Cell-cycle regulated expression of Rap1 in regenerating liver.

Rap1 proteins are capable of competing with Ras p21 for binding to effectors, and of antagonizing some Ras-induced effects, but their participation in normal growth regulation has not been established. The level of Rap1 protein and the expression of the rap1A gene were examined by immunoblotting and Northern analysis during the regenerative growth response in rat liver following partial hepatectomy. Protein and mRNA were significantly down-regulated prior to and during the onset of DNA synthesis. The timing of this effect is consistent with a model in which expression of Rap1 is turned off or down to allow the initiation of proliferation.

Elevated levels of homologous DNA recombination activity in the regenerating rat liver.

We have characterized homologous DNA recombination activity in nuclear protein extracts prepared from quiescent and regenerating rat livers. Activity measured in regenerating liver extracts was elevated approximately 35-fold above control, and its appearance closely mirrored the first wave of DNA synthesis, peaking 24 hours after a regenerative stimulus, and returning fairly rapidly to basal levels. We also identified a strand-transferase protein of approximately 100 kDa whose presence in these extracts correlates with homologous recombination activity. Recent evidence suggests that mammalian somatic cells possess a recombinational DNA repair mechanism analogous to that described in the yeast Saccharomyces cerevisiae. Our results indicate that this recombinational repair process may be regulated in vivo by, or play a role in, progression through the cell division cycle.

Alpha 1-adrenergic receptors in liver regeneration.

The alpha 1-adrenergic receptor mediates the effects of catecholamines on DNA synthesis, as observed in rat liver following a 2/3 partial hepatectomy and in serum-free primary cultures of adult rat hepatocytes exposed to epidermal growth factor. In vitro, norepinephrine action at this receptor heterologously down-regulates epidermal growth factor receptors. In vivo, the alpha 1 receptor's effect on DNA synthesis is restricted to early time points after partial hepatectomy. alpha 1 receptor binding capacity does not vary until 48 hr after liver resection (at which time binding is reduced), but an uncoupling of receptor binding from membrane phosphoinositide turnover occurs between 8 and 16 hr after partial hepatectomy. This change is preceded by a fall in membrane-associated ras p21 detected by radioimmunoassays (46% of control levels by 2 hr after partial hepatectomy). Whether this change represents a loss of p21 protein from membranes or a modification that results in a loss of immunoreactivity is not known.

Regulation of hepatocyte growth: alpha-1 adrenergic receptor and ras p21 changes in liver regeneration.

Catecholamines, acting via the alpha-1 adrenergic receptor, have been demonstrated to influence adult rat hepatocyte DNA synthesis in primary culture and in vivo during liver regeneration following partial hepatectomy (PHX). Earlier investigations have suggested that the alpha-1 effect on DNA synthesis is significant only during the first day following PHX. We examined receptor binding at several early and late time points after surgery, and we observed a significant loss of specific [3H]-prazosin binding to cells isolated from rat livers 48 and 72 hr after PHX. In contrast, the ability of norepinephrine to stimulate inositol phosphate production in isolated cells prelabeled with [3H]-myo-inositol was transiently reduced between 8 and 16 hr, when alpha-1 binding capacity was virtually unchanged. This uncoupling of phosphoinositide turnover from binding was preceded by a drop in hepatic membrane ras p21 content, as assayed by liquid competition radioimmunoassay. The loss of immunoreactive p21 from membranes was significant by 2 hr after PHX. These findings suggest a role for alpha-1 receptors and ras protein in the early events of liver regeneration.

Blockade of alpha-1 adrenergic receptor inhibits hepatic DNA synthesis stimulated by tumor promoters.

Studies with regenerating liver and hepatocyte cultures have shown that the alpha-1 adrenergic receptor (A1AR) is involved in the early events which transmit a mitogenic signal to hepatocytes after 2/3 partial hepatectomy. In this study, we investigated the role of A1AR in DNA synthesis associated with the augmentative hyperplasia stimulated by the xenobiotic hepatic tumor promoters phenobarbital (PB) and alpha-hexachlorocyclohexane (alpha-HCH), and the peroxisome proliferator ciprofibrate. Male F344 rats were treated with each of the three xenobiotics to stimulate hepatic DNA synthesis. When either phenobarbital or alpha-HCH administration was preceded and accompanied by the A1AR antagonist prazosin, DNA synthesis was significantly inhibited, as measured by [3H]thymidine incorporation or 5-bromo-2'-deoxyuridine (BrdU) nuclear labeling index. There was no inhibition of DNA synthesis by prazosin in the ciprofibrate treated group. The inhibition of hepatic DNA synthesis by prazosin was accompanied by non-significant changes in the number of alpha-1 binding sites in the PB and alpha-HCH treated groups, but a significantly reduced number of alpha-1 binding sites in the ciprofibrate treated group. These studies suggest that A1AR is involved in generating the mitogenic signal leading to hepatic DNA synthesis induced by xenobiotic hepatic tumor promoters phenobarbital and alpha-HCH. A1AR is not involved in the mitogenic pathway generated by the peroxisome proliferator ciprofibrate.

Norepinephrine modulates the growth-inhibitory effect of transforming growth factor-beta in primary rat hepatocyte cultures.

TGF-beta is a potent inhibitor of EGF-induced DNA synthesis in primary rat hepatocyte cultures. Norepinephrine (NE) was shown to modulate this inhibition of DNA synthesis. It produced a five-fold increase, from 2.8 pM to 14.4 pM, in the ID50 for TGF-beta. The effect was dose-dependent and was significant at concentrations of 10(-6)M NE and greater. The modulation by NE was mediated by the alpha 1-adrenergic receptor as shown by the ability of the alpha 1 antagonist prazosin to block the activity. This effect might be important during liver regeneration in allowing escape of hepatocytes from negative growth control exerted by TGF-beta.

Early events in the regulation of hepatocyte DNA synthesis: the role of alpha-adrenergic stimulation.

The role of adrenergic agents in DNA synthesis was investigated in two models of stimulated hepatocyte growth: in vitro primary serum-free cultures of adult parenchymal hepatocytes, and in vivo liver regeneration after two-thirds partial hepatectomy. In both systems the alpha 1-adrenergic receptor appeared to be involved in mediating stimulatory effects. In primary hepatocyte cultures norepinephrine acted via this receptor to enhance the DNA synthesis stimulated by epidermal growth factor (EGF), and heterologously downregulated EGF receptors. In liver regeneration the administration of an alpha 1 blocking agent interfered with the first wave of regenerative DNA synthesis, and this effect was preceded by an elevation in EGF receptor number. Measurements of plasma catcholamines demonstrated that elevated levels of norepinephrine and epinephrine were in circulation within 2 h after partial hepatectomy. Surgical hepatic sympathectomy also interfered with early liver regeneration, suggesting that locally delivered adrenergic agents are important to initiation of DNA synthesis. These data suggest that stimulation at the alpha 1-adrenergic receptor is among the early signals for liver regeneration and that heterologous regulation of EGF receptors, similar to that observed in vitro, may be a part of the regenerative response.

Alpha 1-adrenergic effects and liver regeneration.

The effects of several treatments involving alpha-adrenergic mechanisms upon the early stages of rat liver regeneration were examined. Catecholamine concentrations in rat plasma were measured at various times after hepatectomy and were found to be elevated relative to those in plasma from sham-operated rats. Surgical hepatic denervation or injection of an alpha 1-adrenergic receptor antagonist (prazosin) reduced incorporation of [3H]thymidine into liver DNA during the first 24 hr after partial hepatectomy. Chronic guanethidine injections (3 to 6 weeks) reduced liver catecholamine levels, but did not affect its ability to regenerate. The inhibition of regenerative DNA synthesis by prazosin was preceded by an alteration in the binding of epidermal growth factor to regenerating liver, which was apparently the result of an increased number of epidermal growth factor receptors. Thus, alpha 1-adrenergic blockade, which affects both epidermal growth factor receptor binding and subsequent DNA synthesis in hepatocyte primary cultures, can also modulate these processes during liver regeneration in vivo.

Mechanisms of rodent liver carcinogenesis.

Stimulation of DNA synthesis by peroxisome proliferators, including DEHP, should be viewed differently from the stimulation of DNA synthesis by xenobiotic chemicals which stimulate restorative hyperplasia after hepatic necrosis induced by the toxicity of the chemical. The emerging picture of the control mechanisms for hepatocyte proliferation suggests that rather few and distinct factors are involved. The stimulation of DNA synthesis by peroxisome proliferators should be examined in the context of the effects of these factors. Comparisons with other xenobiotics show that induction of DNA synthesis at rates comparable to those of peroxisome proliferators is not sufficient to explain the rates of carcinogenicity associated with peroxisome proliferators. These considerations lead to the conclusion that although DNA synthesis enhances the incidence of neoplasia, it should not be viewed as a complete carcinogen, nor should it be considered as resulting in initiation at rates that can explain the carcinogenic potency of compounds such as peroxisome proliferators.

Norepinephrine decreases EGF binding in primary rat hepatocyte cultures.

Norepinephrine (NE) produced a dose-dependent inhibition of 125I-epidermal growth factor (EGF) binding to adult rat hepatocytes in primary culture. This effect was maximal after 1 hr of incubation with NE and could be blocked by the presence of an alpha 1-specific adrenergic receptor antagonist. The inhibition of binding correlates with the ability of NE to enhance hepatocyte DNA synthesis in the presence of EGF and appears to be mediated by a reduction in EGF receptor number, without a significant change in receptor affinity.

Norepinephrine and epidermal growth factor: dynamics of their interaction in the stimulation of hepatocyte DNA synthesis.

Primary cultures of adult rat hepatocytes are stimulated to enter DNA synthesis by norepinephrine (NE). This stimulation is maximal if the hepatocytes are incubated with NE for more than 12 hr, beginning no later than 2-4 hr after the cells are first plated. After 24 hr in culture, hepatocytes are unresponsive to NE stimulation. A strong synerergistic interaction between NE and epidermal growth factor (EGF) may be observed in cultures incubated with both EGF and NE, or pretreated with NE, then exposed to EGF. This interaction may be related to the finding that NE, in similarity with other factors that enhance EGF stimulation, reduces binding at the EGF receptor during the first 24 hr in culture.

Induction of DNA synthesis in cultured rat hepatocytes through stimulation of alpha 1 adrenoreceptor by norepinephrine.

Addition of norepinephrine to primary cultures of adult rat hepatocytes stimulates the incorporation of [3H]thymidine in a dose-dependent manner. This effect has been observed in serum-free medium containing epidermal growth factor and insulin. Stimulation of DNA synthesis by norepinephrine was strongly antagonized by the alpha 1-adrenergic antagonist prazosin but not by an alpha 2 antagonist or by a beta-adrenergic blocker. The beta agonist isoproterenol did not stimulate significant DNA synthesis. These results indicate that catecholamines interact with the alpha 1 adrenoreceptor to stimulate DNA synthesis in hepatocytes. Since alpha 1 receptors are present in most cells, this receptor may be important in cell growth regulation.