Biomedical subjects

J G Dobson

Publications and source records attributed to J G Dobson.

At least 37 records · Page 2Linked to original sources

PubMed1995

Endogenous adenosine reduces depression of cardiac function induced by beta-adrenergic stimulation during low flow perfusion.

High levels of norepinephrine in the heart are cardiotoxic resulting in contractile dysfunction and arrhythmic activity via beta-adrenoceptor mediated mechanisms. A low flow heart model perfused with physiological saline containing glucose and bubbled with an O2 gas mixture was used to determine whether adenosine, a nucleoside with antiadrenergic properties, could reduce the functional manifestations of catecholamine cardiotoxicity. Isolated rat hearts were treated with dipropylcyclopentylxanthine (DPCPX; 0.1 microM; A1 receptor antagonist) to block endogenous adenosine. In DPCPX-treated hearts stimulated with isoproterenol (ISO; 1 microM) during 45 min of low flow (0.5 ml/min) perfusion, the recovery of contractile function (ConF) at 15 min after the restoration of normal flow was 64% of control (before low flow) values as compared to 110% recovery of ConF in the absence of ISO. The incidence of arrhythmias observed upon restoration of control flow was increased by ISO when the action of endogenous adenosine was blocked with DPCPX. In the absence of DPCPX both the functional depression and arrhythmias induced by ISO were prevented in the presence of phenylisopropyladenosine (PIA; 1 microM; A1 receptor agonist). At 15 min after normal flow was restored. ConF in ISO-treated hearts with PIA was 53% greater than in the absence of PIA and presence of DPCPX. This enhancement of ConF by PIA was significantly reduced by DPCPX. By 30 min after flow restoration, these significant differences were absent. DPCPX reversed the PIA-induced reduction in arrhythmias observed upon restoration of normal flow. PIA and DPCPX alone in the absence of ISO, and ISO in the absence of PIA and DPCPX, did not result in altered ConF upon restoration of normal flow. These findings indicate that intense beta-adrenergic stimulation of the heart during low-flow perfusion in the absence of adenosine A1 receptor activity induces contractile depression and arrhythmicity subsequent to restoration of control perfusion. It is concluded that endogenous adenosine protects the heart against catecholamine toxicity via stimulation of adenosine A1 receptors.

Adenosine↗

PubMed1995

Adenosine enhances nitric oxide production by vascular endothelial cells.

Adenosine per se is a potent vasodilator of vascular smooth muscle. Endothelial cells modulate vascular tone via the release of nitric oxide (NO), which also elicits vasodilation. This study was undertaken to determine whether adenosine could directly stimulate endothelial cells to enhance NO production, which could subsequently reduce vascular tone. NO production was evaluated in porcine carotid artery endothelial cells (PCAEC) and human saphenous vein endothelial cells (HSVEC) seeded on multiwell plates, grown to confluence, and treated with adenosine for 1 h. The bathing medium was collected, and the NO production was determined as reflected by the formation of NO2- and NO3-. NO production by PCAEC was significantly increased by adenosine in a dose-dependent manner, whereas there was only an insignificant tendency for an increase by HSVEC. The addition of the NO synthase competitive inhibitor, NG-monomethyl-L-arginine (NMMA), or the adenosine receptor antagonist, theophylline, prevented the increase in NO production by adenosine. The results suggest that adenosine stimulates, by a receptor-mediated mechanism, the production of NO by arterial, but not by venous, endothelial cells.

Adenosine↗

PubMed1993

Adenosine stimulates proliferation of human endothelial cells in culture.

The effect of adenosine on proliferation of human endothelial cells was investigated by adding adenosine to the medium of cultures derived from human umbilical veins. Cell counts on cultures grown in 10 microM adenosine for 4-7 days were 41-53% greater than counts from control cultures. In contrast, 10 microM adenosine had no effect on growth of a human fibroblast cell strain (IMR-90). Neither inosine nor 2',5'-dideoxyadenosine influenced endothelial cell growth at concentrations of 0.1 or 10 microM. Addition of adenosine deaminase abolished the proliferative effect of added adenosine and inhibited proliferation by 16% in control cultures, suggesting that endogenous adenosine may enhance proliferation in culture. The adenosine receptor antagonist, 8-phenyltheophylline, at 0.1 and 1.0 microM blocked the enhanced proliferation caused by 10 microM adenosine. Addition of 10 microM adenosine enhanced DNA synthesis in endothelial cell cultures as indicated by an increased incorporation of [3H]thymidine into acid-insoluble cell material. The results indicate that addition of physiological concentrations of adenosine to human umbilical vein endothelial cell cultures stimulates proliferation, possibly via a surface receptor, and suggest that adenosine may be a factor for human endothelial cell growth and possibly angiogenesis.

Adenosine↗

PubMed1993

Adenosine inhibition of beta-adrenergic induced responses in aged hearts.

Because adenosine has an antiadrenergic action in the heart, young (3-4 mo) and aged (18-20 mo) adult Sprague-Dawley and Fischer 344 rat hearts were perfused to determine whether interstitial adenosine plays a role in the reduced metabolic and mechanical responsiveness of the aged heart to beta-adrenergic stimulation. Interstitial adenosine was approximately twofold greater in aged hearts compared with young adult hearts, and 10(-8) M isoproterenol (ISO) further increased these levels. ISO increased myocardial adenosine 3',5'-cyclic monophosphate content, glycogen phosphorylase activity, and cardiac contractility by 83, 150, and 130%, respectively, in young hearts but only increased these variables by 45, 74, and 61%, respectively, in aged hearts. Sulfophenyl-theophylline prevented the reduced ISO-induced responsiveness of the above variables in aged hearts. Exogenously administered adenosine deaminase eliminated the reduced ISO-induced contractile responsiveness in aged hearts. The apparent activities of 5'-nucleotidase and adenosine deaminase were not significantly different in ventricular samples from young and aged hearts. These results suggest that the elevated interstitial level of adenosine exerts a greater antiadrenergic effect in the aged heart, rendering it less responsive to beta-adrenergic stimulation. The increased interstitial level of adenosine in the aged heart does not appear to be due to a difference in the activities of either 5'-nucleotidase or adenosine deaminase.

5'-Nucleotidase↗

PubMed1993

Hypoxia enhances isoproterenol-induced increase in heart interstitial adenosine, depressing beta-adrenergic contractile responses.

Endogenous interstitial adenosine may protect the hypoxic heart by attenuating beta-adrenergic-induced contractile and metabolic responses, thereby reducing energy utilization. Constant-flow perfused rat hearts were used to study: 1) the effect of hypoxia on isoproterenol (ISO)-induced increase in interstitial adenosine, as estimated with epicardial surface transudates, and 2) the role of endogenous adenosine in hypoxic depression of ISO-induced contractile responses. ISO (1 nM for 10 minutes) in the normoxic heart increased transudate adenosine 114% from a pre-ISO normoxic value of 343 pmol/ml. ISO administered to the hypoxic heart increased transudate adenosine 357% from a pre-ISO hypoxic value of 797 pmol/ml. The absolute magnitude of the ISO-induced increase in transudate adenosine was 625% greater during hypoxia than during normoxia. This was associated with a reduction in the ISO-induced contractile response during hypoxia. In other experiments, with normoxia ISO (10 nM for 10 seconds) increased developed left ventricular pressure by 140 mm Hg, and the maximum rates of left ventricular pressure development and relaxation by 5,860 and 2,771 mm Hg/sec, respectively, above control values of 90 mm Hg, 2,250 mm Hg/sec, and 1,875 mm Hg/sec. Hypoxia reduced the three ISO-induced contractile responses by 50%, 56%, and 36%. However, 1,3-dipropyl-8-cyclopentylxanthine (5 x 10(-7) M), an adenosine A1-receptor antagonist, added to the hypoxic hearts resulted in only a 31%, 39%, and 9% reduction in the ISO-induced responses in developed left ventricular pressure and the maximum rates of left ventricular pressure development and relaxation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1992

Fluorometric quantitation of adenosine concentration in small samples of extracellular fluid.

Adenosine is a naturally occurring nucleoside which regulates many physiological processes by interacting with adenosine-specific receptors. Knowledge of the extracellular adenosine concentration at the site of adenosine receptors on target cells is required for an understanding of mechanisms involving the action of the nucleoside. Samples of extracellular fluid which reside in close proximity to the surface of target cells are frequently small in volume. This report describes improvements in accuracy and reliability of a fluorometric assay designed for determining the concentration of adenosine in microliter samples of extracellular fluids. The utility of the assay is demonstrated by determining adenosine concentrations in interstitial and coronary effluent samples from normoxic perfused rat hearts. The assay also clearly detects changes in the interstitial and coronary effluent adenosine levels produced by isoproterenol stimulation or hypoxia. Thus, this assay is useful for determining the adenosine concentration in microliter samples of extracellular fluid and should facilitate investigations dealing with the functions of adenosine.

Acetaldehyde↗

PubMed1992

Lack of oscillations in cyclic AMP, cAMP-protein kinase and glycogen phosphorylase during the cardiac cycle in perfused rat hearts.

It is unclear whether reported fluctuations in the level of adenosine 3',5'-cyclic monophosphate (cAMP) during a single cardiac cycle in ventricular muscle are associated with distal changes in cAMP-dependent processes. The degree of cAMP variation and its effect, if any, on biochemical sequelae during the cardiac cycle, were investigated by determining the level of cAMP and the activity ratios of cAMP-dependent protein kinase and glycogen phosphorylase in the rat ventricular myocardium. Isolated perfused hearts contracting at 240 beats/min and free of exogenously administered catecholamines were freeze-clamped, utilizing an automated clamping device capable of freezing the entire heart in less than 50 ms. The cardiac cycle was segmented into phases utilizing three different segmentation schemes. No significant difference was detected between phases regardless of the method of segmentation for cAMP, cAMP-dependent protein kinase, or glycogen phosphorylase levels. These results suggest that the levels of cAMP and the activities of cAMP-dependent protein kinase and glycogen phosphorylase do not vary significantly during a single cardiac cycle in the mammalian myocardium.

Animals↗

PubMed1992

Mechanism of enhanced cyclic AMP stimulation by isoproterenol in aged human fibroblasts.

Human diploid lung fibroblasts (IMR-90) were used to investigate the reported increase in beta-adrenergic-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels in fibroblasts aged in culture. Under basal conditions cellular cAMP was 34.2 +/- 5.6 and 38.4 +/- 9.1 pmol/mg protein in early (PDL 22-24) and late (PDL 47-52) passage fibroblasts, respectively. Net release of cAMP from fibroblasts was 67.8 +/- 8.6 and 18.5 +/- 7.0 pmol/30 min/mg protein in early and late passage cultures, respectively. In confluent, early passage fibroblasts, cellular cAMP and net release of cAMP increased by 2.7-fold and 3.8-fold, respectively, after a 30 min incubation in 2 microM isoproterenol. In confluent late passage fibroblasts, isoproterenol incubation increased cellular cAMP and net release of cAMP by 7.8-fold and 26.1-fold, respectively. Adenosine failed to inhibit isoproterenol-induced stimulation of cAMP in early or late passage fibroblasts. There was no passage-related difference in basal, isoproterenol, or forskolin-stimulated adenylyl cyclase activity in crude fibroblast membrane preparations. The activity of cAMP-phosphodiesterase in sonicates of early and late passage IMR-90 was 9.61 +/- 1.15 and 5.81 +/- 1.11 pmol/min/mg protein respectively. Measurements of cAMP in subconfluent early passage fibroblasts indicated that mechanisms related to the reduced cell density in confluent late passage IMR-90 may, in part, account for the enhanced isoproterenol-induced cAMP levels observed in these cultures. The results suggest that the remainder of the enhanced cAMP response to isoproterenol of in vitro aged fibroblasts may be due to a lower cAMP phosphodiesterase activity in these cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1991

Adenosine and acetylcholine reduce isoproterenol-induced protein phosphorylation of rat myocytes.

Adenosinergic and muscarinic agents have been shown to attenuate the catecholamine-induced augmentation of both protein phosphorylation and contractile state in perfused hearts. The attenuation by phenylisopropyl-adenosine (PIA) and carbamylcholine chloride (CARB) of the isoproterenol (ISO)-induced incorporation of 32P into protein substrates was examined in isolated rat ventricular myocytes. 32P-labelled myocytes exposed to ISO (0.1 microM, 30 s) demonstrated up to an eight-fold increase of 32P incorporation into three protein substrates (155, 31, 6 kD). When myocytes were pre-incubated with either PIA or CARB for 60 s, the ISO-induced 32P incorporation in the 31 kD and the 155 kD substrates was attenuated 37% and 25%, respectively by 1 microM PIA and only 23% and 11%, by 10 microM PIA. A concentration of 1 microM CARB produced a 24% and 17% reduction in these same substrates while 10 microM CARB produced a 44% and 50% reduction. The effects of ISO were antagonized by 10 microM propanolol. The inhibitory effects of PIA were antagonized by the theophylline, sulfophenyltheophylline and dipropylcyclopentylxanthine, whereas atropine antagonized the inhibitory effects of CARB. The 32P incorporation elicited by 1 microM forskolin was reduced more by CARB than PIA. Additionally, while PIA and CARB reduced the ISO-induced increase in cAMP-dependent protein kinase (PKA) activity by 48% and 41% respectively, only CARB attenuated the ISO-elicited increase in cAMP levels, attenuating this response by 58%. The results indicate that PIA was less effective in attenuating ISO-induced 32P incorporation at higher concentrations than at lower concentrations. Moreover, this compound was less potent than CARB at attenuating the effects of ISO. It is conceivable that this difference could be related to activation of stimulatory adenosine receptors (A2) and/or a greater density of muscarinic receptors including multiple inhibitory muscarinic pathways.

Acetylcholine↗

PubMed1991

Adenosine reduces the Ca2+ transients of isoproterenol-stimulated rat ventricular myocytes.

Adenosine in the heart attenuates the contractile and metabolic effects of beta-adrenergic stimulation. The effect of adenosine on changes in intracellular Ca2+ concentration [( Ca2+]i) elicited with electrical stimulation was studied in rat ventricular myocytes in the absence and presence of isoproterenol (ISO). Fura-2 was utilized as a Ca2+ indicator. Autofluorescence was determined, and in vivo calibration was conducted, for each myocyte. Phenylisopropyladenosine (PIA; 10(-7) M; 5 min), an adenosine A1 receptor agonist, had no effect on the Ca2+ transient magnitude (TM) or the rate of Ca2+ transient decline determined at 150 nM Ca2+(i) (RD150). ISO (10(-8) M; 1 min) in the continued presence of PIA resulted in a 16% increase in the TM, but no change in the RD150. Inhibiting the PIA with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10(-7) M; 3 min) in the continued presence of ISO plus PIA resulted in a further 51% increase in the TM and a 57% increase in the RD150. In PIA-treated myocytes, ISO-induced spontaneous high-frequency Ca2+ transients occasionally were observed after the inhibition of PIA by DPCPX. The results of this study suggest that adenosine attenuates myocardial contractile responses to beta-adrenergic stimulation, in part, by reducing the beta-adrenergic-induced changes in the Ca2+ transients occurring in the contracting ventricular myocyte.

Adenosine↗

PubMed1991

Adenosine attenuation of catecholamine-enhanced contractility of rat heart in vivo.

The antiadrenergic action of adenosine was examined in open- and closed-chest preparations of anesthetized rats. The positive inotropic effects of a jugular vein infusion of either isoproterenol or epinephrine were attenuated by phenylisopropyladenosine, an adenosine A1-receptor agonist. 1,3-Dipropyl,8-cyclopentylxanthine, a specific A1-receptor antagonist, inhibited the action of phenylisopropyladenosine. The results indicate that adenosine receptor-mediated mechanisms are functional in the blood-perfused rodent heart and support the possibility of a physiological role for adenosine in modulating cardiac contractility.

Adenosine↗

PubMed1990

Adenosine modulates beta-adrenergic signal transduction in guinea-pig heart ventricular membranes.

The mechanism of the antiadrenergic action of adenosine in the heart was investigated by examining the effects of phenylisopropyladenosine (PIA), an adenosine A1 receptor agonist, on beta-adrenergic receptor and non-receptor elicited increases in adenylyl cyclase activity of guinea-pig ventricular membranes. These membranes contained adenosine A1 receptors (approximately 80 fmol/mg) and at least one ADP-ribosylated G protein with a molecular weight of approximately 40 kDa. PIA attenuated isoproterenol-enhanced adenylyl cyclase activity and [3H]GDP release in this membrane preparation. However, PIA had no significant effect on GPP(NP)P or forskolin activated adenylyl cyclase. Additionally, PIA did not change the sensitivity of the cyclase to either magnesium or GTP in these membranes. The inhibition of isoproterenol-enhanced activity appeared to be dependent on the activation state of the enzyme such that the degree of PIA inhibition decreased with increasing isoproterenol concentration. These data suggest that adenosine inhibition of catecholamine-stimulated adenylyl cyclase activity occurs predominantly by modulating beta-adrenergic receptor signal transduction and that subunits of Gi may be involved in this action.

Adenosine↗

PubMed1990

Influence of beta-adrenergic stimulation and contraction frequency on rat heart interstitial adenosine.

Adenosine (ADO) has an antiadrenergic action in the heart that causes an attenuation of contractile and metabolic responses elicited by beta-adrenergic stimulation. The effect of an increase in oxygen consumption elicited by either beta-adrenergic stimulation or an increase in contraction frequency on interstitial fluid and coronary effluent ADO levels was investigated in isolated perfused isovolumically contracting rat hearts. ADO in left ventricular surface transudates and coronary effluents was rendered fluorescent with chloroacetaldehyde, and the formed ethenoadenosine derivative was quantitated with high-performance liquid chromatography fluorescence detection. Heart preparation integrity was verified by determining the activities of lactate dehydrogenase and ADO deaminase in the transudates. Isoproterenol (10(-8) M) elicited a 45% increase in oxygen consumption and a 54% increase in developed left ventricular pressure in hearts paced at 240 beats/min. With isoproterenol the control transudate ADO concentration (304 pmol/ml) increased 493%, and the control effluent ADO concentration (48 pmol/ml) increased 259%. Increasing the contraction frequency from 180 to 300 beats/min in the presence of 10(-6) M propranolol increased oxygen consumption by 45% and decreased left ventricular pressure by 29%. With the increase in contraction frequency, the transudate ADO concentration did not increase significantly. However, the ADO concentration in the effluent was an average of 269% greater in hearts contracting at the higher frequency. Increasing the contraction frequency of hearts treated with both 10(-6) M propranolol and 10(-5) M atropine also had no significant effect on the level of transudate ADO. The effluent level of ADO increased only 78%. Levels of ADO in transudates were not significantly affected by mesothelial cell metabolism. These results suggest that the beta-adrenergic stimulation the interstitial level of ADO in the heart increases to levels that are sufficient to manifest its antiadrenergic effects. Furthermore, there is not always a correlation between the levels of ADO found in the interstitial and effluent fluid compartments.

Adenosine↗

PubMed1990

Increased myocardial adenosine production and reduction of beta-adrenergic contractile response in aged hearts.

The contractile response of the aged adult heart to beta-adrenergic stimulation is known to be reduced compared with the young adult heart. Since endogenous adenosine exerts an antiadrenergic action in the heart, this study was undertaken to determine if the basal endogenous level of myocardial adenosine increases with age and whether this increase mediates the reduced responsiveness of aged heart to beta-adrenergic stimulation. Young (3-5 months) and aged (12-22 months) Sprague-Dawley adult rat hearts of CD and SD stock were perfused at constant pressure and paced at 270 contractions/min. The two age groups had a similar level of +dP/dtmax (index of contractility) under control conditions. Adenosine release into the coronary effluent was 30 +/- 3 nmol/min/g dry wt from young and 54 +/- 9 nmol/min/g dry wt from aged hearts. Inosine release was also greater from the aged hearts. Isoproterenol (10(-8) M) stimulation increased contractile state by 113% in young hearts and only 69% in aged hearts. Isoproterenol further increased the adenosine and inosine release from both age groups. Theophylline (5 x 10(-5) M), an adenosine antagonist, prevented the difference in the contractile response to isoproterenol stimulation between the young and aged hearts. Elevation of external calcium from 2 to 4 mM increased contractility equally in both age groups without influencing adenosine release. Myocardial oxygen consumption, coronary effluent PO2, oxygen supply-demand ratio, and lactate release were similar for both age groups, indicating that under the conditions studied the elevated release of adenosine by the aged hearts was not due to hypoxia. Aged (10-14 months) adult guinea pig hearts also displayed a reduced responsiveness to the isoproterenol stimulation and released more adenosine compared with young (3-4 months) adult guinea pig hearts. These findings suggest that enhanced adenosine levels that are present in the aged myocardium are responsible, in part, for the reduced contractile responsiveness of the older adult heart to beta-adrenergic stimulation.

Adenosine↗

PubMed1989

Aging increases adenosine and inosine release by human fibroblast cultures.

The effect of in vitro age and donor age on net release of adenosine and inosine was studied in cultures of normal human fibroblasts. Confluent cultures of low-(population doubling level [PDL] 23-25) and high- (PDL 43-45) passage human lung fibroblasts derived from a 16-week-old fetal donor (IMR-90) were incubated for 30 min in physiological saline and the release of adenosine and inosine into the saline was determined by HPLC. Release of adenosine and inosine into the saline bathing low-passage human skin fibroblasts derived from a 16-week-old fetal donor (GM6111) was also determined and compared with two strains of low-passage skin fibroblasts from aged (66-67 years) donors (GM3529 and GM3524). The release of adenosine and inosine by low-passage cultures of fetal lung fibroblasts was 911 and 225 pmol/30 min per mg protein, respectively. In high-passage cultures of lung fibroblasts, release of adenosine and inosine was significantly greater at 1403 and 351 pmol/30 min per mg protein, respectively. The release of adenosine and inosine by low-passage cultures of fetal skin fibroblasts was 250 and 179 pmol/30 min per mg protein, respectively. In low-passage skin fibroblasts from aged donors, release of adenosine and inosine was significantly greater at 583 and 652 pmol/30 min per mg protein, respectively. These results indicate that the net release of adenosine and inosine by cultured human fibroblasts into their extracellular environment is enhanced by in vitro aging of lung fibroblasts and is greater in skin fibroblast from aged donors.

Adenosine↗

PubMed1989

Adenosine receptor coupling to adenylate cyclase of rat ventricular myocyte membranes.

The effects of adenosine analogues on beta-adrenergic receptor and receptor-independent elicited increases in adenylate cyclase activity were investigated using membranes obtained from primary cultures of adult rat ventricular myocytes. Phenylisopropyladenosine, an A1-receptor agonist, at concentrations of 0.1, 1.0, and 10 microM, maximally inhibited isoproterenol-stimulated adenylate cyclase activity by 35, 55, and 41%, respectively. The inhibition by phenylisopropyladenosine was antagonized by 10 microM theophylline. One micromolar phenylisopropyladenosine was much less effective at attenuating forskolin-stimulated activity, such that the maximum inhibition was 26%. Phenylisopropyladenosine had no effect on adenylate cyclase stimulation by 5'-guanylylimidodiphosphate. Phenylaminoadenosine, an A2 agonist, at 10 microM or greater stimulated adenylate cyclase activity. This effect was not significantly inhibited by theophylline or 0.1 microM 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), which antagonized phenylisopropyladenosine inhibition of isoproterenol-stimulated adenylate cyclase activity. Additionally, N-ethylcarboxamidoadenosine, a nonselective adenosine-receptor agonist, had no effect on adenylate cyclase activity in the absence of DPCPX but stimulated adenylate cyclase activity in the presence of DPCPX. These results indicate that both A1 and A2 receptors exist on the ventricular myocyte sarcolemma. More importantly, the findings suggest that adenosine inhibition of catecholamine-stimulated adenylate cyclase activity is primarily due to an alteration in beta-adrenergic receptor-mediated transduction and perhaps in part by a direct inhibition of the catalytic component.

Adenylyl Cyclases↗

PubMed1988

The adenosine Ri agonist, phenylisopropyladenosine, reduces high affinity isoproterenol binding to the beta-adrenergic receptor of rat myocardial membranes.

Adenosine attenuates beta-adrenergic receptor mediated activation of adenylate cyclase in myocardial membranes via adenosine Ri receptors. The effects of adenosine analogs on the binding characteristics of beta-adrenergic receptors were examined in the present study utilizing rat ventricular membranes treated with adenosine deaminase. In 125I-cyanopindolol/isoproterenol competitive binding experiments phenylisopropyladenosine (PIA) significantly increased the IC50 for isoproterenol from 48 +/- 6 nM to 140 +/- 48 nM and steepened the slope of the competition curves from -0.56 +/- 0.03 to -0.90 +/- 0.21. Computer analysis of these curves indicated that binding of isoproterenol to the high affinity state of the beta-adrenergic receptor was eliminated in the presence of PIA. PIA had no effects in the presence of GPP(NH)P. 2-chloroadenosine, a less specific Ri agonist, caused smaller increases in IC50 and slope, without significantly affecting high affinity binding. 2',5'-dideoxyadenosine, a P-site agonist, had no significant effects on isoproterenol binding. During the time course of the competitive binding experiments the membranes displayed isoproterenol-sensitive adenylate cyclase activity in the absence of added GTP. These data suggest that adenosine attenuates catecholamine-induced activation of adenylate cyclase via Ri receptors by decreasing the ability of beta-adrenergic agonists to promote the formation of a high affinity complex composed of the agonist, receptor and stimulatory guanine nucleotide binding protein.

Adenosine↗

PubMed1987

Measurement by fluorescence of interstitial adenosine levels in normoxic, hypoxic, and ischemic perfused rat hearts.

An improved assay was used to investigate the effects of hypoxia or ischemia on interstitial fluid and coronary venous effluent levels of adenosine in isolated perfused nonworking rat hearts. The adenosine in 5- to 10-microliter samples of left ventricular epicardial surface transudates and coronary effluents was reacted with chloroacetaldehyde, and the fluorescent derivative (1,N6-ethenoadenosine) was quantitated using high pressure liquid chromatography and fluorescence detection. Hearts responding to hypoxia could be separated into two groups. In one group of hearts, the control (normoxic) transudate and effluent adenosine concentrations were 94 +/- 24 and 41 +/- 6 pmol/ml, respectively. These values increased by 118 and 96%, respectively, with 5 minutes of hypoxia (30% O2), and returned to control levels 5 minutes after resumption of normoxia. In a second group of hearts, the normoxic control levels of adenosine in the transudates (42 +/- 7 pmol/ml) and coronary effluents (62 +/- 17 pmol/ml) were increased with hypoxia by 174 and 1,178%, respectively. However, the transudate levels continued to rise for 5 minutes after resumption of normoxic perfusion while effluent levels fell. In another series of hearts, global ischemia for 30 seconds elicited an elevation of transudate adenosine levels by 362 to 641% above control (58 +/- 15 pmol/ml) as determined 30 seconds after resumption of perfusion flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗