Adenine nucleotide release from isolated perfused guinea pig hearts and extracellular formation of adenosine.

Abstract

The quantification of adenine nucleotides released from the heart is hampered by their rapid dephosphorylation to adenosine in the extracellular space catalyzed by highly active ectonucleotidases. To determine the total release of adenine nucleotides from isolated Langendorffperfused guinea pig hearts, ecto 5'-nucleotidase was effectively blocked by infusion of a4.-methylene-ADP (AOPCP, 50 ,M). Adenine nucleotides were measured in the coronary venous effluent by the luciferin-luciferase method after enzymatic rephosphorylation to ATP. In hearts perfused at a constant flow rate (10 ml/min) with normoxic buffer (95% 02,5% C02) the release+SEM of adenine nucleotides and adenosine was 0.06±0.01 (n=11) and 0.04+±0.01 (n=13) nmol/min. In the presence of AOPCP, the release of adenine nucleotides increased to 0.43 +0.04 nmol/min (n=9; p<0.05), whereas adenosine remained unchanged. Hypoxic perfusion (10% 02, 85% N2, 5% C02) caused a threefold increase in adenine nucleotide release but a 40-fold increase in adenosine. In contrast, global ischemia (30 seconds) caused adenine nucleotide and adenosine release to rise to similar values of 1.06±0.10 and 0.80+0.14 nmol/min (n=9). Stimulation of hearts with isoproterenol (4 nM) likewise increased the release of adenine nucleotides (0.50±0.04 nmol/min) and adenosine (0.87±0.21 nmol/min) (n=6). To determine the cellular source of adenine nucleotides released from the heart, the coronary endothelial adenine nucleotide pool was selectively prelabeled by [3H]adenosine. Global ischemia increased the specific radioactivity of released adenine nucleotides by 57%. The findings indicate that 1) adenine nucleotides and adenosine are released at the same order of magnitude from the well-oxygenated heart; 2) P-adrenergic stimulation and ischemia stimulate the release of adenine nucleotides and adenosine, both purines reaching vasoactive concentrations in the effluent perfusate; 3) during hypoxic perfusion only the release of adenosine is greatly enhanced; and 4) the coronary endothelium preferentially contributes to the ischemiainduced adenine nucleotide release. (Circulation Research 1991;68:797-806) In hearts adenosine is generally assumed to be formed intracellularly by dephosphorylation of adenine nucleotides' and by hydrolysis of S-adenosyl-L-homocysteine.2 Adenosine production is accelerated when the breakdown of energy-rich adenine nucleotides is increased, for example, by an imbalance in the myocardial supply-to-demand ratio for oxygen.34 After being formed, adenosine is thought to reach the extracellular space by facilitated diffusion5 to finally cause the adaptive changes in coronary blood flow.

Role of A2A‐adenosine receptor activation for ATP‐mediated coronary vasodilation in guinea‐pig isolated heart

“…Similarly, inactivation of ectonucleotidase by monoclonal antibodies reduced the A 2B -AdoRmediated decrease of endothelial permeability caused by adenine nucleotides (Lennon et al, 1998). Furthermore, administration of the ecto-5'-nucleotidase inhibitor AOPCP reduced the formation of adenosine from exogenously administered ATP by the isolated perfused guinea-pig heart (Borst & Schrader, 1991). In our preparation, the inhibitor of 5'-nucleotidase activity AOPCP (50 mM) caused coronary vasodilation by itself, whereas the inhibitor of ectoATPase ARL67156 failed either to prevent the metabolism of ATP or to reduce the vasodilator action of ATP.…”

“…All other compounds were dissolved in either water or saline. AOPCP was incubated with ADA (0.02 U per mmol AOPCP) for 30 min to degrade adenosine that is present as a contaminant in the AOPCP preparation (Borst & Schrader, 1991). AMP deaminase was ®ltered (3 and 0.2 mm pore size, Millipore) to remove particulate material.…”

“…ATP can be released from endothelium in response to increases in shear stress (Bodin et al, 1991) and from perivascular nerve terminals (Ralevic & Burnstock, 1998;Burnstock & Kennedy, 1986). In¯ammatory mediators, ischaemia, and damage to blood vessels also cause ATP release from endothelial cells, neutrophils, cardiomyocytes, erythrocytes and platelets (Borst & Schrader, 1991;Born & Kratzer, 1984;Bodin & Burnstock, 1998;Pearson & Gordon, 1985;Yang et al, 1994;Bergfeld & Forrester, 1992;Gordon, 1986;Ralevic & Burnstock, 1991;1998;Sprague et al, 1996;Olsson & Pearson, 1990). ATP is rapidly broken down to adenosine by endothelial ectonucleotidases (Pearson & Gordon, 1985).…”

“…In the in-situ heart ectoenzymatic conversion of nucleotides to adenosine contributes substantially to the increase of the interstitial adenosine concentration during early regional ischaemia (Kuzmin et al, 1998) and during beta adrenergic stimulation (Headrick et al, 1996). Likewise exogenous ATP is almost completely metabolized to adenosine during a single passage through the heart (Belardinelli et al, 1984;Borst & Schrader, 1991;Fleetwood et al, 1989). In humans, intracoronary administration of ATP caused vasodilation accompanied by an increase in coronary sinus adenosine concentration (Nanto et al, 1997).…”

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia

“…ATP is known to be released into the interstitial spaces within the heart in response to a variety of physiological and pathological stimuli [4], including hypoxia or ischemia [5,6]. In neonatal rat cardiomyocytes, exposure of cultures to ischemic stress mimicked by oxygen-glucose deprivation (OGD) results in the release of ATP via maxi-anion channels [7].…”