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PubMed1992

Activation of protein kinase A is necessary but not sufficient for ethanol-induced desensitization of cyclic AMP production.

Acute addition of EtOH to PC 12 pheochromocytoma cells increases cyclic AMP production, whereas chronic exposure to EtOH results in a decrease in the stimulation of cyclic AMP production in response to 2-chloroadenosine and forskolin. This EtOH-induced desensitization was not observed after chronic EtOH treatment of A126-1B2-1 cells which are a protein kinase A-deficient mutant cell line derived from PC 12 cells. Furthermore, in the parental PC 12 cell line the cell-permeable protein kinase A inhibitor, Rp-isomer of adenosine 3',5'-monophosphorothioate, blocked the development of EtOH-induced desensitization. Thus, activation of protein kinase A is apparently necessary for EtOH-induced desensitization of cyclic AMP production. Chronic treatment of PC 12 cells with forskolin qualitatively mimicked the desensitization observed with chronic EtOH exposure. However, the degree of desensitization induced by forskolin was significantly less than that caused by EtOH even though the acute addition of forskolin caused a greater increase in cyclic AMP production. Furthermore, the acute addition of EtOH inhibited forskolin-stimulated cyclic AMP production, yet inclusion of EtOH during the chronic forskolin treatment of PC 12 cells resulted in a greater degree of desensitization. These findings indicate an obligatory role of protein kinase A in EtOH-induced desensitization of cyclic AMP production in PC 12 cells. However, because protein kinase A activation alone is not sufficient to account for the degree of desensitization, EtOH probably also acts through a mechanism in addition to activation of protein kinase A.

2-Chloroadenosine↗

PubMed1976

Adenosine phosphyorylase activity as distinct from inosine-guanosine phosphorylase activity in Sarcoma 180 cells and rat liver.

Adenosine phosphorylase (EC 2.4.2.-) activity present in Sarcoma 180 cells grown in culture and in rat liver, is shown to be distinct from inosine-guanosine phosphorylase by several criteria: (a) treatment of Sarcoma 180 cell extract with p-chloromercuribenzoate inhibited the two activities to a different extent, (b) adenine selectively protected the adenosine phosphorylase activity of Sarcoma 180 and rat liver extract against heat inactivation, while hypoxanthine selectively protected inosine-guanosine phosphorylase activity, (c) at nearly saturating substrate concentrations and using Sarcoma 180 extract, the rates of ribosylation of a mixture of adenine + hypoxanthine or adenine + guanine, but not of hypoxanthine + guanine, were found to be almost equal to the sum of their individual rates as measured separately, (d) inosine selectively inhibited the ribosylation of hypoxanthine and guanine catalysed by Sarcoma 180 and rat liver extract while 2-chloroadenosine selectively inhibited the ribosylation of adenine and N6-furfuryladenine, (e) pH vs. activity curves were similar with hypoxanthine or guanine as the substrate but they were markedly different from the curve with adenine as the substrate. The potential role of adenosine phosphorylase activity in vivo is discussed.

Adenine↗

PubMed1978

Inhibition of bovine adrenocortical adenylate cyclase activity by adenosine.

Incubation of bovine adrenocortical membranes with corticotropin and 5-guanylylimidodiphosphate produced a state of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) with maximal catalytic activity and an increased sensitivity to inhibition by adenosine. Due to metabolism of adenine nucleotides during adenylate cyclase assays a quantitative assessment of the nature of this inhibition was not possible. However, when determined at 0.2--1.0 mM MgATP2-, half-maximal inhibition of the basal and maximally active states of the enzyme was observed at adenosine concentrations of 210--330 and 70--90 micrometer, respectively. The inhibition appeared to be partially competitive, suggesting that the nucleoside may act as an allosteric negative effector which reduces the affinity of the active site for substrate. Adenosine was 5--6 times more potent as an inhibitor of adrenal adenylate cyclase than 2-chloroadenosine. Adenosine deaminase abolished the inhibitory effect of the nucleoside, whilst theophylline had no effect on activity either in the absence or presence of adenosine.

Adenosine↗

PubMed1979

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↗

PubMed1992

Cross-talk between cAMP and formylmet-leu-phe in human neutrophils: phosphorylation of a 52,000 molecular weight protein.

The mechanism of inhibition of neutrophil phagocytic functions by cAMP-elevating agents has not yet been clarified. In the present work, the effects of adenylate cyclase agonists on protein phosphorylation in the formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated human neutrophils were studied. Before stimulation, 32Pi-labelled cells were incubated with adenosine deaminase to remove the endogenously produced adenosine, an adenylate cyclase agonist itself. A protein of about 52,000 molecular weight was rapidly and transiently phosphorylated when neutrophils were stimulated with fMLP in the presence of isoproterenol, prostaglandin E1, histamine or 2-chloroadenosine. This phosphorylation was blocked by the antagonists of the receptors for the above-listed agents. No phosphorylation of the 52,000 molecular weight protein could be observed if either fMLP or the cAMP-elevating agent were applied alone. A calcium ionophore A23187 and dibutyryl-cAMP could replace fMLP and a cAMP-elevating agent, respectively. Phosphorylation of the 52,000 molecular weight protein was also demonstrated in cell lysates in the presence of cAMP, and in membrane preparations in the presence of the catalytic subunit of cAMP-dependent protein kinase. These data suggest that phosphorylation of the 52,000 molecular weight protein in intact cells is dependent on the cross-talk between the fMLP- and the cAMP-signalling pathways, and may thus be involved in the cAMP-regulatory mechanism.

Bucladesine↗

PubMed1992

In vitro formation and expansion of cysts derived from human renal cortex epithelial cells.

Acquired renal cysts derive from terminally differentiated tubular epithelium in adults as a consequence of increased epithelial cell proliferation, fluid accumulation and extracellular matrix remodelling. To understand better how human epithelial cysts may be initiated and progressively expand, cells from primary cultures of normal human adult renal cortex were dispersed in polymerized type I collagen. The transparent matrix permitted repeated observation by light microscopy of cyst formation from individual renal cells. The cyst cells reacted strongly with distal nephron histochemical markers (cytokeratin antibodies AE1/AE3, epithelial membrane antigen, and Arachis hypogaea lectin) but inconsistently or not at all to markers of proximal tubules (Tetragonolobus purpureas lectin and Phaseolus vulgaris erthroagglutinin lectin). The number of spherical, fluid-filled epithelial cysts that developed in a standardized microscope field quantified cyst initiation. Cyst progression was determined from the increase in the diameter (surface area) of cysts and represents a hyperplastic event. EGF or TGF alpha, were required in serum-free defined medium to cause cysts to develop from individual epithelial cells dispersed in the matrix; insulin was required as a co-factor. The EC50 for EGF was approximately 0.1 ng/ml, and for insulin 1 microgram/ml. Early cultures of normal cortex formed cysts more efficiently when dispersed in collagen matrix than cells passaged several times before suspension in the gel. Agonists of adenylate cyclase (PGE1, AVP, VIP, PTH, forskolin, cholera toxin), methylisobutylxanthine, and 8-Br-cAMP, though incapable of causing cyst formation alone in defined medium, enhanced cyst initiation and progression in the presence of EGF and insulin. Angiotensin II, TNF alpha, beta-estradiol, and pertussis toxin had no effect in the absence or presence of EGF and insulin. Pertussis toxin inhibited cyst initiation and expansion caused by EGF and forskolin but potentiated cyst initiation and expansion caused by EGF and PGE1. Cyst formation and expansion were inhibited by TGF beta 1 and 2-chloroadenosine. Polarized monolayers of human renal cortical cells grown on permeable membranes were used to independently quantify the effects of agonists on the net secretion of solute and water from the basolateral to the apical surface of the cells. PGE1, forskolin, and 8-Br-cAMP stimulated net fluid secretion that was sustained for several days; EGF enhanced forskolin-stimulated fluid secretion. We conclude that the formation and expansion of in vitro cysts derived from solitary human cortex cells depends on the coordinated interplay between cellular proliferation and fluid secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Body Fluids↗

PubMed1992

Inhibition of field stimulation-induced contractions of rat cauda epididymis by purinoceptor agonists but not by adrenoceptor agonists.

1. Adenosine, AMP, ADP, and ATP were approximately equipotent in inhibiting contractions evoked by field stimulation (10 Hz, 60 V, 1 ms, 10 s) of isolated preparations of rat cauda epididymis. Adenosine had no significant effect on contractions induced by the exogenous application of either noradrenaline or ATP. 2. The effects of adenosine and AMP were markedly potentiated by the adenosine uptake inhibitor, dipyridamole. In contrast there was only a two-fold potentiation of the effects of ATP and no potentiation of the effects of ADP by dipyridamole. 3. Inhibitory responses to both adenosine and ATP were blocked by 8-phenyltheophylline. Neither the P2Y-purinoceptor antagonist, reactive blue 2, nor P2X purinoceptor antagonist alpha,beta-methylene ATP, blocked the inhibitory effects of ATP. 4. Several stable analogues of adenosine, namely 5'-(N-ethyl) carboxamidoadenosine (NECA), N6-cyclohexyl-adenosine (CHA), L-N6-(2-phenyl-isopropyl)adenosine (L-PIA), D-N6-(2-phenyl-isopropyl)adenosine (D-PIA) and 2-chloroadenosine (CADO) also inhibited nerve stimulation-induced contractions. NECA was the most potent. L-PIA and D-PIA were approximately equipotent, except in the presence of dipyridamole, when the potency of L-PIA exceeded that of D-PIA. 5. Field stimulation-induced contractions of the rat cauda epididymis were unaffected by the alpha 2-adrenoceptor agonists clonidine and xylazine; isoprenaline in high concentrations produced phentolamine-sensitive inhibition of contractions evoked by field stimulation and exogenous application of noradrenaline. 6. These findings taken together are consistent with the possibility that prejunctional purinoceptors, which are atypical in that they are directly activated by both ATP and adenosine, mediate inhibition of neurotransmission in this tissue. No evidence for the presence of functional alpha 2-adrenoceptors modulating neurotransmission was obtained.

Animals↗

PubMed1992

Direct and indirect stimulations of cyclic AMP formation in human brain.

1. Adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation, by use of an [3H]-adenine prelabelling assay, was measured in fragments of human cerebral cortex, taken in the course of various neurosurgical procedures. 2. Large accumulations of [3H]-cyclic AMP due to isoprenaline, noradrenaline and adenosine and small effects due to forskolin were observed. Histamine, 5-hydroxytryptamine (5-HT) and the excitatory amino acids glutamate and quisqualate were ineffective. 3. The response to noradrenaline consisted of two components; a direct beta-adrenoceptor response and an enhancement mediated by an alpha-adrenoceptor which appears to be similar to that in rat cerebral cortex. 4. The response to isoprenaline was also potentiated by histamine H1 receptor stimulation but the direct effect of 2-chloroadenosine was not altered by histamine, 5-HT or quisqualate. 5. It is concluded that some, but not all, of the indirect modulations of cyclic AMP formation previously observed in experimental animal brain exist in human cerebral cortex.

Adenine↗

PubMed1992

Pharmacological characterization of adenosine A1 and A2 receptors in the bladder: evidence for a modulatory adenosine tone regulating non-adrenergic non-cholinergic neurotransmission.

1. The nerve-evoked contractions elicited by transmural electrical stimulation of mouse urinary bladders superfused in modified Krebs Ringer buffer containing 1 microM atropine plus 3.4 microM guanethidine were inhibited by adenosine (ADO) and related nucleoside analogues with the following rank order of potency: R-phenylisopropyladenosine (R-PIA) greater than cyclohexyladenosine (CHA) greater than 5'N-ethylcarboxamido adenosine (NECA) greater than ADO greater than S-phenylisopropyladenosine (S-PIA). Tissue preincubation with 8-phenyltheophylline (8-PT) displaced to the right, in a parallel fashion, the NECA concentration-response curve. 2. The contractions elicited by application of exogenous adenosine 5'-triphosphate (ATP) were also inhibited by ADO and related structural analogues. The rank order of potency to reduce the motor response to ATP was: NECA greater than 2-chloroadenosine (CADO) greater than R-PIA greater than ADO greater than CHA greater than S-PIA. 3. The ADO-induced ATP antagonism was of a non-competitive nature and was not specific. Tissue incubation with 10 microM NECA not only reduced the motor responses elicited by ATP, but also 5-hydroxytryptamine, acetylcholine and prostaglandin F2 alpha. The action of NECA was antagonized following tissue preincubation with 8-PT. The inhibitory action of NECA was not mimicked by 10 microM CHA. 4. The maximal bladder ATP contractile response was significantly increased by tissue preincubation with 5-30 microM 8-PT. 5. The 0.15 Hz evoked muscular twitch was significantly increased by 8-PT while dipyridamole consistently reduced the magnitude of the twitch response. These results are consonant with the hypothesis that an endogenous ADO tone modulates the bladder neurotransmission. 6. A working model is proposed suggesting the presence of ADO-Al and A2 receptors in the mouse urinary bladder. The A1 receptor subpopulation is probably of presynaptic origin whereas the smooth muscle membranes contain a population of the A2 receptor subtype.

Adenosine↗

PubMed1978

Quantification of the morphological reaction of platelets to aggregating agents and of its reversal by aggregation inhibitors.

1. In rabbit citrated platelet-rich plasma, the changes in shape of the platelets produced by adenosine diphosphate (ADP) or 5-hydroxytryptamine (5-HT) were observed by photometric and volumetric techniques and by measurements of platelet images on electron micrographs either directly or with an image analysing computer. This permitted the indirect manifestations of the shape changes to be correlated with the morphological features responsible for them, i.e. transformation of disks to more spherical forms and extrusion of blebs and spikes. 2. Following the addition of ADP, an initial brief peak in the light scattering records was associated with marked but transient irregularities in the surface of the platelets. These effects were absent when the other shape changes were produced by 5-HT. 3. When the optical manifestations of the shape changes induced by ADP were reversed by the addition of antagonists adenosine triphosphate, 2-chloroadenosine or prostaglandin E1, the morphological changes were reversed by a diminution in the number of spikes and the conversion of spherical platelet bodies to a more discoid form. 4. The volume of extracellular plasma trapped between platelets sedimented by centrifugation was proportional to the number of spikes which they extruded. Under all conditions, the volume of the platelets themselves remained remarkably constant at approximately 5 X 10(-9) microliter./platelet. 5. Addition of a calcium chelating agent alone produced a rapid persistent alteration in the optical properties of platelet-rich plasma. The magnitude of this alteration was proportional to chelator concentration but greater in some plasmas than in others. Similar optical effects were produced when the calcium concentration of platelet-rich plasma was increased by adding calcium. The optical effects produced by calcium or its chelators were unusual in that the changes in transmitted and horizontally scattered light were in opposite directions (transmitted light decreased, scattered light increased) whereas, in all other circumstances so far examined, these changes were in the same direction. The chelators caused the formation of spikes on the platelets without appreciably altering their disk shape, which may explain the unusual nature of the optical effects.

Adenosine↗

PubMed1977

Vascular adenylate cyclase: role of age and guanine nucleotide activation.

Particulate adenylate cyclase activity was examined in broken cell preparations of rat aorta and mesenteric artery from 3- to 5- and 9- to 13-week-old rats. While basal adenylate cyclase activity of the mesenteric artery was 4-fold greater than aortic enzyme activity, there was no difference in enzyme activity with age. GTP and the GTP analogue, 5'-guanylylimidodiphosphate [Gpp(NH)p] stimulated adenylate cyclase activity. Stimulation by Gpp(NH)p did not differ with age for either tissue and occurred without a detectable lag. The vasodilators, isoproterenol, 2-chloroadenosine and prostaglandin E1, were ineffective in increasing adenylate cyclase activity, although marked stimulation was demonstrated with both sodium fluoride and Gpp(NH)p. Even in combination with Gpp(NH)p, isoproterenol did not increase particulate adenylate cyclase activity of these blood vessels; however, with intact arteries, isoproterenol (10(-7)M) did increase aortic and mesenteric cyclic AMP levels. Isoproterenol increased cyclic AMP levels in rats of both ages, at a time when isoproterenol was less effective in maximally relaxing aortic strips from 9- to 13-week-old rats. These data indicate that diminished aortic relaxation with age is not associated with a reduced ability of vascular relaxants to increase aortic cyclic AMP levels. Furthermore, as a first step in establishing that guanine nucleotides are regulators of vascular adenylate cyclase, both GTP and Gpp(NH)p were found to be potent activators of adenylate cyclase from blood vessels.

Adenosine↗

PubMed1979

Production of long term steroid-producing granulosa cell cultures by cell hybridization.

Primary cultures of rat ovarian granulosa cells have been used extensively to study hormonal regulation of cellular function. To date, no long term cultures of ovarian cells which retain their differentiated functions have been developed. Hypoxanthine guanine phosphoribosyl transferase-deficient simian virus 40-transformed rat ovarian granulosa cells were fused with freshly prepared rat granulosa cells using inactivated Sendai virus. Putative hybrid cell strains obtained after selection in medium containing hypoxanthine, aminopterin, and thymidine were analyzed for progesterone synthesis. Neither the original simian virus 40-transformed granulosa cell nor its hypoxanthine guanine phosphoribosyl transferase-deficient derivative produced progesterone, but three of the hybrid strains produced progesterone at basal levels and in response to dibutyryl cAMP. One of these strains produced progesterone in a dose-responsive fashion when exposed to prostaglandin E2, cholera toxin, dibutyryl cAMP, and 2-chloroadenosine. Cell strains obtained by hybridization were remarkably similar to primary cultures of granulosa cells with respect to both the magnitude and temporal aspects of progesterone production in response to dibutyryl cAMP.

Animals↗

PubMed1975

Inhibitory effects of adenine nucleotide analogs on the isolated guinea-pig taenia coli.

The inhibitory actions of adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine and 16 adenine nucleotide and nucleoside analogs on the isolated guinea-pig taenia coli preparation were compared with those of adenosine triphosphate (ATP). Responses were quantitated as magnitude of maximal relaxation, time taken to reach maximal relaxation and activity relative to that of ATP. Inhibitory responses induced by the 5'-di- and triphosphates of 2-chloroadenosine and 2-methylthioadenosine resembled those elicited by ADP and ATP, but the 2-substituted analogs were markedly more potent. AMP and adenosine were less active than ATP; their activities were enhanced by 2-chloro substitution but not by 2-methylthio substitution. 2-Methylthio-AMP and 2-methylthioadenosine were the only analogs which did not elicit maximal relaxation of the taenia coli. 6'-Deoxyhomoadenosine 6'-phosphonic acid was inactive. Adenine nucleotide analogs in which the polyphosphate moiety was modified had steeper log dose-response curves than ATP and induced greater maximal responses than ATP. Analogs in which the polyphosphate alpha, beta-anhydride oxygen was replaced by methylene took up to 5 times longer than ATP to cause maximal relaxation. Other analogs with modified or unmodified polyphosphate side chains caused rapid relaxation of the taenia coli. There was no apparent correlation between relative activity and time to reach maximal response. The findings obtained indicate that di- or triphosphate groupings are of prime importance in binding adenine nucleotides to the putative smooth muscle receptor which mediates their inhibitory responses, and that hydrolysis of the terminal phosphates of adenosine 5'-polyphosphates is not a requirement for inhibitory activity. Reasons for the distinctive inhibitory actions of the phosphate-modified adenine nucleotide analogs are considered.

Adenine Nucleotides↗

PubMed1976

2-Fluoroadenosine 3':5'-monophosphate. A metabolite of 2-fluoroadenosine in mouse cytotoxic lymphocytes.

2-Fluoroadenosine (F-Ado) is a potent inhibitor of lymphocyte-mediated cytolysis studied in vitro. The inhibition of cytolysis by F-Ado was potentiated markedly by an inhibiotr (Ro 20-1724) of adenosine 3':5'-monophosphate (cAMP) phosphodiesterase and, unlike the inhibition caused by adenosine, was irreversible when the cytotoxic lymphocytes were incubated with F-Ado and were then washed free of exogenous nucleoside. Incubation of cytotoxic lymphocytes with F-Ado resulted in the rapid, dose-dependent formation of 2-fluoroadenosine 5'-triphosphate (F-ATP); the build-up of F-ATP within these cells was accompanied by a reciprocal depletion of ATP. Once formed intracellularly, the F-ATP was not diminished during a subsequent 30-min incubation of the cells in F-Ado-free medium. 2-Fluoroadenosine 3':5'-monophosphate (F-cAMP), a novel compound, was synthesized chemically. This cAMP analogue was found to be highly cross-reactive in a radioimmunoassay specific for cAMP and to be equipotent to cAMP in its ability to activate a crude preparation of protein kinase derived from rat brain. A column chromatographic procedure was devised whereby F-cAMP and cAMP could be purified simultaneously from tissue extracts. Treatment of cytotoxic lymphocytes with F-Ado resulted in the formation of presumptive F-cAMP in amounts greater than that of cAMP, as determined by the concentration of F-Ado added to the medium and was not observed when the lymphocytes were incubated with either adenosine or 2-chloroadenosine, two agents which caused large increases in cAMP. The simultaneous presence of Ro 20-1724 enhances greatly the formation of F-cAMP from F-Ado without affecting the pool size of F-ATP. Removal of exogenous F-Ado from cells previously incubated with this drug and subsequent incubation of these cells in drug-free medium did not result in a substantial reduction in intracellular F-Ado (via prior incubation with F-Ado); 2'-deoxyadenosine was also effective in this capacity, while 9-beta-D-arabinofulanosyladenine was without effect. The level of cAMP was elevated transiently, in a dose-dependent manner, by F-Ado, and returned to control value after removal of exogenous F-Ado from the cells. Ro 20-1724 enhanced greatly this transient elevation of cAMP caused by F-Ado.

Adenosine↗

PubMed1978

Characterization of a specific adenosine receptor on human lymphocytes.

We have examined the mechanism of action of adenosine, a naturally occurring nucleoside that has profound effects on lymphocyte function. Adenosine (0.01 micrometer to 10 micrometer) increased lymphocytes cAMP levels in a dose-dependent fashion with a maximal (10 micrometer) increase of about 4-fold, whereas adenine, guanosine, and inosine had no effect on lymphocyte cAMP levels at concentrations of 100 micrometer. Adenosine appears to act on the cell surface since 1) 2-chloroadenosine, a poorly metabolized adenosine analogue, was as active as adenosine and 2) dipyridamole, which markedly inhibited [3H]-adenosine uptake by human lymphocytes, did not affect adenosine-induced accumulation of cAMP. The specificity of the adenosine effect was established by showing that the methylxanthine derivatives, theophylline and 3-isobutyl-1-methylxanthine (IBMX), specifically block the accumulation of cAMP in lymphocytes induced by adenosine. Theophylline is a competitive inhibitor of the effect of adenosine, with an estimated dissociation constant of theophylline-receptor complex of about 6.3 X 10(-7) M. The results suggest that adenosine increases the intracellular cAMP content of lymphocytes as a result of its interaction with a specific membrane receptor which results in the activation of adenylate cyclase.

Adenosine↗

PubMed1979

Adenosine receptor on human basophils: modulation of histamine release.

Adenosine, at physiologic concentrations, inhibits in vitro IgE-mediated human basophil histamine release in a dose-dependent fashion. The inhibition dose-response curve is paralleled by an adenosine-induced increase in cAMP levels of human leukocyte preparations. Further evidence that the adenosine effect is related to changes in cAMP levels is that the nucleoside inhibits only in the first stage of antigen-induced histamine release and fails to inhibit the release caused by ionophore A23187. A poorly metabolized derivative of adenosine, 2-chloroadenosine inhibits as effectively as adenosine; dipyridamole, which blocks adenosine uptake, does not impair the inhibition caused by adenosine. Finally, theophylline, which is a competitive antagonist of adenosine in human lymphocytes also blocks the inhibition of release caused by adenosine. These data suggest that adenosine acts via a specific cell-surface receptor linked to adenylate cyclase. It appears that the human basophil has a specific receptor for adenosine and that this nucleoside may modulate the in vivo release of the mediators of immediate hypersensitivity reactions.

Adenosine↗

PubMed1976

Regulation of adenylate cyclase from cultured human cell lines by adenosine.

Broken cell particulate preparations of adenylate cyclase isolated from the human glioma cell line 132-1N1 were stimulated 2-to 3-fold by 30 muM adenosine. This concentration of adenosine produced a maximal stimulation of the cyclase while 3 to 5 muM adenosine produced half-maximal stimulation. Theophylline, at 40 muM, inhibited the adenosine stimulation of the adenylate cyclase by about 40% while 200 muM produced near complete inhibition. The inhibition by theophylline could be overcome by increasing adenosine to a concentration 10-fold that of theophylline, implying that the inhibition was competitive. Basal activity was not inhibited by even 1.0 mM theophylline, nor was the epinephrine stimulated activity. In contrast, 1.0 muM propranolol essentially completely inhibited the 8-fold stimulation of 1.0 muM epinephrine but had no effect on either basal or adenosine-stimulated activity. Adenosine and 2-chloroadenosine were equipotent in stimulating adenylate cyclase from the 132-1N1 line, whereas neither adenine nor guanosine had any detectable effect. GTP, 10 muM, produced a small variable stimulation of the adenylate cyclase while the GTP analogue, 5'-guanylylimidodiphosphate (Gpp(NH)p), produced a marked stimulation fo the cyclase. Preincubation of the adenylate cyclase preparation with the analogue greatly increased its potency and maximal effect. In contrast, both basal and adenosine-stimulated activity decreased markedly with preincubation. The effects of adenosine or epinephrine in combination with Gpp(NH)p were at least additive and often synergistic in comparison to the effects of the compounds alone. The effects of adenosine on intact and broken cell preparations of the human fibroblast lines WI-38 and VA13-2RA were also examined. In the intact VA13-2RA, adenosine produced rapid and large increases in intracellular and extracellular cyclic adenosine 3':5'-monophosphate (cAMP). In the parental fibroblast line, the WI-38, adenosine slightly elevated basal levels of cAMP, but only produced marked elevations in the presence of non-methylxanthine phosphodiesterase inhibitors. The effect of adenosine on the broken cell particulate preparations of adenylate cyclase from the fibroblasts was similar to its action on the cyclase from the 132-1N1; 30 muM adenosine produced a maximal stimulation of the adenylate cyclase, and the stimulation was inhibited by theophylline.

Adenosine↗

PubMed1976

Spontaneous platelet aggregation in arterial insufficiency: mechanisms and implications.

To investigate the clinical implications and mechanisms of spontaneous platelet aggregation (SPA) in man, 150 normal subjects, 22 patient controls and 130 patients with vascular insufficiency were studied. SPA was negative in normal subjects and patient controls whereas it was positive in 36 of 66 (54%) patients with transient ischemic attacks, 6 of 32 (19%) patients with stable angina, 7 of 10 (70%) patients with acute myocardial infarction and 11 of 14 (80%) patients with acute peripheral arterial insufficiency. The SPA was inhibited with aspirin in vivo, and inhibited competitively in vitro by low concentrations of aspirin, 2-chloroadenosine, prostaglandin E1 or apyrase but only by high concentrations of heparin or hirudin. Addition of platelet-poor plasma from patients with positive SPA did not cause normal platelets to aggregate. Treatment of patients who had acute peripheral arterial insufficiency with aspirin and dipyridamole prevented SPA with notable clinical improvement of the ischemic changes.

Adenosine↗