No abstract

AKR1A, an aldo keto reductase, is involved in the synthesis of ascorbic acid as well as the reduction of a variety of aldehyde compounds. AKR1A −/− mice produce considerably less ascorbic acid (about 10%) compared to AKR1A +/+ mice and require ascorbic acid supplementation in order to breed. To elucidate the roles played by AKR1A in spatial memory, AKR1A −/− male mice were weaned at 4 weeks of age and groups that received ascorbic acid supple mentation and no supplementation were subjected to a Morris water maze test. Juvenile AKR1A −/− mice that received no supple mentation showed impaired spatial memory formation, even though about 70% of the ascorbic acid remained in the brains of the AKR1A −/− mice at day 7 after weaning. To the contrary, the young adult AKR1A −/− mice at 13-15 weeks of age maintained only 15% of ascorbic acid but showed no significant difference in the spatial memory compared with the AKR1A +/+ mice or ascorbic acid supplemented AKR1A −/− mice. It is conceivable that juvenile mice require more ascorbic acid for the appropriate level of formation of spatial memory and that maturation of the neural system renders the memory forming process less sensitive to an ascorbic acid insufficiency.

Previously, we demonstrated that serotonin (5-hydroxytryotamine; 5-HT)-induced hepatocyte DNA synthesis and proliferation are mediated through 5-HT 2B receptor. Moreover, 5-HT 2B receptor-mediated hepatocyte mitogenesis involves activation of the Gq/phospholipase C (PLC) pathway and the epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (RTK)/phosphatidylinositol 3-kinase (PI3K)/extracellular signalregulated kinase (ERK) 2/mammalian target of rapamycin (mTOR) pathway. However, how these two signaling pathways are associated with each other remains unknown. Thus we hypothesized that 5-HT 2B receptor stimulated by 5-HT induces secretion of a putative primary mitogen (e.g., TGF-α, insulin-like growth factor (IGF)-I) via the Gq/PLC pathway in primary cultured hepatocytes, followed by induction of DNA synthesis and proliferation that is mediated by the EGF/TGF-α RTK/PI3K/ERK2/mTOR pathway. Hepatocytes were isolated from normal livers by the two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Isolated hepatocytes were plated onto collagen-coated plastic culture dishes and incubated for 3-h in Williams' medium E supplemented with 5% newborn calf serum, 0.1 nM dexamethasone in 5% CO 2 in air at 37ºC to allow attachment. The medium was changed to Williams' medium E without serum and dexamethasone, and 5-HT with or without test substances was added. We investigated effects of monoclonal antibody against TGF-α (1-100 ng/ml) or IGF-I (1-100 ng/ml) on 5-HTinduced hepatocytes DNA synthesis and proliferation. The TGF-α monoclonal antibody, but not IGF-I monoclonal antibody inhibited hepatocytes DNA synthesis and proliferation induced by 5-HT in a dose-dependent manner. Next, we tested to determine autocrine secretion of TGF-α from cultured hepatocytes. 5-HT (10-6 M) significantly increased TGF-α levels in the culture medium. The maximum TGF-α concentration (30 pg/ml) peaked 10 min after addition of 5-HT. The TGF-α secretion induced by 5-HT was completely blocked by a 5-HT 2B receptor antagonist (LY272015). These results suggest that the proliferative mechanism of action of 5-HT is mediated by 5-HT 2B receptor, and stimulation of 5-HT 2B receptor increases autocrine TGF-α secretion from primary cultured hepatocytes. In conclusion, our data indicate that 5-HT stimulates DNA synthesis and proliferation in primary cultures of adult rat hepatocytes by acting via autocrine secretion of TGF-α induced by the serotonin 5-HT 2B receptor/Gq/PLC pathway.

Background: We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Methods: Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams’ medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Results: Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10−6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10−6 M), RTK (AG538, 10−6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10−6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10−6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. Conclusions: The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

Serotonin (5-hydroxytryptamine; 5-HT) can induce hepatocyte DNA synthesis and proliferation by autocrine secretion of transforming growth factor (TGF)-α through 5-HT 2B receptor/phospholipase C (PLC)/Ca 2 and a signaling pathway involving epidermal growth factor (EGF)/TGF-α receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase 2 (ERK2)/mammalian target of rapamycin (mTOR). In the present study, we investigated whether 5-HT or a selective 5-HT 2B receptor agonist BW723C86, would stimulate phosphorylation of TGF-α RTK and ribosomal p70 S6 kinase (p70S6K) in primary cultures of adult rat hepatocytes. Western blotting analysis was used to detect 5-HT-or BW723C86 (10 6 M)-induced phosphorylation of EGF/TGF-α RTK and p70S6K. Our results showed that 5-HT-or BW723C86 (10 6 M)-induced phosphorylation of EGF/TGF-α RTK peaked at between 5 and 10 min. On the other hand, 5-HT-or BW723C86 (10 6 M)induced phosphorylation of p70S6K peaked at about 30 min. Furthermore, a selective 5-HT 2B receptor antagonist LY272015, a specific PLC inhibitor U-73122, a membrane-permeable Ca 2 chelator BAPTA/AM, an L-type Ca 2 channel blocker verapamil, somatostatin, and a specific p70S6K inhibitor LY2584702 completely abolished the phosphorylation of p70S6K induced by both 5-HT and BW723C86. These results indicate that phosphorylation of p70S6K is dependent on the 5-HT 2B-receptor-mediated autocrine secretion of TGF-α. In addition, these results demonstrate that the hepatocyte proliferating action of 5-HT and BW723C86 are mediated by phosphorylation of p70S6K, a downstream element of the EGF/TGF-α RTK signaling pathway.