Francis L. Belloni

Selective A2a Adenosine Receptor Agonist as a Coronary Vasodilator in Conscious Dogs: Potential for Use in Myocardial Perfusion Imaging

The authors sought to demonstrate the advantages of a selective, potent, short-acting A2A adenosine receptor agonist, CVT-3146 (2-(N-pyrazolyl)Ado derivative), for potential clinical use as a coronary vasodilator during myocardial perfusion imaging. The use of adenosine in a pharmacological stress test during myocardial imaging is limited by side effects mediated by A1 and A2B adenosine receptors and by its ultrashort duration of action. CVT-3146 (0.1–5 &mgr;g/kg) and adenosine (13–267 &mgr;g/kg) were given as peripheral intravenous injections in 10 awake dogs instrumented for measurement of coronary blood flow (CBF). CVT-3146 caused a dose-dependent increase of CBF (ED50 = 0.34 ± 0.08 &mgr;g/kg, maximal increase = 221 ± 18%, n = 6). Adenosine was less potent (ED50 = 51 ± 15 &mgr;g/kg, p < 0.05) but equieffective (maximal increase in CBF = 227 ± 11%). The increase in CBF caused by 2.5 &mgr;g/kg CVT-3146 reached 84 ± 5% of the maximal reactive hyperemia following 20 s of coronary occlusion (n = 4). After a 10-s injection of CVT-3146 (2.5 &mgr;g/kg), the increase in CBF remained at least twofold above baseline for 97 ± 14 s, whereas for adenosine (267 &mgr;g/kg), the twofold increase in CBF lasted only 24 ± 2 s (p < 0.01, n = 6). A 30-s injection of 2.5 &mgr;g/kg CVT-3146 prolonged the twofold increase in CBF up to 221 ± 20 s. No atrioventricular block was noted. At 2.5 &mgr;g/kg, the peak effect of CVT-3146 on CBF was associated with a short-lasting (20 ± 6 s) increase in heart rate (78 ± 9 bpm) and decrease in mean arterial blood pressure (13 ± 6 mm Hg, p < 0.05, n = 6). CVT-3146 is a potent coronary vasodilator. Its short duration of action, minimal and transient systemic hemodynamic effects, and ease of administration may make this agonist suitable for pharmacological coronary vasodilation during myocardial perfusion imaging for noninvasive detection of subcritical arterial stenosis.

The mechanism of adenosine release from hypoxic rat liver cells.

1 Uptake of [14C]‐adenosine into freshly dispersed rat hepatocytes was inhibited 44% by dipyridamole (50 μM) and 60% by nitrobenzylthioinosine (NBTI, 20 μM). The results are consistent with the known ability of these drugs to inhibit adenosine transport in other cell types. 2 The nucleotide analogue, α,β‐methylene adenosine diphosphate (AOPCP, 50 μM), inhibited by 84% the degradation of exogenous 5′ AMP that occurred rapidly when this substrate alone was presented to isolated hepatocytes. This confirms the ecto‐5′‐nucleotidase inhibitory properties of this analogue in isolated hepatocytes. 3 During hypoxic incubation, isolated hepatocytes released adenosine, which accumulated in the extracellular volume. Dipyridamole and NBTI each markedly attenuated this extracellular adenosine accumulation. In contrast, AOPCP had no inhibitory effect on net hypoxic adenosine release. 4 It is concluded that hypoxic rat hepatocytes produce adenosine intracellularly and that this adenosine is released via facilitated diffusion to the extracellular space, based on the inhibition observed with the transport inhibitors. The plasma membrane enzyme ecto‐5′‐nucleotidase does not appear to participate in hypoxic adenosine release from these cells as indicated by the lack of effect of the nucleotidase inhibitor, AOPCP.

Mechanisms of coronary vasodilatation produced by ATP in guinea-pig isolated perfused heart

1 Isolated hearts of guinea‐pigs were perfused in vitro with a physiological salt solution via a retrograde aortic cannulation (Langendorff preparation) at constant perfusion pressure. Bolus intra‐arterial injections of various vasodilator drugs were made and the coronary flow responses were measured with an electromagnetic flow probe placed in the arterial inflow circuit. Inhibitory drugs were infused intra‐arterially. 2 Nitro‐l‐arginine (NLA; 500 μm), an NO synthesis inhibitor, decreased coronary baseline flow by 16 ± 0.8%, converted acetylcholine‐induced coronary vasodilatation to vasoconstriction and had no effect on coronary flow responses to adenosine or papaverine. Sodium nitroprusside‐induced responses were enhanced during NLA infusion by 46 ± 11%. 3 Adenosine 5′‐triphosphate (ATP) increased coronary flow but coronary flow responses to ATP were not altered by infusion of NLA. 4 ATP‐induced coronary dilatation was not significantly attenuated by infusion of the adenosine receptor antagonist XAC, (xanthine amine congener; 2 μm), whereas XAC decreased coronary flow responses to adenosine by 75% ± 5%. 5 ATP‐induced coronary flow responses were reduced by only 31 ± 4% during indomethacin infusion (2.8 μm) whereas indomethacin completely eliminated the initial vasoconstriction phase and greatly attenuated the peak flow and duration of the later vasodilatation phase seen in response to arachidonic acid (0.75 nmol). Indomethacin had no effect on vasodilatations produced by adenosine or prostaglandin I2. 6 These results indicate that ATP‐induced coronary dilatation in the isolated, perfused heart of the guinea‐pig is not dependent upon NO production or upon degradation of ATP to adenosine. The coronary dilator action of ATP may be partially dependent (∼30%) upon the production of vasodilator prostaglandins.

Absence of a role for superoxide anion, hydrogen peroxide and hydroxyl radical in endothelium-mediated relaxation of rabbit aorta

We tested the hypothesis that the endothelium-dependent relaxation of rabbit thoracic aorta in vitro is mediated by reduced metabolites of oxygen. Helical vascular strips were contracted with either norepinephrine or phenylephrine. Oxygen metabolites, generated by the xanthine oxidase reaction, completely relaxed norepinephrine-induced contractile tone but not tone induced by phenylephrine. A mixture of oxygen metabolite scavengers (superoxide dismutase, catalase and mannitol) eliminated the relaxation induced by the xanthine oxidase products. Acetylcholine caused a dose-dependent and endothelium-dependent relaxation of the strips; this was not inhibited by the presence of the scavengers. We conclude that reduced oxygen metabolites have little direct effect on rabbit aortic smooth muscle in vitro, although they indirectly but specifically relax norepinephrine-induced tone, presumably by oxidation of norepinephrine. Oxygen metabolites do not appear to mediate the endothelium-dependent relaxation response of this tissue to acetylcholine.

Effects of Alkylxanthines and Calcium Antagonists on Adenosine Uptake by Cultured Rabbit Coronary Microvascular Endothelium

Adenosine uptake by cultured rabbit coronary microvascular endothelial cells was studied. Radiolabeled [2-3H]-adenosine, present initially in the extracellular space at 10(-6) mol/l, was incorporated into the cell cultures at a steady rate during 30 s-3 h incubations. Incorporated 3H was found mostly (83%) in adenine nucleotides. Incorporation of [3H]-adenosine was attenuated by an adenosine deaminase inhibitor (EHNA) but only at adenosine concentrations of 10(-5) mol/l or higher. Adenosine transport inhibitors (dipyridamole, nitrobenzylthioinosine) attenuated 3H incorporation. Adenosine uptake was also diminished by certain structural analogues of adenosine (e.g., 2-chloroadenosine), by several alkylxanthine drugs (theophylline, isobutylmethylxanthine, enprofylline and 8-phenyltheophylline), and by certain calcium antagonists (verapamil, nifedipine and trifluoperazine). The mechanisms of actions of these agents on adenosine uptake do not appear to be related to phosphodiesterase inhibition, adenosine receptor antagonism or calcium antagonism. The effects of varying adenosine metabolism may contribute to the pharmacologic actions of these agents.

Regulation of Renal A1 Adenosine Receptors in vivo

We have compared renal A₁ adenosine receptor (AR) regulations in rats after chronic agonist and antagonist treatments. In one group, R-phenylisopropyladenosine (R-PIA), a selective A₁ AR agonist, was infused subcutaneously for 7 days. Another group was fed theophylline, a non-selective AR antagonist, for 2 weeks. Renal cortex membrane A₁ AR binding with 1,3-[³H]-dipropyl-8-cyclopentylxanthine demonstrated ∼40% reduction in the B_max for the R-PIA group without any changes in the K_d values. Neither the B_max nor the K_d changed following chronic theophylline treatment. Renal cortex G_iα-proteins from the R-PIA treated rats decreased by ∼30%. Renal G_iα levels did not change in theophylline-treated rats. Consistent with the A₁ AR desensitization, R-PIA-treated rats had significantly higher basal renin release and showed attenuated A₁ AR-mediated inhibition of renin release. These data suggest that prolonged A₁ AR stimulation results in downregulation of renal A₁ ARs and G_iα, accompanied by desensitization of A₁ AR-mediated inhibitory effects on renin release. Unlike cardiac and brain A₁ ARs, renal A₁ receptors are not subject to up-regulation following chronic antagonist treatment.