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Previous studies have suggested that the enzyme microsomal epoxide hydrolase (mEH) is able to mediate sodium-dependent transport of bile acids such as taurocholate into hepatocytes (von Dippe, P., Amoui, M., Alves, C., and Levy, D. (1993) Am. J. Physiol. 264, G528 -G534). In order to characterize directly the putative transport properties of the enzyme, a pCB6 vector containing the cDNA for this protein (pCB6-mEH) was transfected into Madin-Darby canine kidney (MDCK) cells, and stable transformants were isolated that could express mEH at levels comparable with the levels expressed in hepatocytes. Sodium-dependent transport of taurocholate was shown to be dependent on the expression of mEH and to be inhibited by the bile acid transport inhibitor 4,4-diisothiocyanostilbene-2,2disulfonic acid (DIDS), as well as by other bile acids. Kinetic analysis of this system indicated a K m of 26.3 M and a V max of 117 pmol/mg protein/min. The K m value is essentially the same as that observed in intact hepatocytes. The transfected MDCK cells also exhibited sodium-dependent transport of cholate at levels 150% of taurocholate in contrast to hepatocytes where cholate transport is only 30% of taurocholate levels, suggesting that total hepatocyte bile acid transport is a function of multiple transport systems with different substrate specificities, where mEH preferentially transports cholate. This hypothesis is further supported by the observation that a monoclonal antibody that partially protects (26%) taurocholate transport from inhibition by DIDS in hepatocytes provides almost complete protection (88%) from DIDS inhibition of hepatocyte cholate transport, suggesting that taurocholate is also taken up by an alternative system not recognized by this antibody. Additional support for this concept is provided by the observation that the taurocholate transport system is almost completely protected (92%) from DIDS inhibition by this antibody in MDCK cells that express mEH as the only bile acid transporter. These results demonstrate that mEH is expressed on the surface of hepatocytes as well as on transfected MDCK cells and is able to mediate sodiumdependent transport of taurocholate and cholate.

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Structural studies on a very lysine-rich histone show that the carboxyl-terminal half of the molecule is enriched in lysine (and proline). which suggests that it is a site for binding to DNA. The amino-terminal half. containing most of the acidic residues. resembles small, nonhistone proteins and so might have specificity for factors other than DNA.

.49). During the same period, neither overall cell growth nor the concentrations of malate dehydrogenase (EC 1.1.1.37), acid phosphatase (EC 3.1.3.2), or alanine aminotransferase (EC 2.6.1.2) were significantly decreased by the base analog. Addition of thymidine to the growth medium rapidly counteracts the inhibition of tyrosine aminotransferase synthesis but restores the normal concentrations of lactate-, alcohol-, and glucose-6-phosphate dehydrogenases much more slowly. Growth of the cells for only one generation in the presence of bromodeoxyuridine, followed by the addition of thymidine, produces transient decreases in the concentrations of the three "late-responding" dehydrogenases, beginning 2-3 generations after exposure to the analog.It is concluded that the selective inhibitory effects of the analog could result from a mechanism in which bromodeoxyuridine is uniformly incorporated into cellular DNA, but inhibits the transcription of only certain genes into messenger RNA. A mathematical model is derived to account for the observed differences in the kinetics of the inhibition of synthesis of the gene products that are sensitive to the analog.The appearance of certain differentiated functions in embryonic tissues is suppressed in vitro by 5-bromodeoxyuridine, BrdU (1-4), as is the tumorigenicity of mouse melanoma cells (5). We reported earlier that the synthesis of the inducible enzyme tyrosine aminotransferase (TAT) is dramatically ieduced when an established line (HTC) of rat hepatoma cells is grown in culture with BrdU, and we have used this system to study the mechanisms by which BrdU affects specific cellular functions (6). These studies showed that the thymidine analog must be incorporated into DNA to decrease TAT synthesis, and that the effect of BrdU in HTC cells is rather selective, since the rates of cell growth and of general protein and RNA synthesis are relatively unaffected (6). MATERIALS AND METHODSThe procedures described previously (6) were used for growing HTC cells, preparing cell-free extracts by freeze-thawing, and assaying for TAT activity. The same cell lysates were assayed spectrophotometrically for their contents of alcohol (7), lactate (8), glucose-6-phosphate (9), and malate (10) dehydrogenases as described in the references cited. Alanine amiinotransferase was assayed in cell extracts by a method described by Larsson and Tomkins'(to be published). Acid l)hosphatase was determined in uncentrifuged cell lysates prepared by thawing frozen cell pellets in 0.02 M Tris.HCI buffer, pH 7.6. The assa-was performed in 1 ml of 80 mMI sodium acetate buffer, pH's, containing 0.9 mMI p-nitrophenyl phosphate, 2 mMI EDlTA, and 0.4 mg/ml bovine serum albumin. The reacting was terminated with 0.1 ml of 10 N KOH, and p-nitrobphenol was measured spectrophotometrically at 400 nim. A unit of enzyme activity is defined as the production of 1 4mol of product per min at the temperature of the assay (370C for TAT and acid phosphatase, and room teml)erature for the others). RESULTSAs reported previously ...

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Theophylline, a cyclic nucleotide phosphodiesterase inhibitor, increases the rate of tyrosine aminotransferase (TAT) degradation in rat hepatoma tissue culture (HTC) cells. Theophylline (0.1-10 mM) causes a two- to five-fold increase in intracellular cAMP concentration but a 30-60% decrease in cGMP concentration. The decrease in cGMP occurs at doses of theophylline which increase the rate of TAT degradation. When cGMP levels are increased by incubating the cells with either Mn2+, an activator of guanylate cyclase, or 8-bromo-cGMP, an analog of cGMP, the effect of theophylline is reversed and the rate of TAT degradation is slowed. Thus, the rate of TAT degradation is inversely related to the concentration of cGMP in HTC cells. This raises the possibility that a cGMP-dependent event is involved in the control of specific protein degradation.