Actions of adenosine on nitro blue tetrazolium deposition and surface pH during intestinal reperfusion injury

Mesenteric arteries supplying an intestinal segment were occluded for 5 minutes and then released. During reperfusion, two series of measurements were made with various substances topically applied to the extraluminal surface. In the first series, reduced nitro blue tetrazolium (NBT) was extracted from tissue and measured spectrophotometrically, as an index of oxidative damage. In the second series, mucosal and serosal surface pH was measured as an index of the functional ability to maintain ion gradients. In control conditions, NBT deposition averaged 55-63 μg/g tissue. After 60 and 120 minutes of reperfusion, NBT was elevated to 446-479 μg/g, which was approximately half as large as the NBT increment (846 μg/g) produced by a 15-minute application of xanthine plus xanthine oxidase to well-perfused tissue. As expected, NBT levels were significantly lower (299 μg/g) in tissue that was continuously suffused with superoxide dismutase (SOD) plus catalase (CAT) before occlusion and during reperfusion. Similar NBT levels (274 μg/g) were observed after reperfusion in animals that were fed a diet supplemented with the antioxidant vitamin E for 4-6 weeks. These observations affirm that some, but not all, NBT deposition after reperfusion can be attributed to oxyradicals. However, with exogenous adenosine (ADO) applied for the first 30 minutes after occlusion, NBT was elevated to 174 μg/g after 60 minutes, which was only half as large as the increment with SOD plus CAT, even though those substances were continuously applied. The opposite effect was produced by an ADO receptor antagonist, 8-phenyltheophylline; NBT was increased to 516 μg/g. On the other hand, NBT after reperfusion was 294 μg/g with aminoimidazolecarboxamide riboside, an intermediate in adenine nucleotide metabolism that putatively increases endogenous ADO levels during ischemia. This value was similar to the NBT level observed in reperfused tissues treated with SOD plus CAT. With vehicle, mucosal surface pH averaged 7.1, decreased to 6.7 during occlusion, transiently increased during reperfusion, but stabilized at 6.7 after 60 minutes, which was below baseline. However, with ADO, mucosal pH did not decrease during reperfusion. With xanthine plus xanthine oxidase applied to normally perfused tissue for 15 minutes, mucosal pH was reduced to 6.7 after a 60-minutes washout. Overall, these results show that 1) NBT deposition in reperfused intestinal segments is attenuated by exogenous ADO and aggravated by 8-phenyltheophylline, 2) aminoimidazolecarboxamide is approximately as effective as SOD plus CAT or a vitamin E-enriched diet for attenuating NBT deposition, but all are less effective than exogenous ADO, 3) oxygen radicals cause functional alternations that are reflected by changes in mucosal, but not serosal, surface pH, and 4) mucosal pH decreases during reperfusion are prevented by exogenous ADO.