NITRIC-OXIDE FORMATION FROM GLYCERYL TRINITRATE BY RABBIT AORTIC STRIP - DETECTION BY RABBIT TAENIA-COLI CONCURRENT WITH VASORELAXATION

1 The purpose of the present study was to assay NO formation from GTN biotransformation by the rabbit aortic strip (RAS) at times concurrent with its vasorelaxation. Such an assay is an important test of the pr odrug hypothesis where it is postulated that glyceryl trinitrate (GTN) is biotransformed to nitric oxide (NO), the active species that initi ates vascular smooth muscle relaxation. To test such a hypothesis, we propose that a sample of smooth muscle, poorly responsive to GTN, yet sensitive to the effects of NO could be used to detect RAS production of NO from GTN. 2 Muscle strips of rabbit taenia coli (RTCS) and RAS i n close apposition, were mounted in tissue baths, and muscle relaxatio n was recorded with isometric force transducers. Tissues were submaxim ally precontracted with 30-35 mM K+ depolarizing solution and exposed to increasing concentrations of GTN (0.1 nM-10 mu M). 3 EC(25) for GTN -induced relaxation of RTCS in the presence of RAS was significantly d ecreased to that for RTCS in the absence of RAS (5.9 +/- 3.0 x 10(-8) M and 5.5 +/- 3.7 x 10(-6) M, respectively). Mean maximal levels of GT N-induced relaxation of similarly precontracted RTCS also differed in the presence and absence of RAS, viz., 80.8 +/- 2.1% and 29.8 +/- 8.3% respectively. 4 RTCS was found to relax upon administration of NO gas bubbled through the incubation medium. Analysis of tissue bath medium revealed that the NO concentration to which RTCS was exposed attained a maximum of 33 nM. Relaxation of RTCS by NO gas was inhibited by 1 m u M reduced haemoglobin. 5 For GTN-incubation with intestinal and vasc ular smooth muscle preparations, NO formation was greater with RAS com pared to RTCS. Thus, in the two-issue bioassay, the RAS was the predom inant source of NO formation from GTN. 6 Reduced deoxyhaemoglobin (1 m u M), a potent extracellular NO scavenger, was found to decrease the a ugmented GTN-induced relaxation in the RTCS-RAS sandwich preparation f rom 17.3 +/- 1.8% to 8.0 +/- 0.8%. The augmented RTCS response was res tored upon washout and subsequent addition of GTN, in the absence of r educed Hb. 7 These data indicate that nitric oxide or a closely relate d NO-donor is produced by vascular biotransformation of GTN as seen by the increased sensitivity of RTCS to GTN when in the presence of RAS. The results of this bioassay thus support the GTN-NO prodrug hypothes is.