Regional rat brain content of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate after acute and subacute treatment with ethanol

Acute administration of ethanol (1.0, 2.0, and 5.0 g/kg, ip) to naive male rats (Sprague-Dawley) caused a dose-dependent depression of cerebellar guanosine 3′,5′-cyclic monophosphate (cGMP), and a reduction in cortical cGMP at the highest dose of ethanol. The cGMP content was not altered in the anterior hypothalamus, posterior hypothalamus, or striatum. On examining adenosine 3′,5′-cyclic monophosphate (cAMP) levels, only the area of the striatum was reduced (5.0 g/kg, ethanol). In these acute experiments there was a negative correlation between blood ethanol concentrations and body temperatures. An elevated environmental temperature (31 ± 1°C) to prevent hypothermia from ethanol administration indicated that hypothermia was not a contributory factor. In the subacute experiments animals showed at the end of 24 hr of ethanol inhalation less hypothermia than naive animals with similar blood ethanol concentrations, a reduction in cGMP in the cerebellum and cortex, but no alteration in regional brain cAMP. When the animals were injected with ethanol (2.0 g/kg, ip) 48 hr after removal from the chamber (ethanol vapor, 24 hr), ethanol produced no significant reduction in body temperature (tolerance), but a decrease in cerebellar cGMP. The cAMP content of the tissues was similar to control animals. Ethanol administration (2.0 g/kg, ip) 48 hr later to animals which were previously exposed to air only in the chamber (24 hr) demonstrated a reduction in body temperature as compared with tolerant animals, a decrease in cerebellar cGMP, and no depletion of cAMP in regional sections of the brain. From this in vivo study the data seem to suggest that brain cyclic nucleotides, particularly cAMP, may have a limited role in ethanol-induced intoxication and tolerance to the hypothermic effect of ethanol.     

Magnesium sulphate increases lymphocyte adenosine 3':5'-cyclic monophosphate in humans.

We determined the effect of i.v. magnesium sulphate, which is often combined with beta 2-adrenoceptor agonists for tocolytic therapy, on lymphocyte cyclic AMP production, extracellular magnesium and blood calcium concentrations. Sixteen healthy volunteers received i.v. magnesium sulphate 1 g h-1 over 8 h; seven volunteers also had infusion of NaCl 18 mg h-1 as control. Venous blood was taken pre- and post-infusion to determine basal lymphocyte cyclic AMP and the increase evoked by 0.1 mM isoprenaline, as well as serum and plasma concentrations of total and non-protein-bound magnesium and calcium. Following magnesium sulphate there was a significant rise in the isoprenaline-evoked increase in cyclic AMP (P < 0.05) and in the magnesium concentrations (P < 0.01) and a decrease in the free calcium concentration (P < 0.01).     

Effect of glucagon, dibutyryl adenosine 3',5'-cyclic monophosphate and phosphodiesterase inhibitors on rat liver phosphorylase activity and adenosine 3',5'-cyclic monophosphate levels

Theophylline, papaverine and 1-ethyl-4-(isopropylidenehydrazine)-1H-pyrazole-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester HCl (SQ 20,009) were found to inhibit both high K_m (200 μM) and low K_m(3 μM) cyclic 3′,5′-AMP phosphodiesterase activity in 100,000 g supernatant fraction from rat liver. Theophylline was the least potent and SQ 20,009 the most potent inhibitor of both activities. In the liver slice preparation, papaverine (5 × 10^?4), theophylline (10^?3 M) and SQ 20,009 (10^?3 M) failed to elevate cyclic 3′,5′-AMP, while glucagon (10^?6 M) produced a significant (P < 0·05) elevation in the tissue levels of the cyclic nucleotide. All three phosphodiesterase inhibitors significantly (P < 0·05) augmented the ability of glucagon to elevate cyclic 3′,5′-AMP levels. Glucagon and papaverine stimulated phosphorylase activity, while theophylline inhibited both basal and glucagon-stimulated phosphorylase activity. SQ 20,009 was without effect. It is concluded that no cause and effect relationship can be assumed between an ability to inhibit phosphodiesterase and to alter phosphorylase activity.     

Effect of histamine on adenosine 3',5'-cyclic monophosphate levels in granulation tissue

The effect of histamine on cyclic AMP levels in rat granulation tissue was examined in vivo and in vitro. Granulomas formed by subcutaneous implantation of formalin-soaked filter paper disks were used as granulation tissue. Histamine injection (5 mg/kg, i.p.) produced a significant increase in the cyclic AMP levels for 10–30 min after injection. Administration of either burimamide or mepyramine at 1 mg/kg, s.c., 30 min before histamine administration blocked the cyclic AMP increase induced by 5 mg/kg histamine. Burimamide was more effective than mepyramine. Neither of these histamine antagonists at 1 mg/kg blocked the cyclic AMP increase induced by adrenaline (1 mg/kg, i.p.). Propranolol at 1 mg/kg, s.c., reversed the cyclic AMP increase due to adrenaline but partially blocked a similar effect of histamine. Histamine (1 mM and less), added alone to the incubation medium in which the chopped granulation tissue was immersed, had little or no effect on cyclic AMP levels in the granulation tissue. However, when histamine (1 and 10 mM) was added concomitantly with 3-isobutyl-1-methylxanthine (IBMX) (1 mM), a significant increase in cyclic AMP occured, compared to the addition of IBMX alone. IBMX (1 mM) alone caused a significant increase in cyclic AMP compared to controls in which the drug was omitted. 2-Methylhistamine (5 mM) induced a cyclic AMP increase in the presence of IBMX (1 mM). Metiamide (0.01 mM) partially blocked the cyclic AMP increase caused by histamine (1 mM) in the presence of IBMX (1 mM). Mepyramine (0.01 mM) completely blocked this histamine effect. Propranolol (0.01 mM) markedly inhibited the effect of histamine(1 mM). These data suggest that histamine causes the cyclic AMP increase partly by the activation of H₂-receptors on the cells of granulation tissue and partly through the β-receptor action of catecholamines released, although the involvement of H₁-receptors cannot be excluded.     

Catecholaminergic activity and 3',5'-cyclic adenosine monophosphate levels in heart right ventricle after naloxone induced withdrawal

This study evaluated the adaptive changes in noradrenergic neurons and the concomitant production of cAMP during morphine dependence and withdrawal in the right ventricle of the rat. Rats were made dependent on morphine by morphine pellet implantation for 7 days. On the day of sacrifice animals received an acute injection of saline or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. Pretreatment with propranolol 15 min prior to naloxone was conducted to evaluate the possible implication of beta-adrenoceptors. The contents of noradrenaline and dopamine and their metabolites were examined. After naloxone administration to morphine-dependent rats (withdrawal) there was a pronounced increase in the content of normetanephrine and 3,4-dihydroxyphenylacetic acid and increased noradrenaline and dopamine turnover. In addition cAMP levels were increased after naloxone administration to morphine-treated rats. Propranolol did not block the hyperactivity of catecholaminergic neurons or the enhancement of cAMP observed in the heart during withdrawal. The present results indicate that heart catecholaminergic neurons play a significant role in the alterations in heart functions during morphine abstinence syndrome and suggest that those alterations are mediated through cAMP.     

Synthesis and enzymatic activity of some new purine ring system analogues of adenosine 3',5'-cyclic monophosphate.

A series of novel adenosine 3',5'-cyclic monophosphate (cAMP) analogues, as well as their 6-deamino and 6-nitro derivatives, were synthesized where the purine ring was replaced by indazole, benzotriazole, and benzimidazole. The 3',5'-cyclic monophosphates of indazole and benzotriazole ribofuranosides, where the sugar-phosphate moiety is attached to the N-2 nitrogen atoms of the heterocycles, were also prepared. The biological efficiency of the analogues was tested by their ability to activate purified cAMP-dependent protein kinase I (PK-I) from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart. Each cyclic nucleotide is capable of activating both PK isozymes in half-maximum concentrations (Ka) ranging from 2.0 x 10(-8) to 4.8 x 10(-6) M. The cyclic phosphate of N-1-beta-D-ribofuranosylindazole (13) proved to be a very poor activator for both PK-I and PK-II, but when indazole binds by N-2 to ribose or when the hydrogen atom at C-4 is substituted by a nitro or amino group, activities of the analogues increase considerably. The activating potencies of benzotriazole derivatives are similar to that of cAMP, irrespective of the C-4 substituents. The Ka' values of cyclic nucleotides containing benzimidazole were found to be higher for PK-II than for PK-I; e.g. the activity of 4-nitro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphate (32) is nearly 20 times as high for PK-II than for PK-I.     

Relaxation of the guinea-pig tracheal chain preparation by N6,2'-O-dibutyryl 3',5'-cyclic adenosine monophosphate

N⁶,2′-O-Dibutyryl 3′,5′-cyclic adenosine monophosphate (dibutyryl 3′,5′-amp ), isoprenaline and theophylline relax the guinea-pig tracheal chain preparation; whereas 3′,5′-cyclic adenosine monophosphate (3′,5′-amp ) does not. The relaxant effect of isoprenaline, but not that of dibutyryl 3′,5′-amp , was blocked by propranolol. 3′,5′-amp is hydrolyzed rapidly by beef heart phosphodiesterase whereas dibutyryl 3′,5′-amp is not. The presence of equimolar concentrations of dibutyryl 3′,5′-amp does not alter the rate of phosphodiesterase mediated hydrolysis of 3′,5′-amp . These data are consistent with the theories that relaxation of the guinea-pig trachea may be mediated by 3′,5′-amp and that dibutyryl 3′,5′-amp acts by mimicking 3′,5′-amp at its site of action.     

Influence of intracerebroventricular injections of N6, O2'-dibutyryl adenosine 3':5'-cyclic monophosphate on sodium pentobarbital-induced narcosis in rats.

Premedication with dibutyryl cyclic AMP (225 μg, i.c.v.) decreased the responsiveness of the central nervous system and lethality to sodium pentobarbital in the rat. The LD₅₀ of sodium pentobarbital was increased (79.9–116 $\text{solmg}\text{kg}$ i.p.) and the sleep time was decreased from 120min to 76 min in dibutyryl cyclic AMP pretreated animals. The brain and plasma pentobarbital concentrations at the time of awakening were higher in the dibutyryl cyclic AMP group as compared to saline pretreated rats. The threshold dose level of sodium pentobarbital as determined from the dose—response curves and the dose of intravenous infused pentobarbital necessary to suppress EEG activity was increased after administration of the cyclic nucleotide. Pretreatment with gradient doses of dibutyryl cyclic AMP produced a biphasic dose-response of pentobarbital sleep time with the shortest duration observed at 225 μg of dibutyryl cyclic AMP and a progressive increase in duration of sleep time from 250 and 275 μg was produced. Doses of dibutyryl cyclic AMP greater than 275 μg produced death in all animals. The cardiovascular depressant action of pentobarbital was antagonized by dibutyryl cyclic AMP. However, the hypothermic action of pentobarbital was not reversed. The results suggest dibutyryl cyclic AMP produces a generalized stimulation of the CNS and does not specifically antagonize barbiturate-induced toxicity.     

Inhibition of stimulated Jurkat cell adenosine 3',5'-cyclic monophosphate synthesis by the immunomodulatory compound HR325

HR325 (2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'(trifluoromethyl)-phenyl]-propenamide) is an immunomodulatory compound through pyrimidine biosynthesis inhibition with antiproliferative properties which was derived from the isoxazol compound A77 1726 [2-cyano-3-cyclopropyl-3-hydroxy-enoic acid (4-trifluoromethylphenyl)-amide]. During studies of the effects on early signal transduction events of this type of compound, it was found that HR325 dose-dependently inhibited adenosine 3',5'-cyclic monophosphate (cAMP) synthesis by Jurkat cells stimulated with prostaglandin E(2), (PGE(2)), cholera toxin (CTX), or forskolin (FKN). The potency of inhibition by HR325 of FKN-stimulated cells (IC(50) 30.4 microM) was approximately 3-fold higher than that of the other agonists (11.6 and 11.7 microM) and was independent of time of preincubation for both PGE(2) and FKN. Interestingly, A77 1726, an analogue of HR325, displayed a markedly different profile of stimulus-dependent potencies. The inhibition of cAMP synthesis by HR325 when stimulated by both PGE(2) and FKN was unaffected by glucose supplementation, in contrast to HR325-inhibited ATP levels, which were restored under such conditions. Further studies revealed that HR325 reduced intracellular ATP levels by uncoupling oxidative phosphorylation, albeit with a 1000-fold lower potency than the antihelmintic drug niclosamide. In addition, glucose supplementation experiments showed that, in contrast to HR325, the niclosamide-mediated reduction of ATP levels was wholly responsible for its inhibition of PGE(2)- and FKN-stimulated cAMP synthesis.     

The adhesiveness of monocytic U937 cells is stimulated by pro-inflammatory agents and inhibited by adenosine 3':5'-cyclic monophosphate

We investigated the regulation of the adhesiveness of the human promonocytic cell line U-937, differentiated along the monocytic pathway either by 1,25-(OH)2-cholecalciferol or a combination of retinoic acid and dibutyryl cAMP. Adhesion to untreated polystyrene plastic was induced by inflammatory agents like PAF, fMLP or LTB4. The response to PAF first appeared after 48hr of differentiation and was inhibited by PAF antagonists and protein kinase C inhibitors indicating involvement of the phosphatidyl-inositol pathway in the stimulating effect. On the other hand, all the c-AMP raising agents tested inhibited PAF-induced cell adhesion, whatever their target membrane receptors, the Gs transducing protein, the catalytic unit of adenylate cyclase or cAMP phosphodiesterase. Direct stimulation of protein kinase A by Br8-cAMP had a similar effect. Moreover, PAF was able to increase cAMP levels. This suggests the existence of a cAMP based negative control mechanism limiting the action of PAF.     

Effect of heparin and dihydroergotamine on platelet adenosine-3',5'-cyclic monophosphate

Human platelet adenosine-3',5'-cyclic monophosphate (cAMP) levels were determined in platelet rich plasma (PRP) and in washed platelets by a modification of the protein binding assay; the validation of the method is described. Dihydroergotamine (DHE) inhibited epinephrine induced platelet aggregation (ID50 = 2.5 X 10(-7) mol/l), and increased cAMP levels in platelets by an alpha-adrenergic receptor blocking effect, since phentolamine but not propranolol, behaved similarly. The DHE induced cAMP accumulation was correlated to the inhibitory effect on aggregation and showed a characteristic alpha-adrenergic receptor pattern in the presence of alprostadil (PGE1) and epinephrine but not collagen or adenosine diphosphate (ADP). Thrombin induced aggregation was similarly affected by DHE but with 100 times higher concentration. Heparin was found to increase slightly ADP and epinephrine induced aggregation and to decrease cAMP. Also, heparin was found to inhibit thrombin induced platelet aggregation. In washed platelets, the inhibitory effect of thrombin on PGE1 induced cAMP accumulation was counteracted by heparin. This indicates that the binding site of thrombin on platelets is important in the control of adenyl cyclase. Evidence is presented that some of the beneficial synergistic effect of DHE and heparin may consist in the ability of those compounds to produce opposite effects on cAMP system in platelets.     

The effect of intracerebroventricularly administered dibutyryl adenosine 3',5'-cyclic monophosphate on cerebrospinal fluid and serum dopamine-beta-hydroxylase in rabbits

Intracerebrospinal injected or infused dibutyryl adenosine 3′,5′-cyclic monophosphate provoked (a) an initial small decrease of cerebrospinal fluid dopamine-β-hydroxylase activity which lasted for 2 hours, followed by a significant rise of a 4–6 hours duration, together with a rise in total cerebrospinal fluid protein concentration, (b) a significant increase in serum dopamine-β-hydroxylase activity. Pretreatment of rabbits with two intracisternal injections of 6-hydroxydopamine completely abolished the dibutyryl adenosine 3′,5′-cyclic monophosphate induced release of dopamine-β-hydroxylase in the cerebrospinal fluid but only partially the release of dopamine-β-hydroxylase in the serum.