ABSTRACT
We tested whether hypoxia-induced coronary artery dilatation could be mediated by an increase in adenosine concentration within the coronary artery wall or by an increase in adenosine sensitivity. Porcine left anterior descendent coronary arteries, precontracted with prostaglandin F(2alpha) (10(-5) M), were mounted in a pressure myograph and microdialysis catheters were inserted into the tunica media. Dialysate adenosine concentrations were analysed by HPLC. Glucose, lactate and pyruvate were measured by an automated spectrophotometric kinetic enzymatic analyser. The exchange fraction of [(14)C]adenosine over the microdialysis membrane increased from 0.32 +/- 0.02 to 0.46 +/- 0.02 (n = 4, P < 0.01) during the study period. At baseline, interstitial adenosine was in the region of 10 nM which is significantly less than previously found myocardial concentrations. Hypoxia (P(O(2)) 30 mmHg for 60 min, n = 5) increased coronary diameters by 20.0 +/- 2.6% (versus continuous oxygenation -3.1 +/- 2.4%, n = 6, P < 0.001) but interstitial adenosine concentration fell. Blockade of adenosine deaminase (with erythro-9-(2-hydroxy-3-nonyl-)-adenine, 5 microM), adenosine kinase (with iodotubericidine, 10 microM) and adenosine transport (with n-nitrobenzylthioinosine, 1 microM) increased interstitial adenosine but the increase was unrelated to hypoxia or diameter. A coronary dilatation similar to that during hypoxia could be obtained with 30 microM of adenosine in the organ bath and the resulting interstitial adenosine concentrations (n = 5) were 20 times higher than the adenosine concentration measured during hypoxia. Adenosine concentration-response experiments showed vasodilatation to be more pronounced during hypoxia (n = 9) than during normoxia (n = 9, P < 0.001) and the A(2A) receptor antagonist ZM241385 (20 nM, n = 5), attenuated hypoxia-induced vasodilatation while the selective A(2B) receptor antagonist MRS1754 (20 nM, n = 4), had no effect. The lactate/pyruvate ratio was significantly increased in hypoxic arteries but did not correlate with adenosine concentration. We conclude that hypoxia-induced coronary artery dilatation is not mediated by increased adenosine produced within the artery wall but might be facilitated by increased adenosine sensitivity at the A(2A) receptor level.
Subject(s)
Adenosine/metabolism , Coronary Vessels/physiology , Hypoxia/physiopathology , Receptor, Adenosine A2A/physiology , Vasodilation , Acetamides/pharmacology , Adenosine/pharmacology , Adenosine A2 Receptor Antagonists , Adenosine Deaminase/physiology , Adenosine Deaminase Inhibitors , Adenosine Kinase/antagonists & inhibitors , Adenosine Kinase/physiology , Animals , Coronary Vessels/chemistry , Coronary Vessels/drug effects , Dose-Response Relationship, Drug , Glucose/metabolism , Hypoxia/pathology , In Vitro Techniques , Lactates/metabolism , Purines/pharmacology , Pyruvic Acid/metabolism , Receptor, Adenosine A2A/analysis , Receptor, Adenosine A2B/analysis , Receptor, Adenosine A2B/physiology , Swine , Triazines/pharmacology , Triazoles/pharmacologyABSTRACT
A newly available chromatography column packing material that employs hybrid particle technology was used to improve the analysis of adenosine compounds. Using a TBAS buffer/acetonitrile gradient this material permits separation of etheno-adenosine compounds in less than 4 min with excellent resolution and sensitivity (50 fmol). Variability of compound quantification is small (coefficients of variation 0.23+/-0.14% for 50 pmol and 1.70+/-0.53% for 0.5 pmol). The new method is well suited for the analysis of adenosine compounds in small biological samples and permits a high sample throughput in autosampler setups with high precision and reproducibility.
