Effect of the functional state of adrenoreceptors on the mitotic activity of regenerating rat liver

Phentolamine, a blocker of alpha-adrenoceptors, given in a single dose (one hour before or 30 minutes, 8 and 24 hours after liver resection) decreased the mitotic index and the coefficient of mitotic phases, and elevated the glycogen content. The number of two-nucleated cells in rats exposed to phentolamine rose 8 hours after resection (p < 0.05). Obsidan, a blocker of beta-adrenoceptors, administered at the same periods, increased the mitotic index and the coefficient of mitotic phases. It is suggested that phentolamine prolonged the late mitotic phases, inhibiting the entry of the cells into mitosis. Obsidan elicited an opposite effect.     

Species-specific aggregation factor in sponges. V. Influence on programmed syntheses.

Isolated cells from the siliceous sponge Geodia cydonium as well as small primary aggregates (diameter: 70 mum) consisting of them show no increase in rates of programmed syntheses and mitotic activity with time. After addition of a highly purified aggregation factor to a culture with primary aggregates which subsequently form secondary aggregates (diameter: larger than 1000 mum), a dramatic increase of DNA, RNA and protein synthesis occurs. Together with this increase, the cells show a high mitotic activity. The values for the mitotic coefficient reach a first maximum 8 h after the beginning of the secondary aggregation process. The stimulation of the mitotic activity of cells during the aggregation factor induced secondary aggregation process can be suppressed by inhibitors of RNA and protein synthesis as well as by a blocker of DNA synthesis. This finding may indicate that cells from the G0-population enter the proliferating cell pool via the G1-phase.     

Stimulatory and inhibitory effects of catecholamines on DNA synthesis in primary rat hepatocyte cultures: role of alpha 1- and beta-adrenergic mechanisms.

Previous studies suggest that catecholamines may be involved in the regulation of liver growth. Considerable evidence implicates alpha 1-adrenergic mechanisms in the initiation of hepatocyte proliferation, while the role of beta-adrenoceptors is less clear. We have examined further the adrenergic regulation of hepatocyte DNA synthesis, using primary monolayer cultures. In hepatocytes that were also treated with epidermal growth factor and insulin, epinephrine or norepinephrine added early after the seeding strongly accelerated the rate of S phase entry. The beta-adrenergic agonist isoproterenol and the alpha-adrenergic agonist phenylephrine also stimulated the DNA synthesis, but were less efficient than epinephrine and norepinephrine. Experiments with the alpha 1-receptor blocker prazosine and the beta-receptor blocker timolol showed that the stimulatory effect of norepinephrine consisted of both an alpha 1- and a beta-adrenergic component. The alpha 1-component was most prominent in terms of maximal response at high concentrations of the agonist, but the beta-component contributed significantly and predominated at low concentrations (less than 0.1 microM) of norepinephrine. At later stages (about 40 h) of culturing norepinephrine strongly but reversibly inhibited the cells, acting at a point late in the G1 phase. This inhibition was mimicked by isoproterenol and abolished by timolol but was unaffected by prazosine, suggesting a beta-adrenoceptor-mediated effect. The results confirm the alpha 1-adrenoceptor-mediated stimulatory effect, but also show that beta-adrenoceptors may contribute to the growth stimulation by catecholamines. Furthermore, catecholamines, via beta-adrenoceptors and cyclic AMP, inhibit the G1-S transition, and may thus play a role in the termination of hepatic proliferation.     

THE EFFECT OF A MEDIUM DOSE (430 ROENTGENS) OF X-RAY IRRADIATION ON RESTING CELLS OF THE LIVER

The mitotic activity of regenerating liver cells after a single dose (430 r) of x-ray irradiation was studied. In every group of the experimental animals (white rats), the mitotic activity (mitotic index) and the number of abnormal mitotic figures were determined. The results indicated that resting cells irradiated a short time before mitotic activity showed reactions similar to those of cells irradiated during mitotic activity. The disturbances in the irradiated mitotically active cells were only quantitatively different from those in the irradiated resting cells. The disturbances in the irradiated resting cells depended upon the time interval between the irradiation and the beginning of mitotic activity stimulated by partial hepatectomy. It was found that the shorter the time interval, the more pronounced were the disturbances and the more similar they became to those of irradiated mitotically active cells. Conversely, the longer the time interval between the irradiation and the beginning of mitotic activity, the less pronounced were the disturbances and the more similar they became to those of the non-irradiated control cells. A discussion is presented as to whether or not the lesions of resting cells caused by a single medium dose of x-ray irradiation are reversible, and whether such lesions are only brought to light by the process of mitosis or whether the process of mitosis renders it possible for these lesions to develop.     

The possible cyclic AMP-dependence of an early prereplicative event that determines mitosis in regenerating rat liver

The beta-adrenergic blocker dl-propranolol prevented a large proportion of regenerating rat liver cells from entering the mitotic phase of their first cell division cycle without affecting their ability to initiate or complete DNA replication. The drug, at a dose of 20 or 50 mg/kg of body weight, was most effective in reducing mitosis when injected between 1 and 2 hours after the proliferatively activating partial hepatectomy, which was 22 to 23 hours before the peak of DNA-synthetic activity. Propranolol also inhibited the early prereplicative surge of total liver cyclic AMP, which occurs shortly after partial hepatectomy, but this effect was not correlated to the mitosis-inhibiting activity. However, cyclic AMP or dibutyryl cyclic AMP completely reversed propranolol's mitosis-inhibiting action when injected between 1.5 and 2 hours (but not sooner or later) after partial hepatectomy, which was just before the total liver cyclic AMP content began to rise. Thus, there appears to be a transient, propranolol-inhibitable, probably cyclic AMP-initiated event in the early prereplicative development of rat hepatocytes that determines entry into mitosis rather than the initiation of DNA replication.     

Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase.

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous 'dense-vesicle' cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver 'dense-vesicle' cyclic AMP phosphodiesterase.     

12-O-tetradecanoylphorbol-13-acetate induces an immediate, but transient increase in mitotic activity uncoupled from DNA synthesis in the monolayer cultures of rat keratinocyte

A single wave of mitotic activity was observed in a monolayer culture of rat keratinocytes immediately after exposure to 12-O-tetradecanoylphorbol-13-acetate. A peak for cells in prophase, observed at 10 min after the exposure, was followed by a peak for metaphase at 20 min, for anaphase at 25 min and telophase at 30 min after the exposure. Thereafter, the mitotic activity began to subside. This transient stimulation of mitotic activity resulted in an increase of population density in the monolayer culture. There was neither a stimulation of DNA synthesis during this period nor a change of the DNA content after the mitotic activity was completed. This single burst of synchronous mitotic activity which did not require a substantial stimulation of DNA synthesis suggests that the effect was on the initiation process of mitosis among a subpopulation of cells, presumably cells delayed in the G2 phase of the cell cycle.     

Cytological analysis of the intact and regenerating liver of different strains of rats]

Comparative cytological studies were conducted on control and regenerating liver of two strains (August and Cotton) of rats, 2/3 of the liver was resected; 5 or 6 animals were sacrificed at each of the following postoperative periods: in 30 hours, 3, 8, 42 and 120 days. The number of binuclear cells, the size of mononuclear hepatocytes and their nuclei, the mitotic activity, and ploidity of hepatocytes were determined. The intact and regenerating liver of the August rats differed from the intact and regenerating liver of the Cotton rats by a number of cytological indices, excluding the mitotic activity. A conclusion was drawn that the observed interstrain differences in the cytological indices providing regeneration of the liver after resection in the August and Cotton rats depended on the genotype of the given strain.     

Study of the inhibition of mitotic activity in the rat liver]

When affecting the rat liver cells by the alkylating agent dipin or X-ray irradiation in combination with genome induction by phenobarbital, a considerable decrease was observed in the mitotic activity of hepatocytes after hepatoectomy. The decrease in the mitotic activity is preserved for 90 days between introduction of mutagene and phenobarbital. When increasing the time interval to 150 days the inhibition in the mitotic activity is not observed.     

Mitotic activity of regenerating rat liver following stimulation with alpha- and beta-adrenoreceptors

A study was made of the effect of stimulating alpha- and beta-adrenoreceptors on the mitotic activity of the rat regenerating liver following resection. Mesaton, a stimulator of alpha-adrenoreceptors, and isadrin, a stimulator of beta-adrenoreceptors, in a dose of 0.2 mg/kg were injected one hour before liver resection or 30 min, 8 and 24 h after operation. In all experimental groups, mesaton gave rise to an increase in the mitotic index without lowering the coefficient of the mitotic phases. The least pronounced stimulating effect was attained when mesaton was injected 9 hours after partial hepatectomy. Isadrin reduced the mitotic activity as judged from the decrease of the coefficient of the phases and augmentation of the number of binuclear cells. The experiments confirmed a previously advanced assumption that stimulation of alpha-adrenoreceptors favours while that of beta-adrenoreceptors reduces cell proliferation.     

The Circadian Optimal Time for Hepatectomy in the Study of Liver Regeneration

Standardized (light from 0600 to 1800) C3HS mice, hepatectomized at different circadian stages, were killed at 1400 (the peak time of mitotic activity in intact mice). The higher values of mitotic index were those of mice operated at 1400, 48 hr before. The curve of mitotic activity of the regenerating liver of mice operated at 1400 and that of mice operated at 0200 (an opposite time in the circadian stage) are, both, grossly in phase with the curves of mitotic index in young and adult mice liver. The amplitude of the first peak of mitotic activity in mice operated at 0200 was dramatically lower than that of animals operated at 1400. The same applies to hepatocytes as well as to the sinusoid litoral population of cells. It is concluded that 1400 hr, as contrast to 0200 hr, is an optimal time for hepatectomy if one wants to obtain the highest mitotic index first peak during regeneration in a normal phase position (the position of the mitotic index peak in the liver of normal young and adult mice).     

Distribution of mitoses within a lobule of the rat liver in recovery after various types of injuries]

The distribution of mitotic activity inside the rat's liver lobe was found to depend on the time that elapsed after the injury. At the early stages, the maximal quantity of mitotic figures in hepatocytes was observed along the periphery of the lobe, being less in the middle parts. At the later stages, mitotic figures were noted around the central veins. With the prolonged injury of the liver, the maximal amount of dividing hepatic cells was localized in the middle sections of the liver.     

The Circadian Optimal Time for Hepatectomy in the Study of Liver Regeneration

Standardized (light from 0600 to 1800) C3HS mice, hepatectomized at different circadian stages, were killed at 1400 (the peak time of mitotic activity in intact mice). The higher values of mitotic index were those of mice operated at 1400, 48 hr before. The curve of mitotic activity of the regenerating liver of mice operated at 1400 and that of mice operated at 0200 (an opposite time in the circadian stage) are, both, grossly in phase with the curves of mitotic index in young and adult mice liver. The amplitude of the first peak of mitotic activity in mice operated at 0200 was dramatically lower than that of animals operated at 1400. The same applies to hepatocytes as well as to the sinusoid litoral population of cells. It is concluded that 1400 hr, as contrast to 0200 hr, is an optimal time for hepatectomy if one wants to obtain the highest mitotic index first peak during regeneration in a normal phase position (the position of the mitotic index peak in the liver of normal young and adult mice).     

A factor influencing the process of chromatin condensation in hepatocytes of regenerating rat liver]

It was shown with the model supramolecular chromatin systems that the activity of the factor of chromatin condensation changes in the process of liver regeneration. No activity of the factor of chromatin condensation was found 20 hrs after operation. Within 32-33 hrs (in the period of increased mitotic frequency) its activity approaches that prior to the operation. The nature of the activity of the factor of chromatin condensation is discussed with respect to the functional state of chromatin.     

Effect of diaminobutene and diaminopropane on diet-stimulated polyamine synthesis and cell proliferation in rat liver.

The effect of two putrescine analogs were studied on hepatic polyamine synthesis and cell proliferation, both of which were stimulated by food intake. Trans-1, 4-diamino-2-butene (diaminobutene), which is a potent competitive inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), repressed the induction of ODC and effectively inhibited the accumulation of putrescine in rat liver which was induced by the feeding of dietary protein. Unexpectedly, diaminobutene did not suppress DNA synthesis and mitotic activity in rat liver, suggesting that it can mimic the role of putrescine in cell proliferation. 1,3-Diaminopropane effectively repressed the induction of ODC caused by food intake and also suppressed DNA synthesis and mitotic activity without affecting the accumulation of RNA or protein. The suppression of mitotic activity by 1,3-diaminopropane was reversed by a single injection of putrescine, spermidine, spermine, or diaminobutene. It was concluded that rapid accumulation of polyamines, especially putrescine, was a prerequisite for the later enhancement of DNA synthesis and cell proliferation in rat liver caused by food intake.     

Characteristics of liver regeneration and growth in 3-week-old mice of different strains]

Three-week mice of C57BL strain are characterized by low proliferative activity of hepatocytes during normal growth of the liver. Hepatocytes of mice of this strain also had low proliferative activity 44 hours after partial hepatectomy (16%). Mice of the same age, but of other strains (mongrel, CBA, CC57BR) had higher mitotic indices both during normal growth and during regeneration (42; 70 and 60%, respectively). This peculiarity in the mitotic activity of hepatocytes of mice of different strains was also present 7 days after the beginning of the experiment. The data obtained indicated the genetic determination of the level of proliferative activity of hepatocytes.     

A kinase-independent role for Aurora A in the assembly of mitotic spindle microtubules in Caenorhabditis elegans embryos

The assembly of a functional mitotic spindle is crucial for achieving successful mitosis. Aurora A kinase is one of the key regulators of mitotic events, including mitotic entry, centrosome maturation and spindle bipolarity. Caenorhabditis elegans Aurora A (AIR-1) is responsible for the assembly of γ-tubulin-independent microtubules in early embryos; however, the mechanism by which AIR-1 contributes to microtubule assembly during mitosis has been unclear. Here we show by live-cell imaging and RNA-mediated interference (RNAi)-based modulation of gene activity that AIR-1 has a crucial role in the assembly of chromatin-stimulated microtubules that is independent of the γ-tubulin complex. Surprisingly, the kinase activity of AIR-1 is dispensable for this process. Although the kinase-inactive form of AIR-1 was detected along the microtubules as well as on centrosomes, the kinase-active form of AIR-1 was restricted to centrosomes. Thus, we propose that AIR-1 has a kinase-dependent role at centrosomes and a kinase-independent role for stabilizing spindle microtubules and that coordination of these two roles is crucial for the assembly of mitotic spindles.     

Reduced calcium and inhibition of protein kinase C mimic the enhancement of ornithine decarboxylase activity of prolactin in Ambystoma tigrinum tissues

We previously reported that prolactin (PRL) could increase the activity of ornithine decarboxylase (ODC) in liver slices taken from larval tiger salamanders (Ambystoma tigrinum). This action of the hormone was inhibited by oxytocin (OT), the calcium ionophore A23187, and diacyglycerol (DG) and was duplicated by 10 microM verapamil (VML), a calcium channel blocker. Here, we expand these results to show that 1) a higher dose of VML (50 microM) produces an additive effect with PRL; 2) addition of small amounts of calcium (0.1 mM) to the liver culture medium blocks PRL action; 3) neither nifedipine (NIF), a different type of calcium channel blocker, nor EDTA alter PRL action; and 4) gossypol, a reported inhibitor of protein kinase C, mimics PRL action. Additionally, we show that PRL increases ODC activity in tiger salamander tail skin in vitro, a tissue previously demonstrated to be a PRL target tissue in this species. The same set of treatments which we have shown to modify PRL effects on ODC in liver slices affects PRL action in the tail skin in a parallel manner. Thus, the mechanism whereby PRL enhances ODC activity appears to be the same in both these tissues. These results are discussed in conjunction with the findings from similar studies using mammalian tissues in an attempt to assess the current picture of the mechanism of PRL action and the possible role of inositol phospholipid turnover, calcium, and protein kinase C in the action of this hormone.     

Antagonism with dibenamine, D-600, and Ro 3-7894 to estimate dissociation constants and receptor reserves for cardiac adrenoceptors in isolated rabbit papillary muscles

In papillary muscles isolated from reserpinized rabbits, positive inotropic responses to the alpha (alpha)-adrenergic agonist, (-)-phenylephrine in the presence of 10(7) M timolol and the beta (beta)-adrenergic agonist. (-)-isoproterenol were antagonized with the irreversible alpha-adrenergic antagonist, dibenamine, the irreversible beta-adrenergic antagonist. Ro 3-7894, and the calcium blocker, D-600. D-600 was employed as a functional antagonist of both alpha- and beta-adrenoceptor responses. Dissociation constants (Ka values) for drug-receptor interactions were calculated by the method of Furchgott and used to estimate fractional receptor occupancy and agonist efficacies. Comparison of responses showed that the receptor reserve for cardiac beta-adrenoceptors was greater than for alpha-adrenoceptors. D-600 was an effective inhibitor of both cardiac alpha- and beta-adrenoceptor responses; however, estimates of KA and receptor reserves were similar to estimates using an irreversible antagonist for alpha-but not beta-adrenoceptors.     

Regenerative ability of hepatocytes is inhibited in early stages of liver fibrosis.

Female Wistar rats were given three doses of carbon tetrachloride, 10.4 mmol/kg of body weight. The doses were administered within 16 days and another 16 days were allowed to pass before partial (37%) hepatectomy was done. The liver showed very mild fibrosis at that time. DNA synthesis (measured by 3H-thymidine incorporation) was decreased by 53% and mitotic activity of hepatocytes was decreased by 56% when compared to olive oil-pretreated partially hepatectomized controls. The results show that the mitotic potential of hepatocytes in early stages of liver fibrosis is impaired which may influence the course of the disease.