Possible cyclic AMP-dependence of the prereplicative surge of cytosolic calmodulin in proliferatively activated rat liver cells

The infusion of a solution containing triiodothyronine, amino acids, glucagon, and heparin (TAGH solution) triggered rat liver cell proliferation. It also induced a transient prereplicative surge of cytosolic calmodulin (between 6 and 20 hr postinfusion) similar to that observed in liver cells proliferatively activated by partial hepatectomy. The injection of the beta-adrenergic blocker dl-propanolol (20 mg/kg of body weight) at the time of the infusion prevented this transient rise of cytosolic calmodulin and also inhibited the early prereplicative surge of total liver cyclic AMP, which usually occurred between 1 and 4 hr after infusion. Propanolol also inhibited the early prereplicative surge of cyclic AMP and the increase of calmodulin in liver cells proliferatively activated by partial hepatectomy. The infusion of a solution containing cyclic AMP (5 mumoles) and theophylline (10 mg) into normal rats produced an increase of cytosolic calmodulin similar to that observed after infusion of TAGH solution or after partial hepatectomy. Thus it seems that the prereplicative rise of cytosolic calmodulin observed in proliferatively activated liver cells may be regulated by the early prereplicative surge of cyclic AMP.     

The control of liver regeneration by parathyroid hormone and Calcium

Following partial hepatectomy in rats, there were two bursts of hepatocyte DNA-synthetic and mitotic activity which were produced by two subpopulations having different rates of (nearly synchronous) proliferative development. Only about 50% of the cells in both subpopulations could initiate DNA synthesis and enter mitosis when exposed to the hypocalcemic conditions in the parathyroprivic rat for 24 hours before partial hepatectomy. The proliferatively incompetent hepatocytes in these hypocalcemic rats could be induced to initiate their DNA synthetic and mitotic activity by an intraperitoneal injection of the calcium-mobilizing parathyroid hormone (50 USP units/100 g) as late as 12 hours after partial hepatectomy. Single intraperitoneal injections of calcium (0.25 mg/100 g) could also restore the proliferative competence of these hepatocytes, but only when injected at specific periods following partial hepatectomy. The injection of calcium 12 to 15 hours after partial hepatectomy induced hepatocytes in the first subpopulation to finish their development and enter mitosis, but did not affect the second, more slowly developing, subpopulation. Calcium had to be injected 25 hours after partial hepatectomy to stimulate proliferation in this second subpopulation. These data suggest that the hepatocytes which became proliferatively incompetent by prolonged exposure to a hypocalcemic environment are proliferatively activated by partial hepatectomy, but their proliferative development stops at a calcium-dependent stage near the end of the pre-replicative phase of development.     

The control of liver regeneration by parathyroid hormone and calcium.

Following partial hepatectomy in rats, there were two bursts of hepatocyte DNA-synthetic and mitotic activity which were produced by two subpopulations having different rates of (nearly synchronous) proliferative development. Only about 50% of the cells in both subpopulations could initiate DNA synthesis and enter mitosis when exposed to the hypocalcemic conditions in the parathyroprivic rat for 24 hours before partial hepatectomy. The proliferatively incompetent hepatocytes in these hypocalcemic rats could be induced to initiate their DNA synthetic and mitotic activity by an intraperitoneal injection of the calcium-mobilizing parathyroid hormone (50 USP units/100 g) as late as 12 hours after partial hepatectomy. Single intraperitoneal injections of calcium (0.25 mg/100 g) could also restore the proliferative competence of these hepatocytes, but only when injected at specific periods following partial hepatectomy. The injection of calcium 12 to 15 hours after partial hepatectomy induced hepatocytes in the first subpopulation to finish their development and enter mitosis, but did not affect the second, more slowly developing, subpopulation. Calcium had to be injected 25 hours after partial hepatectomy to stimulate proliferation in this second subpopulation. These data suggest that the hepatocytes which became proliferatively incompetent by prolonged exposure to a hypocalcemic environment are proliferatively activated by partial hepatectomy, but their proliferative development stops at a calcium-dependent stage near the end of the pre-replicative phase of development.     

A possible role for cyclic AMP in the initiation of DNA synthesis by isoproterenol-activated parotid gland cells

An intraperitoneal injection of the β-adrenergic agonist dl-isoproterenol hydrochloride (100 mg/Kg body weight) into a rat caused an early, very large (400-fold) cyclic AMP surge (peaking at 10 minutes) in the parotid gland which was followed by a second, much smaller (two-fold) surge 12 to 16 hours later. DNA synthesis began about 16 to 20 hours after the isoproterenol injection and peaked between 24 and 28 hours. The maximum level of DNA-synthetic activity at 24 hours was correlated positively to the magnitude of the small cyclic AMP surge at 12 hours, but not to the size of the much larger cyclic AMP surge at 10 minutes. An intraperitoneal injection of dl-propranolol hydrochloride (59 mg/Kg body weight) at 8 hours after isoproterenol injection abolished the second cyclic AMP surge at 12 hours and markedly (65-75%) reduced the incorporation of [³H]-thymidine into DNA. Injection of dibutyryl cyclic AMP (6.3 mg/Kg body weight) and theophylline (25 mg/Kg body weight) at 8 hours prevented propranolol from inhibiting DNA synthesis. Propranolol appeared specifically to affect the cyclic AMP-dependent pre-DNA-synthetic step because it did not reduce [³H]-thymidine incorporation when injected after the second cyclic AMP surge had passed and DNA synthesis had just begun. Thus, the initial, large cyclic AMP surge following β-adrenergic stimulation may not be necessary for the initiation of prereplicative development, while the much smaller second surge may be needed for the initiation of DNA synthesis.     

Polyamine metabolism in regenerating livers from normal and hypocalcemic rats

There is a marked increase in the concentration of putrescine during the first ten hours following partial hepatectomy in rats. The concentration of spermidine also increases but to a smaller degree. Putrescine levels return to normal between 10 and 24 hours after the operation, whereas the increased spermidine level is maintained. The production of putrescine and spermidine appears to be initiated by the induction of ornithine decarboxylase which shows a single peak of activity at four hours after hepatectomy. The activity of S-adenosylmethionine decarboxylase shows little change following hepatectomy. The changes in polyamine levels and the activities of the enzymes of polyamine metabolism are not affected by thyroparathyroidectomy 72 hours prior to hepatectomy. Thus although these hypocalcemic conditions considerably reduce and delay DNA synthesis and mitosis, the prereplicative changes in polyamine metabolism still occur. These data suggest that the hepatocytes in hypocalcemic animals have become activated and moved to an advanced stage of prereplicative development before being blocked.     

Effect of Kupffer cell blockade at different periods following partial hepatectomy on regeneration of hepatocytes

In male Wistar rats, weighing 140-160 g, the block of the liver mononuclear phagocyte system (MPS) was carried out by means of "carbonyl iron" (type R-100F, particle size 1-1.5 micron). It was induced 2 hours before or 3 and 18 hours after partial hepatectomy. Iron injection previously or at the early prereplicative regeneration period led to a significant delay of the hepatocyte nucleus labeling and mitotic indices peaks against the background of an overall depression of hepatocyte proliferation. The MPS block during the intensive DNA synthesis by regenerating liver hepatocytes was less effective. The facts testify to the importance of Kupffer's cells in the regulation of the reparative liver regeneration.     

Prereplicative enzymic changes in regenerating rat liver.

After partial hepatectomy in rats, the following changes in enzymic activities were observed in the remnant liver during the prereplicative period. In the initial period of the prereplicative process, soon after removal of part of the liver, ornithine decarboxylase [EC 4.1.1.17] and IMP dehydrogenase [EC 1.2.1.14] increase. Subsequently, for entry into the S period, thymidine kinase [EC 2.7.1.75] increases simultaneously with increase in the intracellular cyclic AMP level and decrease in its phosphodiesterase [EC 3.1.4.17].     

Cyclic adenosine 3':5'-monophosphate and the induction of deoxyribonucleic acid synthesis in liver.

A mixture containing glucagon and thyroid hormone was previously devised that enhances markedly nuclear DNA replication and mitosis in the parenchymal liver cells of the unoperated rat. It is now shown that the glucagon of the stimulatory solution can be completely replaced by a mixture of a butyryl derivative of cyclic adenosine 3':5'-monophosphate and theophylline. Cyclic guanosine 3':5'-monophosphate and its butyryl derivatives and insulin and high levels of glucose are inactive. The inactivity of N2-monobutyryl cyclic guanosine 3':5'-monophosphate cannot be ascribed to rapid breakdown in the animal or to the impenetrability of the liver cell since the coumpound elevates the rate of hepatic amino acid transport and the activity of ornithine decarboxylase. The observation of others (MacManus, J.P., Franks, D.J., Youdale, T. & Braceland, B.M. (1972) Biochem. Biophys. Res. Commun. 49, 1201-1207) that the level of cylcic adenosine 3':5'-monophosphate is raised during most of the prereplicative period after 70% hepatectomy is confirmed. The evidence supports a positive role for adenosine 3':5-monophosphate in regulating DNA synthesis in the liver.     

Regulation of dog thyroid epithelial cell cycle by forskolin, an adenylate cyclase activator

Dog thyroid epithelial follicular cells in primary culture are quiescent in an insulin-supplemented serum-free medium. They are induced, after a 16- to 20-h prereplicative phase, to synthesize DNA upon stimulation by forskolin, a general adenylate cyclase activator that mimics all the effects of thyrotropin in these cells. The characteristics of adenylate cyclase activation by forskolin make this drug a convenient tool to enhance cellular cyclic AMP levels for well-defined periods of the cell cycle, allowing determination of which parts of the prereplicative phase are controlled by cyclic AMP. We observe that induction of DNA synthesis by forskolin requires its continuous presence for most of the prereplicative phase until a point that little precedes the initiation of DNA replication. Before this point, interruptions in forskolin presence as short as 2 h delay the onset of DNA synthesis, indicating a rapid regression of the cells to an earlier part of G1 from which they can be rescued by forskolin readdition. Similar delays in the onset of S phase are also induced by reversible protein synthesis inhibitions using pulses of cycloheximide. These data suggest that in dog thyrocytes elevated cyclic AMP levels stimulate the progression into G1 phase until a late commitment point before DNA synthesis. This progression depends on peculiarly labile cyclic AMP-stimulated events which might well be the induction by cyclic AMP of the synthesis of labile proteins.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. III. Effect on DNA synthesis in vivo and on DNA polymerase activity assayed in vitro.

A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro.     

The cyclic AMP-dependent initiation of DNA synthesis by T51B rat liver epithelioid cells.

The brief rise in the cellular cyclic AMP content which occurs late in the prereplicative phases of rat hepatocytes in vivo and T51B rat liver epitheloid cells in vitro seems to be necessary for the initiation of DNA synthesis. Thus, the extracellular calcium-deprivation in T51B rat liver cells in culture which induces a late G-1 block is rapidly reversible (cells surge into S phase within one hour) either by creating a cyclic AMP surge by the addition of calcium or 3-isobutyl-1-methyl xanthine (a cyclic 3',5'-nucleotide phosphodiesterase inhibitor) or by the exogenous addition of low concentrations of cyclic AMP itself (i.e., 10(-8)-10(-5) M). On the other hand, prevention of the calcium-induced cyclic AMP surge by imidazole (a cyclic 3',5'-nucleotide phosphodiesterase activator) blocked the initiation of DNA synthesis by the calcium-deprived T51B cells.     

Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.

Hepatocyte Growth Factor (HGF) is a potent complete mitogen for primary cultures of hepatocytes in vitro. There is strong evidence that this novel growth factor may mediate hepatocyte regeneration after liver damage. We have shown previously that the amount of immunoreactive HGF markedly increases in the serum of rats soon after partial hepatectomy or CCl4 administration. In the present paper, we demonstrate that the level of HGF mRNA in rat liver also dramatically increases from 3 to 6 hours post hepatectomy, peaks at 12 hr and gradually returns to undetectable levels by 72 to 96 hours post hepatectomy. In separate experiments, DNA synthesis (in vivo) was determined in rat liver remnants after partial hepatectomy. DNA synthesis peaked 24 hr after hepatectomy, 12 hr after the peak of HGF mRNA expression. These results suggest that HGF may be one of the major early signals that triggers hepatocyte proliferation during liver regeneration.     

Hormonal control of liver regeneration

Two peaks in cyclic AMP production in rat livers 4 and 12h after partial hepatectomy (MacManus et al., 1972) were confirmed and a third peak established at 22h, which is the peak of DNA synthesis. The increases in cyclic AMP were prevented by beta-adrenergic blocking agents, propranolol and pindolol, without affecting ornithine decarboxylase induction or DNA synthesis. The alpha-blocking agents, phenoxybenzamine and phentolamine, given at the time of partial hepatectomy, delayed the rise in ornithine decarboxylase normally found 4h after operation, but did not affect DNA synthesis. If the alpha-blocking agents were given at 9-12h or 18h, the onset of DNA synthesis was delayed. Phenoxybenzamine did not affect the induction of ornithine decarboxylase in intact rat livers by glucagon or growth hormone, but did inhibit induction by dexamethasone. The induction of ornithine decarboxylase produced by dexamethasone was inhibited by 17alpha-hydroxy-progesterone; this compound also blocked the induction of ornithine decarboxylase in livers of partially hepatectomized rats.     

Characterization and cell cycle kinetics of hepatocytes during rat liver regeneration: in vivo BrdUrd incorporation analysed by flow cytometry and electron microscopy

The incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA has been analysed during hepatocellular regeneration induced by partial hepatectomy in young rats. The kinetic state of the liver has been studied by flow cytometric analysis of the incorporated BrdUrd, while the fine localization of DNA replication sites through the cell cycle has been investigated at the ultrastructural level by the immunogold technique. Eighteen hours after partial hepatectomy flow cytometry revealed an early S phase distribution which corresponded to a specific staining of the interchromatin domains of the hepatocyte nucleus. Thirty-four hours after hepatectomy, on the other hand, when most cells were in late S, a specific staining of heterochromatin domains was observed. The effect of the BrdUrd technique on nuclear aggregation has also been analysed and discussed. The results demonstrate that specific patterns of DNA replication can be recognized during the cell cycle and that flow cytometry and electron microscopy appear to be complementary in the kinetic study of liver regeneration.     

Guanylate cyclase activity and cyclic nucleotide concentrations during liver regeneration after experimental injury

Cyclic nucleotide concentrations and guanylate cyclase activity were measured in regenerating rat liver. Previous work has shown that in livers of partially hepatectomized rats the activity of a membrane-bound guanylate cyclase increases considerably during the early replicative phase [Kimura & Murad (1975) Proc. Natl. Acad. Sci. U.S.A.72, 1965-1969; Goridis & Reutter (1975) Nature (London) 257, 698-700]. Over the same time period after partial hepatectomy, increased tissue concentrations of cyclic GMP were found when the rats were killed under pentobarbital anaesthesia, but not when anaesthesia was omitted. The results obtained on hepatectomized livers were compared with the changes in guanylate cyclase activity and cyclic nucleotide concentrations during the response to galactosamine treatment. Here, a peak of guanylate cyclase activity and of cyclic GMP concentrations occurred at 8h, that is before the beginning of the proliferative response. Both parameters were normal at the time of increased DNA synthesis. There does not, therefore, seem to be a consistent correlation between changes in guanylate cyclase activity or concentrations of cyclic GMP and an increase in liver DNA synthesis. A modest rise in cyclic AMP concentrations was found, however, in livers of galactosamine-treated rats, which was coincident with the time of DNA synthesis.     

Dual effects of glucagon and cyclic AMP on DNA synthesis in cultured rat hepatocytes: stimulatory regulation in early G1 and inhibition shortly before the S phase entry

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP-elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum-free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM-1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24-45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin-induced recruitment of cells to S phase, the rate of G1-S transition was markedly decreased within 1-3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 less than 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8-bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (G0 or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.     

DNA synthesis and mitotic activity in cells of regenerating rat liver following x-irradiation in stages GO and Gl]

Effect of X-rays on DNA synthesis and mitotic activity of regenerating liver cells has been studied. The irradiation was performed at a dose of 630 rad before hepatectomy and 2.5 and 6 hours after the stimulation of liver. With the stimulated liver being irradiated, the number of cells synthetizing DNA and entering into mitosis was seen reduced almost twice, whereas DNA synthesis and entering into mitosis were delayed, resp., by 4 and 6 hours. Irradiation of liver before the stimulation brings about a delay in DNA synthesis and in start of mitosis by 2 and 4 hours, resp., without reducing the numbers of cells capable to synthesize DNA and to enter mitosis.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. II. Alkylation of DNA and inhibition of DNA replication.

Experiments were carried out to determine whether replication of alkylated DNA could be involved in the initiation of hepatocellular carcinoma which results from a single administration of dimethylnitrosamine (DMN) given after partial hepatectomy. The incidence of tumours is higher when DMN is given during the wave of DNA synthesis induced by the operation than when given in the early prereplicative stage. Therefore the alkylation of DNA in the regenerating liver by DMN given at these times and the effect of DMN on DNA synthesis were investigated. The extent, duration and pattern of alkylation of DNA, including the formation of 0-6-methylguanine, were similar whether DMN was given in the early pre-replicative stage (6 h after the operation) or during the period of DNA synthesis (at 24 h). DMN given a 6 h very greatly reduced the wave of DNA replication which would otherwise have ensued. When given at 24 h, by which time DNA synthesis was already taking place, DMN reduced the rate of incorporation of (-3H)thymidine after 1-2 h delay. However, in neither case was DNA synthesis reduced to the level occurring in normal intact liver. Treatment with diethylnitrosamine (DEN) at 6 h or at 24 h had a similar effect to DMN on the wave of DNA replication induced by partial hepatectomy. Methyl methanesulphonate (MMS given in the early pre-replicative stage delayed the wave of DNA synthesis by about 8 h, but when it did take place the extent of synthesis was as great as in untreated animals. When given during the period of DNA replication, MMS rapidly reduced the rate of synthesis. As in the case of the nitrosamines, synthesis was not reduced to the level occuring in normal intact animals. The difference from the nitrosamines lies in the nature of the alkylated bases formed in DNA. The fact that a single treatment with DMN induces cancer in partially hepatectomised animals but not in intact adult animals is not considered to be due to a gross difference in the nature of the alkylation of DNA. The experiments described support the concept that replication of DNA containing bases which are likely to mispair during replication may be necessary to 'fix' the lesion and thus cause a permanent inheritable change in the genetic material.     

Urethane inhibition of DNA synthesis in the hepatocytes of the regenerating liver in mice

Hepatocarcinogen urethane (ethyl carbamate) inhibits DNA synthesis in the regenerating mice liver when administered at the peak of stimulated proliferation--46 hours after partial hepatectomy. The inhibition is temporary and reversible. The maximum inhibition of 3H-thymidine incorporation in the cells is observed 12 hours after urethane administration, and the effect is removed following 20 hours after administration. Another effect of urethane consists in the lengthening of the period of DNA synthesis by 1.38 times, as estimated by the Quastler-Sherman method, though it does not affect the length of G2-period or mitosis. Possible mechanisms of the effect of urethane on the initiation of DNA synthesis and on the rate of DNA replication are discussed.