The individual characteristics of the postradiation reaction of the cyclic nucleotides in rat thymocytes]

It was shown that X-irradiation of rats (4.3 Gy), which were preliminarily divided into two groups by the neutrophilic reaction in the peripheral blood to the effect of a three-hour immobilization, induced different reactions of the cyclic nucleotide system. Thus, in animals hyper-reactive to stress radiation injury to the above system was severe: relative reactivity made 22.8% of the initial value, and adenylate cyclase ability to respond to a hormonal stimulus was drastically inhibited. In hyporeactive animals, relative reactivity of adenylate cyclase system after irradiation made 47% throughout the same period of observation; inhibition of adenylate cyclase activity was also less pronounced.     

In Vivo Evidence that Lithium Inactivates Gi Modulation of Adenylate Cyclase in Brain

In vivo microdialysis of cyclic AMP from prefrontal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 +/- 0.05 mM; n = 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin-catalyzed ADP ribosylation of alpha i (and alpha o) was increased by 20% in prefrontal cortex from lithium-treated rats, but the alpha i and alpha s contents (as determined by immunoblot) as well as the cholera toxin-catalyzed ADP ribosylation of alpha s were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin-stimulated adenylate cyclase activity.     

Changes in brain cyclic AMP metabolism and acetylcholine and dopamine during narcotic dependence and withdrawal.

Effects of morphine administration were studied on cyclic AMP metabolism in several regions of rat brain. In the cortex, cerebellum and thalamus-hypothalamus, morphine dependence did not alter the activity of either adenylate cyclase or phosphodiesterase. However, during withdrawal from the opiate treatment, adenylate cyclase activity declined in all three regions studied. In contrast, the striatal cyclic AMP metabolism was enhanced during morphine treatment as reflected by elevated endogenous cyclic AMP and increased adenylate cyclase. Furthermore, narcotic dependence produced significant increases in acetylcholinesterase activity of rat striatum. Whereas morphine withdrawal reversed the changes in striatal acetylcholine levels and acetylcholinesterase activity, the enhanced striatal dopamine remained unaltered. Although the activity of striatal adenylate cyclase was significantly reduced when compared to the morphine-dependent rats, the drop in cyclic AMP levels was not significant. Methadone replacement did not affect the changes in striatal dopamine seen in morphine-withdrawn rats. Whereas dopamine stimulated equally well the striatal adenylate cyclase from control or morphine-dependent animals, it failed to stimulate the striatal enzyme from rats undergoing withdrawal. The crude synaptosomal fraction of the whole brain from morphine-dependent rats exhibited an increase in cyclic AMP which was accompanied by elevated adenylate cyclase and protein kinase activity. Naloxone administration suppressed this rise in cyclic AMP and reversed the morphine-stimulated increases in the activities of adenylate cyclase and protein kinase. Following the withdrawal of morphine treatment, alterations in cyclic AMP metabolism were similar to those noted in morphine-naloxone group. Furthermore, substitution of morphine with methadone antagonized the observed alterations in cyclic nucleotide metabolism during withdrawal.     

Effects of oestrogen on adenylate cyclase system and glucose output in rat liver.

Effects of chronic oestrogen treatment on catecholamine- and glucagon-sensitive adenylate cyclase activity and glucose output in hepatocytes of castrated male rats were studied. In hepatocytes from male intact or castrated rats, the beta-adrenergic agonist isoprenaline did not stimulate adenylate cyclase activity and glycogenolysis, but glucagon markedly stimulated all these activities. Treatment of castrated animals with 17 beta-oestradiol for 7 days led to the appearance of beta-adrenergic-stimulated increases in both cyclic AMP generation and glucose output. The basal, glucagon- or fluoride-stimulated activities of adenylate cyclase of hepatic membranes prepared from oestrogen-treated rats were similar to those of control animals. Treatment with oestrogen did not influence the number or affinity of beta-adrenergic receptors. In hepatic plasma membranes from control rats, GTP failed to decrease the affinity of beta-adrenergic receptors for agonists, whereas the GTP-induced shift was apparently observed in those from oestrogen-treated animals. These results suggest that oestrogen is able to facilitate the coupling of hepatic beta-adrenergic receptors to the enzyme by increasing the effectiveness of receptor-guanine nucleotide regulation.     

The effects of hormonal and circadian cycles, stress, and activity on levels of cyclic AMP and cyclic GMP in pituitary, hypothalamus, pineal and cerebellum of female rats

Cyclic AMP and cyclic GMP levels were measured in the anterior and posterior pituitary, hypothalamus, pineal and cerebellum of female rats sacrificed during proestrus, metestrus and diestrus. In the first experiment rats were sacrificed by microwave irradiation between 0900 and 1100, between 1600 and 1800 and between 2100 and 2300. Cyclic AMP and cyclic GMP levels did not vary in any region tested as a function of the estrous cycle except for slightly elevated cyclic GMP levels in the posterior pituitary during proestrus. However the time of day at which the animals were sacrificed affected levels of cyclic AMP in the hypothalamus and cerebellum and levels of cyclic GMP in the cerebellum. In a second experiment female rats were all sacrificed between 2130 and 2330 during proestrus and diestrus. In this experiment rats were sacrificed either immediately upon removal from the home cage or after 10 min of immobilization stress, or after 10 min of open field activity. No differences in pituitary cyclic nucleotides were seen between proestrous and diestrous animals. However, stressed animals showed large cyclic AMP increases in the pituitary, and activity increased cyclic GMP levels in the cerebellum and pineal.     

Parathyroid hormone increases sodium/calcium exchange activity in renal cells and the blunting of the response in aging

Na+-dependent Ca2+ efflux was demonstrated in cells isolated from the rat renal cortex, suggestive of the presence of a Na+/Ca2+ exchange carrier in the cells. Parathyroid hormone, when incubated with the cells in vitro, increased Na+-dependent Ca2+ efflux about 60%. The effect of the hormone was specific for biologically active parathyroid hormone analogs and could be mimicked by cyclic nucleotides and forskolin. The effects of parathyroid hormone concentration on Ca2+ efflux and cyclic AMP formation were similar. These findings would be consistent with the view that the cyclic nucleotide might act as the intracellular messenger to increase Na+/Ca2+ exchange activity. Cells isolated from parathyroidectomized rats had decreased Na+-dependent Ca2+ efflux. When these cells were treated in vitro with parathyroid hormone, Na+-dependent Ca2+ efflux was enhanced to the same rate as found with cells from sham-operated animals. Parathyroid hormone-sensitive Na+/Ca2+ exchange activity was markedly blunted in cells from senescent (24 months) rats. Basal Na+-dependent Ca2+ efflux and Na+-independent Ca2+ efflux were not altered in the aged animal. Parathyroid-stimulated adenylate cyclase was also decreased in aging. In contrast, forskolin-stimulated Na+-dependent Ca2+ efflux and adenylate cyclase did not change with senescence. These findings would be compatible with a mechanism of desensitization that occurred at the level of the receptor or hormone-receptor coupling to adenylate cyclase. These results may be of physiological significance in understanding calcium homeostasis and the imbalances in mineral metabolism associated with old age.     

Treatment of streptozotocin-diabetic rats with metformin restores the ability of insulin to inhibit adenylate cyclase activity and demonstrates that insulin does not exert this action through the inhibitory guanine nucleotide regulatory protein Gi.

Insulin caused the inhibition of glucagon-stimulated adenylate cyclase activity in liver plasma membranes, but failed to inhibit this activity in liver membranes from rats made diabetic by treatment with either alloxan or streptozotocin. Treatment of streptozotocin-diabetic rats with insulin, to normalize their blood glucose concentrations, restored this action of insulin. Rats treated with the biguanide drug metformin exhibited a decreased content of the inhibitory guanine nucleotide regulatory protein Gi in liver plasma membranes assessed both structurally, by using a specific polyclonal antibody (AS7), and functionally. Treatment of normal rats with metformin did not alter insulin's ability to inhibit adenylate cyclase in liver plasma membranes; however, metformin treatment of streptozotocin-diabetic rats completely restored this inhibitory action of insulin. Liver plasma membranes from streptozotocin-diabetic animals which either had or had not been treated with metformin had contents of Gi which were less than 10% of those seen in control animals. We conclude that: (i) insulin does not inhibit adenylate cyclase activity through the inhibitory guanine nucleotide regulatory protein Gi; (ii) streptozotocin- and alloxan-induced diabetes elicit a selective insulin-resistant state; and (iii) metformin can exert a post-receptor effect, at the level of the liver plasma membrane, which restores the ability of insulin to inhibit adenylate cyclase.     

Levels of cyclic nucleotides in mouse regional brain following 300 ms microwave inactivation.

Abstract— The uniformity and speed of inactivation of mouse brain adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterase were measured after 6 kW microwave irradiation (MWR). Inactivation of enzymes was uniform throughout the brain during heating and 100% loss of activity was evident after 300 ms. MWR. For comparison of effects of inactivation times on levels of cyclic nucleotides measured in regional brain areas, cyclic AMP and cyclic GMP were estimated after 1.5 kW MWR requiring 4 s of heating and 6 kW MWR requiring 300 ms. Except for corpus striatum, uniformly lower levels of cyclic AMP were measured following 300 ms vs. 4s MWR . There was no change in cyclic GMP levels in regional brain areas after 4s vs. 300 ms MWR . Cyclic AMP and cyclic GMP were measured from the same regional brain tissue samples after 300 ms and ratios calculated. The finding of much lower cyclic AMP:cyclic GMP ratios than had previously been reported suggests that slow inactivation times provide for the measurement of regional brain cyclic nucleotide values which are not consistent with the in-vivo state.     

Mononucleotide Metabolism in the Rat Brain After Transient Ischemia

Nucleotide metabolism was studied in rats during and following the induction of 10 min of forebrain ischemia (four-vessel occlusion model). Purine and pyrimidine nucleotides, nucleotides, and bases in forebrain extracts were quantitated by HPLC with an ultraviolet detector. Ischemia resulted in a severe reduction in the concentration of nucleoside triphosphates (ATP, GTP, UTP, and CTP) and an increase in the concentration of AMP, IMP, adenosine, inosine, hypoxanthine, and guanosine. During the recovery period, both the phosphocreatine level and adenylate energy charge were rapidly and completely restored to the normal range. ATP was only 78% of the control value at 180 min after ischemic reperfusion. Levels of nucleosides and bases were elevated during ischemia but decreased to values close to those of control animals following recirculation. Both the decrease in the adenine nucleotide pool and the incomplete ATP recovery were caused by insufficient reutilization of hypoxanthine via the purine salvage system. The content of cyclic AMP, which transiently accumulated during the early recirculation period, returned to the control level, paralleling the decrease of adenosine concentration, which suggested that adenylate cyclase activity during reperfusion is modulated by adenosine A2 receptors. The recovery of CTP was slow but greater than that of ATP, GTP, and UTP. The GTP/GDP ratio was higher than that of the control animals following recirculation.     

On the Use of Microwave Radiation Energy for Brain Tissue Fixation

Abstract: Focused microwave irradiation (MWR) is an increasingly accepted method of sacrifice of laboratory animals such as the mouse or rat. By fixing the brain within a fraction of a second with heat inactivation, the investigation of fast neurochemical events may be obtained. Even though the technique is widely utilized, its application is inconsistent. This report illustrates some of the requirements necessary for the proper application of MWR for the sacrifice of animals, particularly those related to the length of time MWR is applied and the efficiency with which generated MWR power is coupled to the brain tissue. Studies were performed on the mouse, using either a 2.5 KW or 6.3 KW generator with a focused, closed system waveguide at time intervals of 350 or 500 ms or 1.4 s. During each of these intervals MWR was varied so that core brain temperatures for all groups were held between 83 and 95°C. In contrast with reported studies that used full animal restraint, all animals were minimally restrained for less than 1 s before sacrifice. Tissue content of cyclic AMP, an index of neuronal activity grossly affected by subtle changes in the activity of adenylate cyclase and/or phosphodiesterases, was monitored. No differences in tissue cyclic AMP content in any of 12 brain regions were detected after MWR, either at 350 or 500 ms. A substantial increase in cyclic AMP content occurred in 8 of 12 brain regions examined following microwave irradiation for 1.4 s. On the basis of these experiments, accurate determination of cyclic AMP in rodent brain requires that the maximum time interval of MWR exposure should not exceed 500 ms.     

The cyclic nucleotide content of the blood plasma in mice irradiated at different doses]

Cyclic nucleotide content of plasma was shown to increase after irradiation of mice within a wide range of doses. Compared was the dynamics of changes in the cyclic nucleotide content 24 hr following irradiation with supralethal doses.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

Effect of preincubation irradiation on adenylate cyclase activity and cyclic nucleotide content of the thymus in chick ontogenesis

Adenylate cyclase activity and cAMP and cGMP content of thymus have been studied in intact and irradiated (0.029 Gy, prior to incubation) embryos and chickens. The enzyme activity is stimulated during the postnatal development. The changes in the cyclic nucleotide content are undulatory and oppositely directed. It is suggested that the observed radiation-induced stimulation of adenylate cyclase and the reciprocal changes in the cyclic nucleotide content after hatching are related to the increased specific differentiation of thymus cells.     

Subcellular localization of adenylate cyclase of buffalo spermatozoa

The intracellular localization of adenylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in buffalo sperm was examined. Adenylate cyclase activity is distributed in heads (8.4%), midpieces (16.6%), tails (49.5%) and 5.7% in the soluble supernatant; the total recovery being 81%. A 4-fold increase in specific activity was observed in the tail fraction relative to sonicated suspension. Further fractionation of the tail fraction into plasma membrane and microtubules by dialysis against low ionic strength buffer was followed by marker enzymes (Mg2+ -ATPase, 5'-nucleotidase and alkaline phosphatase) as well as by examination of fractions under electron microscope. The recovered adenylate cyclase (79%) was found in microtubules (45%) and plasma membrane (34%). Cyclic nucleotide phosphodiesterase in tails was distributed in tail plasma membrane (13.7%), microtubules (31.5%) and cytosol (34%) with a total recovery of 80%. Similar results were obtained when the distribution of adenylate cyclase and cyclic nucleotide phosphodiesterase was studied by treatment with Triton X-100; 40% activity of adenylate cyclase present in tails (about 20% relative to sperm sonicate) appeared in the soluble form by this method. The results are discussed in relation to control of cyclic AMP levels in buffalo sperm by adenylate cyclase and cyclic nucleotide phosphodiesterase.     

Influence of parathyroid status of rats on renal tubular handling of adenosine 3'5'-monophosphate: a micropuncture study

In order to correlate cyclic AMP handling by the nephron to the parathyroid status, clearance and micropuncture experiments were performed in rats with intact parathyroid glands, or immediately after parathyroidectomy, or six days after parathyroidectomy. In intact animals cyclic AMP urinary excretion was about twice the filtered load and the tubular addition of the nucleotide was achieved at the end of the accessible proximal tubule. In acutely parathyroidectomized rats cyclic AMP urinary excretion was not different from the filtered load and no proximal tubular addition was detected at the late accessible proximal tubule. In chronically parathyroidectomized animals urinary excretion of cyclic AMP was not different from the filtered load, nevertheless a proximal tubular addition of the nucleotide was observed, similar in magnitude to that of intact rats. The data afford a direct evidence that the convoluted proximal tubule is the major site of cyclic AMP tubular addition, confirm that this addition disappears immediately after parathyroidectomy, but indicate that it re-occurs in chronic parathyroidectomy.     

Elevation of cyclic AMP levels and adenylate cyclase activity in duodenal mucosa from vitamin D-deficient rats by 1alpha,25-dihydroxycholecalciferol (1alpha,25-(OH)2D3).

A single 270 ng dose of 1alpha,25-(OH2D3 rpoduced elevations in cyclic AMP content and adenylate cyclase activity in duodenal mucosa from previously vitamin D-deficient rats. No changes in jejunal or ileal cyclic AMP levels or duodenal cyclic GMP levels were observed. Since 1alpha,25-(OH)2D3 increased both baseline and NaF-stimulated adenylate cyclase activity, it is possible that the vitamin leads to enhanced enzyme synthesis. While parallel changes in duodenal cyclic AMP levels and active calcium absorption in response to 1alpha,25-(OH)2D3 were observed at 6,12,24 and 48 hr after treatment, increases in calcium absorption were observed at 3 hr in duodenum and at 48 hr in ileum in the absence of changes in cyclic AMP levels. Further studies will be required to determine whether or not the changes in duodenal cyclic AMP levels are direct or indirect effects of 1alpha,25-(OH)2D3 administration, and to determine the role, if any, of this nucleotide in the hormones' effect on intestinal calcium absorption.     

A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.     

Postradiation change in the body's reaction to adrenaline

In experiments with albino mongrel rats weighing 220-260 g a study was made of the influence of adrenaline on the cyclic nucleotide content of blood plasma at early times following 60Co-gamma-irradiation (75 Gy). The absence of the organism response to adrenaline is perhaps due to the development of postirradiation desensitization of beta-adrenergic system and inability to induce cAMP formation in a cell. The data are submitted confirming the influence of levamisole on the cyclic nucleotide level. The level of cGMP decreased in thymocytes and increased in blood plasma after the administration of the drug to irradiated animals.     

Disorders in the system of cyclic nucleotides in atherosclerosis: cyclic AMP and cyclic GMP content and activity of related enzymes in human aorta

Cyclic AMP and cyclic GMP content and activities of cyclic nucleotide metabolic enzymes were determined in intima and media of atherosclerotic and unaffected human aorta obtained shortly after death due to myocardial infarction. Cyclic AMP content in fatty streaks and atherosclerotic plaques was lower by three- and five-fold, respectively, as compared with uninvolved intima. Cyclic GMP level in atherosclerotic lesions was estimated to be three-fold higher than in grossly normal area. Basal activity of adenylate cyclase in fatty streaks and plaques was two- to six-fold lower than in unaffected intima. Besides, the ability of adenylate cyclase to be stimulated by the stable analogue of prostacyclin, carbacyclin, was suppressed in plaques. Guanylate cyclase activity in fatty streaks was 1.5- to three-fold higher than in normal tissue. The thiol-reducing agent, dithiothreitol, decreased the enzyme activity to normal level, suggesting the oxidative nature of guanylate cyclase activation in the lesion zone. There were no significant changes in cyclic AMP phosphodiestease activity in the regions of the atherosclerotic lesion. Cyclic GMP phosphodiesterase activity in atherosclerotic plaques was two-fold lower than in the intima of unaffected areas. We did not find differences in the content of cyclic nucleotides or related enzyme activities in the media of uninvolved areas of human aorta nor in the media underlying atherosclerotic lesions. Our findings suggest that development of human atherosclerotic lesions is accompanied by dramatic changes in the cyclic nucleotide metabolism featuring gradual hormonal receptor uncoupling from adenylate cyclase, activation of guanylate cyclase in fatty streaks and inhibition of cyclic GMP phosphodiesterase in plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.