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T Christoffersen

Publications and source records attributed to T Christoffersen.

At least 73 records · Page 4Linked to original sources
PubMed1987

Temporal requirement for epidermal growth factor and insulin in the stimulation of hepatocyte DNA synthesis.

Primary monolayer cultures of adult rat hepatocytes were used to study the temporal interaction of epidermal growth factor (EGF) and insulin in their stimulation of DNA synthesis. The hepatocytes were cultured both under defined conditions and with serum. EGF and insulin interacted synergistically. The entry into S phase (G1 exit) followed first-order kinetics both in untreated and hormone-stimulated cells. Addition of EGF and insulin at the time of plating did not alter the lag period before the DNA synthesis started (25-26 h), but the rate constant for the S phase entry increased five- to sixfold. Experiments where the time of hormone addition was varied indicated that insulin exerted its strongest effect at the time of plating, whereas the cells became more responsive to EGF after being cultured for up to 40-50 h. The responsiveness to EGF at these later stages required an early exposure of the hepatocytes to insulin. When the administration of EGF to insulin-pretreated hepatocytes was postponed for 44 h after plating in serum-free medium, the cellular sensitivity was increased as compared to EGF treatment at 0 h (a one-log shift of the dose-effect curve), the rate of S phase entry was more rapid, and the lag period for the onset of the EGF effect (i.e., shift of rate constant) was shortened (6-7 h vs. 26 h).

Animals↗
PubMed1986

Oral contraceptive use and breast cancer in young women. A joint national case-control study in Sweden and Norway.

The possible association between oral contraceptive (OC) use and the risk of breast cancer developing before the age of 45 was investigated by means of a population based case-control study in Sweden and Norway. Information was obtained by personal interview from 422 (89.2%) of all eligible patients with a newly diagnosed breast cancer from May, 1984, to May, 1985, and from 722 (80.6%) of all contacted age-matched controls. A multivariate analysis, which accounted for several possible confounding factors, revealed a significant (p = 0.03) association between total duration of OC use and breast cancer risk. The relative risk (RR) of breast cancer after 12 or more years of OC use was 2.2 (1.2-4.0). OC use for more than 7 years before first full-term pregnancy entailed an increased breast cancer risk (RR = 2.0 [1.0-4.2]) which was of borderline significance. When total duration of use was considered, the risk of breast cancer was virtually unrelated to age at first OC use and latency from first use. The results suggest that long-term use of OCs may increase the risk of breast cancer in young women.

Adult↗
PubMed1986

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase.

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.

Adenylyl Cyclases↗
PubMed1986

Increased number of beta-adrenoceptors in hepatocytes from rats treated with 2-acetylaminofluorene.

Treatment of rats with chemical carcinogens, including 2-acetylaminofluorene (2-AAF), leads to a strong increase in the hepatic catecholamine-sensitive adenylate cyclase activity. The present study was undertaken to investigate the mechanism for the development of this increase. We report that hepatocytes isolated from rats which had been fed 2-AAF (0.025% w/w) for 8-12 weeks had an increased number of beta-adrenoceptors, as determined by [3H]dihydroalprenolol binding to whole cells and [125I]iodocyanopindolol binding to washed particles. For both ligands the number of binding sites was about 4-fold higher in hepatocytes from 2-AAF-treated rats than in those from controls. The adenylate cyclase activity of the carcinogen-fed animals showed both a general increase manifested in the basal level (2-fold) and in the activities obtained by stimulation with guanine nucleotides (2-3-fold), cholera toxin (1.5-fold), and glucagon (1.3-fold) and a selective, larger increase in the beta-adrenoceptor-linked activity (7-fold increment of the isoproterenol-sensitive activity). The results indicate that the number of hepatocyte beta-adrenoceptors increases during 2-AAF carcinogenesis. This may, at least in part, explain the rise in catecholamine-sensitive adenylate cyclase activity.

2-Acetylaminofluorene↗
PubMed1985

The separate estimation of cAMP intracellularly bound to the regulatory subunits of protein kinase I and II in glucagon-stimulated rat hepatocytes.

A method is described for the separate determination of cAMP intracellularly bound to the regulatory moieties (RI and RII) of protein kinase I and II. The cAMP endogenously bound to RI or RII in hepatocyte extract was adsorbed to protein A-agarose beads coated with antibodies against RI or RII. The endogenously bound cAMP was eluted from the washed beads with dilute acetic acid before being assayed. By all criteria tested, the present method did not perturb the intracellularly established equilibrium between bound and free cAMP. Stabilization of R X cAMP complexes was achieved by including sulfate in the extraction medium and sulfate/glycerol during the subsequent steps. Hepatocytes were isolated from fed male rats and contained about 0.25 pmol of RI and 0.2 pmol of RII per 10(5) cells. An intracellular titration of the cAMP binding sites of RI and RII was achieved by incubating the cells with various concentrations (1 pM to 10 nM) of glucagon. The fractional saturation of RI and RII was always similar, being 20% in nonstimulated cells. 50% saturation occurred when free cAMP was 0.46 pmol/10(5) cells. A Scatchard plot of the data for the endogenous cAMP binding suggested that cAMP interacted with RI and RII in a slightly positively cooperative manner. About 5% of the intracellularly bound cAMP was sedimentable at 10,000 X g. The apparent affinity of these particulate-associated binding sites was similar to that of soluble RI and RII. Under the conditions used no evidence was obtained for cAMP binding to other proteins than RI and RII.

Animals↗
PubMed1985

Effect of serum on isoproterenol-induced cyclic AMP accumulation in human lymphocytes.

The effects of autologous serum on basal and isoproterenol (IPR) or prostaglandin E1 (PGE1) stimulated adenosine 3',5' cyclic monophosphate (cAMP) levels were investigated in human lymphocytes. For all blood donors, serum (25% (v/v)) lowered the basal cAMP content. In contrast, the responsiveness of the lymphocyte cAMP accumulation to (-)-IPR was increased. This effect was most clearly demonstrable in bicarbonate buffered incubation medium (40-50% increase of maximal response), but was also seen in phosphate buffered medium (10-20% increase). Serum did not alter the sensitivity of the lymphocytes to IPR. The response to PGE1, which was a considerable more effective stimulator of cAMP accumulation than IPR, was not affected in any consistent way by serum. The results indicate that serum influences the regulation of lymphocyte cAMP and that this effect may partly be exerted at the level of the beta-adrenoceptors.

Alprostadil↗
PubMed1985

Quantitative aspects of the effects of insulin, epidermal growth factor and dexamethasone on DNA synthesis in cultured adult rat hepatocytes.

Epidermal growth factor (EGF) and insulin in combination have previously been shown to initiate S-phase in primary cultures of adult rat hepatocytes. We here describe the detailed time course and dose-dependency of the effects of EGF and insulin on DNA synthesis in cultured hepatocytes. The DNA synthesis was assessed either biochemically or autoradiographically with a fairly good correlation between the two methods. DNA synthesis started 24-30 h after plating of the cells and peaked at approximately 70 h. Up to 70% of the cells entered DNA synthesis during this period. EGF and insulin acted synergistically on the DNA synthesis. Dexamethasone raised the DNA synthesis slightly, maximal effect occurred at concentrations above 2.5 nM and this agent was routinely used in the experiments with EGF and insulin. In the presence of 0.4 microM insulin from the time of plating, EGF dose-dependently increased the DNA synthesis with maximal effect at 5-15 nM. When added in combination with 1.7 nM EGF, insulin enhanced the DNA synthesis over the concentration range from 0.1 to 3 nM. These studies show that primary cultures of hepatocytes are useful in assessing the quantitative aspects of the interactions between the growth stimulating effects of hormones.

Animals↗
PubMed1984

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone.

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

A comparison of the binding characteristics of the beta-adrenoceptor antagonists 3H-dihydroalprenolol and 125I-iodocyanopindolol in rat liver.

The binding characteristics of 3H-dihydroalprenolol and 125I-iodocyanopindolol have been compared in a particulate fraction from regenerating rat liver. When total 3H-dihydroalprenolol binding and inhibition of total 3H-dihydroalprenolol binding by (-)isoprenaline, (-)alprenolol and (+/-)cyanopindolol was investigated, it was found that all agents were bound to two classes of saturable binding sites. In the inhibition studies, the presence of two binding components was not obvious until the data were transformed into Hofstee plots and these were decomposed, except in the case of (+/-)cyanopindolol. Only (+/-)cyanopindolol was found to distinguish clearly between the two saturable binding sites identified by 3H-dihydroalprenolol, as indicated by a broad plateau in the inhibition curve. When 125I-iodocyanopindolol was used as radioligand, only one saturable binding site was identified, even in the presence of less selective inhibiting ligands. The lower affinity component of 3H-dihydroalprenolol binding could be inhibited by 10 microM phentolamine. However, binding experiments with 3H-prazosin indicated that the lower affinity component was not identical with the alpha-adrenoceptor. Phentolamine did not influence 125I-iodocyanopindolol binding. Thus, due to its higher specific activity and a high degree of selectivity, 125I-iodocyanopindolol appears to be the ligand of choice.

Adrenergic beta-Antagonists↗
PubMed1983

Mechanisms for the emergence of catecholamine-sensitive adenylate cyclase and beta-adrenergic receptors in cultured hepatocytes. Dependence on protein and RNA synthesis and suppression by isoproterenol.

Adult male rat hepatocytes, which normally respond poorly to beta-adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine-sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the beta-adrenoceptor ligand [125I]cyanopindolol. The emergence of beta-adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydrotestosterone to the cultures, singly or in combination, did not prevent the augmented beta-adrenergic responsiveness. The increase in catecholamine-sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3-hourly intervals led to a dose-dependent suppression of the rise in isoproterenol-responsive adenylate cyclase and prevented the increase in beta-adrenoceptor binding.

Adenylyl Cyclases↗
PubMed1983

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes.

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.

Animals↗
PubMed1982

The proliferation response of rat liver parenchymal cells after partial hepatectomy. A methodological study comparing flow cytometry of nuclear DNA content and in vivo and in vitro uptake of thymidine.

DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [3H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [3H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination. The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [3H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [3H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G1-fractions was only 2 and 7%, respectively.

Animals↗
PubMed1981

Inhibitory effect of prostaglandins on the stimulation by glucagon and adrenaline of formation of cyclic AMP in rat hepatocytes.

Several prostaglandins were found to inhibit hormone-induced cyclic AMP accumulation in suspensions of intact rat hepatocytes. Prostaglandin E1 in concentrations of 0.05--25 micrometers inhibited the cyclic AMP response to glucagon. Maximal inhibition was about 50%. The effect was rapid, being evident within 30 s. Prostaglandins E2, F1 alpha, F2 alpha, A1 and A2 also inhibited the glucagon effect on cyclic AMP in hepatocytes. In cells made highly responsive to adrenaline, by pretreatment of the animals with the carcinogen 2-acetylaminofluorene, and inhibitory effect of prostaglandin E1 was seen also on adrenaline-induced cyclic AMP accumulation. The mechanism of the inhibitory effect of prostaglandins on hormone-stimulated cyclic AMP accumulation was not clarified. Prostaglandin E1 did not inhibit glucagon binding to intact hepatocytes, and so far we have not been able to demonstrate any effect of the prostaglandins on the adenylate cyclase or phosphodiesterase in broken cell preparations. It is concluded that while several previous studies have shown that stimulatory effects of prostaglandins on cyclic AMP are only marginal or lacking in parenchymal liver cells the present data indicate that several prostaglandins exert strong inhibitory interference with hormone-induced cyclic AMP accumulation.

3',5'-Cyclic-AMP Phosphodiesterases↗