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The human p53 gene mutated at position 249 per se is not sufficient to immortalize human liver cells
(1999)
For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.
In the perfused rat liver the anaphylatoxin C5a enhanced glucose output, reduced flow, and elevated prostanoid overflow. Because hepatocytes (HCs) do not express C5a receptors, the metabolic C5a actions must be indirect, mediated by e.g. prostanoids from Kupffer cells (KCs) and hepatic stellate cells (HSCs), which possess C5a receptors. Surprisingly, the metabolic C5a effects were not only impaired by the prostanoid synthesis inhibitor, indomethacin, but also by the thromboxane A(2) (TXA(2)) receptor antagonist, daltroban, even though HCs do not express TXA(2) receptors. TXA(2) did not induce prostaglandin (PG) or an unknown factor release from KCs or sinusoidal endothelial cells (SECs), which express TXA(2) receptors, because (1) daltroban did neither influence the C5a-induced release of prostanoids from cultured KCs nor the C5a-dependent activation of glycogen phosphorylase in KC/HC cocultures and because (2) the TXA(2) analog, U46619, failed to stimulate prostanoid release from cultured KCs or SECs or to activate glycogen phosphorylase in KC/HC or SEC/HC cocultures. In the perfused liver, Ca(2+)-deprivation inhibited not only flow reduction but also glucose output elicited by C5a to similar extents as daltroban. Similarly, in the absence of extracellular Ca(2+), flow reduction and glucose output induced by U46619 were almost completely prevented, whereas glucose output induced by the directly acting PGF(2alpha) was only slightly lowered. Thus, in the perfused rat liver PGs released after C5a- stimulation from KCs and HSCs directly activated glycogen phosphorylase in HCs, and TXA(2) enhanced glucose output indirectly mainly by causing hypoxia as a result of flow reduction.
Expression of pyruvate kinase M2 in preneoplastic hepatic foci of N-nitrosomorpholine-treated rats
(1999)