Inhibition of late sodium current to reduce electrical and mechanical dysfunction of ischaemic myocardium.

This commentary on the review by DA Saint in the current issue of the British Journal of Pharmacology focuses on the pathological role of late I(Na) in the heart, the evidence supporting inhibition of late I(Na) as a therapeutic target in ischaemic heart disease, and the therapeutic applications and challenges for development of new late I(Na) inhibitors. Recent reports from a large clinical outcome trial (MERLIN) of ranolazine, a drug known to inhibit late I(Na), indicated that it was safe and reduced recurrent ischaemia and arrhythmic activity. In combination with other results indicating that inhibition of late I(Na) reduces ischaemia, myocardial Ca(2+) overload, and electrical and mechanical dysfunction when late I(Na) is increased, the new clinical trial results suggest that reduction of cardiac late I(Na) is safe and therapeutically beneficial.

Inhibition of the late sodium current as a potential cardioprotective principle: effects of the late sodium current inhibitor ranolazine.

Pathological conditions linked to imbalances in oxygen supply and demand (for example, ischaemia, hypoxia and heart failure) are associated with disruptions in intracellular sodium ([Na(+)](i)) and calcium ([Ca(2+)](i)) concentration homeostasis of myocardial cells. A decreased efflux or increased influx of sodium may cause cellular sodium overload. Sodium overload is followed by an increased influx of calcium through sodium-calcium exchange. Failure to maintain the homeostasis of [Na(+)](i) and [Ca(2+)](i) leads to electrical instability (arrhythmias), mechanical dysfunction (reduced contractility and increased diastolic tension) and mitochondrial dysfunction. These events increase ATP hydrolysis and decrease ATP formation and, if left uncorrected, they cause cell injury and death. The relative contributions of various pathways (sodium channels, exchangers and transporters) to the rise in [Na(+)](i) remain a matter of debate. Nevertheless, both the sodium-hydrogen exchanger and abnormal sodium channel conductance (that is, increased late sodium current (I(Na))) are likely to contribute to the rise in [Na(+)](i). The focus of this review is on the role of the late (sustained/persistent) I(Na) in the ionic disturbances associated with ischaemia/hypoxia and heart failure, the consequences of these ionic disturbances, and the cardioprotective effects of the antianginal and anti-ischaemic drug ranolazine. Ranolazine selectively inhibits late I(Na), reduces [Na(+)](i)-dependent calcium overload and attenuates the abnormalities of ventricular repolarisation and contractility that are associated with ischaemia/reperfusion and heart failure. Thus, inhibition of late I(Na) can reduce [Na(+)](i)-dependent calcium overload and its detrimental effects on myocardial function.

Effects of dipyridamole on coronary collateralization and myocardial perfusion in patients with ischaemic cardiomyopathy.

AIMS: There is evidence that oral dipyridamole, a nucleoside uptake blocker that increases myocardial adenosine levels, lessens myocardial ischaemia by inducing coronary collateral growth in animal models of ischaemic heart disease. However, whether dipyridamole can exert a similar effect in humans with coronary artery disease is controversial. METHODS AND RESULTS: We studied 30 male patients (mean age 55+/-9 years) with coronary artery disease and left ventricular systolic dysfunction (ejection fraction >40%). Patients were randomized into three matched groups. Group A patients (n=10) received dipyridamole alone at a dose of 75 mg t.i.d. orally for 8 weeks. Group B patients (n=10) underwent exercise training at 60% of peak .VO(2)three times a week for 8 weeks, and received dipyridamole. Group C patients (n=10) had neither exercise testing nor dipyridamole. On study entry and after 8 weeks all patients underwent an exercise test with gas exchange analysis, dobutamine stress echocardiography, 201-thallium planar myocardial scintigraphy, and coronary angiography. Peak .VO(2)increased significantly only in trained patients. Thallium uptake of the collateral-dependent myocardium, coronary collateral score and wall thickening score increased significantly only in groups receiving dipyridamole, the greatest improvement being in group B patients. Plasma adenosine levels were also the highest in group B (P<0.001 vs A and C). Correlations were found between changes in adenosine levels and increases of both thallium uptake (r=-0.70;P=0.001) and collateralization (r=0.72;P=0.001). CONCLUSION: Exercise training potentiates the effects of dipyridamole on coronary collateralization and myocardial perfusion in humans with ischaemic cardiomyopathy.

Selective attenuation by adenosine of arrhythmogenic action of isoproterenol on ventricular myocytes.

We examined whether adenosine equally attenuated the stimulatory effects of isoproterenol on arrhythmic activity and twitch shortening of guinea pig isolated ventricular myocytes. Transmembrane voltages and whole cell currents were recorded with patch electrodes, and cell twitch shortening was measured using a video-motion detector. Isoproterenol increased the action potential duration at 50% repolarization (APD50), L-type Ca2+ current [I(Ca(L))], and cell twitch shortening and induced delayed afterdepolarizations (DAD), transient inward current (I(Ti)), and aftercontractions. Adenosine attenuated the arrhythmogenic actions of isoproterenol more than it attenuated the effects of isoproterenol on APD50, I(Ca(L)), or twitch shortening. Adenosine (0.1-100 micromol/l) decreased the amplitude of DADs by 30 +/- 6% to 92 +/- 5% but attenuated isoproterenol-induced prolongation of the APD50 by only 14 +/- 4% to 59 +/- 4% and had no effect on the voltage of action potential plateau. Adenosine (30 micromol/l) inhibited I(Ti) by 91 +/- 4% but decreased isoproterenol-stimulated I(Ca(L)) by only 30 +/- 12%. Isoproterenol-induced aftercontractions were abolished by adenosine (10 micromol/l), whereas the amplitude of twitch shortening was not reduced. The effects of adenosine on twitch shortenings and aftercontractions were mimicked by the A1-adenosine receptor agonist CPA (N6-cyclopentyladenosine) and by ryanodine. In conclusion, adenosine antagonized the proarrhythmic effect of beta-adrenergic stimulation on ventricular myocytes without reducing cell twitch shortening.

Inhibition of phosphodiesterase type III before aortic cross-clamping preserves intramyocardial cyclic adenosine monophosphate during cardiopulmonary bypass.

UNLABELLED: Inotropes are often used to treat myocardial dysfunction shortly after cardiopulmonary bypass (CPB). beta-Adrenergic agonists improve contractility, in part by increasing cyclic adenosine monophosphate (cAMP) production, whereas phosphodiesterase type III inhibitors prevent its breakdown. CPB is associated with abnormalities at the beta-receptor level and diminished adenyl cyclase activity, both of which tend to decrease cAMP. These effects may be increased in the presence of preexisting myocardial dysfunction. We tested the hypothesis that inhibition of phosphodiesterase type III before global myocardial ischemia and pharmacologic arrest results in the preservation of intramyocardial cAMP concentration during CPB. Twenty adult patients undergoing coronary artery bypass grafting with CPB were studied. After CPB was instituted, a myocardial biopsy was obtained from the apex of the left ventricle. Patients were randomized to receive either placebo or milrinone (50 micro/kg) through the bypass pump 10 min before aortic cross-clamping. Another myocardial biopsy was performed adjacent to the left ventricular apex just before weaning from CPB. Myocardial cAMP concentration was determined by radioimmunoassay. Myocyte protein content was determined by the Bradford method by using a commercial kit. There were no significant demographic differences between the groups; however, patients in the Milrinone group had a lower left ventricular ejection fraction than placebo (41% +/- 13% vs 53% +/- 7%; P < 0.05). Patients who received milrinone had larger cAMP concentrations at the end of CPB compared with placebo (21 +/- 12.5 pmol/mg protein versus 12.8 +/- 2.2 pmol/mg protein; P < 0.05). The administration of milrinone before aortic cross-clamping is associated with increased intramyocardial cAMP concentration at the end of CPB. IMPLICATIONS: The administration of a single dose of milrinone before aortic cross-clamping resulted in significantly larger intramyocardial cyclic adenosine monophosphate concentration in myocardial biopsy specimens compared with controls.

A partial agonist of the A(1)-adenosine receptor selectively slows AV conduction in guinea pig hearts.

The use of full agonists of the A(1)-adenosine receptor (A(1)-ADOR) as antiarrhythmic agents is limited by their actions to cause high-grade atrioventricular (AV) block, profound bradycardia, atrial fibrillation, and vasodilation. It may be possible to avoid these undesired actions by use of partial agonists. We determined the effects of CVT-2759, a potential partial agonist of A(1)-ADORs, on guinea pig hearts. CVT-2759 (0.1-100 microM) increased the S-H interval of the isolated heart from 45 +/- 1 to 60 +/- 3 ms (P < 0. 01) with a half-maximal effect at 3.1 microM. CVT-2759 did not cause second-degree AV block. CVT-2759 significantly attenuated the actions of the full agonists N(6)-cyclopentyladenosine and adenosine. CVT-2759 caused a moderate slowing of atrial rate by 10 microM. In contrast, CVT-2759 was a full agonist to decrease cAMP content of rat adipocytes and Fischer rat thyroid line 5 cells. Results of radioligand binding assays indicated that CVT-2759 stabilized a high-affinity, G protein-coupled state of the A(1)-ADOR in membranes prepared from rat adipocytes but not in membranes prepared from the guinea pig brain. The results suggest that a weak A(1)-ADOR agonist, such as CVT-2759, may be useful to slow AV nodal conduction and thereby ventricular rate without causing AV block, bradycardia, atrial arrhythmias, or vasodilation.

Synthesis and biological effects of a new series of 2-amino-3-benzoylthiophenes as allosteric enhancers of A1-adenosine receptor.

New derivatives of PD 81,723, an allosteric enhancer of agonist binding to the A1-adenosine receptor, have been synthesized and evaluated in an intact cell assay. Compounds 3a, 3o and 3p appeared to be more potent than PD 81,723 and at a concentration of 0.1 microM caused significant reductions of cAMP content of CHO cells expressing the human A1-adenosine receptor. Compounds 4e and 4o appeared to be allosteric enhancers at a low concentration and antagonists at a higher concentration, whereas compounds 3c, 3g, 3s and 4l appeared to be weak antagonists that are also allosteric enhancers at the higher concentration of 10 microM.

Role of A(2A)-adenosine receptor activation for ATP-mediated coronary vasodilation in guinea-pig isolated heart.

1. Adenosine-5'-triphosphate (ATP) and adenosine are potent coronary vasodilators. ATP is rapidly converted to adenosine by ectonucleotidases. We examined whether coronary vasodilation caused by exogenous ATP is mediated by P(2) receptor activation or by A(2A)-adenosine receptor activation. 2. Effects of interventions on coronary conductance were determined by measuring coronary perfusion pressure in guinea-pig isolated hearts perfused at a constant flow of 10 ml min(-1). 3. ATP and adenosine both caused sustained, concentration-dependent increases of coronary conductance. Maximal responses to both agonists were equivalent. The values of pD(2) (+/-s.e.mean) for ATP and adenosine were 6.68+/-0.04 and 7.06+/-0.05, respectively. Adenosine was significantly more potent than ATP (P<0. 0001, n=10). 4. The values of pIC(50) for the selective A(2A)-adenosine receptor antagonist SCH58261 to antagonize equivalent responses to ATP and adenosine were 8.28+/-0.08 and 8.28+/-0.06 (P=0.99, n=6), respectively. 5. The non-selective adenosine receptor antagonists xanthine amine congener (XAC) and CGS15943 antagonized similarly the equivalent vasodilations caused by ATP (pIC(50) values 7.48+/-0.04 and 7.45+/-0.06, respectively) and adenosine (pIC(50) values 7. 37+/-0.13 and 7.56+/-0.11). 6. In contrast to ATP and adenosine, the two P(2) agonists 2-methylthio-ATP and uridine-5'-triphosphate failed to cause stable increases of coronary conductance, caused desensitization of vasodilator responses, and were not antagonized by SCH 58261, 8-parasulphophenyltheophylline, or XAC. 7. Glibenclamide attenuated coronary vasodilations caused by ATP and adenosine by 88 and 89%, respectively, but failed to attenuate those caused by 2-methylthio-ATP. 8. These results strongly suggest that sustained, submaximal coronary vasodilation caused by exogenous ATP is entirely mediated by adenosine acting upon A(2A)-adenosine receptors.

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

A2A-adenosine receptor reserve for coronary vasodilation.

BACKGROUND: Adenosine is a potent coronary vasodilator and causes an increase of coronary blood flow by activation of A2A-adenosine receptors (A2A-AdoRs). The purpose of this study was to test the hypothesis that the high potency of adenosine and adenosine analogues to cause coronary vasodilation is explained by the presence of a large A2A-AdoR reserve ("spare receptors"). METHODS AND RESULTS: A novel, irreversible antagonist of A2A-AdoRs was used to inactivate receptors and reduce the response to agonist. Agonist-induced increases of coronary conductance before and after exposure of hearts to the irreversible antagonist were compared. Three agonists were studied: 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS21680), adenosine, and 2-chloro-N6-cyclopentyladenosine (CCPA). Data were analyzed to determine agonist KA (equilibrium dissociation constant) and EC50 values. Values of KA for activation of A2A-AdoRs by CGS21680, adenosine, and CCPA were 105, 1800, and 2630 nmol/L, respectively. In contrast, values of EC50 for CGS21680, adenosine, and CCPA to increase coronary conductance were 1.5, 85, and 243 nmol/L, respectively. By use of the law of mass action, it was calculated that half-maximal responses to CGS21680, adenosine, and CCPA occurred when only 1.3%, 5%, and 9%, respectively, of A2A-AdoRs were occupied by agonist. CONCLUSIONS: Receptor reserves for 3 A2A-AdoR agonists were large. The receptor reserve for A2A-AdoRs to cause an increase of coronary conductance can explain both the high potency of adenosine to cause coronary vasodilation and the observation that an A2A-AdoR agonist can cause coronary vasodilation without systemic effects.

Potentiating effect of acetylcholine on stimulation by isoproterenol of L-type Ca2+ current and arrhythmogenic triggered activity in guinea pig ventricular myocytes.

INTRODUCTION: The objective of this study was to determine whether the effect of isoproterenol (Iso) to increase L-type Ca2+ current [I(Ca(L))] and action potential duration (APD) was potentiated in ventricular myocytes following termination of an exposure of these cells to acetylcholine (ACh), and whether this potentiating effect of ACh could be arrhythmogenic. METHODS AND RESULTS: Transmembrane currents and potentials of guinea pig isolated ventricular myocytes were measured using the whole cell, patch clamp technique. Stimulation of I(Ca(L)) and prolongation of APD caused by Iso (10 nmol/L) were attenuated in the presence of ACh (10 micromol/L), but were transiently enhanced by 111% +/- 20% and 214% +/- 44%, respectively, following termination of a 2- to 4-minute exposure of myocytes to ACh. No changes were observed in the absence of Iso. Both the amplitude and incidence of Iso-induced transient inward current, afterdepolarizations, and sustained triggered activity were greater immediately after termination of exposure to ACh than before application of ACh. CONCLUSION: Stimulation by Iso of I(Ca(L)) is transiently enhanced in guinea pig ventricular myocytes following termination of exposure of these cells to ACh. The rebound increase of Iso-stimulated I(Ca(L)) is associated with an increase of APD and induction of arrhythmogenic triggered activity.

Inverse agonists and neutral antagonists of recombinant human A1 adenosine receptors stably expressed in Chinese hamster ovary cells.

Receptor antagonists can be classified as neutral antagonists or antagonists with inverse agonist activity based on their effectiveness to reduce the spontaneous agonist-independent activity of receptors. The goals of this study were to (1) demonstrate that A1-adenosine receptors (A1AdoRs) expressed at high density (4000-8000 fmol/mg of protein) in Chinese hamster ovary (CHO) cells cause constitutive activation of inhibitory G proteins and inhibition of adenylyl cyclase activity and (2) identify both neutral A1AdoR antagonists and antagonists with inverse agonist activity. The activity of A1AdoR agonists and antagonists was determined by assays of both specific binding of [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) to membranes and cAMP content of intact cells in the presence of adenosine deaminase (2-5 units/ml). The A1AdoR agonist N6-cyclopentyladenosine (CPA) significantly increased binding of [35S]GTPgammaS by 241 +/- 7% compared with control. The A1AdoR antagonists N-0861, N-0840, and WRC-0342 did not alter binding of [35S]GTPgammaS, whereas the antagonists 8-cyclopentyl-1, 3-dipropylxanthine (CPX), CGS-15943, xanthine amine congener, and WRC-0571 significantly reduced binding of [35S]GTPgammaS by 28-53% from control, respectively. The effects of both the agonist N6-cyclopentyladenosine (CPA) and the antagonist CPX to alter binding of [35S]GTPgammaS were attenuated by 1 micro M N-0861. CPA reduced cAMP content of forskolin-stimulated CHO:A1AdoR cells, and N-0861 and WRC-0342 did not alter cAMP content, but the antagonists CPX and WRC-0571 increased the cAMP content of CHO:A1AdoR cells. The effects of both CPX and WRC-0571 to increase cAMP content of forskolin-stimulated CHO:A1AdoR cells were attenuated by either N-0861 or WRC-0342. The results indicate that both N-0861 and WRC-0342 are neutral antagonists, whereas both CPX and WRC-0571 are antagonists with inverse agonist activity.

The A2A adenosine receptor mediates coronary vasodilation.

The coronary vasodilation caused by adenosine is due to activation of A2 adenosine receptors (A2AdoRs), but the subtype or subtypes of A2AdoR (A2A and/or A2B) that mediate this action are uncertain. The purpose of this study was to test the hypothesis that A2AAdoRs mediate coronary vasodilation caused by exogenous or endogenous adenosine in the guinea pig isolated perfused heart. The newly described A2AAdoR antagonist SCH58261 was used to selectively block A2AAdoRs. Attenuations by SCH58261 of increases in coronary conductance (A2 response) and of atrioventricular nodal conduction time (A1 response) caused by exogenous and endogenous adenosine and by agonists with relative selectivity for A2A and A1AdoRs were measured. The CGS21680-induced increase of coronary conductance was antagonized by SCH58261 in a concentration-dependent and competitive manner with a KB value of 5.01 nm. Also reversed by SCH58261 (60 nmol/L) were the increases in coronary conductance caused by the relatively selective A1AdoR agonists CCPA (70 nM), and (R)-(-)N(b)-(2-phenyl-isopropyl)adenosine (60 nM) but not those caused by sodium nitroprusside (1.2 microM) and diltiazem (0.4 microM). SCH58261 (< or = 100 nM) did not attenuate the A1AdoR-mediated prolongations of S-H interval caused by either adenosine or CCPA. SCH58261 attenuated the coronary vasodilation caused by 50 nM adenosine with an IC50 value of 6.8 +/- 0.6 nM. The coronary vasodilations caused by the nucleoside uptake inhibitor draflazine and the adenosine kinase inhibitor iodotubercidin were completely reversed by 60 nM SCH58261 or adenosine deaminase (7 U/ml). Thus, the A2AAdoR plays a major role as mediator of coronary vasodilation caused by exogenous and endogenous adenosine and by AdoR agonists.

Differential A1 adenosine receptor reserve for two actions of adenosine on guinea pig atrial myocytes.

Adenosine activates adenosine-induced inwardly rectifying K+ current (IKAdo) and inhibits isoproterenol (100 nM)-stimulated L-type Ca2+ current (beta-ICa,L) of guinea pig atrial myocytes with EC50 values of 2.17 and 0.20 microM, respectively. We determined whether this 11-fold difference in potency of adenosine is due to the existence of a greater A1 adenosine receptor reserve for the inhibition of beta-ICa,L than for the activation of IKAdo. Atrial myocytes were pretreated with vehicle (control) or the irreversible A1 adenosine receptor antagonist 8-cyclopentyl-3-[3-[[4-(fluorosulfonyl)benzoyl]oxy]propyl]-1-propylxa nthine (FSCPX) (10 and 50 nM) for 30 min, and after a 60-min washout period, concentration-response curves were determined for the adenosine-induced activation of IKAdo and inhibition of beta-ICa,L. Pretreatment of atrial myocytes with 10 nM FSCPX reduced the maximal activation of IKAdo by 60% (7.9 +/- 0.2 to 3.2 +/- 0.1 pA/pF). In contrast, a higher concentration of FSCPX (50 nM) was required to reduce the maximal inhibition of beta-ICa,L by 39% (95 +/- 4% to 58. 7 +/- 5.6%) and caused a 15-fold increase in the EC50 value of adenosine. Values of the equilibrium dissociation constant (KA) for adenosine to activate IKAdo and inhibit beta-ICa,L, estimated according to the method of Furchgott, were 2.7 and 5.6 microM, respectively. These values were used to determine the relationship between adenosine receptor occupancy and response. Half-maximal and maximal activations of IKAdo required occupancies of 40% and 98% of A1 adenosine receptors, respectively. In contrast, occupancies of only 4% and 70%, respectively, of A1 adenosine receptors were sufficient to cause half-maximal and maximal inhibitions of beta-ICa, L. Consistent with this result, a partial agonist of the A1 adenosine receptor SHA040 inhibited beta-ICa,L by 60 +/- 3.5% but activated IKAdo by only 18.1 +/- 2.5%. The results indicate that the A1 adenosine receptor is coupled more efficiently to an inhibition of beta-ICa,L than to an activation of IKAdo.

Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology.

Cardiomyocytes and vascular cells readily form, transport, and metabolize the endogenous adenine nucleoside adenosine and act to regulate both interstitial and plasma adenosine concentrations. Cardiovascular cells also have membrane adenosine receptors. Cell and tissue distributions, signal transduction pathways, and pharmacology of each of the four subtypes of adenosine receptors are subjects of intense investigation. The A1-adenosine receptors mediate the negative dromotropic, chronotropic, inotropic, and the anti-beta-adrenergic actions of adenosine. Activation of A(2A)- and perhaps A(2B)-adenosine receptors causes vasodilation. Evidence of novel actions mediated by A(2B)- and A3-adenosine receptors is accumulating. Adenosine is cardioprotective during episodes of cardiac hypoxia/ischemia; several potential mechanisms may be involved. Pharmacologic tools are currently available for laboratory investigation of the actions of adenosine, and the development of adenosine receptor subtype-selective agonists and antagonists for therapeutic purposes is beginning.

Agonist-independent effect of an allosteric enhancer of the A1 adenosine receptor in CHO cells stably expressing the recombinant human A1 receptor.

The allosteric enhancer PD 81,723, a 2-amino-3-benzoylthiophene derivative, has been shown to potentiate agonist binding to A1 adenosine receptors (A1AdoRs) and to enhance the functional effects of adenosine and adenosine analogs. The objective of this study was to determine whether the apparent agonist-independent effect of PD 81,723 observed in CHO cells stably expressing the recombinant human A1AdoR was due to the potentiation of the action of endogenous adenosine, to the presence of constitutive receptor activity and/or to the binding of PD 81,723 to the agonist binding site of the A1AdoR. The allosteric enhancer PD 81,723, the A1AdoR agonist (R)-N6-(2-phenylisopropyl)adenosine and adenosine all significantly inhibited forskolin-stimulated cAMP accumulation in intact cells and increased [35S]-5'-(gamma-thio)triphosphate binding to cell membranes. The effects of adenosine on cAMP formation and [35S]-5'-(gamma-thio)triphosphate binding were attenuated by adenosine deaminase, but the effects of PD 81,723 were not. In the presence of ADA, the A1AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine increased forskolin-stimulated cAMP accumulation in cells expressing the recombinant human A1AdoR but not in nontransfected CHO cells. In binding experiments, the agonist (R)-N6-(2-phenylisopropyl)adenosine, but not PD 81,723, significantly displaced the specific binding of the A1AdoR agonist [3H]-N6-cyclohexyladenosine and the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine. The results of this study demonstrate that in CHO cells stably expressing the recombinant human A1AdoR, the agonist-independent effect of PD 81,723 is not due to potentiation of the action of endogenous adenosine or mediated by the binding of the allosteric enhancer to the agonist binding site of the recombinant human A1AdoR. It is possible that these effects are due to potentiation of constitutive receptor activity by PD 81,723.

Binding of the novel nonxanthine A2A adenosine receptor antagonist [3H]SCH58261 to coronary artery membranes.

This study demonstrates quantification of A2A adenosine receptors (A2AAdoRs) in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells. Radioligand binding assays were performed using the new selective A2AAdoR antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-epsilon]-1,2,4-triazolo[1,5-c)pyrimidine ([3H]SCH58261). Binding of the radioligand to membranes was rapid, reversible, and saturable. The densities of A2AAdoRs in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells were 900 +/- 61, 892 +/- 35, and 959 +/- 76 fmol/mg protein, respectively. Equilibrium dissociation constants (Kd values) calculated from results of saturation binding assays were 2.19, 1.20, and 0.81 nmol/L, and Kd values calculated from results of association and dissociation assays were 2.42, 1.01, and 0.40 nmol/L for [3H]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells, respectively. The specific binding of [3H]SCH58261 as a percentage of total binding at a radioligand concentration equal to the Kd value was 65% to 90% in the three membrane preparations. The order of ligand potencies determined by assay of competition binding to sites in porcine coronary membranes using [3H]SCH58261, unlabeled antagonists (SCH58261, 8-(3-chlorostyryl)caffeine [CSC], and xanthine amine congener [XAC]), and unlabeled agonists ([3H]2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoaden osine [CGS 21680], 2-hexynyl-5'-N-ethylcarboxamidoadenosine [HE-NECA], [3H]5'-N-ethylcarboxamidoadenosine [NECA], and R(-)N6-(2-phenylisopropyl)adenosine [R-PIA]) was SCH58261 > HE-NECA = CSC = CGS 21680 = XAC > NECA = R-PIA. The Hill coefficients of displacement by A2AAdoR ligands of [3H]SCH58261 binding were not significantly different from unity, indicating that [3H]SCH58261 bound to a group of homogeneous noninteracting sites in all membrane preparations. The order of ligand potencies to compete for [3H]SCH58261 binding sites in porcine striatal and PC12 cell membranes was, in part, different from that for porcine coronary arterial membranes. The different rank orders of potencies for agonists and antagonists at A2A receptors of porcine coronary arteries, striatum, and PC12 cells and significant differences in absolute values of potency of ligands in the three preparations may indicate the existence of different subtypes of A2AAdoRs. The antagonist radio-ligand [3H]SCH58261 should be of value for pharmacological characterization of A2A adenosine receptors in other preparations.

A novel irreversible antagonist of the A1-adenosine receptor.

We determined the effects of 8-cyclopentyl-3-[3-[[4-(fluorosulfonyl)benzoyl]oxy]propyl]-1-propylxanth ine (FSCPX), a putative irreversible antagonist of the A1-adenosine receptor, on cardiac A1-adenosine receptor-mediated responses and on the specific binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) to guinea pig cardiac and brain membranes. FSCPX (5 microM) completely reversed the increase in K+ current of guinea pig atrial myocytes caused by 100 microM adenosine (259 +/- 30 to 20 +/- 7 pA) but had no significant effect on K+ currents caused by either 0.5 microM carbachol or 100 microM GTP gamma S. The attenuation of K+ current by FSCPX was both time and concentration dependent and persisted after washout of the antagonist. Pretreatment of atrial myocytes with FSCPX (50 nM) markedly attenuated the activation of K+ current and the inhibition of isoproterenol-stimulated I(Ca,L) caused by adenosine by 90.1% and 84.2%, respectively, but did not alter the responses of atrial myocytes to carbachol. FSCPX (1 microM) irreversibly antagonized the A1-adenosine receptor-mediated increase in atrioventricular nodal conduction time of isolated perfused guinea pig hearts from 10.5 +/- 0.5 to 0.7 +/- 0.6 msec but did not significantly alter the A2-adenosine receptor-mediated decrease in coronary resistance. Preincubation of guinea pig cardiac membranes with 0.1, 1.0, or 3.0 microM FSCPX for 30 min reduced the Bmax of [3H]CPX binding by 41 +/- 10%, 67 +/- 6%, and 80 +/- 1% (mean +/- standard error, three experiments), respectively, with no significant change in the Kd. Similarly, 0.1 and 1.0 microM FSCPX irreversibly reduced the binding of [3H]CPX to guinea pig forebrain membranes by 65 +/- 5% and 83 +/- 2% (four experiments), respectively, but did not reduce the binding of [3H]CGS 21680, an A2a-adenosine receptor agonist, to striatal membranes. FSCPX did not affect the potency of 5'-guanylylimidodiphosphate to inhibit the binding of [3H]CCPA, an A1-adenosine receptor agonist, to brain membranes. The results indicate that FSCPX is a specific, irreversible, A1-adenosine subtype-selective receptor antagonist.

Cellular basis for the negative dromotropic effect of adenosine on rabbit single atrioventricular nodal cells.

The effects of adenosine on action potentials, rate-dependent activation failure (the cellular basis for second-degree atrioventricular [AV] block), and the recovery of excitability in rabbit isolated single AV nodal cells were studied using the whole-cell patch-clamp technique. Adenosine (1 micromol/L) shortened the duration, depressed the amplitude, and reduced the rate of rise of the AV nodal cell action potential. Adenosine (10 micromol/L) caused a significant hyperpolarization (7 +/- 1 mV) of AV nodal cells. Adenosine increased the occurrence and the rate dependence of activation failure (Wenckebach periodicity) of AV nodal cells: this effect was concentration dependent and mediated by A1 adenosine receptors. The rate-dependent activation failure caused by adenosine was associated with a prolongation of the effective refractory period by 18 +/- 2 ms (P < .05), an increase in the duration of activation delay, and an elevation (from 0.22 +/- 0.04 to 0.30 +/- 0.03 nA, P < .05) of the threshold current amplitude required to activate AV nodal cells. The results suggest that the slowed recovery of excitability of AV nodal cells caused by adenosine forms the cellular basis for adenosine-induced second-degree AV block. Adenosine decreased ICa,L and activated IK,ADO of AV nodal cells. These actions of adenosine on ion currents may contribute to the effect of this nucleoside to depress excitability of AV nodal cells. The enhancement by adenosine of rate-dependent activation failure of AV nodal cells implies that the negative dromotropic effect of adenosine should be more pronounced during an episode of supraventricular tachycardia than during normal rhythm.

1,3-Dipropyl-8-[2-(5,6-epoxy)norbornyl]xanthine, a potent, specific and selective A1 adenosine receptor antagonist in the guinea pig heart and brain and in DDT1MF-2 cells.

The objective of this study was to characterize the adenosine receptor (AdoR) antagonistic properties of a newly synthesized alkylxanthine, 1,3-dipropyl-8[2-(5,6-epoxy)norbornyl]xanthine (ENX), and compare them to those of 1,3-dipropyl-8-(cyclo-pentyl)xanthine (CPX), 1,3-dipropyl-8-(3-noradamantyl)xanthine (NAX) and (+/-)-N6-endo-norbornan-2-yl-9-methyladenine (N-0861). The potencies and selectivities of ENX, CPX, NAX and N-0861 were determined by functional studies of guinea pig isolated perfused hearts, and by radioligand binding assays for A1 and A2a AdoRs in the guinea pig forebrain and striatum. ENX competitively antagonized A1 AdoR-mediated prolongations of atrioventricular nodal conduction time caused by Ado or by 2-chloro-N6-cyclopentyladenosine, but not those caused by carbachol (0.14 microM) or MgCl2 (3 mM). Schild analysis of 2-chloro-N6-cyclopentyladenosine-antagonist competition curves yielded pA2 values for ENX, CPX and NAX of 8.45 +/- 0.19, 8.55 +/- 0.28 and 8.79 +/- 0.15, respectively. ENX (30 microM) and N-0861 (30 microM) did not attenuate the A2 AdoR-mediated increase in coronary conductance caused by adenosine. CPX and NAX attenuated the coronary vasodilation caused by adenosine with IC50 values of 1.5 and 7.1 microM, respectively. Radioligand binding assays revealed that ENX, CPX and NAX and N-0861 had a 400-, 209-, 110- and 10-fold greater affinity, respectively, for A1 than for A2a AdoRs of guinea pig brain membranes. Thus, ENX was equipotent with CPX and NAX and more potent than N-0861 (pA2 = 6.2) as an antagonist at A1 AdoRs, but had lower affinity for A2 AdoRs in guinea pig hearts and brain striatum than did either CPX or NAX. In DDT1 MF-2 cells, all three alkylxanthines had similar affinities for A1 AdoRs, whereas the affinity of N-0861 for A1 AdoRs was significantly lower. ENX appears to be the most A1 AdoR subtype-selective of the alkylxanthine class of AdoR antagonists reported to date.