Role of adenosine A1 and A2 receptors in the regulation of aldosterone production in rat adrenal glands

Summary To investigate the roles of adenosine A₁ and A₂ receptors in the regulation of aldosterone production, we examined the effects of adenosine and adenosine agonists (N⁶-cyclohexyl adenosine; selective adenosine A₁ receptor agonist and 5-N-ethylcarboxamine adenosine; selective adenosine A₂ receptor agonist) on aldosterone and cyclic AMP production in rat adrenal capsular cells. Neither adenosine nor 5-N-ethylcarboxamine adenosine caused significant effects on basal aldosterone or cyclic AMP production. Also, adenosine (10^–3M) showed no consistent effects on aldosterone and cyclic AMP production induced by ACTH. On the other hand, N⁶-cyclohexyl adenosine exhibited a significant inhibition of basal aldosterone and cyclic AMP production at doses of 10^–4 M and 10^–3 M; furthermore, 10^–3 M N⁶-cyclohexyl adenosine inhibited aldosterone and cyclic AMP production stimulated by ACTH. These results suggest that adenosine A₁ receptors are coupled to and inhibit adenylate cyclase and may be involved in the inhibition of aldosterone production.     

Accumulation of purine catabolites in solid tumors exposed to therapeutic hyperthermia

Intensified adenosine triphosphate (ATP) degradation following therapeutic hyperthermia is often observed in solid tumors. As a result, accumulation of purine catabolites can be expected together with formation of protons at several stages during degradation to the final product, uric acid. Proton formation in turn can contribute to the development of heat-induced acidosis. Furthermore, oxidation of hypoxanthine and xanthine may result in generation of reactive oxygen species, which may lead to DNA damage, lipid peroxidation and protein denaturation, thus also contributing to heat-induced cytotoxicity. In hyperthermia experiments a tumor-size-dependent, significant increase in the levels of the following catabolites has been demonstrated: [IMP+GMP] (sum of guanosine and inosine monophosphate levels), inosine, hypoxanthine, xanthine and uric acid, along with a drop in ATP and guanosine triphosphate (GTP) levels. These data suggest that formation of reactive oxygen species and protons during purine degradation may indeed play a significant role in the antitumor effect of hyperthermia.     

Enzymatic formation of potential anticancer and antiviral inosine analogues

Theoretically, inosine analogues should act as effective inhibitors of tumor cell proliferation and viral replication. To acquire a broad spectrum of new candidate inosine analogues, a rapid, facile, quantitative and stereoselective method for deaminating potential antitumor and antiviral adenine analogues previously synthesized in our laboratory was developed. A novel 5-adenylic acid deaminase, with relaxed substrate requirements, fromAspergillus species was utilized to deaminate four hexofuranosyladenine nucleosides and five adenine nucleoside dialdehydes to their corresponding inosine analogues. The fastest rates of deamination for the hexofuranosyl nucleosides were for the compounds where the vicinal hydroxyl groups on the sugars are oriented in the erythro configuration. For rapid deamination of the adenine nucleoside dialdehydes, theR configuration at the proximal carbon atom is preferred, while the nature of the group on the distal carbon atom has no significant effect on the rate or extent of deamination.     

Neuronal responses to extracellularly applied cyclic AMP: Role of the adenosine receptor

Summary At low doses, theophylline blocks the neuronal depressant effects of 5-AMP, but not cyclic AMP. Higher doses (100 mg/kg) block cyclic AMP responses and reduce the effects of noradrenaline and GABA. It is concluded that cyclic AMP does not depress neurones via the adenosine receptor.We are grateful to the Wellcome Trust and Royal Society for travel grants.     

AMP deaminase, 5′-nucleotidase and adenosine deaminase in rat myocardial tissue in myocardial infarction and hypothermia

Summary AMP deaminase, 5-nucleotidase and adenosine deaminase have been estimated in skeletal muscle and myocardial tissue in normal rats and in rats subjected to experimental myocardial infarction or hypothermia. A difference in the enzyme distribution was found between the right and left ventricles in the normal rat. A decrease in the activity of 5-nucleotidase and an increase in the activity of adenosine deaminase were observed in infarcted myocardial tissue. The activity of all 3 enzymes was found to be depressed in the myocardium in rats subjected to hypothermia. These results are discussed in relation to adenosine production and its beneficial effects.     

Platelet aggregation is inhibited by phycolectins

Lectins from four marine algal species were examined for interaction with human platelets. The lectin designated hypnin A, from the red algaHypnea japonica, inhibited adenosine diphosphate (ADP)-or collagen-induced human platelet aggregation in a dose-dependent manner. Complete inhibition was observed at concentrations of 100 and 5 g/ml of the lectin with, ADP (2 M) and collagen (0.2 g/ml)-induced platelet aggregation, respectively. At the inhibitory concentration of 0.5 to 100 g/ml, the lectin did not induce aggregation of resting platelets. Lectins from the other three algal species also inhibited ADP-induced human platelet aggregation. These results indicate that the algal lectins are a new group of inhibitors and may be useful to study glycoconjugates on platelet membranes and to design novel platelet aggregation inhibitors.     

Brain cyclic nucleotide and energy metabolite responses to subanesthetic and anesthetic concentrations of halothane

Summary Adults rats were exposed to 0.5, 1.0 and 1.5% halothane, delivered in air, for 1 h. Whole brain 3,5-cyclic adenosine monophosphate (cAMP) of halothane-exposed rats showed only a slight increase relative to control values. 3,5-Cyclic guanosine monophosphate (cGMP) was increased significantly in halothane-exposed rats, and the response was directly related to the halothane concentrations. Adenosine triphosphate (ATP) and phosphocreatine (PC) remained unchanged relative to control values. Correspondence of these values to apparent discrepancies in the literature is discussed.Acknowledgments. This research was supported by Public Health Service Grant NS-14355.     

Chemical modification of genetic damage from continuous irradiation in mice

Summary It has been demonstrated that Adeturon (S-2-aminoethyl-iso-thiuronium adenosine triphosphate) showed a marked protective effect against translocations induction in mice germ cells after chronic -or neutron-irradiation.     

Changes in the activities of protein kinase modulators in the cerebellum of mice due to ethanol,caffeine, or phenobarbital administration

Summary Decreased activities of both the inhibitory modulator of adenosine 35-monophosphate (cAMP)-dependent protein kinase (A-PK) as well as the stimulatory modulator of guanosine 35-monophosphate (cGMP)-dependent protein kinase (G-PK) from the mouse cerebellum were noted due to the administration of excessive doses of ethanol, caffeine, and phenobarbital for up to 28 days. The dose-dependence of the inhibition of A-PK or the stimulation of G-PK was observed as a function of the amount of protein kinase modulators in the cerebellum of mice receiving different doses of ethanol.Acknowledgments. This work was supported by a grant (RR 08119-06-PK modulator project) from NIH, USA. J.L. Williams, T. Floyd-Jones, C.F. Duggans, D.L. Boone, and S.O. Smith were prebaccalaureate trainees of MBS program. Authors thank Dr J.F. Kuo of Emory University for encouragement in this project.     

May K+ ions stimulate the formation of cyclic AMP in the brain independently on their depolarizing action?

Summary The effect of potassium ions on the formation of adenosine 3,5-monophosphate (cAMP) in the rat cerebral cortex in vivo was studied under conditions where development of spreading depression had been blocked by pretreatment of the cerebral cortex by topically applied magnesium ions. A linear relationship between potassium concentrations applied to the cortical surface and levels of cAMP has been found. Moreover, potentiation of the K⁺-effect by magnesium ions has been observed.     

Multiple flavonoid-binding sites within multidrug resistance protein MRP1

Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N₃-[-³²P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C₄ (LTC₄) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N₃-[-³²P]ADP trapping, weakened inhibition of LTC₄ transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.Received 1 May 2003; received after revision 18 June 2003; accepted 24 June 2003     

Influence of cyclic nucleotides on protein synthesis in vascular smooth muscle

Summary The incorporation of leucice-¹⁴C into protein in bovine mesenteric arteries was augmented by cyclic GMP (10^–3 M) and decreased by cyclic AMP (10^–3 M). There was no effect of 5 AMP (10^–3 M). The phosphodiesterase inhibiting drugs theophylline (10^–3 M) and papaverine (5×10^–5 g/ml) both decreased the leucine-¹⁴C incorporation.We are indebted to Mrs.Lena Burlin for hear assistance. Finacial support has been provided by the Swedish State Medical Research Council (No. 04X-101X-4498).     

Metabolic pathways of δ-aminolaevulinic acid inRhodopseudomonas spheroides

Summary Incorporation of -aminolaevulinic acid 5¹⁴C and 4¹⁴C into the inosine monophosphate pool and into porphyrins, was studied in cell suspensions ofR. spheroides. The results contradict a direct incorporation of -aminolaevulinic acid into the purine ring of nucleotides through -dioxovaleric acid. It would suggest a nonspecific incorporation after degradation of -aminolaevulinic acid without a transamination as a first reaction.     

Flying insects: model systems in exercise physiology

Insect flight is the most energy-demanding exercise known. It requires very effective coupling of adenosine triphosphate (ATP) hydrolysis and regeneration in the working flight muscles.³¹P nuclear magnetic resonance (NMR) spectroscopy of locust flight muscle in vivo has shown that flight causes only a small decrease in the content of ATP, whereas the free concentrations of inorganic phosphate (P _i ), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were estimated to increase by about 3-, 5- and 27-fold, respectively. These metabolites are potent activators of glycogen phosphorylase and phosphofructokinase (PFK). Activation of glycolysis by AMP and P _i is reinforced synergistically by fructose 2,6-bisphosphate (F2,6P₂), a very potent activator of PFK. During prolonged flight locusts gradually change from using carbohydrate to lipids as their main fuel. This requires a decrease in glycolytic flux which is brought about, at least in part, by a marked decrease in the content of F2,6P₂ in flight muscle (by 80% within 15 min of flight). The synthesis of F2,6P₂ in flight muscle can be stimulated by the nervous system via the biogenic amine octopamine. Octopamine and F2,6P₂ seem to be part of a mechanism to control the rate of carbohydrate oxidation in flight muscle and thus function in the metabolic integration of insect flight.Dedicated to Dr. Ernst Zebe, Emeritus Professor of Zoology (University of Münster) on the occasion of his 70th birthday.     

Effects of several preoperative medications on fat cell lipolysis,and activity of adipose tissue cyclic AMP phosphodiesterase

Summary Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a clinical dose, omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 g ml^–1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compartible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.The authours are grateful to Dr D. C. Williams for his continued support and encouragement.     

Cyclic AMP binding proteins in saliva

Summary Cyclic adenosine 3,5-monophosphate (cyclic AMP)-binding proteins which have the characteristics of cyclic AMP-dependent protein kinase (E.C. 2.7.1.37) regulatory subunits, have been identified in rat and human saliva. Concentration of these proteins was increased in rat saliva after stimulation with isoproterenol, suggesting that they were released during exocytosis. Cyclic AMP-dependent protein kinase catalytic activity was not measurable in saliva of either species.     

Adenosine uptake in pyrimidine 5′-nucleotidase deficient human erythrocytes via a high affinity transport system

Summary Using a pulse-labeling technique,¹⁴C-adenosine uptake into pyrimidine 5-nucleotidase (P5N) deficient erythrocytes (RBC) was found to be impaired. The Lineweaver-Burk plot showed K_m values of 2.0×10^–3 mM and 0.2 ×10^–3 mM for normal RBC and P5N deficient RBC, respectively. These results indicate that P5N is one of regulators of the adenosine transport system and/or is associated with adenosine carrier protein.     

Antiproliferative effects of 4′, 5′-unsaturated adenine nucleosides on leukemia L 1210 cells in vitro

Summary Several 4, 5-unsaturated adenine nucleosides were shown to have antiproliferative activity against L 1210 leukemia cells in vitro. The active nucleosides were cytotoxic to the L 1210 cells as demonstrated by Trypan Blue uptake. The cytotoxicity was not induced by alterations in the ribonucleoside and deoxyribonucleoside triphosphate levels of the L 1210 cells.     

Effect of dibutyryl cAMP and theophylline on cultured rat embryonic shields

Summary Effects of N⁶, O² dibutyryl adenosine 3,5-cyclic monophosphate (db-cAMP) and theophylline (Th) on cultured rat embryonic shields were studied. After addition of db-cAMP to the culture medium, an increase of the weight of explants and of the incidence of the skeletal muscle was observed. Theophylline seems to be ineffective.     

Pineal-related changes in cyclic AMP levels of rat medial basal hypothalamus

Summary Pinealectomy (Px) in adult male rats resulted in increased cyclic AMP accumulation by medial basal hypothalamic (MBH) explants 3 and 7 days after surgery. 24 h after superior cervical ganglionectomy (Gx) an augmented MBH cyclic AMP accumulation was observed. The effects of Px and Gx were additive, as revealed in animals subjected to Gx 3 days after Px.This study was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (N^o 6638) and from the Fundación Alberto J. Roemmers, Argentina.