study, the effects of Ca 2+ and cyclic adenosine monophosphate (cAMP) on microsomal triglyceride (TG) transfer protein (MTP) activity were investigated in rat liver. MTP activity was high when liver contained low levels of cAMP, which was induced by administration of glucose, or high levels of total Ca

In this study, we investigated the effect of rice bran water extract fermented with Lactobacillus plantarum KCCM-12116 (RBLp) on ADP (20 μM)-, collagen (10 μg/mL)-, and thrombin (0.2 U/mL)-stimulated platelet aggregation. RBLp dose-dependently inhibited ADP-, collagen-, and thrombin-induced platelet aggregation, with IC 50 values of 501.1, 637.2, and > 2,000 μg/mL, respectively. The platelet aggregation induced by ADP plus RBLp (750 μg/mL) was increased by the adenylate cyclase inhibitor, SQ22536, and the cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS. Treatment with RBLp increased the phosphorylation of VASP (Ser 157), an A-kinase substrate, which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that the RBLp-induced increases in cAMP contributed to the phosphorylation of VASP (Ser 157), which in turn resulted in an inhibition of ADP-induced platelet aggregation, thereby indicating that RBLp has an antiplatelet effect via cAMP-dependent phosphorylation of VASP (Ser 157). Thus, RBLp may have therapeutic potential for the treatment (or prevention) of platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

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