THE INFLUENCE OF SECRETIN, GLUCAGON AND OTHER PEPTIDES, OF AMINO ACIDS, PROSTAGLANDIN ENDOPEROXIDE ANALOGUES AND DIAZEPAM ON THE LEVEL OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN NEUROBLASTOMA GLIOMA HYBRID CELLS

Abstract— Neuroblastoma glioma hybrid cells display many properties of neurons. A series of compounds, among them a number of amino acids, peptides and peptide hormones were tested for their ability to influence the level of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in the hybrid cells. Two prostaglandin endoperoxide analogues exhibit a weak stimulatory action, if applied in at least micromolar concentrations. At nanomolar concentrations, only the gastrointestinal hormones secretin and glucagon stimulate the formation of cyclic AMP, as detected in the presence of a phosphodiesterase inhibitor. The effect of secretin but not that of glucagon is antagonized by secretin-(5–27), suggesting that secretin and glucagon act on the cells via different receptors. These results appear to be noteworthy since (a) an effect of secretin or glucagon on a cell with neuronal characteristics has not yet been described, and (b) many peptide hormones have been detected both in the gastrointestinal tract and the nervous system.     

Synergistic Effects of Adenosine and Compounds Related to Adenosine 3': 5'-cyclic Monophosphate on Dedifferentiating Iris Epithelial Cells in Culture

Addition of adenosine or 5' AMP to the primary culture of newt iris epithelial cells produces an extensive array of fine cytoplasmic processes in a fraction of the cell population. Dibutyryl cyclic AMP, monobutyryl cyclic AMP, cyclic AMP and theophylline have the same effect, but the minimum effective concentrations of the latter four compounds are higher than those of the former two. When the primary cultures are treated simultaneously with two effective compounds at various concentrations a synergis-tic effect is observed between dibutyryl cyclic AMP and theophylline; cyclic AMP and theophylline; adenosine and theophylline; adenosine and cyclic AMP. The results support the interpretation that an increase in cellular level of cyclic AMP, which is produced by exogenous adenosine or 5' AMP as well as by exogenous cyclic AMP, its derivatives or theophylline, is responsible for the formation of radial cytoplasmic processes. The morphogenetic response does not depend on the presence of serum in the culture medium. The possibility is discussed that the cellular level of cyclic AMP is involved as one link of the sequential events which control the dedifferentiation of iris epithelial cells in cell-type conversion.     

Physiological roles of adenosine derivatives which are released during neurotransmission in mammalian brain.

1. Experiments using synaptosome beds suggested that ATP was released from presynaptic sites and degraded to adenosine in the synaptic cleft and that the resulting adenosine was taken up again into nerve endings where it was re-phosphorylated to ATP. 2. Adenosine derivatives in the synaptic cleft inhibited the postsynaptic potentials in olfactory cortex slices in vitro, presumably by the inhibition of Ca2+ influx into nerve endings which resulted in the reduction of transmitter release. 3. The adenosine derivatives also increased the level of cyclic AMP in the slices under the same conditions as above. 4. Although the nature of the "adenosine receptors" for both functions was remarkably similar, the increase of cyclic AMP did not mediate the inhibitory action, but the presynaptic increase of cyclic AMP induced by adenosine derivatives might mediate the facilitation observed in the olfactory cortex. 5. Possible physiological roles of extracellular adenosine derivatives in mammalian brain were classified, at different sites of action around the synapses, with different time courses and modes of action, directly or via the increase of intracellular cyclic AMP.     

Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.     

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20-1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5'-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.     

Nucleotide release provides a mechanism for airway surface liquid homeostasis

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca(2+) -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A(2b) adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N(6)-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A(2b) adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A(2b) receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.     

Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation.

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2--3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low Km (0.17 muM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.     

Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2–3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low K_m (0.17 μM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study. Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.     

Adenosine receptors, cyclic AMP accumulation, and DNA-synthesis in aortic smooth muscle cell cultures of adult and neonatal rats.

The effects of two adenosine analogs on cyclic AMP (cAMP) accumulation and DNA synthesis were studied in cultured smooth muscle cells (SMCs) isolated from adult and neonatal rat arteries. N-ethylcarboxamido adenosine (NECA) dose-dependently increased intracellular cAMP levels and appeared to be more potent in adult than in neonatal SMCs. R-phenylisopropyl adenosine (R-PIA), in nanomolar concentrations, counteracted the increase in cAMP evoked by 10 microM forskolin in adult but not in neonatal SMCs, indicating that the enhanced "A2" response seen in adult SMCs was not due to a lack of "A1" receptors in these cultures. Binding experiments performed using the adenosine antagonist XAC did not reveal any differences in the number or affinity of the adenosine receptors between neonatal and adult SMCs. This indicates effects presumably on the G-protein level. A high capacity to spontaneously synthesize DNA and a weak response to platelet-derived growth factor (PDGF) were seen in the neonatal SMCs. Furthermore, NECA had no effect on PDGF-induced DNA synthesis in these cells. In contrast, adult SMCs presented a low rate of spontaneous DNA synthesis and a marked proliferative response to PDGF, which was inhibited by NECA. This inhibition paralleled the increase in cAMP elicited by NECA. Our findings suggest that neonatal and adult SMCs differ both in their response to growth factors and growth inhibitors.     

Regulation of Depolarization-Dependent Release of Neurotransmitters by Adenosine: Cyclic AMP-Dependent Enhancement of Release from PC12 Cells

We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2-chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM-dependent phospholipid N-methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2-chloroadenosine did not alter the release of [3H]norepinephrine [(3H]NE) in the absence of depolarization. However, depolarization-dependent release of [3H]NE was markedly elevated by short (1-20 min) pretreatments with adenosine or 2-chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2-chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus-secretion coupling process.     

Specificity of adenosine inhibition of cAMP-induced responses in Dictyostelium resembles that of the P site of higher organisms

Adenosine acts as a cyclic AMP antagonist in Dictyostelium discoideum. It inhibits the binding of cyclic AMP to cell surface receptors and the induction of postaggregative differentiation by cyclic AMP. We investigated the nucleoside specificity and dose dependency of both inhibitory effects of adenosine. It was found that adenosine inhibits cyclic AMP binding and cyclic-AMP-induced differentiation with a K_i of about 300 μM. Alterations in the purine moiety of adenosine generally decrease the inhibitory effect of the molecule, whereas alterations in the ribose moiety are tolerated and in most cases even increase the inhibitory effect of the molecule on both cyclic AMP binding and differentiation induction. A strong correlation (r = 0.996, P < 0.01%) between the specificities for adenosine derivatives of these two inhibitory processes is demonstrated. The nucleoside specificity for the inhibition of cyclic AMP action in D. discoideum resembles that of the P site of higher organisms. In contrast to effects mediated by the P site of higher organisms, the effects of adenosine mediated by the Dictyostelium receptor cannot be prevented by inhibiting adenosine uptake; this makes it very likely that the adenosine receptor, which is involved in the effects of adenosine on cyclic AMP binding and differentiation induction, is located at the cell surface.     

The dose dependent effect of cyclic AMP on ribonucleotide reductase in mitogen stimulated mononuclear cells

Cyclic adenosine monophosphate arrests proliferating T lymphocytes in the G1 phase of the cell cycle. Here we demonstrate that exogenous and endogenous elevations in cyclic AMP concentration result in diminished mitogen stimulation, cell cycle arrest, and decreased ribonucleotide reductase messenger RNA concentrations in peripheral blood mononuclear cells. At lower concentrations (less than 1mM) of dibutyryl cyclic AMP that do not generate cell cycle arrest there is inhibition of ribonucleotide reductase activity without decreased messenger RNA concentration for the M2 subunit of ribonucleotide reductase. However, at higher concentrations of dibutyryl cyclic AMP there is G1 cell cycle arrest and reduced M2 specific messenger RNA concentration. Thus, cyclic AMP inhibition of lymphocyte activation may occur by different mechanisms that are dose dependent.     

Regulation of calcium-activated nonselective cation channel activity by cyclic nucleotides in the rat insulinoma cell line,CRI-G1

The regulation of a calcium-activated nonselective cation (Ca-NS⁺) channel by analogues of cyclic AMP has been investigated in the rat insulinoma cell line, CRI-G1. The activity of the channel is modulated by cyclic AMP in a complex way. In the majority of patches (83%) tested concentrations of cyclic AMP of 10 μm and above cause an inhibition of channel activity which is immediately reversible on washing. In contrast, lower concentrations of cyclic AMP, between 0.1 and 1.0 μm, produce a transient activation of channel activity in most patches (63%) tested. One group of analogues, including N⁶-monobutyryl cyclic AMP and N⁶, 2′-O-dibutyryl cyclic AMP reduced the activity of the Ca-NS⁺ channel at all concentrations tested and 2′-O-Monobutyryl cyclic AMP produced inhibition in all patches tested except one, at all concentrations. A second group produced dual concentration-dependent effects on Ca-NS⁺, low concentrations stimulating and high concentrations inhibiting channel activity. 6-Chloropurine cyclic AMP and 8-bromo cyclic AMP produced effects similar to those of cyclic AMP itself. In contrast, 8-[4-chlorophenylthio] cyclic AMP also showed a dual action, but with a high level of activation at all concentrations tested up to 1mm. Ca-NS⁺ channel activity was also predominantly activated by low concentrations of S_p-cAMPS. The activating effects of both S_p-cAMPS and cyclic AMP are antagonized by R_p-cAMPS, which by itself only produced a weak inhibition of Ca-NS⁺ channel activity even at concentrations of 10 μm and above. The results are discussed in terms of a model in which cyclic AMP, and other cyclic nucleotides, modulate the activity of the Ca-NS⁺ channel by binding to two separate sites.     

Cyclic AMP-Dependent Induction of Serotonin N-Acetyltransferase Activity in Photoreceptor-Enriched Chick Retinal Cell Cultures: Characterization and Inhibition by Dopamine

The activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme in the melatonin biosynthetic pathway, was examined in low-density monolayer cultures of chick embryo retinal cells prepared with three levels of photoreceptor enrichment. In cultures prepared from embryonic day 8 retinas (E8), photoreceptors represented approximately 30% of the total cell population, whereas in those prepared from embryonic day 6 retinas (E6), approximately 70% of the cells were photoreceptors. In E8 retinas treated with kainic acid to destroy neurons (E8K), the relative content of photoreceptors was increased to approximately 50%. NAT activity was detectable in the cultures under all conditions studied, and was markedly increased by drugs that increase intracellular cyclic AMP levels and cyclic AMP-dependent protein kinase activity: 8-bromocyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). Consistent with the hypothesis that NAT is localized in photoreceptors, the effects of the stimulatory treatments were significantly greater in E6 and E8K cultures than in E8 cultures. The stimulation of NAT activity in E6 cultures was inhibited by actinomycin D and cycloheximide, suggesting the involvement of RNA and protein synthesis. Dopamine inhibited the induction of NAT activity by forskolin and IBMX, but not that elicited by 8-bromocyclic AMP. The dopamine-mediated suppression of activity was significantly inhibited by pertussis toxin and by spiperone and sulpiride, both D2-dopamine receptor antagonists, but not by SCH 23390, a D1-dopamine receptor blocker, or antagonists of alpha-adrenergic, beta-adrenergic, or serotonergic receptors. Because the inhibitory effect of dopamine on E6 and E8K cultures was at least as great as that on E8 cultures, the results suggest that dopamine acts on D2-like receptors on photoreceptors. The receptors appear to be coupled to adenylate cyclase through an inhibitory GTP-binding protein and to mediate inhibition of cyclic AMP synthesis and consequent induction of NAT activity.     

Effects of adenosine, 2-deoxyadenosine and N6-phenylisopropyladenosine on rat islet function and metabolism

Adenosine (1.0-100 mum). N(6)-phenylisopropyladenosine (0.1-10 mum) and 2-deoxyadenosine (10 mm) all produced a dose-dependent inhibition of glucose-stimulated insulin release. The inhibition of glucose-stimulated insulin release by adenosine and N(6)-phenylisopropyladenosine was abolished by 3-isobutyl-1-methylxanthine (0.1 mm), whereas 2-deoxyadenosine inhibited insulin release even in the presence of 3-isobutyl-1-methylxanthine. These adenosine nucleosides also inhibited the release of insulin induced by 4-methyl-2-oxopentanoate (20 mm), dl-glyceraldehyde (30 mm) and l-leucine (20 mm). Adenosine (10 mum). N(6)-phenylisopropyladenosine (10 mum) and 2-deoxyadenosine (10 mm) did not inhibit insulin biosynthesis or [U-(14)C]glucose oxidation at concentrations of the nucleosides that gave maximal inhibition of insulin release. However, adenosine, 2-deoxyadenosine and N(6)-phenylisopropyladenosine produced marked inhibition of the glucose-stimulated increases seen in islet cyclic AMP accumulation. Similar to its effects on insulin release, 3-isobutyl-1-methylxanthine (0.1 mm) antagonized the inhibitory effects of cyclic AMP accumulation produced by adenosine and N(6)-phenylisopropyladenosine, but had no effect on the inhibition of cyclic AMP accumulation seen with 2-deoxyadenosine. These results show that adenosine and its specifically modified analogues, 2-deoxyadenosine and N(6)-phenylisopropyladenosine, are strong inhibitors of insulin release from rat islets, a function that appears to be the consequence of their ability to inhibit the accumulation of cyclic AMP. It is proposed that the B cells, in common with many other tissues, may possess two different sites at which adenosine nucleosides interact to produce their biological effects; these are the so-called ;P' and ;R' sites first described by Londos & Wolff [(1977) Proc. Natl. Acad. Sci. U.S.A.74, 5482-5486].     

Regulation of cyclic AMP and cyclic GMP levels by adrenocorticotropic hormone in cultured neurons

The effect of adrenocorticotropic hormone (ACTH) on the intracellular concentration of cyclic nucleotides was studied in cultures of neurons from embryonic chick cerebral hemispheres. Incubation of neurons with ACTH(1-24) in the presence of phosphodiesterase inhibitor isobutylmethylxanthine resulted in a sustained increase in cyclic AMP while rise in cyclic GMP level was transient. The values obtained for half-maximal stimulation were 0.5 microM and 0.03 nM for cyclic AMP and cyclic GMP respectively. Concomitantly, ACTH(1-24) stimulated guanylate cyclase activity (half-maximal stimulation at 0.02 nM). These results suggest the existence of two distinct populations of ACTH receptors in neurons and provide the first evidence that cyclic GMP does mediate the action of ACTH in neurons.     

Separation of mitogen-induced suppressor and helper cell activities during inhibition of interferon production by cyclic AMP.

The effect of exogenous cyclic AMP on mitogen-induced suppression and enhancement of the in vitro plaque-forming cell (PFC) response and on mitogen induction of immune interferon (also called type II) in cultures was examined. Mitogen induction of immune interferon was quantitatively associated with mitogen-induced suppressor activity, and cyclic AMP blocked both the suppressor activity and the production of immune interferon in mouse (C57B1/6) spleen cell cultures. The evidence is as follows: (a) The concentrations of dibutyryl cyclic AMP that blocked T-cell mitogen (staphylococcal enterotoxin A) suppressor activity were the same as those that blocked mitogen induction of immune interferon. (b) The blocking action of dibutyryl cAMP on both the suppressor and interferon effects of mitogen was a function of the time of dibutyryl cAMP addition to cultures relative to mitogen addition. (c) A dramatic immunoenhancing effect of mitogen occurred in the presence of dibutyryl cAMP under conditions that blocked production of immune interferon. Specifically, mitogen-induced helper cell function is dramatically enhanced in the presence of dibutyryl cyclic AMP, if the mitogen is added to cultures 24 to 48 hr after SRBC and dibutyryl cyclic AMP. Dibutyryl cyclic GMP did not affect the mitogen- or cyclic AMP-induced effects under the conditions of our test system. Under the conditions described here, then, cyclic AMP appears to selectively block suppressor cell activity while allowing or aiding mitogen-induced helper cell activity. It is possible that the immune response is a reflection of the ratio of helper to suppressor activities in the system.     

The influence of sex steroids on calcium- and magnesium-induced mitogenesis in isolated rat thymic lymphocytes

Elevated calcium and magnesium concentrations promoted mitotic activity in rat thymic lymphocyte cultures. Oestradiol inhibited calcium- but not magnesium-induced mitogenesis. One prerequisite for the mitogenic action of calcium is a raised intracellular concentration of cyclic adenosine 3′5′ monophosphate (cyclic AMP) but cyclic AMP-induced mitogenesis was insensitive to oestradiol. This suggests that the steroid blocks the mitogenic process at a stage preceding the endogenous cyclic AMP elevation. Furthermore the mitogenic actions of adrenaline, which stimulates adenylate cyclase (the enzyme responsible for cyclic AMP biosynthesis), and caffeine, which inhibits phosphodiesterase (the enzyme which degrades cyclic AMP) were also insensitive to oestradiol inhibition. This precludes a direct effect of the steroid on these enzymes. However, oestradiol did inhibit the mitogenic action of parathyroid hormone (PTH). Since the mitogenic action of PTH probably involves increased calcium entry to the cell, oestradiol may block this ion influx. The inhibition of calcium- and PTH-induced mitogenesis must be attributable to some structurally specific action of oestradiol. The steroids cholesterol, progesterone and testosterone all failed to reduce calcium-induced mitogenesis, whereas both α and β oestradiol were effective. In addition to its insensitivity to oestradiol inhibition, magnesium-stimulated mitosis was unaffected by both imidazole and calcitonin at concentrations which significantly reduced calcium-stimulated proliferation. These findings are compatible with the thesis that magnesium-induced mitogenesis does not involve the elevation of cyclic AMP concentrations.     

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.     

Expression of A1 Adenosine Receptors Modulating Dopamine-Dependent Cyclic AMP Accumulation in the Chick Embryo Retina

Dopamine and 2-chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16-day-old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine-dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12-day-old chick embryo retinas, with an IC50 of approximately 1 microM. This effect was not blocked by dipyridamole. N6-(l-Phenylisopropyl)adenosine, (l-PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 microM which was two orders of magnitude lower than its stereoisomer d-PIA (10 microM). The maximal inhibition of the dopamine-elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3-isobutyl-1-methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective; whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11- to 12-day-old embryos and 2-chloroadenosine did not interfere with the apomorphine-mediated shift in cyclic AMP levels. In the retinas from 5-day-old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)