Normothermic and hypothermic models for studying the deleterious effects of hypoxia-reoxygenation on EDHF-mediated relaxation in isolated porcine coronary arteries.
BACKGROUND
The vasomotor response of the coronary artery is altered by hypoxia-reoxygenation (H-R) induced damage. The aim of our study was to compare and evaluate normothermic and hypothermic models which are suitable for future drug studies of vasoprotective action against H-R injury.


METHODS
Porcine coronary arterial rings were isolated and placed in Krebs-Henseleit (K-H) solution. Rings were exposed to normoxic conditions (control group) and two different H-R conditions: the first induced by a 95% N(2)-5% CO(2) gas mixture (40- and 60-min hypoxia) in a normothermic protocol, and the second induced by hypothermic (4 degrees C) hypoxia-reoxygenation in an air-tight beaker filled with K-H solution (24- and 48-hours hypoxia). Reoxygenation was applied by introducing K-H solution aerated with a 95% O(2)-5% CO(2) mixture under normothermic (37 degrees C) conditions. To test the EDHF-mediated relaxation by substance P, rings were first incubated in L-NNA, nitric oxide synthase inhibitor, and indomethacin, cyclooxygenase inhibitor, and then pre-contracted with thromboxane analogue U-46619. Analysis of the maximum relaxation of the arterial rings was performed by one-way ANOVA, followed by Bonferroni's post-test.


RESULTS
Distal segments of the coronary artery responded faster to contraction induced by U-46619 and were relaxed by substance P to a greater extent than proximal segments. Maximal relaxations of arterial rings induced by a 10 nM solution of substance P were significantly reduced (p<0.001) from the values for normoxic rings (81.0+/-1.0%, n=30) after 40-min H-R (50.5+/-5.3%, n=30), 60-min H-R (32.1+/-3.5%, n=30), 24-hours hypothermic H-R (56.0+/-2.3%, n=30) and after 48-hours hypothermic H-R (38.5+/-5.1%, n=30).


CONCLUSIONS
The model employing 40-min normothermic H-R is as effective as 24-hours hypothermic H-R, and 60-min normothermic H-R as 48-hours hypothermic H-R for studying the deleterious effects of H-R on EDHF-mediated relaxation.
Institute of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. lovro.ziberna@mf.uni-lj.si
Lovro
L
Ziberna
Mojca
M
Lunder
Jernej
J
Kuzner
Gorazd
G
Drevensek
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000818:Animals; D001685:Biological Factors / Q000494:pharmacology*; D002245:Carbon Dioxide / Q000009:adverse effects; D015687:Cell Hypoxia / Q000187:drug effects; D003331:Coronary Vessels / Q000187:drug effects* / Q000503:physiopathology*; D004305:Dose-Response Relationship, Drug; D007035:Hypothermia / Q000209:etiology* / Q000503:physiopathology; D000860:Hypoxia / Q000209:etiology* / Q000503:physiopathology; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000503:physiopathology; D018377:Neurotransmitter Agents / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D019335:Nitroarginine / Q000494:pharmacology; D009924:Organ Culture Techniques; D010100:Oxygen / Q000009:adverse effects / Q000494:pharmacology; D015203:Reproducibility of Results; D013373:Substance P / Q000494:pharmacology; D034421:Sus scrofa; D013696:Temperature; D013997:Time Factors; D014661:Vasoconstriction; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects*
D001685:Biological Factors; D018377:Neurotransmitter Agents; D014662:Vasoconstrictor Agents; C067410:endothelium-dependent hyperpolarization factor; D002245:Carbon Dioxide; D019335:Nitroarginine; D013373:Substance P; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D019001:Nitric Oxide Synthase; D010100:Oxygen