Combined effect of adenosine and papaverine on the metabolism of adenine nucleotides in rat thymocytes

It is shown that in [14C]adenine-labelled thymocytes adenosine increases the content of adenine nucleotides and simultaneously accelerates their catabolism. Papaverine induces acceleration of splitting and a decrease of the specific ATP radioactivity but increases the AMP content and its specific radioactivity. The both effectors intensify considerably the outlet of total radioactive label from cells. If the papaverine effect in the extracellular medium results in accumulation mainly of hypoxanthine in the extracellular medium then the adenosine presence causes accumulation of inosine and hypoxanthine approximately in equal amounts. The release of labelled adenosine from thymocytes in all cases is an insignificant part of extracellular radioactivity. A conclusion is drawn that under conditions of the combined action of the substances under study papaverine removes the adenosine effect caused by its under study papaverine removes the adenosine effect caused by its phosphorylation with the formation of ATP and exerts the dose-depended action on adenine nucleotide metabolism in thymocytes.     

Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat.

Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3':5'-monophosphate (cyclic AMP) or guanosine 3':5'-monophosphate (cyclic GMP) at an initial concentration of 0.1 mM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate. The metabolism of the cyclic nucleotides was studied by 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-labeled cyclic nucleotide. Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold. The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo experiments.     

Effect of papaverine on the absorption and metabolism of 14C- adenosine and 14C-adenine in thymocytes

Some peculiarities of adenosine and adenine nucleotide metabolism in rat thymocytes were investigated. It was shown that the uptake of labelled adenosine or adenine by thymocytes is markedly inhibited by papaverine due to the decrease of the adenylate kinase activity, on the one hand, and to the acceleration of ATP catabolism and inosine and hypoxanthine release into the environment, on the other. ATP catabolism occurs in a special compartment which in [14C] adenosine and [14C] adenine prelabelled thymocytes has a higher specific radioactivity as compared with the whole cell. In [14C] adenine-prelabelled thymocytes and extracellular medium, papaverine does not influence the content but increases the specific radioactivity of adenosine.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.     

Hypoxanthine-guanine exchange by intact human erythrocytes

The uptake and release of [14C]hypoxanthine by human erythrocytes, suspended in a tris(hydroxymethyl)aminomethane (Tris)-glucose-NaCl isotonic medium (pH 7.4), have been studied at 37 degrees C. The uptake of hypoxanthine, mediated by its incorporation into inosine 5'-monophosphate (IMP), was markedly stimulated by preincubating the cells in phosphate-buffered saline. After a lag time, [14C]IMP-enriched erythrocytes released [14C]hypoxanthine in the medium. Formycin B, at concentrations known to inhibit purine nucleoside phosphorylase in intact erythrocytes, affected hypoxanthine uptake and release and led to an increase in the intracellular concentration of inosine, suggesting that the main catabolic path of IMP is the sequential degradation of the nucleotide to inosine and hypoxanthine. The addition of guanine to a suspension of [14C]IMP-enriched erythrocytes led to an increase in the rate of [14C]hypoxanthine release, which was unaffected by the presence of formycin B. During the guanine-induced hypoxanthine release, guanine was taken up by the cells as GMP. These results suggest that the presence of guanine in the incubation medium activates a catabolic path in human erythrocytes leading to IMP degradation without formation of inosine.     

Cyclic adenosine 3',5'-monophosphate and prostacyclin inhibit membrane phospholipase activity in platelets.

[¹⁴C]-Arachidonic acid is incorporated mainly into phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of horse platelet membranes. Treatment of washed platelets with thrombin leads to a rapid loss of radioactivity from these phospholipids. The liberated [¹⁴C]-arachidonate is immediately transformed into hydroxyacids and thromboxanes. Treatment with dibutyryl cyclic AMP, cyclic AMP phosphodiesterase inhibitors or prostacyclin, a newly discovered prostaglandin that stimulates platelet adenylate cyclase, prevents the action of thrombin on phospholipid break-down as well as on platelet aggregation. Dibutyryl cyclic AMP does not affect the metabolism of exogenous [¹⁴C]-arachidonic acid. Cyclic AMP may thus play a crucial role in the regulation of platelet phospholipase acitivity, and this could explain at least in part the inhibition of aggregation caused by substances which, like prostacyclin, raise the levels of cyclic AMP.     

Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.     

Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.     

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats.

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.     

Regulation of carbohydrate metabolism in mouse liver. Effect of glucagon on gluconeogenic/glycolytic flux in isolated perfused livers.

1. Glucagon stimulated gluconeogenesis from both [U-14C]lactate and [14C]xylitol in isolated perfused mouse liver. 2. Addition of cyclic AMP also stimulated gluconeogenesis from [U-14C]lactate. 3. Glucagon caused a rapid (2.5 min) 12-fold increase in hepatic cyclic AMP but not cyclic GMP concentration. 4. Glucagon caused a rapid and stable decrease in hepatic fructose 1,6-diphosphatase activity measured in vitro. 5. The results are interpreted to indicate that glucagon stimulates hepatic gluconeogenesis in mice via cyclic AMP by two different mechanisms: (a) increased substrate uptake (i.e. utilization) and (b) increased gluconeogenic efficiency (i.e. inhibition of alternate substrate fates).     

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⁺.     

Extracellular Cyclic AMP—Adenosine Pathway in Isolated Adipocytes and Adipose Tissue

Objective: Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)–adenosine pathway in adipose tissue. Research Methods and Procedures: Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 μL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 μM isoproterenol, or 10 μM isoproterenol plus 1 mM α,β‐methylene adenosine 5′‐diphosphate (AMPCP), a 5′‐nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 μM isoproterenol, or 1 μM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Results: Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP‐provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. Discussion: These data suggest the existence of a cyclic AMP—adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.     

Stimulatory effect of gonadotropins on the synthesis of adenosine 3′ :5′-cyclic monophosphate and progesterone by suspensions of rat ovarian interstitial cells

A cell suspension was prepared from immature rat ovaries by treatment with trypsin and collagenase. The isolated cells were capable of converting [8-¹⁴C]adenine to cyclic [¹⁴C]AMP and the rate of this conversion was stimulated in vitro by luteinizing hormone and human chorionic gonadotropine, but not by prolactin, norepinephrine, dopamine or albumin. The accumulation of progesterone was also measured in these cells by radioimmunoassay. In vitro addition of luteinizing hormone stimulated the accumulation of radioimmunoassayable progesterone. The conversion of [8-¹⁴C]adenine to cyclic [¹⁴C]AMP showed a rapid increase during the first 30 min of the incubation period when luteinizing hormone was added to the incubation medium. Progesterone accumulation in response to the same dose of luteinizing hormone showed a lag period for the first 30 min of incubation after which there was an increase up to 2 h. The luteinizing hormone-induced progesterone accumulation was sensitive to puromycin, but there was no effect on the luteinizing hormone-induced increase in cyclic [¹⁴C]AMP formation from [8-¹⁴C]-adenine. Actinomycin D also inhibited the luteinizing hormone-induced progesterone accumulation, with no effect on cyclic AMP formation. The results suggest that the luteinizing hormone-induced progesterone accumulation in rat ovarian interstitial cell suspension is preceded by an increased accumulation of cyclic AMP and that the accumulation of steroid under the influence of luteinizing hormone involve processes sensitive to puromycin and antinomycin D.     

Responsiveness to glucagon by isolated rat hepatocytes controlled by the redox state of the cytosolic nicotinamide--adenine dinucleotide couple acting on adenosine 3'':5''-cyclic monophosphate phosphodiesterase.

1. The effects of changes in the cytoplasmic [NADH]/[NAD+] ratio on the efficacy of glucagon to alter rates of metabolism in isolated rat hepatocytes were examined. 2. Under reduced conditions (with 10mM-lactate), 10nM-glucagon stimulated both gluconeogenesis and urea synthesis in isolated hepatocytes from 48h-starved rats; under oxidized conditions (with 10mM-pyruvate), 10nM-glucagon had no effect on either of these rates. 3. The ability of glucagon to alter the concentration of 3':5'-cyclic AMP and the rates of glucose output, glycogen breakdown and glycolysis in cells from fed rats were each affected by a change in the extracellular [lactate]/[pyruvate] ratio; minimal effects of glucagon occurred at low [lactate]/[pyruvate] ratios. 4. Dose-response curves for glucagon-mediated changes in cyclic AMP concentration and glucose output indicated that under oxidized conditions the ability of glucagon to alter each parameter was decreased without affecting the concentration of hormone at which half-maximal effects occurred. 5. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.05 mM) significantly reversed the inhibitory effects of pyruvate on glucagon-stimulated glucose output. 6. For exogenously added cyclic [3H]AMP(0.1 mM), oxidized conditions decreased the stimulatory effect on glucose output as well as the intracellular concentration of cyclic AMP attained, but did not alter the amount of cyclic [3H]AMP taken up. 7. The effects of lactate, pyruvate, NAD+ and NADH on cyclic AMP phosphodiesterase activities of rat hepatocytes were examined. 8. NADH (0.01--1 MM) inhibited the low-Km enzyme, particularly that which was associated with the plasma membrane. 9. The inhibition of membrane-bound cyclic AMP phosphodiesterase by NADH was specific, reversible and resulted in a decrease in the maximal velocity of the enzyme. 10. It is proposed that regulation of the membrane-bound low-Km cyclic AMP phosphodiesterase by nicotinamide nucleotides provides the molecular basis for the effect of redox state on the hormonal control of hepatocyte metabolism by glucagon.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

Aminopyrine uptake by guinea pig gastric mucosal cells. Mediation by cyclic AMP and interactions among secretagogues

The role of cyclic nucleotides in regulating acid secretion by dispersed mucosal cells from guinea-pig stomach was examined by measuring first the ability of histamine and carbachol to stimulate [dimethylamine-14C]aminopyrine uptake and cyclic nucleotide metabolism and secondly, the effect of exogenous cyclic nucleotides on basal and stimulated [14C]aminopyrine uptake. The [14C]aminopyrine was found in an acidic, osmotically sensitive compartment, probably associated with the initial steps in acid secretion by these cells. Although histamine increased [14C]aminopyrine uptake and cyclic AMP synthesis as expected, histamine was approx. 10-fold more potent in inducing [14C]aminopyrine uptake. This dissociation of [14C]aminopyrine uptake and cyclic AMP metabolism process was further manifested by the observation that prostaglandin E1 failed to increase [14C]aminopyrine uptake, although it did cause a rise in cellular cyclic AMP. Furthermore, prostaglandin E1 did not alter the [14C]-aminopyrine uptake caused by histamine. Carbachol was found to increase the [14C]aminopyrine uptake and also to potentiate the ability of histamine to increase [14C]aminopyrine uptake. Carbachol, however, affected neither the histamine-induced increase in cyclic AMP nor the binding of [3H]histamine to the cells. Cimetidine, a histamine H2 receptor antagonist, blocked the [14C]aminopyrine uptake induced either by histamine alone or by the potentiating combination of histamine plus carbachol. These results suggest that cyclic AMP is mediating the action of histamine on [14C]aminopyrine uptake but changes in cyclic AMP per se are not necessarily the cause for the potentiated increase in [14C]aminopyrine uptake. Furthermore, the potentiated response observed with histamine plus carbachol on [14C]aminopyrine uptake occurs at a biochemical step distal to and not obviously related to cyclic AMP generation.     

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrrenal cells of vitamin E-deficient rats

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3′,5′-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5′-triphosphate [¹⁴C]ATP to cyclic [¹⁴C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [¹⁴C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induce cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [¹⁴C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [¹⁴C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [¹⁴C]AMP continued to be formed. The in vitro addition of α-tocopherol reduced the inhibition of ACTH-induced cyclic [¹⁴C]AMP formation by ascorbate in Vitamin E-deficient rats.These studies suggest that α-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with membrane-bound enzyme adenylate cyclase.     

Direct action of somatostatin on dispersed mucosal cells from guinea-pig stomach

In dispersed mucosal cells from guinea-pig stomach, somatostatin inhibited in a noncompetitive fashion (Ki, 2 x 10(-8) M) the increase in cellular cyclic AMP caused by histamine but not by prostaglandin E1 or phosphodiesterase inhibitors. Somatostatin also inhibited the increase in [14C]aminopyrine uptake caused by low concentrations of histamine probably by interfering with the synthesis of cellular cyclic AMP.     

3-Isobutyl-1-Methylxanthine decreases renal cortical interstitial levels of adenosine and inosine

The purpose of this study was to test the hypothesis that endogenous cyclic AMP, via metabolism by phosphodiesterase, contributes to interstitial levels of adenosine in the renal cortex in vivo. This hypothesis was tested by determining the effects of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, on renal cortical interstitial levels of adenosine and inosine. Changes in renal cortical interstitial adenosine and inosine levels were assessed in rats by implanting microdialysis probes into the renal cortex and measuring adenosine and inosine levels in the dialysate exiting the kidney using high performance liquid chromatography. When added to the dialysate entering the kidney at concentrations of 0.5, 1 and 2.5 mM, 3-isobutyl-1-methylxanthine significantly and dose-dependently decreased interstitial levels of both adenosine and inosine. The percentage changes from baseline of interstitial levels of adenosine and inosine were: −39 ± 6% and −19 ± 6%, respectively, with 0.5 mM 3-isobutyl-1-methylxanthine; −45 ± 7 % and −24 ± 8 %, respectively, with 1 mM 3-isobutyl-1-methylxanthine; and −56 ± 12 % and −38 ± 8 %, respectively, with 2.5 mM 3-isobutyl-1-methylxanthine. These data suggest that in the renal cortex, cyclic AMP metabolism via phosphodiesterase is an important source of renal interstitial adenosine.     

Adenine mononucleotides and their metabolites liberated from and applied to isolated tissues of the mammalian brain

Preincubation with [¹⁴C] adenine labeled the nucleotide fraction of isolated cerebral tissues, which subsequently released 0.18% of their¹⁴C content per minute, a proportion increased threefold by electrical excitation. Of the¹⁴C released, 2–3% was as 5-adenine nucleotides and about 2% as cyclic adenosine 35-monophosphate (cAMP). Among the 5-nucleotides AMP greatly preponderated, and ATP and ADP were detected. When added to (unlabeled) incubating neocortical tissue, ATP and AMP yielded adenosine as the major product, with smaller quantities of inosine and hypoxanthine, to effluent fluids. cAMP so added yielded 5-nucleotides and the other compounds named; adenosine yielded mainly inosine and hypoxanthine. Results from these reactions and others in which theophylline was included led to the conclusion that an appreciable proportion of the effluent [¹⁴C] adenosine, inosine, and hypoxanthine derived from cAMP.