Essential Role of Neuron-Enriched Diacylglycerol Kinase (DGK), DGKβ in Neurite Spine Formation, Contributing to Cognitive Function

BACKGROUND: Diacylglycerol (DG) kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Of the 10 subtypes of mammalian DGKs, DGKbeta is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We, therefore, developed DGKbeta KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKbeta. In addition, overexpression of DGKbeta in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKbeta, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKbeta but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that membrane-localized DGKbeta regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory

Characterization of the transport properties of organic anion transporting polypeptide 1 (oatp1) and Na(+)/taurocholate cotransporting polypeptide (Ntcp) : comparative studies on the inhibitory effect of their possible substrates in hepatocytes and cDNA-transfected COS-7 cells

In the present study, we compared the inhibitory effects of organic anions (including bile acids) on the uptake of taurocholate (TC) and estradiol 17beta-D-glucuronide (E(2)17betaG), typical substrates for sodium taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide (oatp1), respectively, using primary cultured rat hepatocytes and Ntcp- or oatp1-transfected COS-7 cells. The Na(+)-dependent uptake of TC was inhibited by nine bile acids and five nonbile acid organic anions in a concentration-dependent manner, and their inhibitory effects were similar in both primary cultured rat hepatocytes and Ntcp-transfected COS-7 cells. BQ-123 (1 microM) and indomethacin (10 microM), both of which exhibit no Ntcp-mediated transport, significantly inhibited the Na(+)-dependent uptake of TC mediated by Ntcp. In addition, the Na(+)-independent uptake of E(2)17betaG was inhibited by 15 organic anions in a concentration-dependent manner, and their inhibitory effects were similar between primary cultured rat hepatocytes and oatp1-transfected COS-7 cells. BQ-123 (1 microM), pravastatin (1 microM), and indomethacin (10 microM), all of which do not undergo oatp1-mediated transport, significantly inhibited the Na(+)-independent uptake of E(2)17betaG mediated by oatp1. These results are consistent with the hypothesis that the hepatic uptake of TC and E(2)17betaG is predominantly mediated by Ntcp and oatp1, respectively. In addition, it was clearly demonstrated that we cannot refer to the substrate specificity of transporters based on inhibition studies

Transport of temocaprilat into rat hepatocytes : role of organic anion transporting polypeptide

The mechanism for hepatic uptake of temocaprilat, an angiotensin-converting enzyme inhibitor that is predominantly excreted into bile was studied using isolated rat hepatocytes and COS-7 cells expressing the organic anion transporting polypeptide (oatp1). The uptake of temocaprilat into isolated rat hepatocytes exhibited saturation with a Km of 20.9 microM and a Vmax of 0.21 nmol/min/mg protein. This uptake was temperature sensitive and was significantly reduced by metabolic inhibitors, a sulfhydryl-modifying reagent and an anion-exchange inhibitor, although the replacement of Na+ with Li+ in the medium did not affect the uptake. [3H]Temocaprilat uptake was inhibited by estradiol-17beta-D-glucuronide and dibromosulphophthalein, typical substrates for the Na+-independent organic anion transporter, in a concentration-dependent manner, whereas excess estradiol-17beta-D-glucuronide did not completely inhibit the uptake. Temocaprilat uptake into COS-7 cells transfected with oatp1 cDNA revealed a concentration-dependency with a Km of 46.7 microM, a value comparable with that obtained in isolated hepatocytes. The contribution of oatp1 to carrier-mediated hepatic uptake of temocaprilat was less than 50% by correcting the uptake clearance with that of estradiol-17beta-D-glucuronide. A good linear correlation was observed for the inhibitory effect of angiotensin-converting enzyme inhibitors (benazeprilat, cilazaprilat, delaprilat and enalaprilat) between isolated hepatocytes and oatp1-expressing cells. These data suggest that oatp1, along with another transporter(s), mediates the uptake of angiotensin-converting enzyme inhibitors into rat hepatocytes

Carrier-mediated hepatic uptake of peptidic endothelin antagonists in rats

The endothelin antagonist BQ-123, an anionic cyclopentapeptide, is taken up by rat hepatocytes through active transport systems. Here, we have examined the hepatocellular uptake mechanism for several BQ-123 derivatives with anionic charges using isolated rat hepatocytes. BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, were taken up by hepatocytes in a concentration-dependent manner. The uptake of BQ-485 was most efficient, whereas compound A showed comparable uptake with BQ-123. The uptake of these peptides was Na(+)- and energy-dependent, suggesting that active transport mechanisms are involved in their uptake into hepatocytes. BQ-485, BQ-518, and compound A can almost completely inhibit both the Na(+)-dependent and -independent uptake of [(3)H]BQ-123, with inhibition constants (K(i)) that are comparable to the Michaelis-Menten constants (K(m)) for their Na(+)-dependent and -independent uptake, respectively. Inhibition by BQ-485 was competitive, and the uptake of BQ-485 can be inhibited by BQ-123, with K(i) values that are comparable with the K(m) values for BQ-123 uptake. The uptake of BQ-123 by COS-7 cells transfected with either Na(+)-dependent taurocholate-cotransporting polypeptide (Ntcp) or Na(+)-independent basolateral organic anion-transporting polypeptide (oatp1) was minimal. Thus, these three peptides share the transporters that also recognize BQ-123 but appear to differ from Ntcp and oatp1