James G. Dobson

Mechanism of adenosine inhibition of catecholamine-induced responses in heart.

The properties of adenosine inhibition of catecholamine-induced responses were investigated, using an isolated rat heart preparation. Perfusion of hearts with 0.1 (μM isoproterenol increased myocardial cAMP content 2.8-fold, activation of cAMP-dependent protein kinase 4.4-fold, phosphorylase a formation 3.4-fold, left ventricular pressure 1.8-fold, rate of ventricular pressure development 2.1-fold, and rate of ventricular relaxation 2.2-fold within 1 minute.

Role of Adenine Nucleotides, Adenosine, and Inorganic Phosphate in the Regulation of Skeletal Muscle Blood Flow

Experiments were performed on isolated frog sartorius muscle and in situ dog skeletal muscle to determine whether adenine nucleotides and their degradation products are released during contraction in concentrations capable of producing arteriolar dilation ATP was not detectable (>10−8M) in the bathing solution of the resting or contracting frog sartorius muscle. Inorganic phosphate (P1) in the muscle bath increased from 9 × 10−5M to 28 × 10−5M with 30 minutes of contraction (2 Hz) or with rest. With the dog hindlirnb preparation, ATP, ADP, and AMP were not detectable (>5 × 10−8M in the venous blood collected after 5 minutes of ischemic contraction whereas P1 was present at a concentration of 3.7 × 10×8M. Arterial blood levels required to elicit detectable vasodilation for ATP, ADP, AMP, and P1 were 28.7 × 10−8M, 27.1 × 10−8M 31.4 × 10−8M and 7.2 × 10−4M respectively. The adenosine concentration in dog muscle increased from 0.7 to 1.5 nmole/g with ischemic contraction, and hypoxanthine and inosine increased from 4.5 to 8.5 nmole/g and 2.0 to 5.5 nmole/g, respectively. The adenosine concentration in venous plasma collected from the hiodlimb immediately after termination of the irchemic contraction period was 2.2 × 10−7MM as compared to 0.4 × 10−7M in control venous and arterial blood samples. Hypoxanthine and inosine concentrations in venous blood increased 22- and 270-fold, respectively, foflowing ischemic contraction. The calculated interstitial fluid adenosine concentration was twice the arterial concentration of adenosine required to elicit maximal arteriolar dilation. These findings suggest that adenosine may play a role in the metabolic regulation of skeletal muscle blood flow, whereas ATP, ADP, AMP, and P1 may not.

Reduction by adenosine of the isoproterenol-induced increase in cyclic adenosine 3',5'-monophosphate formation and glycogen phosphorylase activity in rat heart muscle

The effect of adenosine on the increase in cardiac cyclic adenosine 3′,5′-monophosphate (cyclic AMP) concentration and glycogen phosphorylase activity produced by isoproterenol was investigated. Slices of rat ventricular myocardium 0.5 mm thick weighing 15-20 mg were cut, washed, and incubated at 37 °C in physiological saline gassed with either O2 (oxygenated) or N2 (anoxia). The concentration of cyclic AMP declined as the time of incubation increased in both oxygenated and anoxic muscle. In oxygenated muscle, isoproterenol, 10 fiM, produced a 2.2-fold increase in cyclic AMP concentration and phosphorylase activity. Adenosine at 1 fiM caused a 35% and 75% reduction in the isoproterenol-produced increase in cyclic AMP concentration and phosphorylase activity, respectively, without affecting basal levels. Reduction of the isoproterenol-elicited increase in cyclic AMP occurred within 2 minutes. Adenosine alone only at a high concentration of 1 nw increased cyclic AMP by 38% in oxygenated muscle. Adenine and inosine did not mimic the effect of adenosine on the isoproterenol-induced augmentation of cyclic AMP. Addition of adenosine deaminase to the physiological saline prevented the effects of adenosine but did not affect basal cyclic AMP. In anoxic tissue, isoproterenol failed to produce an increase in cyclic AMP and phosphorylase. Addition of adenosine deaminase to anoxic tissue resulted in an isoproterenol-produced increase in cyclic AMP, indicating that adenosine may inhibit an isoproterenol-induced increase in cyclic AMP during anoxia. These results suggest that adenosine attenuates the catecholamine-induced increase in cyclic AMP concentration and phosphorylase activity in oxygenated cardiac muscle, whereas, in the anoxic myocardium, adenosine may be responsible for preventing an increase in cyclic AMP upon β-adrenergic stimulation. Thus, adenosine may antagonize catecholamine elicited glycogenolysis.

Adenosine A2 receptor function in rat ventricular myocytes

OBJECTIVE This study was undertaken to investigate the functional significance of adenosine A2 receptor stimulation in a mammalian ventricular myocyte preparation. METHODS Isolated contracting rat ventricular myocytes were employed to assess the contractile, adenylyl cyclase and cyclic AMP responses to adenosine receptor stimulation. RESULTS In single myocytes the presence of A1 receptors was confirmed, as indicated by the A1 receptor agonist, phenylisopropyladenosine (PIA), reducing by 60 and 74% the inotropic response and activation of adenylyl cyclase, respectively, elicited by the beta-adrenergic agonist, isoproterenol. An A1 receptor antagonist, dipropylcyclopentylxanthine (DPCPX), prevented the antiadrenergic action of PIA. The A2 receptor agonist, carboxyethylphenethyl-aminoethyl-carboxamido-adenosine (CGS-21680; 0.01-10 microM) increased myocyte inotropy in a concentration-dependent manner, reaching a maximum of 41-45%. Ethylcarboxamidoadenosine (NECA), naphthyl-substituted aralkoxy-adenosine (SHA-082) and adenosine in the presence of DPCPX also increased myocyte inotropy, as evidenced by increases in myocyte shortening, duration of shortening, time-to-peak shortening, time-to-75% relaxation and rate of maximal shortening. The agonists, however, did not effect the maximal rate of relaxation. The A2 receptor antagonists, chlorofuranyldihydrotri-azoloquinazolinimine (CGS-15943) and chlorostyrylcaffeine (CSC), the latter selective for the A2a receptor, prevented the contractile responses elicited by the A2 agonists. Compared to the concentrations of A2 receptor agonists necessary to increase myocyte contractile variables, 3-12 times greater concentrations of the agonist were required to increase myocyte adenylyl cyclase activity and cAMP levels. CONCLUSIONS The results suggest the presence of adenosine A2a receptors in the rat ventricular myocyte that appear to be responsible for an increase in inotropy via cAMP-dependent and -independent mechanisms.

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.

Mechanisms of Activation of Cardiac Glycogen Phosphorylase in Ischemia and Anoxia

The effects of ischemia and anoxia on cardiac adenosine 3′, 5′-monophosphate (cyclic AMP) concentration, glycogen phosphorylase activity ratio (−5′-AMP: +5′-AMP), phosphorylase kinase activity ratio (pH 6.8:8.2), and myocardial contractility (left ventricular dP/dt) were studied in an open-chest rat heart preparation. Ischemia produced by termination of coronary blood flow increased cyclic AMP from 0.55 to 0.77 μmoles/kg in 5 seconds and phosphorylase from 0.14 to 0.57 in 20 seconds. Anoxia induced by breathing N2 increased cyclic AMP from 0.50 to 0.62 μmoles/kg in 10 seconds and phosphorylase from 0.14 to 0.65 in 30 seconds. Phosphorylase kinase increased with ischemia but did not change with anoxia. Beta-receptor blockade with practolol prevented the rise in cyclic AMP and phosphorylase kinase but blocked the increase in phosphorylase only in ischemia. Myocardial contractility declined precipitously during the first 20 seconds of anoxia. Epinephrine (0.1 μg/kg) caused an increase in cyclic AMP comparable to that elicited by anoxia, and it produced an increase in dP/dt during N2 breathing. These results suggest that in the intact working heart ischemia induces phosphorylase a formation through a cyclic AMP-dependent transformation of phosphorylase kinase; however, in anoxia phosphorylase a formation depends only on the regulation of the catalytic activity of phosphorylase kinase without conversion of this enzyme to its activated form. An increase in cyclic AMP during anoxia is not associated with a positive inotropic response even though such a response is obtained with epinephrine. Factors other than the elevation of myocardial cyclic AMP may be limiting in the control of both cardiac glycogenolysis and inotropic state.

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)

The role of cyclic adenosine 3', 5'-monophosphate and calcium in the regulation of contractility and glycogen phosphorylase activity in guinea pig papillary muscle.

We studied the relationships between the positive inotropic effects of isoproterenol, increased frequency of contraction or paired electrical stimulation, and cyclic AMP concentration and phosphorylase activity in isolated guinea pig papillary muscles. The minimum concentration of isoproterenol (10 hm) that augmented isometric force development increased cyclic AMP concentration. However 100 avi isoproterenol was required to increase the phosphorylase activity ratio (-AMP/+AMP) from 0.15 ± 0.03 to 0.25 ± 0.03. After addition of 1 &mgr;m isoproterenol to the bath, cyclic AMP increased within 0.5 minute from 0.58 ± 0.03 to 1.04 ± 0.13 mol/kg (wet weight), peak contractile force was elevated 2-foW at 1 minute, and the phosphorylase activity ratio rose to 0.40 ± 0.02 in 4 minutes. Although an increase in contraction frequency (6/min to 36/min)and paired stimulation produced more than a 3-fold increase in peak contractile force, there were no changes in cyclic AMP and phosphorylase activity. The cydic AMP concentration during diastole was 0.60 ± 0.04 and in midsystole, 0.55 ± 0.03 &mgr;mol/kg. Anoxia increased the phosphorylase activity ratio from 0.19 ± 0.02 to 0.41 ± 0.04 without elevation of cyclic AMP concentration. Removal of Ca2+ from the bathing medium prevented active force development and the anoxic increase in phosphorylase activity, but did not prevent the isoproterenol-induced increase in cyclic AMP and phospborylase. These results suggest that cyclic AMP is a factor in the catechola-mine-induced enhancement of inotropic state. However, it does not appear to play a role in the maintained augmentation of inotropic state produced by increased contraction frequocy and paired stimulation, nor does the concentration of tbe cydic nudeotide appear to vary during the contraction cyde or during anoxia. Extracellular Ca2+ is required for contraction, the positive inotropic action of catecbolamines and phosphorylase b to a conversion by anoxia.

Effects of chronic adenosine uptake blockade on adrenergic responsiveness and left ventricular chamber function in pressure overload hypertrophy in the rat

Background Increased sympathetic activity contributes to the progression of heart failure. Adenosine counteracts sympathetic activity by inhibition of presynaptic norepinephrine release and attenuation of the metabolic and contractile responses to β-adrenergic stimulation. In this study, we tested the hypothesis that the adenosinergic effects (uptake blockade) of dipyridamole may retard the progression of pressure overload hypertrophy in the rat. Methods and results To verify that the administration of dipyridamole increases myocardial adenosine levels in the rat, epicardial adenosine concentrations were measured from 12 isolated, perfused rat hearts exposed to 10−7 and 10−6 mol/l dipyridamole. Adenosine concentrations were increased with both doses of dipyridamole. Also, 9 weeks of dipyridamole treatment resulted in decreased sensitivity to the adenosine A1-receptor agonist, 2-chloro-N6-cyclopentyl adenosine, suggesting that dipyridamole increases adenosine levels in the intact rat. In the second part of the study, rats were divided into either abdominal aortic-banded or shamoperated groups and were treated with either dipyridamole or saline. After 9 weeks of treatment, two-dimensional Doppler echocardiographic studies were performed and the adrenergic responsiveness to 10−8 mol/l isoproterenol was assessed in vitro. The saline-treated banded group demonstrated concentric left ventricular hypertrophy, abnormal diastolic filling, increased wet lung weights and attenuation of adrenergic responsiveness. In contrast, the dipyridamole-treated banded rats exhibited more concentric geometry (higher relative wall thickness with similar left ventricular mass), normal left ventricular filling characteristics and preserved adrenergic responsiveness. Systolic left ventricular chamber and myocardial function, as assessed by stress-endocardial and midwall shortening relationships, were not significantly altered by banding or dipyridamole treatment. Conclusions Dipyridamole treatment prevented the development of abnormal left ventricular chamber filling, preserved adrenergic responsiveness and appeared to attenuate detrimental chamber remodeling in rats with pressure overload hypertrophy.

Adenosine A1 receptor-mediated antiadrenergic effects are modulated by A2a receptor activation in rat heart.

Presently, the physiological significance of myocardial adenosine A2a receptor stimulation is unclear. In this study, the influence of adenosine A2a receptor activation on A1 receptor-mediated antiadrenergic actions was studied using constant-flow perfused rat hearts and isolated rat ventricular myocytes. In isolated perfused hearts, the selective A2a receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) potentiated adenosine-mediated decreases in isoproterenol (Iso; 10-8 M)-elicited contractile responses (+dP/d t max) in a dose-dependent manner. The effect of ZM-241385 on adenosine-induced antiadrenergic actions was abolished by the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10-7 M), but not the selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191, 10-7 M). The A2a receptor agonist carboxyethylphenethyl-aminoethyl-carboxyamido-adenosine (CGS-21680) at 10-5 M attenuated the antiadrenergic effect of the selective A1 receptor agonist 2-chloro- N 6-cyclopentyladenosine (CCPA), whereas CSC did not influence the antiadrenergic action of this agonist. In isolated ventricular myocytes, CSC potentiated the inhibitory action of adenosine on Iso (2 × 10-7 M)-elicited increases in intracellular Ca2+concentration ([Ca2+]i) transients but did not influence Iso-induced changes in [Ca2+]itransients in the absence of exogenous adenosine. These results indicate that adenosine A2areceptor antagonists enhance A1-receptor-induced antiadrenergic responses and that A2a receptor agonists attenuate (albeit to a modest degree) the antiadrenergic actions of A1 receptor activation. In conclusion, the data in this study support the notion that an important physiological role of A2a receptors in the normal mammalian myocardium is to reduce A1 receptor-mediated antiadrenergic actions.Presently, the physiological significance of myocardial adenosine A2a receptor stimulation is unclear. In this study, the influence of adenosine A2a receptor activation on A1 receptor-mediated antiadrenergic actions was studied using constant-flow perfused rat hearts and isolated rat ventricular myocytes. In isolated perfused hearts, the selective A2a receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2, 4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) potentiated adenosine-mediated decreases in isoproterenol (Iso; 10(-8) M)-elicited contractile responses (+dP/dtmax) in a dose-dependent manner. The effect of ZM-241385 on adenosine-induced antiadrenergic actions was abolished by the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-7) M), but not the selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191, 10(-7) M). The A2a receptor agonist carboxyethylphenethyl-aminoethyl-carboxyamido-adenosine (CGS-21680) at 10(-5) M attenuated the antiadrenergic effect of the selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), whereas CSC did not influence the antiadrenergic action of this agonist. In isolated ventricular myocytes, CSC potentiated the inhibitory action of adenosine on Iso (2 x 10(-7) M)-elicited increases in intracellular Ca2+ concentration ([Ca2+]i) transients but did not influence Iso-induced changes in [Ca2+]i transients in the absence of exogenous adenosine. These results indicate that adenosine A2a receptor antagonists enhance A1-receptor-induced antiadrenergic responses and that A2a receptor agonists attenuate (albeit to a modest degree) the antiadrenergic actions of A1 receptor activation. In conclusion, the data in this study support the notion that an important physiological role of A2a receptors in the normal mammalian myocardium is to reduce A1 receptor-mediated antiadrenergic actions.