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.     

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.     

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N⁶-ethano[¹⁴C]adenosine nor 1,N⁶-ethanol[¹⁴C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-³H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N⁶-Benzyl[¹⁴C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N⁶-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8-¹⁴C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Intracellular formation of analogs of cyclic AMP. Studies with brain slices labeled with radioactive derivatives of adenine and adenosine.

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N6-etheno[14C] adenosine nor 1,N6-etheno[14C] adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-3H] adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chloro-cyclic AMP was not obtained. N6-Benzyl[14C] adenosine was converted only to intracellular monophosphates and significant formation of radioactive N6-benzylcyclic AMP was not detected during a subsequent incubation. 2'-Deoxy-[8-14C] adenosine was converted to both intracellular radioactive 2'-deoxy-adenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2'-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

Modulation by Unsaturated Fatty Acids of Norepinephrine- and Adenosine-Induced Formation of Cyclic AMP in Brain Slices

Abstract: The effect of linoleic acid on the formation of cyclic AMP in the slices of guinea pig cerebral cortex was examined. Treatment of the slices with linoleic acid resulted in an increase of basal and of norepinephrine-stimulated formation of cyclic AMP. The stimulatory effect on the basal level of cyclic AMP was not specific for linoleic acid: the potency of the fatty acid was related to the magnitude of unsaturation. In contrast, the enhancement of norepinephrine-stimulated formation of cyclic AMP seemed relatively specific for linoleic acid and arachidonic acid. Linoleic acid markedly enhanced the stimulated formation of cyclic AMP by histamine and adenosine, as well that by norepinephrine, without affecting that by excitatory amino acids and veratridine. Theophylline, adenosine deaminase, and 2'-deoxyadenosine antagonized the effect of linoleic acid. Linoleic acid enhanced the maximum responses to norepinephrine and adenosine without altering the ED₅₀ values for these agonists. When linoleic acid-treated slices were washed with Krebs-Ringer containing defatted bovine serum albumin, both enhancement of the response to norepinephrine and the amount of [¹⁴C]linoleic acid incorporated in a free form significantly diminished.     

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.     

UPTAKE AND RELEASE OF [14C]ADENINE DERIVATIVES AT BEDS OF MAMMALIAN CORTICAL SYNAPTOSOMES IN A SUPERFUSION SYSTEM

(1) Synaptosomal fractions from guinea pig neocortical dispersions prepared in sucrose solutions were deposited from saline media as ‘beds’ on nylon bolting cloth. When incubated with 0.5–10 μm -[¹⁴C]adenine or adenosine in glucose bicarbonate salines, uptake of ¹⁴C from adenosine proceeded at about four times the rate of uptake of [¹⁴C]adenine. This contrasted with the relative uptake of the two compounds to neocortical tissue slices or to beds made from mitochondrial fractions, where uptake was similar with the two precursors. Uptake of both precursors to synaptosome beds was much greater than uptake of inosine. (2) Synaptosome beds, [¹⁴C]adenosine-loaded, contained 88 per cent of the ¹⁴C as 5′-adenine nucleotides, the remainder being present as cyclic AMP, inosine, hypoxanthine and adenosine. When superfused, the ¹⁴C output consisted mainly of adenosine, inosine and hypoxanthine, with some 7 per cent of 5′-nucleotides and 4 per cent of cyclic AMP. (3) Electrical pulses and the addition of 50 mm -KCl each increased the efflux of ¹⁴C from superfused [¹⁴C]adenosine-loaded beds. The superfusates issuing after excitation contained the same ¹⁴C-labelled compounds as issued before, with a small increase in the proportional yield of adenosine. The additional output of ¹⁴C following electrical pulses was diminished by about 50 per cent by 0.5 μm -tetrodotoxin while that following KCl was not affected; it was however prevented when the superfusing fluids were free of Ca²⁺.     

CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE FORMATION IN GUINEA-PIG BRAIN SLICES: EFFECT OF H1- AND H2-HISTAMINERGIC AGONISTS

—A variety of histamine analogs elicit accumulations of radioactive cyclic AMP in guinea-pig neocortical and hippocampal slices labelled during a prior incubation with [¹⁴C]adenine. The H₁agonist, 2-aminoethylthiazole, elicits accumulation of cyclic AMP in neocortical and hippocampal slices both in the absence or presence of adenosine. The presence of adenosine increases the maximum response to 2-aminoethylthiazole and decreases the EC₅₀ by nearly 10-fold. In the absence of adenosine the effects of 2-aminoethylthiazole are antagonized in hippocampal slices by both d-brompheniramine and metiamide, while in the presence of adenosine only d-brompheniramine is an effective antagonist. The H₂-agonist, 4-methylhistamine, elicits a somewhat smaller accumulation of cyclic AMP than does 2-aminoethylthiazole in both cortical and hippocampal slices. In the presence of adenosine the response to 4-methylhistamine is enhanced, but is markedly lower than that seen with the combination of adenosine and 2-aminoethylthiazole. The dose-response relationship for 4-methylhistamine in the presence of adenosine appears in hippocampal slices to consist of two components. The response to 4-methylhistamine in the absence of adenosine is blocked by metiamide, while in the presence of adenosine the response is partially blocked by both H₁ and H₂-antagonists. The accumulation of cyclic AMP elicited by histamine is greatly increased by adenosine but the EC₅₀ is not significantly decreased. The results suggest that (i) both H₁- and H₂-receptors regulate cyclic AMP-formation in the central nervous system, (ii) the synergism between adenosine and histamine is mediated primarily by interaction with H₁-receptors and (iii) that adenosine greatly increases the affinity of the H₁-receptors for both H₁ and H₂-agonists without affecting its affinity for histamine.     

Neuroblastoma cell adenylate cyclase: direct activation by adenosine and prostaglandins.

—Adenylate cyclase activity of permeabilized neuroblastoma cells was measured by the conversion of [α³²P]ATP into labelled cyclic AMP. Adenosine (10^?6 - 10^?4m ) induced a dose-dependent increase in cyclic AMP formation. This effect could not be accounted for either by an adenosine-induced inhibition of the phosphodiesterase activity present in the enzyme preparation, or by a direct conversion of adenosine into cyclic AMP. This indicates that the observed increase in cyclic AMP accumulation reflected an activation of adenylate cyclase. Adenosine is partially metabolized during the course of incubation with the enzyme preparation. However, none of the identified non-phosphorylated adenosine metabolites were able to induce an adenylate cyclase activation. This suggests that adenosine itself is the stimulatory agent. The apparent K_m of the adenylate cyclase for adenosine was 5 ± 10^?6-10^?5m . Maximal activation represented 3-4 times the basal value (10-100 pmol cyclic AMP formed/10 min/mg protein). The adenosine effect was stereospecific, since structural analogues of adenosine were inactive. Adenosine increased the maximal velocity of the adenylate cyclase reaction. The stimulatory effect of adenosine was inhibited by theophylline. Prostaglandin PGE₁ had a stimulatory effect much more pronounced than that of adenosine (6-10-fold the basal value at 10^?6m ). Dopamine and norepinephrine induced a slight adenylate cyclase activation which was not potentiated by adenosine. It is concluded that adenosine is able to activate directly neuroblastoma cell adenylate cyclase. It seems very likely that such a direct activation is also present in intact nervous tissue and account, at least partly, for the observed cyclic AMP accumulation in response to adenosine.     

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.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

The interaction of norepinephrine and prostaglandin E1 on the adenyl cyclase system of human and rabbit blood platelets

Prostaglandin E₁ markedly increased the formation of cyclic [³H]AMP from labeled adenine in human and rabbit blood platelets. Norepinephrine alone had no stimulatory effect, but it reduced cyclic AMP levels elevated by prostaglandin E₁. Phentolamine overcame the inhibitory effect of norepinephrine, whereas propranolol did not. Homogenization of platelets reduced, but did not abolish, the inhibitory effect of norepinephrine on adenyl cyclase activity induced by prostaglandin E₁.     

Cyclic AMP-induced release of [14C]taurine from pinealocytes.

Pinealocytes were prelabelled with [¹⁴C]taurine. Twenty-four hours later they were treated with derivatives of cyclic AMP. It was found that dibutyryl cyclic AMP and ?-chloro-phenyl-thio cyclic AMP treatment caused a large increase in the release of [¹⁴C]taurine. The effect of dibutryl cyclic AMP on [¹⁴C]taurine release was near maximal fifteen minutes after treatment started. In view of the known stimulatory effects of norepinephrine on pineal cyclic AMP and the recent discovery that norepinephrine causes the release of taurine from pinealocytes, one can conclude that norepinephrine stimulates [¹⁴C]taurine release from pinealocytes by acting through a cyclic AMP mechanism.     

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 ATP pool associated with adenyl cyclase of brain tissue

R adioactive adenine has been frequently used to label cyclic adenosine 3', 5'-monophosphate (cAMP) in diverse cells and tissues (K uo and D e R enzo , 1969; S himizu , D aly and C reveling , 1969, B rooker , 1971). We have previously reported that cAMP from the guinea-pig cerebral cortex is derived from a precursor pool of adenine nucleotides which is more highly labelled than the bulk cellular ATP (S himizu , C reveling and D aly , 1970a). The present study was carried out to throw additional light on the property of the precursor pool. Levels of cAMP and ATP and specific activities of [¹⁴C]cAMP and [¹⁴C]ATP were determined simultaneously in cerebral slices which were prelabelled with [¹⁴C]adenine and subsequently incubated with veratridine, histamine or adenosine.     

ACTIONS OF NEUROHUMORAL AGENTS AND CEREBRAL METABOLITES ON OUTPUT OF ADENINE DERIVATIVES FROM SUPERFUSED TISSUES OF THE BRAIN

—Adenine nucleotides of guinea-pig neocortical tissues were labelled by prior incubation with [¹⁴C]adenine and excess of adenine was then removed by superfusion with precursor-free media. During continued superfusion labelled adenine derivatives were released at a stable rate of about 0·05 per cent of the tissue ¹⁴C/min and this rate was increased about five-fold by electrical stimulation. Various compounds, including some known to increase the cyclic AMP content of cerebral tissues, were examined for action on the release of [¹⁴C]adenine derivatives from the tissue and also on the rates of lactate production by the tissue, both before and during electrical excitation. The tissue content of adenine nucleotides following exposure of the tissue to these compounds was also determined. Noradrenaline, γ-aminobutyrate and acetylcholine together with carbamoylcholine at the concentrations examined were without effect on the release of ¹⁴C compounds from the tissue. Also, noradrenaline and γ-aminobutyrate caused no alteration in lactate production but brought about some decrease in the adenylate energy charge of the tissue. Histamine, 100 μm , brought about a small but consistent increase (35 per cent) both in release of ¹⁴C-compounds and lactate output, while reducing the adenylate energy charge of the tissues. l -Glutamate at 5 mm decreased the tissue adenylate energy charge to a greater extent than did histamine; it increased the release of ¹⁴C-compounds seven to eight-fold and similarly increased the tissues' rates of lactate production. Lower concentrations of glutamate had smaller effects. In those cerebral tissues whose cyclic AMP content is increased by l -glutamate, the increase is probably brought about by intermediation of released adenosine.     

NUCLEOTIDE METABOLISM IN RAT BRAIN

Abstract— The uptake, the conversion to nucleotides, and their incorporation into RNA for labelled glycine, aspartate, the free bases and nucleosides of purines and pyrimidines were investigated with cortical slices of rat cerebrum. At the end of a 1-hr incubation time the slice-to-medium ratio of the radioactivities for labelled aspartate, glycine, adenine and adenosine were 34, 26, 20 and 5, respectively, while the slice-to-medium ratios for hypoxanthine, inosine, guanine, guanosine, xanthine, orotate, cytidine, cytosine, uridine, and uracil ranged from 1.3:1 to 2:1. Over 99 per cent of the total radioactivity taken up by the cortical slices was present in the TCA supernatant and 86, 82, 65, 50, 34, 23, 20 and 1.6 per cent of this radioactivity was in the form of nucleotides at the end of a 1-hr incubation with labelled adenine, adenosine, hypoxanthine, inosine, uridine, orotate, cytidine, and glycine, respectively. The incorporation of various radioactive precursors into RNA of cortical slices suggests that nucleotides originating from either de novo synthesis or preformed purine derivatives enter the same nucleotide pool utilized for RNA synthesis. The supernatant fraction from homogenized cerebrum was investigated for the presence of various anabolic and catabolic enzymes associated with nucleotide metabolism. These results were correlated with the data from the RNA incorporation studies, and a possible role for AMP: pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, I.U.B. 2.4.2.7) to achieve intercellular transfer of AMP is discussed.     

Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain

—The accumulation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) was studied in cell-free homogenates of guinea pig brain. Homogenates, prepared in Krebs-Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [³H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [³²P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg²⁺ and Ca²⁺, resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH)₂-ZnSO₄ mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell-free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain.