Cyclic AMP-Generating Systems in Cell-Free Preparations from Guinea Pig Cerebral Cortex: Loss of Adenosine and Amine Responsiveness Due to Low Levels of Endogenous Adenosine

The accumulations of radioactive cyclic AMP elicited by adenosine, norepinephrine, and histamine in adenine-labeled vesicular entities of a particulate fraction from guinea pig cerebral cortex are greatly reduced as a result of prolonged preincubation. The presence of adenosine deaminase during preincubations largely prevents the loss of adenosine, norepinephrine and histamine responses. Adenosine deaminase was inactivated by deoxycoformycin prior to stimulation of cyclic AMP accumulation by adenosine or amines. If adenosine deaminase is not inactivated, responses to norepinephrine are not significant and histamine responses are reduced by 50%. Adenosine deaminase cannot restore responsiveness of the cyclic AMP-generating systems. It is proposed that, in particulate fractions of guinea pig cerebral cortex, low levels of adenosine cause a slow loss of receptors and/or coupling of receptors to cyclic AMP-generating systems.     

ACCUMULATION OF CYCLIC ADENOSINE 3', 5'-MONOPHOSPHATE IN CEREBRAL CORTICAL SLICES FROM RAT AND MOUSE: STIMULATORY EFFECT OF α- AND β-ADRENERGIC AGENTS AND ADENOSINE

Abstract— Norepinephrine, epinephrine, isoproterenol, and adenosine elicit enhanced accumulations of cyclic AMP in incubated slices of rat cerebral cortex. Combinations of norepinephrine, epinephrine, isoproterenol, or histamine with adenosine have a greater than additive effect on cyclic AMP levels. The effects of isoproterenol appear to be mediated via a classical β-adrenergic receptor whereas the effects of norepinephrine appear due to interactions with both α- and β-adrenergic receptors. The presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiates the effects of the catecholamines and reveals a histamine-mediated increase in cyclic AMP levels. After an initial stimulation of cyclic AMP formation with norepinephrine, followed by washing of the slices, the cyclic AMP-generating system is unresponsive to norepinephrine but does respond to an adenosine-norepinephrine combination. In mouse cerebral cortical slices, catecholamines appear to elicit an accumulation of cyclic AMP primarily via interaction with a β-adrenergic receptor.     

Activation of Cyclic AMP-Generating Systems in Brain Membranes and Slices by the Diterpene Forskolin: Augmentation of Receptor-Mediated Responses

The diterpene forskolin markedly activates adenylate cyclase in membranes from various rat brain regions and elicits marked accumulations of radioactive cyclic AMP in adenine-labeled slices from cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, hypothalamus, thalamus, and medulla-pons. In cerebral cortical slices, forskolin has half-maximal effects at 20-30 microM on cyclic AMP levels, both alone and in the presence of the phosphodiesterase inhibitor ZK 62771. The presence of a very low dose of forskolin (1 microM) can augment the response of brain cyclic AMP-generating systems to norepinephrine, isoproterenol, histamine, serotonin, dopamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin does not augment responses to combinations of histamine-norepinephrine adenosine-norepinephrine, or histamine-adenosine. For norepinephrine and isoproterenol in rat cerebral cortical slices and for histamine in guinea pig cerebral cortical slices, the presence of 1 microM-forskolin augments the apparent efficacy of the amine, whereas for adenosine, prostaglandin E2, and vasoactive intestinal peptide, the major effect of 1 microM-forskolin is to increase the apparent potency of the stimulatory agent. In rat striatal slices, forskolin reveals a significant response of cyclic AMP systems to dopamine and augments the dopamine-elicited activation of adenylate cyclase in rat striatal membranes. The activation of cyclic AMP systems by forskolin is rapid and reversible, and appears to involve both direct activation of adenylate cyclase and facilitation and/or enhancement of receptor-mediated activation of the enzyme.     

An activator of protein kinase C (phorbol-12-myristate-13-acetate) augments 2-chloroadenosine-elicited accumulation of cyclic AMP in guinea pig cerebral cortical particulate preparations

Norepinephrine and histamine markedly augment accumulations of cyclic AMP elicited by 2-chloroadenosine in a guinea pig cerebral cortical vesicular preparation. In addition, these biogenic amines stimulate phosphatidylinositol turnover. Phosphatidylinositol turnover is associated with mobilization of internal calcium and with stimulation of protein kinase C. Phorbol-12-myristate-13-acetate (PMA), a known activator of protein kinase C, has no effect on cyclic AMP levels alone, but in a concentration-dependent manner enhances accumulations of cyclic AMP elicited by 2-chloroadenosine. PMA, like norepinephrine, also enhances accumulations of cyclic AMP elicited by histamine. PMA has no effect on the synergistic accumulations of cyclic AMP elicited by combinations of amines and 2-chloroadenosine. PMA also augments accumulations of cyclic AMP elicited by forskolin. The results suggest that activation of phosphatidylinositol turnover by biogenic amines may lead via stimulation of protein kinase C to enhanced responsiveness of cyclic AMP-generating systems.     

Depolarization-evoked accumulation of cyclic AMP in brain slices: inhibition by exogenous adenosine deaminase

In guinea pig cerebral cortical slices labeled during a prior incubation with radioactive adenine, electrical stimulation or the presence of depolarizing agents such as veratridine, ouabain, and high concentrations of K⁺ elicit a marked accumulation of radioactive cyclic AMP. This accumulation is reduced in all cases by the presence of theophylline, a compound that antagonizes the stimulatory effects of adenosine on cyclic AMP accumulation in brain slices. Exogenous adenosine deaminase also reduced the accumulation of cyclic AMP elicited by electrical stimulation, veratridine, and high concentrations of K⁺. Thus, adenosine formed in neuronal compartments under depolarizing conditions appears to be released into the extracellular medium as a prerequisite to stimulation of the cyclic AMP-generating system. Adenosine deaminase does not prevent the reduction in levels of ATP under depolarizing conditions, nor does it antagonize the accumulation of cyclic AMP elicited by a combination and norepinephrine. Adenosine deaminase does not, however, prevent the accumulations of cyclic AMP elicited by the depolarizing agent, ouabain.     

Involvement of Adenosine-Sensitive Cyclic AMP-Generating Systems in Cobalt-Induced Epileptic Activity in the Rat

An injection of cobalt chloride solution into the unilateral sensorimotor cortex of rats induced electrographic epileptic activity, which was followed by a peripheral motor disturbance. Brain slices were prepared from the cortical region including the injection site and from the other cortical regions of rats between 8 and 50 days after the injection. In the cortical slices, we examined cyclic AMP accumulations elicited by adenosine and its stable analogue 2-chloroadenosine. Adenosine and 2-chloroadenosine at their maximal dose increased cyclic AMP accumulation six- to 10-fold and 10–15-fold, respectively, and the elicitation was markedly inhibited by the adenosine antagonist 8-phenyltheophylline. The cyclic AMP accumulation was increased in the primary epileptic region of the cortex adjacent to the injection site of cobalt chloride solution, whereas it was unchanged in the other cortical regions. The increase in cyclic AMP accumulation was observed regardless of the presence or absence of the adenosine uptake inhibitor dipyridamole, the phosphodiesterase inhibitor DL-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, and adenosine deaminase. Such an increased accumulation of cyclic AMP in the primary epileptic cortex was detected as early as 8 days after the injection. The cyclic AMP accumulation continued to increase and reached a peak level 17–19 days after the injection, and it returned to the control levels after 40–50 days, in correspondence with the electrographic and behavioral findings. It is concluded that alterations in adenosine receptormediated generation of cyclic AMP in the primary epileptic cortex are closely associated with the central process of cobalt-induced epilepsy. In general, the adenosine-sensitive cyclic AMP-generating systems may serve as a common mechanism in experimental models of epilepsy.     

The accumulation of cyclic AMP and cyclic GMP in guinea pig brain slices: Effect of calcium ions,norepinephrine and adenosine

The effect of Ca²⁺ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca²⁺ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E₁ and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca²⁺, norephinephrine and adenosine.     

Vasopressin inhibition of cyclic AMP accumulation and effects on the learned response in inbred mouse strains

The effects of a single injection of arginine vasopressin on the cyclic AMP responses of cerebral cortex slices to noradrenaline, forskolin and 2-chloro-adenosine were tested in six inbred mouse strains. The noradrenaline response was reduced in one strain and that to 2-Cl-adenosine in 2 strains. There was a positive correlation between the differences in the cyclic AMP response to 2-Cl-adenosine between control and AVP-treated mice and the differences in latencies during the extinction period of conditioned avoidance response learning.     

Adenosine and Cyclic AMP in Rat Cerebral Cortical Slices: Effects of Adenosine Uptake Inhibitors and Adenosine Deaminase Inhibitors

The endogenous levels of adenosine functionally linked to cyclic AMP systems in rat cerebral cortical slices are regulated by both adenosine deaminase and adenosine uptake systems. 2'-Deoxycoformycin (2'-DCF), an adenosine deaminase inhibitor, slightly increased basal, adenosine, and norepinephrine-elicited accumulations of cyclic AMP, whereas dipyridamole, an uptake inhibitor, had an even greater effect on cyclic AMP accumulations under the same conditions. Combinations of 2'-DCF and dipyridamole elicited a greater effect than either compound alone. Neither 2'-DCF nor dipyridamole significantly augmented accumulations of cyclic AP elicited by a depolarizing agent, veratridine, suggesting that the adenosine "released" during neuronal depolarization of brain slices is not as subject to inactivation by uptake or deamination as endogenous adenosine in control brain slices. The accumulation of cyclic AMP elicited by a combination of norepinephrine and veratridine was greater than additive. The response to a pure beta-adrenergic agonist, isoproterenol, was not potentiated by 2'-DCF, dipyridamole, or veratridine, consonant with minimal interaction of endogenous adenosine with beta-adrenergic systems.     

Accumulations of Cyclic AMP in Adenine-Labeled Cell-free Preparations from Guinea Pig Cerebral Cortex: Role of α-Adrenergic and H1-Histaminergic Receptors

Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine-labeled nucleotides in particulate fractions from Krebs-Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac-like entities, which apparently are associated with a significant portion of the membranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine-labeled cyclic AMP-generating systems by norepinephrine is by means of alpha-adrenergic receptors, while activation by histamine is through H1- and H2-histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via "release" of endogenous adenosine. It is proposed, based on the virtual absence of an alpha-adrenergic or H1-histaminergic response in the presence of a combination of potent adenosine and H2-histaminergic antagonists, that alpha-adrenergic and H1-histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs-Ringer particulate fractions, but merely facilitate activation by beta-adrenergic, H2-histaminergic, or adenosine receptors.     

INTERRELATIONSHIPS AMONG THE LEVELS OF ATP, ADENOSINE AND CYCLIC AMP IN INCUBATED SLICES OF GUINEA-PIG CEREBRAL CORTEX: EFFECTS OF DEPOLARIZING AGENTS, PSYCHOTROPIC DRUGS AND METABOLIC INHIBITORS

—Adenine nucleotides of guinea-pig cerebral cortical slices were labelled during a 40 min incubation with [¹⁴C]adenine. Subsequent incubation of cortical slices with depolarizing agents, such as veratridine, ouabain, batrachotoxin and high concentrations of potassium ions, or with certain psychotropic drugs such as chlorpromazine, chlorimipramine or prenylamine resulted in a reduction in both endogenous and radioactive ATP, accompanied by a marked increase in levels of both endogenous and radioactive cyclic AMP. Reduction of ATP levels during incubation with depolarizing agents, such as veratridine, is probably associated with increased activity of membranal Na⁺-K⁺-activated ATPase, while the reduction elicited by psychotropic drugs is proposed to be due to inhibition of mitochondrial synthesis of ATP. With both classes of compounds reduction of ATP levels results in enhanced formation and efflux of adenosine which stimulates formation of cyclic AMP from intracellular ATP in the compartments of brain slices which contain the cyclic AMP-generating systems. Certain classical metabolic inhibitors such as 2,4-dinitrophenol, azide, 1,2-naphthoquinone-8-sulfonate and cyanide also reduce ATP levels and in the case of 2,4-dinitrophenol, cyanide, and azide elicit small but significant accumulations of cyclic AMP. With certain metabolic inhibitors reduction of ATP within the cyclic AMP generating compartments would appear to prevent or reduce the accumulation of cyclic AMP elicited by amines, adenosine or veratridine.     

FUNCTIONAL COMPARTMENTS OF ADENINE NUCLEOTIDES SERVING AS PRECURSORS OF ADENOSINE 3'',5''-MONOPHOSPHATE IN MOUSE CEREBRAL CORTEX

Cyclic AMP accumulates in cerebral cortical slices from the C57B1/6J mouse incubated with the following stimulatory agents: norepinephrine, adenosine, veratridine and adenosine-biogenic amine combinations. The results with slices labelled with radioactive adenine or adenosine provide evidence for the existence of distinct functional compartments of adenine nuclcotides which serve as precursors of cyclic AMP on stimulation with specific agents. Thus, in slices labelled with [¹⁴C]adenine or [³H]adenosine the ratio of [¹⁴C] to [³H]cyclic AMP was dependent on the stimulatory agent; with veratridinc the ratio was 1.4 while with adenosine the ratio was 3.0. In addition, a greater than 2-fold difference in the ratio of endogenous/radioactive cyclic AMP was observed in adenine or adenosine-labelled slices after incubation with veratridine, norepinephrine, adenosine or adenosine-amine combinations; the lowest ratios after stimulation with veratridine and the highest after adenosine or adenosine-amine combinations. The high ratio observed with adenosine was in part due to a quite marked incorporation of the stimulant, adenosine, into the accumulating cyclic AMP. Such distinct functional compartments of cyclic AMP precursors may represent different cell types and/or morphological entities within one cell type.     

1-Isoamyl-3-isobutylxanthine: a remarkably potent agent for the potentiation of norepinephrine, histamine, and adenosine-elicited accumulations of cyclic AMP in brain slices.

1-Isoamyl-3-isobutylxanthine (EC₅₀ t 5 μM) potentiates by 2 to 6-fold the accumulations of cyclic AMP elicited in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. In addition, the xanthine derivative causes a 2 to 3-fold elevation of basal levels of cyclic AMP. 1-Isoamyl-3-isobutylxanthine has no effect on accumulations of cyclic AMP elicited by histamine or adenosine in the presence of a potent phosphodiesterase inhibitor, ZK 62771. The xanthine derivative retards the disappearance of cyclic AMP after a prior stimulation by adenosine. The results indicate that 1-isoamyl-3-isobutylxanthine is an extremely potent and effective inhibitor of phosphodiesterases involved in the regulation of cyclic AMP levels in guinea pig cerebral cortical slices. The 1-benzyl, 1-isoamyl, and 1-isobutyl derivatives of 3-isobutylxanthine potentiate the accumulation of cyclic AMP elicited by adenosine, while the 1-methyl derivative and 1-isoamyl-3-methylxanthine are inhibitory undoubtedly because of blockade of adenosine-receptors by these compounds. Xanthines with bulky 1- and 3- substituents appear to be relatively weak adenosine-antagonists and relatively specific and potent agents for inhibition of phosphodiesterases involved in cyclic AMP metabolism in brain tissue.     

Role of Neuronal Signal Input in the Down-Regulation of Central Noradrenergic Receptor Function by Antidepressant Drugs

Rats with unilateral lesions of the locus coeruleus were used to study the role of norepinephrine (NE) signal input in the down-regulation by antidepressants of the noradrenergic cyclic AMP-generating system in the cortex. Chronic administration of both desipramine (blockade of NE reuptake) and iprindole (no blockade of NE reuptake) reduced the cyclic AMP response to NE on the nonlesioned side, but had little or no effect on the lesioned side. The results indicate that NE signal input and thus the formation of the NE-receptor complex are prerequisites for inducing noradrenergic subsensitivity.     

Regulation of adenosine 3':5'-monophosphate-dependent protein kinase in cerebral cortex

Alterations in the cyclic AMP-dependent protein kinase activity ratio in response to putative neurotransmitters and other cyclic AMP-elevating agents in intact cerebral cortical slices and Krebs-Ringer particulate preparations from cerebral cortex were examined. Both norepinephrine (30 microM) and forskolin (20 microM) produced a time-dependent increase in intracellular levels of cyclic AMP in cerebral cortical slices which was paralleled by an increase in both cyclic AMP and the protein kinase activity ratio. The increases were maximal at 5 min. and remained elevated for at least 15 min. Forskolin, norepinephrine, adenosine and isoproterenol produced a concentration-dependent increase in both cyclic AMP and the protein kinase activity ratio, however, the degree of increase observed was dissimilar. Thus, a 5-fold change in intracellular cyclic AMP resulted in only a 2-fold increase in the activity ratio. Of the agents examined, forskolin produced the most marked change in the activity ratio (from 0.23 to 0.78 at 100 microM) while isoproterenol at 100 microM produced only a 50% increase in the activity ratio. The half-time for the decline in forskolin elicited elevations of either the activity ratio or cyclic AMP was about 4-6 min. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, both were significantly prolonged being 60-70% of the maximum observed immediately after forskolin stimulation, at 15 min. Potentiation of forskolin elicited increases in the activity ratio by Ro 20-1724 were also observed but the increase in the activity ratio was maximal at 7.5 min. while cyclic AMP accumulations continued to rise during the entire 15 min. incubation. Particulate preparations from cerebral cortex were found to contain a cyclic AMP-dependent protein kinase which could be activated 2 to 3-fold with either forskolin, norepinephrine, or adenosine. Unlike the intact brain slice the changes in protein kinase activity ratio and intracellular levels of cyclic AMP in cell-free particulate preparations were similar in both time and degree.     

Accumulation of adenosine 3'',5''-monophosphate in incubated slices from discrete regions of squirrel monkey cerebral cortex: effect of norepinephrine, serotonin and adenosine

THE ROLE of adenosine 3′5′-monophosphate (cyclic AMP) in the central nervous system is as yet unknown. However, a variety of putative neurotransmitters such as norepinephrine, serotonin and histamine have been found to elicit enhanced accumulations of cyclic AMP in cerebral cortical slices from a variety of species (SHIMIZU and DALY, 1972; FORN and KRISHNA, 1971; SHIMIZU, TANAKA, SUZUKI and MATSUKADO, 1971; FUMAGALLI, BERNAREGGI, BERTI and TRABUCCHI, 1971). In addition, depolarization of membranes in cortical slices elicits a marked accumulation of cyclic AMP, probably mediated through the action of adenosine (KAKIUCHI, RALL and MCILWAIN, 1969; SHIMIZU. CREVELING and DALY, 1970). These results, obtained with grey matter from the entire cerebral cortex, suggest an intimate relationship between neuronal activity, release of putative neurotransmitters, and enhanced accumulation of cyclic AMP in brain tissue. Since innervation and electrical activity in the cerebral cortex differs among functionally distinct neocortical areas and in the histologically distinct limbic cortex, it appeared possible that these differences would also be manifest in the regional control of cyclic AMP levels. We now report that the response of the cyclic AMP generating system to putative neurotransmitters and adenosine does differ among discrete functional regions of the squirrel monkey cerebral cortex.     

Blockade of central beta-adrenergic receptors by tazolol (1-isopropylamino-3-(2-thiazoloxy)-2-propanol).

Tazolol, a β₁-adrenergic agonist in heart, had no intrinsic β-adrenergic agonist activity with respect to cyclic AMP-generating systems in rat cerebral cortical slices or with respect to firing of rat cerebellar Purkinje cells. Instead, tazolol proved to be a relatively potent and specific β-adrenergic antagonist. The IC₅₀ for (±) tazolol in antagonizing (-) isoproterenol-elicited accumulation of cyclic AMP in rat cortical slices was 7 × 10^?7M. The IC₅₀ in antagonizing [³H] dihydroalprenolol-binding in rat cortical homogenates was 2.9 × 10^?7 M. Tazolol was about 10 fold more potent in both cases than the β-antagonist, (±) sotalol. Tazolol antagonized the inhibitory, β-adrenergically mediated effects of iontophoretically applied norepinephrine on firing of cerebellar Purkinje cells. The inhibitory effects of γ-aminobutyric acid on firing of Purkinje cells were not altered by tazolol. Tazolol appeared to lack significant local anesthetic activity as evidenced by its lack of effect on spike height in spontaneous firing Purkinje cells.     

The nature of receptors regulating the formation of cyclic AMP in brain tissue.

Extensive studies of the past seven years have provided fundamental information on the pharmacological properties of receptors controlling formation of cyclic AMP in brain slices and homogenates. Receptors for adenosine, α- and β-adrenergic agonists, dopamine, serotonin, H₁- and H₂-histaminergic agonists, and prostaglandins of the E series have been defined and evidence for a glutamate receptor has been presented. Extrapolation of such pharmacological data to studies with whole animals should provide important information as to the physiological significance of specific cyclic AMP-generating systems to the function of the intact brain.     

Pertussis toxin effects on adenylate cyclase activity, cyclic AMP accumulation and lipolysis in adipocytes from hypothyroid, euthyroid and hyperthyroid rats

Adipocytes from hypothyroid rats have a decreased responsiveness to agents that activate adenylate cyclase, whereas cells from hyperthyroid rats have an increased responsiveness as compared to the controls. This is reflected in cyclic AMP accumulation as well as lipolysis. Administration of pertussis toxin to rats or its in vitro addition to adipocytes increased basal lipolysis and cyclic AMP accumulation as well as the response to norepinephrine or forskolin. The effects of thyroid status was not abolished by toxin treatment. Pertussis toxin-catalyzed ADP ribosylation of Ni was increased in adipocyte membranes from hypothyroid rats as compared to those from euthyroid rats. However, no change in sensitivity to N6-(phenylisopropyl)adenosine was observed. The data suggest that the amount of Ni might not be rate-limiting for the inhibitory action of adenosine. A consistent decrease in maximal lipolysis was observed in freshly isolated adipocytes from hypothyroid animals as compared to those from the controls. Such defective maximal lipolysis was not corrected by adenosine deaminase or in vivo administration of pertussis toxin. The relationship between cyclic AMP levels and lipolysis suggests that in fat cells from hypothyroid rats either the cyclic AMP-dependent protein kinase or the lipase activity itself may limit maximal lipolysis. There appears to be multiple effects of thyroid status on lipolysis involving factors other than those affecting adenylate cyclase activation.     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.