Treatment of cholestasis of pregnancy with peroral activated charcoal. A preliminary study.

Elevated serum bile acid levels may play a role in the symptoms associated with cholestasis of pregnancy. Nineteen women (20 pregnancies) with cholestasis of pregnancy were randomized to receive either activated peroral charcoal (9 women, 10 pregnancies) with a dose of 50 g 3 times a day for 8 days or only normal follow-up (n = 10). Serum total bile acids, aminotransferases, alkaline phosphatase, albumin, total cholesterol, and bilirubin (total and conjugated) were evaluated after overnight fasting at the start of the study and on days 4 and 8 of follow-up. By day 8 of treatment serum total bile acid concentrations were lower in patients of the charcoal group than in the control group (P < 0.05). A decrease of total bile acids was observed in seven patients but in only one of the controls (P < 0.05). No other observations (including pruritus) were changed significantly by charcoal. The outcome of pregnancy was good in both groups. This preliminary study suggests that peroral activated charcoal may be considered an alternative in the treatment of intrahepatic cholestasis of pregnancy.

Expression and function of beta-adrenergic receptors in human hematopoietic cell lines.

We investigated the expression and functional characteristics of beta-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [125I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of beta-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimal or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of beta-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32P-labelled beta 2-adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their beta-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage.

Effect of apolipoprotein E polymorphism and XbaI polymorphism of apolipoprotein B on response to lovastatin treatment in familial and non-familial hypercholesterolaemia.

Despite the well-documented efficacy of lovastatin, a wide inter-individual variation in treatment responses has been observed. The aim of the present study was to investigate the possible roles of apolipoprotein E (apo E) phenotype and apolipoprotein B (apo B) XbaI genotype on this variation. The apo E phenotype was determined in 232 subjects (78 cases of familial hypercholesterolaemia [FH] and 154 cases of non-familial hypercholesterolaemia [non-FH]) and the apo B XbaI genotype was determined in 211 subjects (67 cases of FH, 144 cases of non-FH). Depending on their baseline total serum cholesterol levels, these patients used a starting dose of lovastatin of either 20 or 40 mg nightly. After 6 weeks of therapy, slightly but significantly smaller reductions in LDL-cholesterol were observed in patients with the E4/3 phenotype compared with those with the E3/3 phenotype in non-FH with lovastatin 20 mg (-20 vs. -28%; P = 0.043) and in total cholesterol in FH with lovastatin 40 mg (-23 vs. -27%; P = 0.023). No significant differences were found in non-FH patients starting with lovastatin, 40 mg. After doubling of the lovastatin doses, all treatment responses became similar among apo E phenotypes. Moreover, when all patients using lovastatin 40 mg either at 6 or 12 weeks were pooled (n = 224), no differences in treatment responses were observed between the E3/2, E3/3, E4/3 and E4/4 phenotypes. The apo B XbaI genotype did not affect the hypocholesterolaemic efficacy of lovastatin in any of the patient groups. Thus our results indicate that inter-individual variation in the treatment response to lovastatin in both familial and non-familial hypercholesterolaemia is mainly due to factors other than the apo E phenotype or apo B XbaI genotype.

Two ornithine decarboxylase mRNA species in mouse kidney arise from size heterogeneity at their 3' termini.

Ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) mRNA present in mouse kidney comprises two species with molecular sizes of approximately 2.2 and approximately 2.7 kilobases (kb). cDNA clones prepared from murine kidney OrnDCase mRNA were used to determine the reason for the size heterogeneity of these mRNAs. Two of the cDNA clones (pODC16 and pODC74) that differed at the 3' termini were isolated and sequenced. DNA sequencing indicated that each cDNA had a poly(A) tail; however, pODC74 was 429 nucleotides longer than pODC16 at the 3' end and contained two AATAAA signals for poly(A) addition. That the longer cDNA corresponded to the larger mRNA was confirmed by hybridization of a unique Pst I/Pst I fragment from the 3' terminus of pODC74 only to the 2.7-kb OrnDCase mRNA. The two cDNAs did not represent full-length copies of OrnDCase mRNAs and were 1199 (pODC16) and 1204 base pairs (bp) (pODC74) long. There were five mismatches in their 759-bp-long overlapping nucleotide sequence, suggesting that the 2.2- and 2.7-kb OrnDCase mRNAs may be products of two separate, yet very similar, OrnDCase genes. Androgen regulation of the accumulation of these two OrnDCase mRNAs appeared to occur coordinately, as testosterone administration brought about comparable increases in their concentrations in mouse kidney.

Difluoromethylornithine-induced amplification of ornithine decarboxylase genes in Ehrlich ascites carcinoma cells.

Stepwise increments of the concentration of 2-difluoromethylornithine, a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (EC 4.1.1.17), resulted in a selection of cultured Ehrlich ascites carcinoma cells capable of growing in the presence of up to 50 mM difluoromethylornithine. Dialyzed extracts of drug-resistant tumor cells exhibited a very high ornithine decarboxylase activity and contained large excess of immunoreactive ornithine decarboxylase protein. Hybridization analyses with cloned complementary DNA revealed that the difluoromethylornithine-resistant tumor cells also expressed mRNA of the enzyme at greatly enhanced rate. The overproduction of ornithine decarboxylase by the tumor cells grown under the pressure of difluoromethylornithine was at least partly attributable to a 10 to 20-fold increase in the total gene dosage of ornithine decarboxylase involving an amplification of several genes of the gene family. The gene amplification developed appeared to be stable, as the gene dosage only slowly (during a period of several months) returned towards the normal level upon the removal of difluoromethylornithine. The overproduction of ornithine decarboxylase was accompanied by an enhanced resistance of the enzyme towards difluoromethylornithine in vitro.

Effect of a nonsteroidal antiandrogen, flutamide, on androgen receptor dynamics and ornithine decarboxylase gene expression in mouse kidney.

The mechanisms by which nonsteroidal antiandrogens such as flutamide (alpha, alpha, alpha-trifluoro-2-methyl-4'-nitro-m-propionotoluidide) influence androgen receptor distribution and androgen-regulated gene expression are poorly understood. Therefore, we studied acute and long-term effects of flutamide, administered alone or in combination with testosterone, on androgen receptor dynamics in mouse kidney. Nuclear androgen receptors were measured using 5 mM pyridoxal 5'-phosphate extracts of renal nuclei isolated with the hexylene glycol method. Androgen-regulated ornithine decarboxylase (ODC) and ODC-messenger RNA were used as biological markers for hormone action. A single dose of flutamide increased the measurable concentration of renal nuclear androgen receptors in a dose-dependent manner by 1 h after treatment, although to a lesser extent than a comparable dose of testosterone. When 5 mg flutamide was given concomitantly with a submaximal dose of testosterone (0.1 mg), nuclear androgen receptor concentration was similar to that achieved with flutamide alone; this inhibitory effect of the antiandrogen was reversed by a 10-fold higher dose of testosterone. The influence of flutamide on the steady-state receptor levels in renal nuclei achieved by continuous androgen administration was investigated by giving a single dose of this compound to mice with testosterone-releasing implants. In these animals, flutamide administration decreased nuclear androgen receptor concentration with an initial half-life of about 3.3 h. This half-life was similar to that after cycloheximide administration, but significantly longer than that measured (1.3 h) upon removal of the implant. During treatment of female mice for 8 days with testosterone-releasing implants (40 micrograms/day), both the immunoreactive and catalytically active ODC concentration increased about 300-fold. In contrast, there was no stimulation of ODC during the prolonged administration of flutamide, although this treatment resulted in a dose-dependent increase in the nuclear androgen receptor concentration. However, flutamide (up to 650 micrograms/day) given concomitantly with testosterone (40 micrograms/day) almost completely abolished the testosterone-induced increase in ODC. The changes in ODC-messenger RNA concentration, as measured by hybridization to a complementary DNA probe, paralleled those of the enzyme protein suggesting that flutamide action involves inhibition of transcription of androgen-regulated gene(s). We conclude that 1) nuclear androgen receptor turnover in mouse kidney is a relatively rapid process and 2) nonsteroidal antiandrogens such as flutamide have an intrinsic ability to form

Androgen induction of ornithine decarboxylase mRNA in mouse kidney as studied by complementary DNA.

To investigate the mechanisms by which androgens regulate ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) in mouse kidney, a cDNA clone encoding OrnDCase mRNA was prepared. Purification of OrnDCase mRNA from kidneys of androgen-treated mice was accomplished by immunoadsorption of renal polysomes to a protein A-Sepharose column and enrichment for poly(A)-containing RNA by oligo(dT)-cellulose. Double-stranded cDNA synthesized from this mRNA was inserted into the Pst I site of plasmid pBR322 by using oligo(dG . dC)-tailing and was propagated in Escherichia coli. Plasmids containing cDNA sequences coding for OrnDCase were identified by differential colony hybridization, by radioimmunological detection of OrnDCase-like antigens in bacterial cultures, and by cell-free translation of hybrid-selected mRNA followed by immunoprecipitation with monospecific OrnDCase antiserum. A restriction endonuclease fragment of the selected plasmid DNA (pODC54) was labeled by nick-translation and used to study changes in OrnDCase mRNA concentration. After a single dose of testosterone, renal OrnDCase mRNA concentration increased as soon as 6 hr and peaked 24 hr after steroid injection, as measured by RNA blot hybridization. Continuous androgen treatment for 4 days resulted in a 10- to 20-fold increase in OrnDCase mRNA concentration in normal animals, but no induction of this mRNA was detected in mice that have an inherent defect of the androgen receptor (testicular feminization). These results indicate that androgens regulate OrnD-Case synthesis in mouse kidney, at least in part, by increasing OrnDCase mRNA accumulation.

Ornithine decarboxylase mRNA in mouse kidney: a low abundancy gene product regulated by androgens with rapid kinetics.

We have used ODC gene expression in mouse kidney as the biological marker for studies of early androgen action. Some of the characteristics of this regulation involve its strict androgen specificity, the dependency on functional androgen receptors, the lack of requirement for pituitary hormones, and the ability of physiological androgens to bring about activation of the ODC gene. Some recent findings have revealed an additional intriguing feature in the regulation of ODC gene expression in that androgen sensitivity of ODC stimulation is genetically regulated in the mouse kidney (unpublished observations). One of the mechanisms by which androgens regulate renal ODC synthesis is to increase the concentration of ODC mRNA. Increased accumulation of this mRNA was seen as soon as 6 hr after testosterone administration, and it peaked 24 hr posttreatment. In general, acute changes in immunoreactive ODC concentration and ODC mRNA accumulation had very similar kinetics, suggesting that androgens induced de novo synthesis of ODC by increasing the rate of ODC gene transcription. In addition, there was always a highly significant correlation between the catalytic enzyme activity and immunoreactive enzyme protein concentration indicating that androgens do not specifically regulate the active site of ODC by either activating or inhibiting the enzyme by posttranslational modifications. A typical feature of ODC in virtually all eukaryotic tissues is the extremely rapid turnover rate of the enzyme with a biological half-life of 10-30 min. However, no direct information on the turnover rate of ODC mRNA is currently available, although indirect experiments have assigned a half-life of about seven hours for this mRNA. The availability of cDNA clones for ODC mRNA measurements will now permit us to address this question more directly, and also to investigate a possible role of androgens in the stabilization of ODC mRNA. In this regard it is of interest to note that chronic treatment of mice with pharmacological doses of testosterone prolongs the half-life of ODC protein four- to tenfold.

Hormone receptors and target cell responsiveness.

The present article deals with some basic principles in the mechanism of hormone action. All classes of hormones elicit the majority of their physiological effects via specific receptors which are located in three separate compartments of target cells: cell membranes (peptide and glycopeptide hormones), cytoplasm (steroid hormones) and nucleus (thyroid and steroid hormones). These receptors, although distinctively different in their subcellular localizations share some mutual chemical characteristics which play important roles in the regulation of hormone action. Target cell receptor concentration seems to be one variable by which the magnitude and duration of hormone action is regulated, and is subject to both homologous and heterologous hormonal control. In addition, ligand-receptor interaction and occupancy of the receptors by their respective hormones are important factors that are responsible for regulation of hormone action. A major step towards understanding the physiological role of hormone receptors in man are findings that altered receptor function is involved in pathogenesis of and therapeutic approaches to a variety of diseases.

Estrogen receptor in human myoma tissue.

The occurrence and characteristics of an estrogen receptor in the cytosol of myoma samples from human uteri were investigated employing dextran-coated charcoal and density gradient centrifugation techniques. Receptor binding site concentrations in 24 myoma specimens ranged from 23 to 515 fmol/mg cytosol protein (98+/-108, mean+/-S.D.). In one myoma sample no receptor was found. The apparent equilibrium dissociation constant (Kd) was 1.3 X 10(-10) mol/l for estradiol-17beta. On sucrose density gradient centrifugation, [3H]estradiol was bound by macromolecules with sedimentation rates of 4 and 8 S. The latter component was specific for estrogens, whereas the former contained specific and nonspecific binding sites. Ligand specificity studies were carried out utilizing 30 different steroidal compounds. A good correlation was found between the in vitro binding affinity and the in vivo estrogenic potency of the compounds tested. The cytosol estrogen receptor from myoma had a ligand specificity which closely resembled that of the corresponding receptor in normal human myometrium and endometrium as well as in human breast carcinoma. The myoma estrogen receptor level was compared to that in normal myometrium and endometrium in 13 uterine specimens. The receptor concentrations in cytosol fractions from myoma and myometrium correlated significantly (P less than 0.05), whereas no correlation existed between the receptor levels in endometrial and myoma cytosols. Furthermore, the estrogen receptor content in myoma samples did not correlate to estradiol-17beta levels in the myoma cytosol or serum of the same patient.