Cyclic nucleotide responses and radiation-induced mitotic delay in Physarum polycephalum

The response of the plasmodial levels of cyclic AMP and cyclic GMP in Physarum polycephalum to several putative phosphodiesterase inhibitors and to ionizing radiation has been measured. Isobutylmethylxanthine (2 mM) induces a rapid transient threefold elevation of cyclic AMP alone, with maximum response in about 10 min and return to the base line in about 30 min. Theophylline (2 mM) induces a rapid, sustained twofold elevation of cyclic GMP only. Caffeine (2 mM) and Ro-20-1724 (18 microM) both elicit a rapid transient rise in cyclic AMP, resembling the isobutylmethylxanthine response, and a slow transient elevation of the cyclic GMP level. Of particular interest is the rapid threefold transient elevation of the cyclic AMP, but not of the cyclic GMP, level by gamma radiation. These results (1) emphasize the organism and agent specificity of cyclic nucleotide responses, (2) indicate that the kinetics and apparent refractory response are compatible with the function of previously identified membrane receptor systems, i.e., a blue light receptor and a choleragen receptor mediating cyclic AMP responses, and (3) suggest a relationship between the agent-specific cyclic nucleotide responses and the modification of gamma-radiation-induced delay of mitosis by the same agents, i.e., a decrease in the delay by those which elevate cyclic GMP levels and an increase in the delay by those which elevate only cyclic AMP levels.     

Modification of radiation-induced division delay by caffeine analogues and dibutyryl cyclic AMP

The mitotic selection procedure for cell cycle analysis was utilized to investigate the concentration-dependent modification of radiation-induced division delay in Chinese hamster ovary (CHO) cells by methyl xanthines (caffeine, theophylline, and theobromine) and by dibutyryl cyclic AMP. The methyl xanthines (concentrations from 0.5 to 1000 micrograms/ml) all reduced radiation-induced division delay with the effect being linear between approximately 100 and 1000 micrograms/ml. After doses of 100-300 rad, delay was reduced by 75, 94 or 83 per cent at 1000 micrograms/ml for each drug, respectively. However, the addition of dibutyryl cyclic AMP had an opposite effect: radiation-induced delay was increased by the concentration range of 0.3 to 300 micrograms/ml. These results indicate that in mammalian cells the control of cell cycle progression and the modification of radiation-induced division delay are not simply related to intracellular levels of cyclic AMP. Rather, there appear to be at least two competing mechanisms which are differentially affected by caffeine analogues or by direct addition of dibutyryl cyclic AMP. The direct effect of caffeine and the methyl xanthines on membrane calcium permeability is considered.     

The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum

In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.     

Reduction of ultraviolet-induced mitotic delay by caffeine in G2-phase irradiated plasmodia of Physarum polycephalum

Synchronously mitotic surface Plasmodia ofPhysarum polycephalum were ultra-violet-irradiated at different times during G2-phase (—4 h to —20 min with respect to metaphase), and treated immediately thereafter with varying concentrations of caffeine. It was observed that ultraviolet-induced mitotic delay is reduced significantly by this methylxanthine. In plasmodia irradiated between —4 and —1 h with respect to metaphase, the effect was concentration-dependent and the need for a certain threshold dose for obtaining the reduction in delay was apparent. However, higher doses than this were fairly toxic when applied at this part of the cycle and led to more mitotic delay than that obtained with UV alone. The most striking observation made during this study was the phase-specific precipitous effect seen in those plasmodia irradiated at about 20 min before mitosis which almost eliminated the long delay due to ultraviolet-irradiation. These results are discussed in the context of some of the known effects of ultraviolet and caffeine on a mitosis-promoting factor. It is proposed that the significant reduction of ultraviolet-induced mitotic delay reported here is due to the reactivation of the ultraviolet-inactivated mitosis-promoting factor by caffeine. Alternatively, it is possible that caffeine may prevent the inactivation of this factor by ultraviolet.     

Urinary excretion of cyclic nucleotides, creatinine prostaglandin E2 and thromboxane B2 from mice exposed to whole-body irradiation from an enhanced neutron field

Urine volume and excretion of cyclic AMP, cyclic GMP, prostaglandin E2 (PGE2), thromboxane B2 (TxB2) and creatinine were evaluated as potential indicators of radiation damage in mice given 2-5 Gy to the whole body from an enhanced neutron field. In general, urinary cyclic AMP, cyclic GMP, creatinine and urine volumes were positively correlated across time postexposure, for each radiation dose. TxB2 levels positively correlated with urine volume and cyclic AMP excretion only in animals given 2.0 Gy. None of these parameters suggests their use as a prognostic indicator of the extent of radiation damage. Urinary excretion of PGE2 was negatively correlated with other urinary parameters. Biphasic increases in urinary PGE2 were also observed. The initial transient elevation 2-3 days postexposure was not correlated with the dose (2-5 Gy). The second elevation of PGE2 excretion occurred at 6-10 days. The magnitude of the latter increase suggests that urinary PGE2 excretion may be a useful indicator of whole-body or kidney exposure to neutron fields.     

Evidence for a messenger function of cyclic GMP during phosphodiesterase induction in Dictyostelium discoideum

Chemotactic stimulation of vegetative or aggregative Dictyostelium discoideum cells induced a transient elevation of cyclic GMP levels. The addition of chemoattractants to postvegetative cells by pulsing induced phosphodiesterase activity. The following lines of evidence suggest a messenger function for cyclic GMP in the induction of phosphodiesterase: (i) Folic acid and cyclic AMP increased cyclic GMP levels and induced phosphodiesterase activity. (ii) Cyclic AMP induced both cyclic GMP accumulation and phosphodiesterase activity by binding to a rate receptor. (iii) The effects of chemical modification of cyclic AMP or folic acid on cyclic GMP accumulation and phosphodiesterase induction were closely correlated. (iv) A close correlation existed between the increase of cyclic GMP levels and the amount of phosphodiesterase induced, independent of the type of chemoattractant by which this cyclic GMP accumulation was produced. (v) Computer simulation of cyclic GMP binding to intracellular cyclic GMP-binding proteins indicates that half-maximal occupation by cyclic GMP required the same chemoattractant concentration as did half-maximal phosphodiesterase induction.     

Synergistic effect of heat-shock and UV-irradiation on mitosis and protein synthesis in G2-phase plasmodia of Physarum polycephalum Schw--reduction in UV-induced mitotic delay in a preheat-shocked system

The synergistic effect of UV irradiation and heat-shock during the last 3 hr of G2 phase of the cell cycle in the plasmodia of P. polycephalum, in terms of mitotic delay and inhibition of protein synthesis, has been evaluated. The mitotic delay due to both perturbers coordinately increased closer to mitosis. Maximum mitotic delay was obtained in plasmodia heat-shocked after UV irradiation, indicating the presence in this system of either a heat-labile mitogenic substance which is comparatively less susceptible to UV or a substance which is made more susceptible to hyperthermia by UV. A protective role for heat-shock applied before irradiation has been observed in that, radiation-induced mitotic delay is significantly reduced in this combination. There was severe inhibition of translation in all the perturbed classes. Organelle level effects which are independent of major protein synthetic activities or different levels of heat-shock protein production could be the reason for the lack of correlation between percentage inhibition of general protein synthesis and the extent of mitotic delay with respect to the two double-perturbed systems.     

Cyclic AMP in the cell cycle of Physarum polycephalum

Cyclic AMP, [³H]thymidine incorporation, and DNA content were measured in the cell cycle of Physarum polycephalum. A sensitive radioimmunoassay was employed to assay cyclic AMP so that plasmodia could be assayed individually. In contrast to previously published results (Lovely, J.R. and Threlfall, R.J. (1976) Biochem. Biophys. Res. Commun. 71, 789–795), no pre-mitotic peak of cyclic AMP was detected. In seven experiments levels of cyclic AMP showed only small changes in individual experiments and ranged from 1–6 pmol/mg protein in different experiments. When plasmodia in the immediate premitotic period were collected on the basis of nuclear mitotic morphology, no evidence of a peak of cyclic AMP was found. Light was found to increase plasmodial cyclic AMP in a rapid, transient fashion. However, the brief exposure of cell cycle samples to light during collection did not induce any apparent cell cycle specific peaks of cyclic AMP. Although the occurrence of extremely rapid transient peaks of cyclic AMP in the cell cycle cannot be ruled out, it appears likely that the P. polycephalum cell cycle can proceed normally without major changes in cyclic AMP.     

Oppositional effects of acetylcholine and isoproterenol on isometric tension and cyclic nucleotide concentrations in rabbit atria.

The effects of acetylcholine chloride and isoproterenol on myocardiial cyclic GMP, cyclic AMP and on isometric tension were studied in isolated electrically driven rabbit atria. Acetylcholine (0.5 muM) produced a significant decrease in isometric force that was associated with a significant elevation in atrial cyclic GMP. Cyclic AMP was significantly lowered at 15 seconds after the addition of acetylcholine, but was only slightly decreased at earlier time periods. Both the negative inotropic action and increase in cyclic GMP after addition of acetylcholine were blocked by atropine. Isoproterenol (0.1 muM) produced a significant increase in isometric tension that was associated with a significant elevation in atrial cyclic AMP levels, whereas cyclic GMP levels were not changed. These effects were blocked by practolol. The increases in atrial cyclic GMP and cyclic AMP following addition of acetylcholine and isoproterenol, respectively, preceded the changes in isometric tension in response to these agents. These data support the hypothesis that changes in intracellular levels of cyclic AMP and cyclic GMP may mediate the positive and negative inotropic effects of adrenergic and cholinergic agents.     

Effects of exogenous cyclic AMP on growth characteristics and radiation response of Reuber H35 hepatoma cells

Reuber H35 rat hepatoma cells, clone KRC, were used to study the effect of cyclic AMP on radiation-induced cell death. Treatment of logarithmically growing cultures with 0.5 mM cAMP for 17 hr prior to irradiation resulted in a decreased cell survival. Similar results were obtained with cultures irradiated after treatment with Bt2cAMP. Treatment of H35 cells with cAMP or Bt2cAMP caused inhibition of their proliferation and resulted in an accumulation of cells in early S phase and a depletion of G2-phase cells. In synchronized cultures cells were relatively radioresistant during their S phase. In addition to single-dose treatment with X rays, the effect of Bt2cAMP on radiation-induced cell death was studied during fractionated irradiation with 2.5 Gy per day. This fractionated irradiation resulted in a dose-reduction factor of 1.6 at the 10% survival level and a 10-fold decrease in the surviving cell population due to the cooperative effects of Bt2cAMP on growth rate and radiation survival. The effect of cAMP on radiation-induced mitotic delay was also studied. It appeared that whereas cAMP had no effect on the progression of G2 cells into mitosis, it prevented cells from recovery from the X-ray mitotic delay in G2.     

Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide- dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.     

Changes in plasma cyclic nucleotides levels during various acute physical stresses

Studies were performed with healthy volunteers to determine the effects of various stresses known to increase sympathetic nerve activity on the plasma concentration of cyclic AMP and cyclic GMP. Plasma cyclic AMP rose promptly in response to exercise; the elevation was completely abolished by a simultaneous injection of propranolol. Plasma cyclic GMP rose slightly after exercise; the elevation was completely abolished by a simultaneous injection of atropine. No significant changes occurred during tilting and cold pressor stress. The increase in plasma concentrations of cyclic AMP and cyclic GMP may serve as putative indices for beta-adrenergic, and cholinergic functions respectively during exercise of humans.     

Effects of Caffeine on Radiation-Induced Phenomena Associated with Cell-Cycle Traverse of Mammalian Cells

Caffeine induced a state of G₁ arrest when added to an exponentially growing culture of Chinese hamster cells (line CHO). In addition to its effect on cell-cycle traverse, caffeine ameliorated a number of the responses of cells to ionizing radiation. The duration of the division delay period following X-irradiation of caffeine-treated cells was reduced, and the magnitude of reduction was dependent on caffeine concentration. Cells irradiated during the DNA synthetic phase in the presence of caffeine were delayed less in their exit from S, measured autoradiographically, and the radiation-induced reduction of radioactive thymidine incorporation into DNA was lessened. Cells synchronized by isoleucine deprivation, while being generally less sensitive to the effects of ionizing radiation than mitotically synchronized cells, were equally responsive to the effects of caffeine. The X-ray-induced reduction of phosphorylation of lysine-rich histone F1 was less in caffeine-treated cells than in untreated cells. Finally, survival after irradiation was only slightly reduced in caffeine-treated cells. A possible role of cyclic AMP in cell-cycle traverse of irradiated cells is discussed.     

Ethanol inhibition of antigen-induced histamine release from guinea pig lung: correlation with intracellular levels of cyclic nucleotides.

The effect of ethanol on histamine release from lungs of sensitized guinea pigs was studied in conjunction with measurements of tissue concentrations of cyclic AMP and cyclic GMP. Addition of antigen $\text{in vitro}$ elicited a rapid increase in cyclic AMP and cyclic GMP and stimulated release of histamine. Ethanol (2%) inhibited antigen-induced release of histamine over 95% and completely inhibited the increase in both cyclic nucleotides. The activity of cyclic AMP-dependent protein kinase was only slightly affected by ethanol.Metiamide blocked the ovalbumin stimulated increase in cyclic AMP but not cyclic GMP. Pyrilamine did not prevent the rise in either cyclic nucleotide. This suggests that the antigen-induced rise in cyclic AMP is an indirect result of histamine released from the tissue. The inability of H₁ and H₂ receptor antagonists to affect antigen-induced elevation of cyclic GMP in sensitized lung fragments suggests that an elevation in cyclic GMP might be either a primary event in the mediator release sequence or secondary to the release of a mediator other than histamine. The ability of ethanol to inhibit mediator release might be due to its capacity to attenuate the antigen-induced elevation of cyclic GMP in sensitized lung.     

The influence of sex steroids on calcium- and magnesium-induced mitogenesis in isolated rat thymic lymphocytes

Elevated calcium and magnesium concentrations promoted mitotic activity in rat thymic lymphocyte cultures. Oestradiol inhibited calcium- but not magnesium-induced mitogenesis. One prerequisite for the mitogenic action of calcium is a raised intracellular concentration of cyclic adenosine 3′5′ monophosphate (cyclic AMP) but cyclic AMP-induced mitogenesis was insensitive to oestradiol. This suggests that the steroid blocks the mitogenic process at a stage preceding the endogenous cyclic AMP elevation. Furthermore the mitogenic actions of adrenaline, which stimulates adenylate cyclase (the enzyme responsible for cyclic AMP biosynthesis), and caffeine, which inhibits phosphodiesterase (the enzyme which degrades cyclic AMP) were also insensitive to oestradiol inhibition. This precludes a direct effect of the steroid on these enzymes. However, oestradiol did inhibit the mitogenic action of parathyroid hormone (PTH). Since the mitogenic action of PTH probably involves increased calcium entry to the cell, oestradiol may block this ion influx. The inhibition of calcium- and PTH-induced mitogenesis must be attributable to some structurally specific action of oestradiol. The steroids cholesterol, progesterone and testosterone all failed to reduce calcium-induced mitogenesis, whereas both α and β oestradiol were effective. In addition to its insensitivity to oestradiol inhibition, magnesium-stimulated mitosis was unaffected by both imidazole and calcitonin at concentrations which significantly reduced calcium-stimulated proliferation. These findings are compatible with the thesis that magnesium-induced mitogenesis does not involve the elevation of cyclic AMP concentrations.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a pea a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2–3 fold increase at 15–30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E₂ also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise incyclic AMP. These results suggest that the relative amount of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells.

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

Effect of inhibition of the catalytic activity of cyclic AMP-dependent protein kinase on mitosis in PtK1 cells

Evidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP-dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP-dependent kinase during mitosis, dividing PtK1 cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP-dependent protein kinase (PKI) and an affinity-purified polyclonal antiserum (anti-C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early- to mid-prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti-C during early- to mid-prophase also caused a significant delay in the completion of mitosis, with many cells becoming "hung up" in prometaphase. Anti-C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti-C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP-dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic spindle microtubules is unclear.     

Changes in the content of cyclic AMP and cyclic GMP in eggs of Mactra solidissima during the second cleavage cycle.

The levels of cyclic AMP and cyclic GMP during the second cleavage cycle of eggs of Mactra solidissima have been measured. Cyclic GMP levels vary reciprocally with cyclic AMP levels, the cyclic GMP level increasing up to 4 to 5-fold and the cyclic AMP level falling to 50 percent at the onset of mitosis.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.