Metabolism of bilirubin by a clonal strain of rat hepatoma cells.

These studies demonstrate that the MH(1)C(1) strain of rat hepatoma cells has the ability to take up and conjugate bilirubin and then excrete the conjugated pigment into the culture medium. On incubation with unconjugated bilirubin, the average rate of appearance of conjugated bilirubin in the medium was 4.4 +/- 0.20 microg per mg of cell protein per hour (mean +/- SE). The products formed from bilirubin by MH(1)C(1) cells were chromatographically identical to those found in normal rat bile. Assay of bilirubin UDP glucuronyl transferase activity in homogenates of MH(1)C(1) cells gave a value of 3.3 +/- 0.50 microg of conjugated pigment formed per mg protein per hour, only moderately less than the enzyme activity of liver from normal rats. Rat fibroblasts in culture did not conjugate bilirubin, nor did they contain bilirubin UDP-glucuronyl transferase activity. As in living animals, flavaspidic acid inhibited bilirubin metabolism by MH(1)C(1) cells, suggesting that the mechanism for bilirubin uptake is similar to that of normal liver. In contrast to the findings in animals, however, preincubation of MH(1)C(1) cells with phenobarbital led to only minimal enhancement of pigment conjugation. MH(1)C(1) cells represent the first example of a clonal strain of cells in culture in which many of the pathways of hepatic bilirubin metabolism remain intact. They should, therefore, serve as a useful model for studies of bile pigment metabolism which are not easily performed in the living animal.

Transfer of bilirubin uridine diphosphate-glucuronyltransferase to enzyme-deficient rats.

Cells from a clonal strain (MH(1)C(1)) of rat hepatoma were transplanted subcutaneously into two homozygous Gunn rats, which are jaundiced because the enzyme bilirubin uridine diphosphate-glucuronyltransferase is absent from the liver. Because of the enzyme activity present in the transplanted cells, the recipient rats developed the capacity to conjugate bilirubin and reverted in large part to a normal pattern of bilirubin excretion.

Metallothionein: a protein conferring resistance in vitro to tumor necrosis factor.

The role of metallothionein (MT) in the cytotoxicity of tumor necrosis factor (TNF) was investigated in vitro. A human epithelial cell line (HE100) and a mouse fibroblast line (CI 1D100) had previously been cultured to become resistant to 100 microns CdCl2 and were cultured routinely in cadmium-containing medium. The MT content of these cells and the nonresistant parent cell lines (HE and CI 1D) was determined both qualitatively and quantitatively by immunochemical techniques. Immunofluorescence microscopy revealed the cadmium-resistant cell lines to be intensely rich in MT in both the nuclear and cytoplasmic compartments. This finding was confirmed by immuno-electron microscopy which also showed the labeling to be freely distributed and not membrane-bound. In comparison, a very weak labeling of the parent cell lines was observed. MT concentration, as determined by enzyme-linked immunosorbant assay, was found to be 4.05 +/- 1.13% and 3.91 +/- 0.7% of the total protein for HE100 and CI 1D100 cells, respectively. We were unable to detect MT in the parent cell lines by this technique. Dose-survival curves obtained after 3 days treatment of the cells with TNF (0.125-500 ng/ml) revealed that the MT-rich substrains were significantly resistant compared to the parent strains (P < 0.001; t tests). In growth rate studies, where cells were exposed to TNF over a dose range of 0.25-250 ng/ml for 6 days, the resistance of the MT-rich cell lines was confirmed (P < 0.002). These data indicate that MT confers resistance to the cytotoxic effects of TNF in vitro and that sensitivity to TNF may be related to the MT content of the cell.

Increased resistance to chlorambucil in cultured cells with a high concentration of cytoplasmic metallothionein.

Cultured cells with elevated levels of cytoplasmic metallothionein (MT), a small-molecular-weight protein extremely rich in cysteine, showed resistance against the alkylating drug chlorambucil. One human epithelial line (HE) and one mouse fibroblast line (Cl 1D) were used, as well as substrains of both cell lines (HE100 and Cl 1D100). The substrains contain large amounts of MT. Dose-survival curves obtained from cloning studies with the murine cells after exposure to chlorambucil for 1 hr revealed that Cl 1D100 was approximately 3-fold more resistant than were Cl 1D cells (D0 = 32.9 and 11.6 micrograms/ml, respectively); this difference was highly significant (p less than 0.001; t test). In growth rate studies, both MT-containing strains showed resistance against chlorambucil over the total dose range of 0.5 to 100 micrograms/ml compared to the parent strains. After 4 days treatment with 25 micrograms/ml, the growth inhibition of Cl 1D was 67%, but only 13% in Cl 1D100. For HE and HE100, 50 micrograms/ml gave growth inhibitions of 83 and 65%, respectively. Following 1- and 24-hr incubations of cells with [14C]chlorambucil and subsequent ultracentrifugation and gel filtration of the cytosols, about 20 to 40% of the 14C-derived radioactivity coeluted with the MT from the resistant strains. The results indicate that intracellular MT sequesters chlorambucil or its toxic metabolites and that the protein thus contributes to the resistance against this drug.

The synthetic hepatic peptides pyroglutamylglutamylglycylserylasparagine and pyroglutamylglutamylglycylserylaspartic acid inhibit growth of MH1C1 rat hepatoma cells transplanted into Buffalo rats or athymic mice.

Repeated i.p. injections of the synthetic peptides pyroglutamylglutamylglycylserylasparagine and pyroglutamylglutamylglycylserylaspartic acid inhibited the long-term growth of MH1C1 rat hepatoma cells by 50-70% in three in vivo models: metastatic colony growth in the lungs of young Buffalo rats; s.c. tumor growth in young Buffalo rats; and s.c. tumor growth in athymic mice. The amide free peptide pyroglutamylglutamylglycylserylaspartic acid which inhibited the tumor growth in all the models showed a curvilinear dose-response relationship with a maximal effect at 1000 pmol/animal in mice and at 100 pmol/animal in rats. The amidated peptide pyroglutamylglutamylglycylserylasparagine, which was only tested in the lung model, showed growth inhibition with 2, 20, or 200 pmol/animal, but 200 pmol/animal was most effective. We have recently reported that these peptides show cochromatography with hepatic growth inhibitory peptides, isolated from mouse liver.

Growth inhibition, cytokinesis failure and apoptosis of multidrug-resistant leukemia cells after treatment with P-glycoprotein inhibitory agents.

The multidrug transporter P-glycoprotein (Pgp), which is frequently overexpressed in multidrug resistant leukemia, has many proposed physiological functions including involvement in transmembraneous transport of certain growth-regulating cytokines. Therefore, we studied cell growth of three pairs of drug resistant and sensitive leukemia cell lines (KG1a, K562 and HL60) exposed to three different inhibitors of Pgp. The resistant KG1a and K562 sublines, which expressed high levels of Pgp, responded to low doses of the cyclosporin SDZ PSC 833, the cyclopeptolide SDZ 280-446, and the cyclopropyldibenzosuberane LY335979 with a dose-dependent growth inhibition. In the resistant variants of KG1a and K562 cells the mean half-maximal growth inhibitory doses (GI50) of SDZ PSC 833 were 312 (SE 41) and 414 (SE 50) nM, those of SDZ 280-446 were 685 (SE 51) and 578 (SE 54) nM, and those of LY335979 were 66 (SE 1) and 48 (SE 8) nM, respectively. Exposure to 1 microM SDZ PSC 833 resulted in tetraploidization, cytokinesis failure and apoptosis of the KG1a and K562 MDR variants. Conversely, parental cells with no or low levels of Pgp and the non-Pgp resistant variant of HL60 cells were not receptive to these cytotoxic effects. We conclude that inhibition of Pgp may exercise selective cytotoxicity in Pgp-rich leukemia cells indicating a possible therapeutic target in multiresistant leukemia.

The cyclosporin PSC 833 increases survival and delays engraftment of human multidrug-resistant leukemia cells in xenotransplanted NOD-SCID mice.

Circumvention of chemoresistance in cancer may involve several modulator drugs with high affinity for the multidrug transporter P-glycoprotein (Pgp), which is expressed in a number of multi-resistant malignancies. Pgp acts as a membrane efflux pump with broad substrate specificity including antineoplastic drugs and endogenous substances such as certain cytokines and sphingolipids. Therefore, the consequence of Pgp blockade could be far more complex than intracellular drug retention. In the present study exposure of the Pgp inhibitor, PSC 833 (1200 ng/ml), to Pgp expressing KG1a/200 human leukemia cells provoked cell cycle arrest and apoptosis in vitro. This finding was put to test in vivo using a xenotransplant model of KG1a/200 human cells intravenously inoculated into non-obese diabetic severe combined immunodeficient (NOD-SCID) mice. The animals were randomly allocated to receive treatment with PSC 833 (n = 32) or placebo (n = 24). PSC 833 (30 mg/kg) was subcutaneously injected six or 12 times separated by 48-96 h. The overall mean whole blood concentration of PSC 833 was 1191 +/- 60 ng/ml (s.e.m.) at 20 h after administration. Tumor engraftment was significantly reduced in the treatment group (P = 0.037), which also had prolonged survival compared to control animals (P = 0.0016). This is the first study that demonstrates antileukemic effects of a Pgp inhibitor as single agent therapy in vivo, and the present data raise the possibility of alternative exploitation of modulators in cancer chemotherapy.

The Norway study: plasma concentrations, efficacy, and adverse events.

In the Norwegian comparative trials of piroxicam and naproxen in osteoarthritis, conducted by Husby and co-workers and Rugstad et al, relationships between plasma drug concentrations and the following were examined: efficacy in patients with osteoarthritis; all adverse reactions; and serious adverse events, which included gastrointestinal ulceration or bleeding and congestive heart failure. Increased plasma concentrations were correlated with age and female sex, but efficacy, overall side effects, and serious adverse events were not correlated with increasing plasma levels.

Monitoring of azathioprine treatment by determination of 6-thioguanine nucleotide concentrations in erythrocytes.

Thioguanine nucleotides (6-TGN) are intracellular metabolites that may contribute to the antiproliferative effects of AZA. The objectives of our study were to describe the variability of 6-TGN concentrations during AZA therapy and to investigate possible correlations between 6-TGN levels and subsequent myelosuppression. We measured 6-TGN concentrations in RBC of 65 renal transplant recipients from day 0 until 11-64 days after transplantation. High 6-TGN concentrations were observed in relation to elevated S-creatinine. In 15 patients, 6-TGN concentrations above 200 pmol/8 x 10(8) RBCs were measured (high 6-TGN group: mean maximal 6-TGN = 552 pmol/8 x 10(8) RBCs, SE = 91). In the remaining 50 patients, mean maximal 6-TGN was 82 pmol/8 x 10(8) RBCs, SE = 6.1 (low t-TGN group). In the former group, mean S-creatinine measured on the day of maximal 6-TGN was 466 mumol/L (SE = 62.3), while in the latter it was 190 (SE = 14.7). In the high 6-TGN group, we observed a lower mean nadir neutrophil count than in the low 6-TGN group (3.4 vs. 5.1 x 10(9) neutrophils/L). The nadir neutrophil count occurred, on the average, 12.7 days after maximal 6-TGN in the high 6-TGN group, with no such delay in the low 6-TGN group. This study demonstrates for the first time that 6-TGN in RBCs may rise to very high levels during impaired renal function. Furthermore, the results support the hypothesis that myelosuppressive side effects of AZA therapy correlate with 6-TGN concentrations. Renal transplant recipients may benefit from the monitoring of AZA through RBC 6-TGN measurements.

Monitored high-dose azathioprine treatment reduces acute rejection episodes after renal transplantation.

BACKGROUND: Azathioprine (AZA) is widely used in organ transplantation. Common practice is to adjust dose according to body weight only, despite documented pharmacokinetic variability. The purpose of this study was to investigate whether high-dose AZA treatment monitored by 6-thioguanine nucleotides (6-TGN) levels reduces the incidence of rejection episodes in renal transplantation without a corresponding increase in myelotoxicity. METHODS: Patients receiving cyclosporine, steroids, and AZA were randomized into either the low-dose AZA group (3 mg/kg on day 0, then 2 mg/kg/day the first week and 1 mg/kg/day thereafter) or the high-dose AZA group. In the latter, AZA was started at 5 mg/kg/day and then adjusted to keep 6-TGN concentrations (measured twice weekly) between 100 and 200 pmol/8 x 10(8) RBCs. RESULTS: A total of 360 transplant recipients were included in the final analysis. The cumulative incidence of first rejection episodes was reduced by 21%, from 62.8% in the low-dose group to 49.4% in the high-dose group (difference: 13.3%; 95% confidence interval: 3.2-23.5). Similar results were found in subgroups according to HLA-DR match. The 6-TGN concentration was significantly higher in the high-dose AZA group during the first month, and the reduction in rejection episodes was achieved in the same period. A larger proportion of patients in the high-dose group had nadir white blood cell count below 2.0 x 10(9) leukocytes/L (13.3% vs. 4.4%; difference: 8.9%; confidence interval: 3.1-14.7). CONCLUSIONS: High-dose AZA therapy in a triple-drug regimen, monitored by 6-TGN, will keep myelotoxicity within acceptable limits with the benefit of a reduction in acute rejection episodes.

Clinical pharmacokinetics of methyldopa.

Absorption of methyldopa from the gastrointestinal tract is incomplete and variable; bioavailability after oral administration is about 25% (range 8 to 62%). The average time to reach maximum plasma concentration (tmax) [chemically determined] is 2 hours, when the maximum plasma concentration of active drug accounts for 50% of the radioactivity, the remainder representing various metabolites. Physicochemical determination of methyldopa shows that bi-phasic elimination occurs after both intravenous and oral administration, the half-life of the alpha-phase being 0.21 hours (range 0.16 to 0.26 hours) and of the beta-phase 1.28 hours (range 1.02 to 1.69 hours) in normal subjects. Methyldopa is less than 15% protein bound, whereas the primary metabolite, which most probably is the O-sulphate, is about 50% protein bound. The apparent volume of distribution in the central compartment is about 0.23L/kg (range 0.19 to 0.32L/kg), and the total volume of distribution (calculated as Vdarea) is about 0.60L/kg (range 0.41 to 0.72L/kg) in healthy volunteers. Acid-labile conjugates are formed after oral administration. These acid-labile conjugates, in particular the O-sulphate, are probably formed in the intestinal cells, since they are detected in very small amounts after intravenous administration. Additionally, there is a rapid formation of partly unidentified metabolites after both intravenous and oral administration. After intravenous administration the quantitatively most prominent metabolites are methyldopamine and the glucuronide of dihydroxyphenylacetone, but traces of 5 or 6 other metabolites have also been found and identified. These metabolites are probably formed in the liver, but the complete metabolic pattern is still unknown. The renal clearance of methyldopa (95 ml/min/m2) is more than 50% higher than the endogenous creatinine clearance. Renal excretion of some metabolites is slower. Extrarenal elimination accounts for about 50% of the total body clearance of the drug. Renal excretion is very low in patients with renal failure, resulting in accumulation of both active drug and, in particular, its metabolites. There is a marked accumulation of unidentified metabolites in renal failure patients, which possibly explains the strong and prolonged hypotensive action of methyldopa in these patients.

Metallothionein in cultured human epithelial cells and synovial rheumatoid fibroblasts after in vitro treatment with auranofin.

Radioimmunoassay (RIA) and reversed-phase high-pressure liquid chromatography (HPLC) were used to investigate gold-binding proteins of possible metallothionein (MT) nature occurring upon auranofin exposure of cultured human cells. An epithelial cell line (HE) and two sub-strains were examined. The HEAF sub-strain had been made resistant to 2 mumole auranofin/l culture medium. The resistance was associated with the appearance of gold-binding substances with gel filtration characteristics like MT. The HE100 sub-strain had been made resistant to 100 mumole CdCl2/l and contained high amounts of cytosolic Cd-induced MT. In addition, cultured synovial fibroblasts, derived from normal (SN) and rheumatoid (SRA) synovial tissues, were investigated. Evidence was obtained by RIA that the low molecular weight (mol.wt. 6000-7000) gold-binding proteins occurring in the HEAF cells and SRA cells following auranofin exposure, were of MT nature. The relative amounts of MT in the epithelial cell lines were: HE:HEAF:HE100 = 1:18:100. The relative amounts in the synovial fibroblasts were: SN:SRA:SRA treated with auranofin = 1:3:10. The HPLC methods used were found suitable for isolation of Cd-MT in the HE100 cells, but not for the Au-MT in the HEAF cells. By HPLC, the Cd-MT in the HE100 cells was resolved into 3 MT-1 and 1 MT-2 iso-proteins exhibiting the amino acid composition typical of MT. Judged by HPLC, the MT in these cells constituted 0.4% of the cytosolic proteins.

Cadmium uptake and metabolism in cultured cells.

Cultured cells have been made resistant to otherwise lethal concentrations of the toxic Cd ion, probably by induction of metallothionein (MT) synthesis and binding of Cd to the MT. One human epithelial cell line (HE) and two enzyme-deficient mutants of mouse fibroblasts (L-cells) (Cl 1D and A9) and their Cd-resistant substrains with a high content of MT, have been used to study cellular Cd uptake and metabolism. For cell survival of "wild type" cells, the critical level of intracellular Cd is determined to be around 6 nmole Cd/mg cell protein. Resistant cells can tolerate Cd levels several times above this concentration, if the major part of Cd is bound to MT. The technique of perturbed angular correlation spectroscopy (PAC) has been applied to living Cd-resistant cells. It was shown that greater than 66% of Cd in the resistant strains was bound to MT, and that MT is apparently freely suspended in the cell cytoplasm. Chelating agents differ in toxicity and Cd-releasing effect on the cells, but apparently remove the non-MT-bound Cd pool. After various periods of Cd omission, either in vitro or in vivo, growing the cells as tumors in athymic nude mice, the stability of Cd resistance in these cells seems to be dependent on the capacity of cells for de novo synthesis of MT shortly after re-exposure to the metal.

Epidemiology of NSAID-induced gastrointestinal problems and the role of cimetidine in their prevention.

It is difficult to ascertain the incidence of gastrointestinal side-effects associated with intake of non-steroidal anti-inflammatory drugs (NSAIDs). In retrospective studies, some NSAIDs have been reported to be associated with a higher incidence of gastrointestinal side-effects than others. However, this has not been verified either in a prospective case-review study or in a large double-blind study. Serious side-effects, such as bleeding, perforation and heart failure, occur in approximately 1% of patients using NSAIDs. One-third of all patients receiving NSAIDs will have gastrointestinal complaints. Since at least 10% of patients terminate treatment with NSAIDs as a result of side-effects, even reduction of those that are not life-threatening would be of great benefit. H2-receptor antagonists have proved effective in ulcer treatment, and their use as prophylaxis against the side-effects of NSAIDs is being widely studied. In a recent study, 63 patients who had experienced serious upper gastrointestinal side-effects were given cimetidine while continuing their NSAID therapy. All but 4 of the 47 who had gastric or duodenal ulcer on first admission were healed at 8 weeks, and none of the remaining 16 with diffuse bleeding gastritis experienced further clinical episodes of bleeding or ulcer-related dyspepsia.

The influence of partial hepatectomy on the pharmacokinetics of preoperatively injected 4'-epidoxorubicin in rats.

Preoperative administration of 4'-epidoxorubicin (Epi-A) has been suggested as adjuvant therapy in patients undergoing liver resection for hepatocarcinoma. To assess the influence of partial hepatectomy on the pharmacokinetics of Epi-A, an experimental study in rats was undertaken in which 5 mg/kg Epi-A was given i.v. 10 min prior to a 2/3 hepatic resection or sham operation. Epi-A levels in liver tissue and plasma were determined using a sensitive and specific HPLC method. A marked uptake of Epi-A in liver tissue was found at 10 min after injection. The partially hepatectomized rats showed a 2-fold increase in AUC between 4 and 72 h as compared with the sham-operated controls. The terminal half-life from 24 to 72 h was not significantly changed by the partial hepatectomy. The plasma binding of Epi-A was measured at 4 h post-surgery. The fraction of unbound Epi-A was 0.16 in partially hepatectomized animals and 0.20 in sham-operated rats. The results indicate that when Epi-A is given prior to liver resection, a dose reduction might be necessary to avoid increased side effects due to the rise in AUC.

Human hepatoma cells rich in P-glycoprotein display enhanced in vitro invasive properties compared to P-glycoprotein-poor hepatoma cells.

Whether the phenotypes of drug resistance and metastatic activity in cancer are dependent on each other or not is controversial. We compared in vitro invasive properties of human hepatoma cells resistant to epirubicin and rich in P-glycoprotein (Pgp) (HB8065/R) with the parental epirubicin-sensitive, Pgp-poor cells (HB8065/S). The HB8065/R cells displayed elevated capacity to migrate in a transwell chamber assay (three- to fourfold compared to the HB8065/S cells), both in the absence and presence of a reconstituted basement membrane extract (Matrigel). In the presence of the P-gp inhibitor PSC 833 (1.5 micrograms/ml) the capacity of the HB8065/R cells to cross Matrigel-coated filters was attenuated by approximately 25%. Compared to the HB8065/S cells, the resistant cell line expressed higher level of plasminogen activator inhibitor (PAI)-1 mRNA (approximately threefold), which was reflected by a approximately fivefold increase in secreted PAI-1 immunoactivity (approximately 50 ng/10(6) HB8065/R cells). Furthermore, treatment with PSC 833 was associated with upregulation of PAI-1 mRNA (approximately 3.5-fold) and immunoactivity (approximately twofold) in the HB8065/R cells. Level of tissue inhibitor of metalloproteinases (TIMP)-1 was also significantly increased in the HB8065/R cells compared to the HB8065/S cells, whereas both cell lines showed low constitutive expression of TIMP-2. Levels of TIMPs were not altered by PSC 833. These data suggest that overexpression of Pgp in these hepatoma cells may covariate with the phenotypes of both enhanced in vitro invasiveness and high PAI-1 expression, whether randomly acquired or not.

Determination of free concentration of piroxicam and naproxen in plasma. The influence of experimental conditions in equilibrium dialysis.

An equilibrium dialysis method was established in order to investigate possible relationships between free drug concentrations of piroxicam and naproxen and clinical events. Therefore the influence of variations in pH, phosphate concentration and sodium azide concentration of the dialysis buffer on the free concentrations of piroxicam and naproxen was investigated. Piroxicam was found to have a pH-dependent protein binding. Therefore a good control of pH during the dialysis process is necessary. This has been achieved by increasing the buffer capacity of the dialysis buffer, by adding an antibacterial agent to the dialysis buffer and by cleansing the dialysis cells with 70% ethanol before use to prevent bacterial growth. Addition of 0.03% sodium azide as an antibacterial agent and the use of a 0.09 mol/l phosphate buffer gave good pH control. A method to correct for deviations of pH in measurements of free concentrations of piroxicam by a simple mathematical correction has been found. As naproxen was found to have a protein binding independent of pH, a pH-correction is not necessary for this drug. Standardized conditions in determination of protein binding of drugs by equilibrium dialysis are important, as composition of the dialysis buffer and pH of plasma compartment at equilibrium may influence the free concentration measurements. Comparisons of data from experiments using different methods are therefore difficult; the importance of pH-control is stressed. With the methods used in the present investigation, equilibrium dialysis in connection with HPLC, the coefficients of variation for piroxicam and naproxen free concentrations are 5.5% and 7.4%, respectively.

Induction of in vitro resistance to 4'-epidoxorubicin and cis-dichlorodiammineplatinum in hepatoma cells.

We have developed in vitro resistance to 4'-epidoxorubicin (Epi-A) and cis-dichlorodiammineplatinum (cis-DDP) in one rat (MH1C1) and one human hepatoma cell line (HepG2). When compared to their parental cells, the Epi-A resistant rat cells were 17 times and the resistant human cells 27 times more resistant to Epi-A in terms of GI50 in the cell growth inhibition assay. The cis-DDP resistant rat cells were 20 times and the resistant human cells 12 times more resistant to cis-DDP. Cross-resistance to cis-DDP was observed in the Epi-A resistant rat cells but not in the human cells. The multidrug resistant gene product, GP 170, was markedly expressed in both Epi-A resistant substrains compared with their parent lines, suggesting a role of this protein in the development of resistance to Epi-A. Cadmium-binding proteins of metallothionein (MT) size bound 52% of cytosolic 109cadmium in the cis-DDP resistant human cells compared with 8% in the parental cells. This may indicate that these proteins contribute to the observed cis-DDP resistance.

The regulation of cyclic AMP levels in cultured MH1C1 rat hepatoma cells and in solid tumours derived from MH1C1 cell inoculates.

Several studies have found high cAMP content in hepatomas in vivo, while hepatoma cells in vitro have very low levels. To explore this discrepancy and the regulation of cAMP in hepatomas, we have examined the cell line MH1C1 from Morris hepatoma 7795. These cells in culture contained low intracellular cAMP concentrations (approximately 0.5 pmol/mg protein at confluency), and were unresponsive to glucagon and prostaglandins (PG) E1 and E2. In contrast, solid hepatomas in rats developed from inoculates of MH1C1 had a 40-fold higher basal cAMP concentration and were stimulated by PGE1 and PGE2. Fibroblasts cultured from these tumours also contained high cAMP levels and responded strongly to PGE1. This may suggest that the difference in cAMP regulation between hepatomas in vivo and hepatoma cells in vitro results from the presence of other cells in the solid tumour rather than from selection of low-cAMP cells during the cloning procedure. Low-Km and intermediate-Km cAMP phosphodiesterase activity was high in MH1C1, compared to normal hepatocytes. This might contribute to the low cAMP level. The ability of MH1C1 to form cAMP was not defective, as the level could be increased more than 200-fold by beta-adrenergic activation in the presence of the phosphodiesterase inhibitor methylisobutylxanthine.

Renal side effects of high and low cyclosporin A doses in patients with rheumatoid arthritis.

Thirty-nine patients with classical or definite rheumatoid arthritis (RA) entered a randomized double-blind placebo controlled study with low dose cyclosporin A (CyA) (mean whole blood CyA level after 26 weeks 282 +/- 33 micrograms/l) or placebo treatment for 48 weeks. The placebo treatment had to be withdrawn before 48 weeks in 9 out of 19 patients because of a deterioration of their arthritis symptoms. All 20 patients in the CyA group completed 26 weeks of treatment. Fourteen of them were followed up for 48 weeks of treatment on CyA and then 12 weeks after CyA treatment was withdrawn. The side effects in the CyA group were compared with the results from another study with high CyA doses (mean whole blood CyA level 419 +/- 71 micrograms/l) for 26 weeks in 11 other patients with RA. Serum creatinine increased 19.7% (p less than 0.01) in the low CyA dose group after 26 weeks as compared with 59.5% in the high CyA dose group. Creatinine clearance was reduced by 17.9% (low dose) (p less than 0.01) and 26.2% (high dose), respectively. Twelve weeks after withdrawal of the low dose CyA treatment, serum creatinine values were still higher than before treatment (p less than 0.05), but creatinine clearance had normalized. Eight of the 20 patients treated with low CyA doses also started with nonsteroidal anti-inflammatory drugs (NSAIDs) after 20-36 weeks of CyA treatment. Serum creatinine was 16.6% above baseline 4 weeks before NSAIDs compared with an increase of 40.4% (p less than 0.05) 4 weeks after start of NSAID treatment.(ABSTRACT TRUNCATED AT 250 WORDS)