Beneficial role of adenosine in myocardial ischemic and reperfusion injury

Adenosine has been recognized as an important regulator of myocardial function and coronary vascular tone in the ischemic myocardium. 5′-Nucleotides, which converts 5′-adenosine monophosphate to adenosine, is responsible for adenosine production in the ischemic myocardium. We have found that α₁-adrenergic receptors activated in ischemic hearts increase both 5′-nucleotidase activity and adenosine production. The primary role of endogenous adenosine is to increase coronary blood flow through adenosine A₂ receptors. This adenosine-induced coronary vasodilation is amplified by α₂-adenosine stimulation. Stimulation of adenosine A₂ receptors also attenuates both free radical generation by activated leukocytes and the aggregation of platelets. In turn, adenosine A₁ receptor activation attenuates β-adrenoceptor-mediated increases in myocardial contractility, Ca²⁺ influx into myocytes and norepinephrine release form the presynaptic nerves. Any or all of these effects may attenuate ischemic and reperfusion injury. Indeed, endogenous and exogenous adenosine appears to ameliorate contractile dysfunction and infarct size following ischemia and reperfusion. Therefore, we believe that adenosine plays an important role as a modulator of ischemic and reperfusion injury. © 1993 Wiley-Liss, Inc.     

A positive inotropic effect of adenosine in cardiac preparations of right atria from diseased human hearts

The actions of adenosine in the human atrium are of clinical relevance, for instance, to stop supraventricular arrhythmias. The purpose of the present study was to re-evaluate the inotropic effect of adenosine in the human atrium. We studied the effect of adenosine (cumulatively applied) on force of contraction in isolated electrically driven right atrial preparations from patients with coronary heart disease undergoing cardiac surgery. It has been known for some time that adenosine via A₁-adenosine receptors can elicit a negative inotropic effect in human atrial preparations. We report here that in 25% of the patients studied, a positive inotropic effect to adenosine was detectable. The A₁-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine attenuated this effect. Hence, we conclude that at least in some patients, adenosine can lead to a hitherto overlooked positive inotropic effect.     

Mechanism of the negative dromotrophic effect of adenosine 5′-triphosphate in the guinea pig heart in vivo

To test the hypothesis that the vagus nerve does not play a major mechanistic role in the cardiac electrophysiologic action of adenosine 5′-triphosphate (ATP), the negative dromotropic actions of the nucleotide and adenosine on atrioventricular (AV) nodal conduction were quantitated in an in vivo guinea pig model. Anesthetized animals were divided into three groups. In group 1, ATP and adenosine (0.1–10.0 μmol/kg, iv) were given at random as a rapid bolus during right atrial pacing (paced cycle length 220–270 msec). This protocol was repeated following the administration of atropine (0.2 mg/kg, iv) followed by propranolol (1 mg/kg, iv). In group 2, ATP and adenosine (1.6 μmol/kg, iv) were given in the absence and presence of the selective A₁-adenosine receptor antagonist, N⁶-endonorbornan-2-yl-9-methyladenine (N-0861, 30 μmol/kg, iv), and in group 3, ATP and adenosine were given as in group 2 but instead of N-0861, an adenosine transport blocker, dipyridamole (250 μg/kg, iv), was used. Standard lead I and II electrocardiogram, right atrial, right ventricular, and His bundle electrogram as well as systemic arterial blood pressure were continuously monitored and recorded. AV nodal conduction time was quantitated as AH interval. ATP and adenosine were equipotent in suppressing AV nodal conduction. For example, maximal AH interval following the highest dose of ATP and adenosine was 102 ± 10 and 112 ± 7 msec, respectively. These effects were not significantly altered by either muscarinic cholinergic or β-adrenergic blockade. Furthermore, N-0861 attenuated and dipyridamole enhanced the effects of ATP and adenosine similarly. It was concluded that the electrophysiologic action of ATP in the guinea pig AV node in vivo is independent of the vagus nerve and mediated by its degradation to adenosine and the action of the latter on A₁-adenosine receptors. © 1993 Wiley-Liss, Inc.     

Purinergic responses in the feline pulmonary vascular bed

Adenosine and ATP produce dose-dependent, tone-dependent response in the pulmonary vascular (PV) bed, under conditions of controlled pulmonary blood flow and constant left atrial pressure in vivo in intact-chest, spontaneously breathing cats. At low, resting PV tone, adenosine produces dose-dependent vasoconstrictor responses by acting on “A₁-like” adenosine receptors which activate a phospholipase and thromboxane release. ATP Produces dose-dependent vasoconstrictor responses, in part, following its metabolism to adenosine, by acting on “A₁-like” adenosine receptors and by acting on P_2x receptors. At elevated PV tone, data to date support that adenosine and ATP produce dose-dependent vasodilator responses by acting on A₂ and P_2Y receptors, respectively. Mechanisms to explain how a change in vascular tone alters the response of the vascular bed to a substance are not known. Moreover, the role of purinoceptors in pulmonary vascular disease is not known. Identification of selective pharmacological probes for purinoceptors in the pulmonary vascular bed is necessary to investigate their role in the development of pulmonary hypertension of acute and chronic lung disease. © 1993 Wiley-Liss, Inc.     

Potential of adenosine receptor agonists for the prevention and treatment of coronary artery disease and acute myocardial infarction

Adenosine is an ubiquitously produced autocoid which mediates its effects via four receptor subtypes (A₁, A_2A, A_2B and A₃) which show a relatively widespread tissue distribution. Adenosine itself is upregulated in vascular tissue in response to hypoxia and induces vasodilation as a homeostatic response. The vasodilator effects of adenosine are exploited clinically, where it is used as a pharmacological vasodilator during stress testing. More recently, it has become clear that adenosine and adenosine agonists possess significant potential for cardioprotection. Thus, given prior to an ischaemic insult, adenosine and adenosine A₁ receptor agonists can reduce infarct size, reduce arrhythmia and improve post-ischaemic cardiac function. In addition, when given during myocardial ischaemia just prior to reperfusion, adenosine and adenosine A_2A receptor agonists can inhibit neutrophil adhesion, activation and infiltration into post-ischaemic myocardium, thereby inhibiting lethal reperfusion injury and further salvaging myocardial tissue. There is also some evidence that stimulation of adenosine A₃ receptors can protect myocardium. The realisation that adenosine analogues possess cardioprotective activity has stimulated research to identify subtype selective analogues with the optimal profile for use in acute myocardial infarction (MI) and cardiac surgery. Several compounds have reached the stage of early clinical development and hold significant promise as future therapies. In addition to direct cardioprotective effects, adenosine A₁ selective agonists may also indirectly benefit the heart via their metabolic effects. Thus, adenosine A₁ receptor stimulation results in a marked suppression of lipolysis in adipose tissue, which in turn leads to a reduction in the levels of circulating free fatty acids, triglycerides and very low density lipoproteins. Triglycerides are now accepted as an independent risk factor for mortality from coronary artery disease. Thus, the potential exists for the development of orally-active selective A₁ receptor agonists for the treatment of hypertriglyceridaemic patients to reduce the incidence of primary or secondary coronary events. Progress is being made in the design of such A₁ selective agonists.Therefore, the next decade should see advances in the therapeutic application of adenosine agonists in the porphylactic treatment of coronary artery disease and in acute MI.     

REFLEXES MEDIATED BY CARDIAC SYMPATHETIC AFFERENTS DURING MYOCARDIAL ISCHAEMIA: ROLE OF ADENOSINE

1. Myocardial ischaemia and infarction activate vagal and sympathetic sensory endings in the ischaemic myocardium, resulting in powerful reflex effects. The vagal afferents are either mechano- or chemosensitive, whereas sympathetic afferents may be mechano-, chemosensitive or both. 2. Activation of vagal afferents results in sympathoinhibitory, cardioinhibitory, vasodepressor responses. Cardiac sympathetic afferents activated during myocardial ischaemia mediate sympathoexcitatory, vasoconstrictor cardioaccelerator responses. 3. The focus of the present review is on the activation of sympathetic afferents by myocardial ischaemia and on the resulting reflex responses that they mediate. 4. These endings are more likely to be activated as the degree of ischaemia progresses from subendocardial towards transmural. They are evenly distributed between the anterior and inferoposterior wall. Although it has been suggested that these endings are activated by bradykinin, recent evidence indicates that they are activated by adenosine released from the ischaemic myocardium. Results from our laboratory indicate that this effect is due to the activation of adenosine A₁, but not adenosine A₂ receptors. 5. Activation of ventricular vagal and sympathetic afferent fibres during myocardial ischaemia in humans is responsible for the autonomic changes observed and, in the case of the sympathetic afferents, for the sensation of angina pectoris.     

Antagonism of novel inotropic agents at A1 adenosine receptors and m-cholinoceptors in human myocardium

Summary The effects of the new inotropic agents saterinone, sulmazole, UD-CG 212.C1 and milrinone at A₁ adenosine receptors and m-cholinoceptors were evaluated in human myocardium from patients with heart failure. At A₁ adenosine receptors, all compounds inhibited ³H-DPCPX-binding to ventricular membrane preparations at micromolar concentrations. As judged from the K_i-values, the rank order of potency was saterinone > sulmazole > UD-CG 212.C1 > milrinone. The new inotropic agents also displaced the binding of ³H-QNB at m-cholinoceptors. Except for saterinone, the concentration ranges of mean K_i-values were considerably higher at m-cholinoceptors than at A₁ adenosine receptors. The rank order of potency was saterinone > sulmazole > UD-CG 212.Cl > milrinone. Competition of the A₁ adenosine receptor agonist R-PIA to ³H-DPCPX-binding showed a biphasic curve with a shallow slope (Hill coefficient n_H = 0.63) and revealed two affinity states of the A₁ adenosine receptor. In the presence of guanine nucleotides [Gpp(NH)p], the competition curve showed one low affinity class of binding sites and was shifted to the right. In contrast, the competition curves of the new inotropic agents were characterized by a monophasic, steeper slope (mean Hill coefficient n_H = 0.98). Guanine nucleotides had no effect. Similar results were obtained with saterinone and carbachol at m-cholinoceptors. Competition with carbachol revealed three affinity states of the m-cholinoceptor, the superhigh affinity binding was reversed by Gpp(NH)p. Competition with saterinone revealed one class of binding sites which was not influenced by Gpp(NH)p. Accordingly, in isolated, electrically driven human atrial trabeculae, the negative inotropic effect of adenosine was antagonized concentration-dependently by saterinone, sulmazole and UD-CG 212.Cl. Similarly the negative inotropic effect of carbachol was antagonized concentration-dependently by saterinone. It is concluded that the new inotropic agents bind to A₁ adenosine receptors and that their interaction is of antagonist nature. This mechanism might contribute to their capacity to enhance force of contraction by stimulation of cAMP-formation in addition to phosphodiesterase inhibition. The effects of saterinone may be partially due to antagonism at m-cholinoceptors. This is presumably not the case with the other inotropic agents studied given their low affinity for this receptor.Send offprint requests to M. Böhm at the above addressSupported by the Deutsche Forschungsgemeinschaft     

Evidence against a role of nitric oxide in the indirect negative inotropic-effect of M-cholinoceptor stimulation in human ventricular myocardium

Nitric oxide (NO) has been reported to mediate several effects in response to muscarinic cholinergic stimulation in cardiovascular tissues. Recently, an attenuation of guinea pig cardiac myocyte contraction by NO has been described. The aim of the present study was to determine whether the indirect negative inotropic effect of M-cholinoceptor stimulation in human myocardium is in part due to an effect of endogenous NO. Therefore, the effect of carbachol was studied under control conditions and during inhibition of NO-synthase by pretreatment with N^G-monomethyl-l-arginine (NMMA). Functional experiments were performed in isolated, electrically driven (1 Hz, 37°C) left ventricular papillary muscle strips of human myocardium. Since cytokines have been reported to be increased in the serum of patients with heart failure and could induce NO-synthase activity in failing myocardium, we compared samples from nonfailing and terminally failing (classified as NYHA IV) hearts. The indirect negative inotropic effect of carbachol (10 mol/l) was studied in the presence of the \-adrenoceptor agonist isoprenaline (0.03 mol/l).After stimulation with isoprenaline, carbachol significantly (P < 0.05) reduced force of contraction. This effect was diminished in failing myocardium compared to nonfailing, probably due to the diminished inotropic response most likely due to the lower cAMP levels in response to \-adrenoceptor stimulation in the former condition. Pretreatment with NMMA (100 mol/l) altered the antiadrenergic effect of carbachol neither in nonfailing nor in failing preparations. Furthermore, inhibition of guanylyl cyclase, the target enzyme of NO, by preincubation with methylene blue (10 mol/l) for 30 min had no effect on the carbachol-induced decrease in force of contraction. Basal force of contraction, as well as the positive inotropic effect of isoprenaline remained unaffected by NMMA or methylene blue.The present study provides evidence that the indirect negative inotropic effect of M-cholinoceptor agonists is not due to an effect of NO in the human myocardium. Furthermore, the well known enhancement of cGMP in response to M-cholinoceptor stimulation appears not to be involved in this antiadrenergic effect.     

The coexistence of adenosine A1 and A2 receptors in guinea-pig aorta

The effects of adenosine, 5'-(N-ethyl)carboxamidoadenosine (NECA), 2-chloroadenosine (2-CA), No-cyclohexyladenosine (CHA) and N6(R-2-phenylisopropyl)-adenosine (R-PIA) on the tone of phenylephrine-constricted guinea-pig isolated aorta have been examined. For aortic relaxation the analogues exhibited the following rank order of potency: NECA > adenosine > 2-CA > R-PIA > CHA. This is consistent with previous reports that relaxation of this tissue is mediated by the adenosine A₂ receptor. An unexpected finding was that R-PIA, 2-CA and CHA all induced contractions at concentrations lower than were required for relaxation, giving a biphasic dose-response curve. Neither NECA nor adenosine contracted the aorta. This is consistent with activation of vascular A₁ receptors. An A₁-selective concentration of the antagonist l,3-dipropyl-8-cyclopentyl xanthine abolished the contraction elicited by R-PIA in the guinea-pig aorta. This further suggests that the contraction is mediated by a₁ receptors.     

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

Background and purpose:

Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A₁, A_2A, A_2B and A₃) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes.

Experimental approach:

mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB activation, tumour necrosis factor α (TNF-α) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis.

Key results:

mRNA and protein for A₁, A_2A, A_2B and A₃ adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A_2A and A₃ adenosine receptors inhibited p38 MAPK and NF-κB pathways, an effect abolished by selective adenosine antagonists. A_2A and A₃ receptor agonists decreased TNF-α and IL-8 production. The phosphoinositide 3-kinase or G_s pathways were involved in the functional responses of A₃ or A_2A adenosine receptors. Synoviocyte A₁ and A_2B adenosine receptors were not implicated in the inflammatory process whereas stimulation of A_2A and A₃ adenosine receptors was closely associated with a down-regulation of the inflammatory status.

Conclusions and implications:

These results indicate that A_2A and A₃ adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.     

Prolonged agonist exposure induces imbalance of A1 and A2 receptor-mediated functions in rat brain slices

Agonist-induced desensitization of A₁ and A₂ adenosine receptors was studied in brain slices obtained from either rat striatum or cortex, exposed to the adenosine analogue N⁶-cyclopentyl adenosine (CPA) for selected time periods (15–60 min), and repeatedly washed at the end of agonist exposure. Adenosine receptor function in control and agonist-exposed slices was evaluated by measuring coupling with the cAMP producing system in striatum and with membrane phosphoinositide metabolism in cortex. In rat striatum, exposure to the adenosine analog resulted in a rapid, time-and dose-dependent desensitization of cyclase-linked A₁ receptors, as shown by a gradual loss of A₁ receptor-mediated inhibition of basal cAMP formation. Binding studies with [³H]cyclo-hexyl-adenosine (CHA) showed no changes of A₁ receptors for the shortest agonist exposure periods utilized (i.e., 15 and 30 min), indeed suggesting an initial uncoupling of receptors from their transduction system. After a 60 min exposure to the adenosine analogue, a significant change of [³H]CHA higher affinity component was instead detected, probably reflecting later agonist-induced receptor changes related to irreversible receptor loss. The initial agonist-induced loss of A₁ receptor function was also confirmed in rat cortex, where A₁ receptor-mediated modulation of phosphoinositide breakdown was dose-dependently impaired by pre-exposure of slices to the adenosine analogue. A₂ receptors seemed resistant to agonist-induced desensitization, at least for the exposure periods utilized. In striatum, changes of A₁ receptor function were indeed accompanied by a concomitant increase of A₂ receptor mediated stimulation of adenylate cyclase activity, suggesting an “unmasking” of A₂ stimulatory receptors as a consequence of inhibitory receptors. These result therefore indicate that the over-exposure to adenosine analogues can result in a functional unbalance between A₁ and A₂ adenosine receptors, which might be at the bases of changes of adenosine neuroprotective activity in brain pathological conditions characterized by an increased release of the nucleoside. © 1993 Wiley-Liss, Inc.     

Aza-Analogs of 8-Styrylxanthines as A2A-Adenosine Receptor Antagonists

In the present study we synthesized aza-analogs of 8-styrylxanthines, in which the ethenyl bridge is replaced by an imine, amide, or azo function, in order to investigate structure-activity relationships of the 8-substituent of A_2A-selective xanthine derivatives. Thus, various 8-substituents were combined with theophylline or caffeine, respectively, and affinities of the novel compounds for adenosine A₁- and A_2A-receptors were determined and compared with those of analogous 8-styrylxanthine derivatives. 8-(Benzylideneamino)caffeine derivatives exhibited high affinity and selectivity for A_2A-adenosine receptors, but were unstable in aqueous buffer solution at physiological pH values. 8-(Phenylazo)caffeine derivatives were less potent than corresponding 8-styrylcaffeine derivatives at adenosine receptors. The most potent azo compound of the present series was 8-(m-chlorophenylazo)caffeine ( 14b ) exhibiting a K_i value of 400 nM at A_2A-adenosine receptors and 20-fold selectivity versus A₁-receptors. Due to the facile synthetic access to 8-(phenylazo)xanthine derivatives, which are obtained by coupling of 8-unsubstituted xanthines with phenyldiazonium salts, 14b may be an interesting new lead compound for the development of more potent and selective A_2A-antagonists with azo structure.     

Adenosine inhibits the L-type calcium current in human atrial myocytes

The effects of adenosine on the L-type Ca ²⁺ current (I _Ca) were studied in human atrial myocytes using the whole-cell voltage clamp technique. I _Ca was recorded under physiological calcium concentrations (1.8 mmol/l) at 37?°C. Under these conditions the current density of basal I _Ca averaged 4.0 pA/pF. Isoprenaline (1 µmol/l) increased basal I _Ca to 249.7%. Adenosine (100 µmol/l) in the presence of isoprenaline (1 µmol/l) decreased I _Ca from the level obtained with isoprenaline to 87.5% of basal I _Ca. Adenosine (0.1 to 100 µmol/l) also reduced basal I _Ca, maximally to 64.5% of control. Activation and inactivation parameters of basal I _Ca were not significantly different between adenosine (100 µmol/l) and control recordings. Our results show that adenosine affects both basal and isoprenaline stimulated I _Ca in human atrial myocytes. Although a considerable decrease of basal I _Ca was seen, we conclude that the action of adenosine on L-type Ca ²⁺ current in human atrial myocytes is mainly antiadrenergic. Both effects may contribute to the antiarrhythmic properties of adenosine.     

Species-dependent effects of adenosine receptor agonists on contractile responses of vas deferens to ATP

1 Experiments were carried out to examine the postjunctional actions of adenosine receptor agonists on the smooth muscle of the vas deferens of the guinea-pig and rabbit. 2 Although they produced neither contraction nor relaxation by themselves, adenosine analogues enhanced contractions of the guinea-pig vas deferens induced by 10 μm ATP. The rank order of potency was N⁶-cyclopentyladenosine (CPA) > 5′-N-ethylcarboxamidoadenosine (NECA) > adenosine > CGS 21680. Dose–response curves for NECA were shifted to the right by the nonselective adenosine receptor antagonist 8(p-sulphophenyl)theophylline (8-SPT; 100 μm ) and by the selective A₁-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 m m ). 3 In the rabbit vas deferens, contractions induced by ATP (1 m m ) were inhibited rather than facilitated by NECA. Neither CPA, R(–)-N⁶-(2-phenyl isopropyl)-adenosine (R-PIA) nor CGS 21680 had any effect. 4 The results indicate that the smooth muscle of the guinea-pig vas deferens expresses facilitatory adenosine A₁ receptors but not adenosine A₂ receptors. In contrast, in rabbit there are postjunctional inhibitory adenosine A_2A receptors but not adenosine A₁ receptors.     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A_2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 0.85 nM and 35 fmol mg⁻¹ protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [³H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A_2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments.
3. Thermodynamic data indicate that [³H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A_2A-adenosine receptors.
4. It is concluded that in human lymphocyte membranes [³H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A_2A-receptor. The presence of A_2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

     

Adenosine receptor agonists: from basic medicinal chemistry to clinical development

Adenosine is a physiological nucleoside which acts as an autocoid and activates G protein-coupled membrane receptors, designated A₁, A_2A, A_2B and A₃. Adenosine plays an important role in many (patho)physiological conditions in the CNS as well as in peripheral organs and tissues. Adenosine receptors are present on virtually every cell. However, receptor subtype distribution and densities vary greatly. Adenosine itself is used as a therapeutic agent for the treatment of supraventricular paroxysmal tachycardia and arrhythmias and as a vasodilatatory agent in cardiac imaging. During the past 20 years, a number of selective agonists for A₁, A_2A and A₃ adenosine receptors have been developed, all of them structurally derived from adenosine. Several such compounds are currently undergoing clinical trials for the treatment of cardiovascular diseases (A₁ and A_2A), pain (A₁), wound healing (A_2A), diabetic foot ulcers (A_2A), colorectal cancer (A₃) and rheumatoid arthritis (A₃). Clinical evaluation of some A₁ and A_2A adenosine receptor agonists has been discontinued. Major problems include side effects due to the wide distribution of adenosine receptors; low brain penetration, which is important for the targeting of CNS diseases; short half-lifes of compounds; or a lack of effects, in some cases perhaps due to receptor desensitisation or to low receptor density in the targeted tissue. Partial agonists, inhibitors of adenosine metabolism (adenosine kinase and deaminase inhibitors) or allosteric activators of adenosine receptors may be advantageous for certain indications, as they may exhibit fewer side effects.     

Subepicardial steal and reduction of myocardial oxygen consumption by adenosine

The direct effects of adenosine on cardiac oxygen balance, hemodynamics and regional myocardial perfusion were studied in isolated canine hearts. Intracoronary infusion of adenosine produced a dose-dependent shift in the transmural distribution of coronary blood flow away from left ventricular subendocardium and a reduction in myocardial oxygen consymption (MVO₂). The data suggest that adenosine may partially decrease MVO₂ by a redistribution of flow from a high to low oxygen-extracting region of the myocardium.     

Patent Update Cardiovascular & Renal: Recent progress in modulators of purinergic activity

As knowledge of the molecular biology of purinergic receptors continues to evolve, the identification of novel compounds that mimic or block the actions of the purine nucleoside and its nucleotide, ATP, at P₁ and P₂ receptor subtypes, respectively, has continued to be an area of active interest in the pharmaceutical industry. Adenosine A₁ receptor selective agonists with reduced cardiovascular liabilities have been recently described, as have novel A₃ receptor selective agonists; both classes of agents showing potential as anti-ischaemic agents in animal models. A number of potent and receptor subtype selective agonists for P₂ receptors have also been developed that are being used to characterise the functional role of this receptor class in various mammalian tissues. On the antagonist front, selective A₁ receptor antagonists are being targeted as cognition enhancers while selective A_2a receptor antagonists have been developed with potential use in Parkinson's disease therapy. Despite the clinical failure of Gensia's “site and event specific” adenosine potentiating agent, Acadesine?, there is continued interest in compounds that enhance the availability of endogenous adenosine, specifically novel inhibitors of adenosine transport and adenosine kinase.     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.