Extracellular metabolism of cyclic AMP

Intravenously administered cyclic [8-³H]AMP to rats was quickly eliminated from the circulation. After 2 min 93% of the administered radioactivity disappeared from the plasma, and most of it was recovered in the kidney, liver and muscles. The label in the tissues was recovered mainly in the form of nucleotides, ATP, ADP, AMP and IMP.In vitro contact of cyclic AMP with perfused liver, isolated liver cells and adipose tissue resulted in a rapid breakdown of the nucleotide, presumably on the outer surface of the cells. The degradation products have been identified mainly as adenosine and inosine.Incubation of adipose tissue and isolated liver cells with [³H]AMP also resulted in the breakdown of the nucleotide in the medium. The rate of AMP degradation by these tissues was faster than that for cyclic AMP degradation.The data suggest that cyclic AMP is readily metabolized on the outer surface of cells to products which may be converted within the cells to nucleotides. These findings seem of importance for the quantitative assessments of cellular cyclic AMP outflow during hormonal stimulation.     

Formation of cyclic AMP from exogenous ATP by isolated hepatocytes and adipocytes

Suspensions of isolated rat hepatocytes and adipocytes converted exogenous ATP to cyclic AMP at a rate which was about 30--50% of that observed with homogenates of isolated cells. Formation of cyclic AMP was stimulated by hormones (isoprenaline in the case of adipose tissue and glucagon in the case of liver) and sodium fluoride. Experiments with [alpha-32P]ATP indicated that the conversion of exogenous ATP to cyclic AMP did not occur within the cells. It is proposed that in isolated hepatocytes ad adipocytes some catalytic units of adenylate cyclase are exposed on the outer surface of the cell membrane.     

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.     

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.     

Uptake and Metabolism of [3H] Adenosine by Aplysia Ganglia and by Individual Neurons

[3H]Adenosine was taken up and metabolized by isolated ganglia of the marine mollusc Aplysia californica. After 2 h, most of the radioactivity was recovered as metabolites, including ATP, ADP, and AMP, as well as the deaminated products, inosine, hypoxanthine, and uric acid. Little remained in the form of adenosine. These pathways were not uniformly distributed among various tissue elements. In most individual neurons, inosine and its breakdown products were the principal metabolites of [3H]adenosine, whereas ATP and other nucleotides predominated in the connective tissue sheath. Endogenous levels of ATP, ADP, AMP, and adenosine in ganglia, sheath, and individual neurons were also determined using a fluorimetric-HPLC assay. The concentrations of the nucleotides were quite uniform in sheath and among the individual neurons assayed (1-5 pmol/microgram of protein); however, concentrations of adenosine were considerably higher in neurons than in the sheath.     

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.     

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.     

Adenine nucleotide metabolism in isolated chicken hepatocytes.

The turnover of the adenine nucleotide pool, the pathway of the degradation of AMP and the occurrence of recycling of adenosine were investigated in isolated chicken hepatocytes, in which the adenylates had been labelled by prior incubation with [14C]adenine. Under physiological conditions, 85% of the IMP synthesized by the 'de novo' pathway (approx. 37 nmol/min per g of cells) was catabolized directly via inosine into uric acid, and 14% was converted into adenine nucleotides. The latter were found to turn over at the rate of approx. 5 nmol/min per g of tissue. Inhibition of adenosine deaminase by 1 microM-coformycin had no effect on the formation of labelled uric acid, indicating that the initial degradation of AMP proceeds by way of deamination rather than dephosphorylation. Inhibition of adenosine kinase by 100 microM-5-iodotubercidin resulted in a loss of labelled ATP, demonstrating that adenosine is normally formed from AMP but is recycled. Unexpectedly, 5-iodotubercidin did not decrease the total concentration of ATP, indicating that the loss of adenylates caused by inhibition of adenosine kinase was nearly completely compensated by formation of AMP de novo. Anoxia induced a greatly increased catabolism of the adenine nucleotide pool, which proceeded in part by dephosphorylation of AMP. On reoxygenation, the formation of AMP de novo was increased 8-fold as compared with normoxic conditions. The latter results indicate the existence of adaptive mechanisms in chick liver allowing, when required, channelling of the metabolic flux through the 'de novo' pathway, away from the uricotelic catabolic route, into the synthesis of adenine nucleotides.     

Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions.

Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP --> ADP --> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.     

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.     

Lack of correlation between hormonal effects on cyclic AMP and glycogenolysis in rat liver.

Portions of liver were obtained by biopsy from rats infused with various concentrations of glucagon or epinephrine and analyzed for cyclic AMP, glycogen, phosphorylase activity, and glycogen synthetase I activity. The response of tissue cyclic AMP to glucagon or epinephrine was far less sensitive than other metabolic parameters; at certain lower doses of glucagon or epinephrine, glycogen decomposed without a simultaneous increase in the hepatic level of cyclic AMP. It is probable that hormonal activation of adenylate cyclase results in an increase of cyclic AMP only in its small “active” pool without detectable changes in its much larger inactive or bound pool. Though the active cyclic AMP is expected to be released into the circulation or to be labeled with [³H]adenine in preference to the inactive nucleotide, neither the increase of cyclic AMP in the vena cava in vivo nor the incorporation of [³H]adenine into tissue cyclic AMP in liver slices in vitro exhibited more sensitivity to glucagon than the hepatic level of cyclic AMP as a whole. Thus, it remains to be settled whether cyclic AMP is compartmentalized in the cell or plays no essential role in the stimulation of hepatic glycogenolysis induced by small doses of hormones.     

Correlations between cyclic AMP binding and chemoreception in Paramecium

Paramecium tetraurelia is attracted to cyclic AMP, which probably, as other attractants, signifies the presence of food. Attraction to cyclic AMP was specific, saturable, and, therefore, likely to be receptor-mediated. In these studies, we measured the binding of cyclic [3H]AMP to whole cells and found it to be saturable, reversible, and displaying specificity similar to that of attraction. An HPLC method of separating nucleotides was devised and used to determine that external cyclic AMP was degraded in the absence of IBMX, a phosphodiesterase inhibitor, and that cyclic AMP was taken into the cells in small amounts. Since binding and attraction were subsequently measured in the presence of IMBX, it was cyclic AMP and not a degradation product that served as the attractant stimulus for Paramecium.     

Cyclic nucleotide metabolism in rat colonic epithelial cells with different proliferative activities

Cyclic nucleotide metabolism was examined in rat distal colonic epithelial cells with different proliferative activities. Lower crypt cells had DNA synthetic rates 7-10-fold higher than surface cells. Without a phosphodiesterase inhibitor proliferative cells had reduced basal cyclic AMP-, cyclic GMP-, and cyclic AMP-dependent protein kinase activity ratios, as well as blunted cyclic AMP responses to prostaglandin E2 and vasoactive intestinal peptide compared to superficial cells. In the presence of 3-isobutyl-1-methylxanthine, basal cyclic AMP and responses to prostaglandin E2 and vasoactive intestinal peptide of proliferative cells exceeded values in superficial cells. This correlated with higher membrane adenylate cyclase activity in the proliferative cells. By contrast, particulate and soluble guanylate cyclase activities of superficial cells were higher than in proliferative cells. The apparent high Km soluble and particulate cyclic AMP and cyclic GMP phosphodiesterase activities of proliferative cells were 4-7-fold higher than those in superficial cells. Moreover, the apparent low Km soluble activity was absent in superficial cells. Thus, an altered rate of nucleotide degradation may mediate reduced cyclic AMP and cyclic GMP in proliferative versus superficial cells. Dibutyryl cyclic AMP, prostaglandin E2 or vasoactive intestinal peptide inhibited [3H]thymidine incorporation into DNA of colonic segments. Thus, reduced cyclic AMP in lower crypt cells may be a determinant of their greater proliferative activity.     

A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates.

A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.     

Assay for adenylate cyclase and cyclic nucleotide phosphodiesterases and the preparation of high specific activity 32-P-labeled substrates.

Simple one step assay methods for adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) and cyclic nucleotide phosphodiesterases (3',5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) have been developed. [alpha-32-P] ATP is used as the substrate for adenylate cyclase. Acid-heat destruction of [32-P] ATP remaining after the cyclase reaction followed by Zn-Ba treatment quantitatively leaves cyclic [32-P] AMP in the supernatant essentially free from other 32-P-containing compounds. This assay method requires no corrections for recovery and routinely yields blank values less than 0.03 per cent. If higher sensitivity is desired, a simple 5 min alumina column step can be introduced into the procedure which quantitatively elutes cyclic [32-P] AMP directly into a liquid scintillation vial and lowers the blank values to less than 0.002 per cent. This method is rapid and easily performed, without sacrificing high reliability, specificity, or sensitivity. One step phosphodiesterase assays are easily accomplished using 32-P-labeled cyclic nucleotides as substrates. Descending paper chromatography of the reaction mixture on individual 2 cm wide paper strips gives a complete and quantitative separation of all possible products including [5'-32-P] AMP and [5'-32-P] GMP from their respective 32-P-labeled 3',5'-cyclic nucleotides in 1-2 h. The paper strips are cut, inserted in scintillation vials without scintillant and the 32-P-products determined by Cerenkov counting. Low blank values of less than 0.5 per cent and the use of high specific activity 32-P-labeled cyclic nucleotide substrates make this method the most reliable and most sensitive phosphodiesterase assay described to date. Because of the simplicity, specificity, and high sensitivity obtainable with these assay methods using 32-P-labeled substrates, we have also devised simple conditions for the preparation and purification of [alpha-32-P] ATP, cyclic [32-P] AMP and cyclic [32-P] GMP with specific activities in excess of 100 Ci/mmol. These high specific activity 32-Plabeled cyclic nucleotides are important for these new assay methods and are also useful to follow purification recovery of endogenous cyclic AMP and cyclic GMP from biological materials before protein binding or radioimmunological isotope displacement assays when performed in the femtomole range.     

Beta-adrenergic activity of (+/-)-hydroxybenzylisoproterenol in isolated rat fat cells and hepatocytes

The pharmacology of (+/-)-hydroxybenzylisoproterenol with respect to stimulation of cyclic AMP accumulation by isolated rat fat cells and liver cells was examined. (+/-)-Hydroxybenzylisoproterenol was found to be a full agonist and twice as potent as (-)-isoproterenol in liver cells, and equipotent to (-)-isoproterenol in fat cells with regard to stimulating cyclic AMP accumulation. A study of the ability of this catecholamine to stimulate adenylate cyclase activity of broken-cell preparations revealed that (+/-)-hydroxybenzylisoproterenol was equipotent to (-)-isoproterenol in liver cell homogenates, while 3- to 4-fold more potent than (-)-isoproterenol in fat cell ghost membranes. (+/-)-Hydroxybenzylisoproterenol was also found to be as potent as (-)-isoproterenol in stimulating cyclase activity of S49 mouse lymphoma cell membranes. Competition studies of specific [125I]iodohydroxybenzylpindolol binding to liver cell membranes revealed a Kd of 10 nM for (+/-)-hydroxybenzylisoproterenol and 25 nM for (-)-isoproterenol binding to the liver beta-adrenergic receptor. Competition studies of specific (-)-[3H]dihydroalprenolol binding to fat cell membranes indicated a similar affinity of these sites for both (+/-)-hydroxybenzylisoproterenol and (-)-isoproterenol. The guanyl nucleotide Gpp(NH)p induced a shift in the curve for competition of (-)-[3H]dihydroalprenolol binding by (-)-isoproterenol to the right, but failed to do so when (+/-)-hydroxybenzylisoproterenol was the competing agonist. Properties of (+/-)-[3H]hydroxybenzylisoproterenol binding to fat cell or liver cell membranes were inconsistent with those expected of adenylate cyclase coupled beta-adrenergic receptors.     

Systematic variations in the content of the purine nucleotides in the steady-state perfused rat heart. Evidence for the existence of controlled storage and release of adenine nucleotides

1. The contents of the major purine nucleotides in the isolated non-working perfused rat heart varied systematically during 80min of perfusion. In particular the amounts of ATP, ADP, GTP, cyclic AMP and cyclic GMP in the well-oxygenated myocardium showed changes ranging from 25 to 60% of the mean concentrations. The apparent periodicity was about 30min for some and about 60min for other nucleotides. 2. These data are in contrast with measurements of parameters reflecting heart performance, which remained constant over this period of perfusion. 3. The ATP/ADP ratio, the cyclic AMP content, the GTP content and the GTP/GDP ratio in the tissue bore a constant relationship to one another, and all showed the same temporal variation. 4. Increasing the energy demand on the heart by administration of bovine somatotropin (1μg/ml) tended to damp the variations, and generally lower the content of all the nucleotides. 5. The total extractable adenine nucleotide pool also showed systematic temporal variations of as much as 1.3μmol/g wet wt. of tissue within 10min. 6. These variations could not be accounted for as inter-conversion with adenosine, other purine nucleotides, nucleosides or purine-degradation products either in the tissue or in the perfusion medium. No evidence was found in this preparation of the purine nucleotide oscillations described by Lowenstein and his co-workers [see Tornheim & Lowenstein (1975) J. Biol. Chem. 250, 6304–6314]. 7. Further, the pool size increases cannot be satisfactorily explained by either synthesis de novo or the breakdown of any purine macromolecular species in the cell. Thus it is suggested that an unsuspected substantial storage form of purine nucleotide may exist in heart.     

Diadenosine polyphosphates and the control of cyclic AMP concentrations in isolated rat liver cells.

Extracellular diadenosine polyphosphates (Ap(n)A), through their interactions with appropriate P(2) receptors, influence a diverse range of intracellular activities. In particular, Ap(4)A stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that, like ATP, Ap(4)A and other naturally occurring diadenosine polyphosphates attenuate glucagon-stimulated accumulation of cyclic AMP in isolated rat liver cells. The characteristics of Ap(4)A- and ATP-dependent modulation of glucagon-stimulated cyclic AMP accumulation are similar. These results are discussed in the context of the repertoire of intracellular signalling processes modulated by extracellular nucleotides.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.