In a recent study, we demonstrated that in vitro balloon injury rendered the pig coronary artery more sensitive to relaxation by SIN-1 (Kennedy et al in press). Superoxide is generated by the vessel wall and, in the presence of nitric oxide, superoxide can rapidly combine with NO to produce peroxynitrite (Guzik et al 2002). Damage to endothelium of the artery caused by balloon injury will alter the balance between superoxide/nitric oxide and peroxynitrite. Reduced production of superoxide by the injured vessel may increase the response to nitric oxide donor drugs by protecting the NO liberated. The aim of the current study was to study the mechanisms by which in vitro balloon injury makes the vessel more sensitive to NO donor drugs.

Porcine hearts were obtained fresh from a local abbatoir. The left anterior descending (LAD) coronary artery was injured with a 3.5mm balloon catheter by inflating 3 times to a pressure of 10atm. for 30s. Injured and control left circumflex (LCx) rings (3-4mm) were set up in 10ml baths (37^oC) containing gassed Krebs solution (95%0₂/5%CO₂) for measurement of isometric contraction and relaxation. Rings were equilibrated for 1 hour then contracted with 0.5mM U46619. Relaxation to cumulative addition of S-nitroso-N-acetyl-penicillamine (SNAP; 1nM-10µM) and spermine NONOate (1nM-10µM) was measured. To examine the effect of in vitro balloon injury on the endothelium, arteries were opened longitudinally and stained with 0.5% toluidine blue or silver nitrate. The effect of balloon injury and nitric oxide donors on superoxide generation was measured using lucigenin-enhanced chemiluminescence (CL). Lucigenin was used at a concentration of 250µM (Nunes et al 1997) and CL measured over 15 mins. SNAP, NONOate and SIN-1 were added at a concentration of 2µM. Results were presented per mg of tissue. CL generation was measured immediately after injury or after 2 hours storage at 37^oC in aerating Krebs' solution. All statistical comparisons were made using a Student's Paired t-test.

The contraction to U46619 was significantly enhanced after in vitro balloon injury (9.3±0.9g in control vs. 15.3±2.5g in injured; n=6, p<0.05). As we observed previously with SIN-1, the dose response curves to SNAP and NONOate were shifted to the left after artery injury (EC50 was 1.302µM in control vs. 1.116µM in injured vessels for SNAP and 1.323µM vs. 1.081µM for NONOate; n=6; p<0.05). Staining with toluidine blue revealed that balloon injury removed approximately 65-70% of the endothelium. The non-injured artery had only small areas of artefactual endothelium damage. Control and balloon injured vessels both produced a low level of CL which was unchanged both immediately (33.3±3.0U/mg in control vs. 27.0±2.8U/mg in injured; n=6, p=ns) and 2 hours after balloon injury. Addition of SIN-1, SNAP and NONOate did not affect the CL signal. SOD (1000U) significantly reduced the CL signal (39.7±10U/mg in control vs. 7.7±1.0 with SOD; n=4, p<0.05).

This study has demonstrated that in vitro injury enhances sensitivity to several nitric oxide donor drugs. The underlying mechanism may be due to denudation of the endothelium but is not due to alterations in vascular generation of superoxide.

Guzik, T.J. et al (2002) Hypertension 39:1088-1094.
Kennedy, S. et al (2003) Eur. J. Pharmacol. In press.
Nunes, G. et al (1997) Circulation 96:3593-3601.