304A AASLD ABSTRACTS HEPATOLOGY October 1995

789 MOLECULAR CLONING; EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF A HEPATOCYTE PROSTAGLANDIN F2a RECEPTOR. C de Vries, F Nensch/q:fer-Rube, K l-lanecke. K Jungermann GP Piisehel Institut f. Biochemie, Georg-August-Universitat, Humboldtallee 23, D- 37073 Gottingen, Germany

Prostaglandins have been shown to play an important role as locally acting signal substances during intercellular communication between non-parenehymal liver cells, where they are produced, and hepatocytes. They mediate the increase in glycogenolysis in hepatocytes by e.g. zymosan, endotoxin, complement factors, nnmunoglobulins and partially after stimulation of hepatic nerves. So far the prostaglandin receptors on hepatoeytes have been characterized only by binding assays and in functional studies (reviewed in 1). Here, for the first time, the characterization of a rat hepatocyte prostaglandin. F2a receptor on the molecular level is shown: Hepatocyte eDNA was prepared from purified isolated rat hepatocytes without significant contamination with non-parenehymal cells. Overlapping PCR- fragments of the rat hepatocyte prostaglandin F2a (FP) receptor were generated using primers deduced from the previously published sequence of the mouse ovary FP receptor. The PCR-produCts were cloned and sequenced. From this sequence two new primers covering 25 bp of the 5' and 3' end of the coding region were designed and a PCR-product containing the entire coding region was generated by PCR using the proofreading PWO-polymerase. The PCR-product was cloned into the euearyotie expression vector pSVL and expressed in COS7 cells. The resulting receptor protein bound PGF2a specifically with an apparent K d of 10 nM. PGD 2 and PGE 2 competed for binding with decreasing affinity. Stimulation of transfeeted COS7 cells with PGF2a increased the formation of inositol phosphates with an ED50 of about 50 nM. Thus, the cloned FP-receptor expressed in COS7 resembles the functionally characterized FP-receptor in hepatocytes, however, it caused an increase in inositol phosphates at a lower PGF2a concentration than had been observed in hepatocytes.

1) GP Piisehel, K Jongermann (1994) Prog Liv Dis 12, 19-46.

790 CLONING, FUNCTIONAL CHARACTERIZATION AND TISSUE EXPRESSION OF THE RAT LIVER P2U RECEPTOR. AI Sharara. JM-J Romac. RA Liddle and JG Fitz. Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC.

Extracellular nueleotides including A'rP and UTP regulate liver cell transport and metabolism through activation of purinergie receptors. This P2U class of receptors is G-protein coupled, and activates signal transduction systems involving the breakdown of membrane phospholipids and the elevation of cytoplasmic free Ca 2+. Based on recent cloning of P2U receptors from mouse neuroblastoma and human cells, the purpose of these studies was to identify and functionally characterize P2U receptors in rat liver. A kZAPII rat liver eDNA library was screened by plaque hybridization with a 700 bp BsrD1 fragment encompassing the open reading frame from the cloned murine P2U receptor eDNA. A single clone (rP2u R] was identified and sequenced by the dideoxy chain termination method revealing a 2.2 kb eDNA with an open reading frame encoding a 374 aa. protein that was >95% homologous to the murine P2U receptor. Northem blot analysis performed at high stringeney with a probe containing a fragment of the open reading frame indicated the presence of a 3.4 kb mRNA in liver tissue only, suggesting that this clone was specific for liver tissue. Functional characterization was performed using two-electrode voltage clamping (TEVC) after the microinjection of Xenopus laevis oocytas with the receptor cRNA obtained by in vitro'transcription. Superfusion of oocytes expressing the rP2u receptor with 1-100 I.tM ATP or UTP induced a large inward current. This current was inhibited by niflumie acid or by injection of EGTA or heparin, consistent with a Ca 2+ activated chloride current. The rank order of potency was UTP>ATI~ATPyS>ADP>>AMP=adenosine and the UTP concentration required for half-maximum current was 0.35-0.5 !ttM. Expression and activation of rP2uR was not affected by preincubafion with pertussis toxin suggesting that the rP2u receptor does not couple to Gi.

The rat liver P2U receptor exhibits siguifieant homology with the mouse and human clones at its stmcmral and functional levels. Identification of the eDNA will allow localization and functional characterization of purmergie signalling in liver using in sita hybridization and other techniques.

791 DEMONSTRATION AND PARTIAL CHARACTERISATION OF PHOSPHOL1PID METHYLTRANSFERASE : ACTIVITY IN BILE CANALICULAR MEMBRANE FROM HAMSTER LIVER A Varma. T Davis, HA Ahmed, RP Jazxawi, TC Northfield. Division of Biochemical Medicine, St George's Hospital, London.

Despite the fact that phosphatidyleholine (PC) constitutes only -30% of the phospholipids (PL) in the bile eanalieular membrane (BCM), it accounts for >95% of the PL in bile. The origin of biliary PC is largely unknown. One possibility is that billary PC is derived from that in BCM, which in turn has its own PC replenished through new synthesis in the endoplasmie retieuinm. An alternative mechanism is through methylation of phosphatidylethanolamine (PE) in BCM. PL methyltransfemses eatalyse the conversion of PE to PC using S-adenosyl methionine (SAMe) as the methyl donor. Aims: To investigate and partially eharacterise methyltrensferase activity in BCM. Methods: Hamster liver was fraetionated and organelles (mierosomes, BCM, basolateral membrane (BLM)) separated using sucrose density gradient ultraeentifugatinn. Purity of fractions was assessed by measuring the specific activity of marker

1 4 enzymes. Methyltransferase activity was assessed Using C-methyl-SAMe and PE as substrates innabated with organdies. PL was extracted and separated by thin layer chromatography. The radioactivity in the PC and in the mono- and di-methyl intermediates formed were measured by scintillation counting. Enzyme activity was measured in BCM in comparison to mierosomes and BLM, and the effect of incubation time, pH and substrate concentration on specific activity was assessed. Results: The purity of BCM rich fraction, as assessed by NADPH eytoehrome C reduetase activity, was confirmed. PL methyltransferase specific activity (nmol of SAMe utilized/mg protein/hour) in microsomes, BCM and BLM was 0.18, 0.35 and 0.02 respectively. The kinetics are shown below. Discussion: PL methyltransferase was demonstrated in BCM

Amount of v SAMe utilized (nmonl/mg) in BCM

. . . . . , . . ,0 , ,

Time(min) pH Cone of SAMe (nM) rich fraction. Its tunction remains speculative, but may include synthesis of PC for enrichment of bile or BCM PL. Conclusion: PL methyltransferase in BCM may play an important role in biliary PL secretion and in maintaining the integrity of BCM. They may be amenable to therapeutic intervention.

792 I N D U C T I O N O F R E T I N O I C A C I D R E C E P T O R I~' IS I N - VOLVED IN I N H I B I T I O N OF P R O L I F E R A T I O N OF C U L - TURED RAT H E P A T O C Y T E S BY R E T I N O I C ACID. H Ikedal~ M lnao 2. and K Fuiiwara 2. 3First Dept. of Internal Medicine, Faculty of MediCine, University of Tokyo, Tokyo and 2Third Dept. of Internal Medicine, Saitama Medical School, Saitama, JAPAN

Retinoic acid regulates differentiation and proliferation of a variety of ceUs. We have demonstrated that retinoic acid inhibits proliferation of cultured rat hepatocytes (BBRC 1993;191:675). Retinoic acid has two types of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which act as transcriptional factors. Thus, retinoic acid exerts its effect by modulating gene expression. There are three subtypes (aft,y) of RARs and RXRs, and each Subtype has a different distribution in tissues and a specific pattern of induction by retinoic acid in various ceils. Although the distribution of RARs and RXRs has been studied in the liver, l i t t le is known about the expression and modulation of these receptors in hepatocytes. D,B/E.CI]_YL To exam- ine the expression-and induction of RARs and a possible role of these receptors in inhibition of proliferation by retinoic acid in cultured rat hepatocytes. METHODS & RESULTS; 1) Retinoic acid was added to the culture medium of hepatocytes isolated from normal rats, 2h after p la t ing and HGF was added 24h later. RARs mRNA levels were determined after another 24h incubation: In quiescent cells, 10-7M retinoic acid increased steady state level of RARI3 mRNA by 3-fold, whereas levels of RARa and RARy mRNA were not changed. Similar result was obtained when the cells were exposed to HGE Time course study showed that retinoic acid increased RARI3 mRNA level gradually up to 3h after its addition and formed a plateau of the level thereafter. 2) The ceils were pre-incubated with retinoic acid for various time before HGF addition and DNA synthesis was examined 48h after plat- ing. DNA synthesis was reduced by 56.3%, 55.5% and 40.5% com- pared to the controls with the pre-incubation for 24h, 3h and 2h, r e - spect ively, but not changed with the p re - incuba t ion for lh . These inhibitory effects were correlated with the extent of RAR[3 expression at the time of HGF addition. CONCLUSIONS: In cultured rat hepato- cytes, retinoic acid induced RAR[5 expression. Such induction may play an important role in inhibition of proliferation of rat hepatocytes.