Interaction of hypoxanthine/xanthine oxidase with nitrergic relaxation in the porcine gastric fundus

Abstract

The influence of hypoxanthine (HX)/xanthine oxidase (XO) on short-term [electrical field stimulation (EFS; 4 Hz) for 10 s and 3 min; bolus of exogenous NO (10(-5) M)] and long-term [EFS (4 Hz) and continuous NO-infusion for 20 min] nitrergic relaxations was investigated in circular muscle strips of the pig gastric fundus. HX (3x10(-4) M) / XO (64 mu ml(-1)) did not affect EFS for 10 s and 3 min; the short-lasting relaxation in response to a bolus of exogenous NO (10(-5) M) was changed into a biphasic relaxation with a small and short first phase followed by a larger and prolonged second phase. Cu/Zn superoxide dismutase (Cu/Zn SOD; 1000 u ml(-1)) and uricase (100 mu ml(-1)) respectively enhanced the amplitude of the first phase and diminished the amplitude of the second phase. Ascorbate (5x10(-4) M) and bilirubin (2x10(-4) M) prevented the prolonged component. Exposure to HX/XO during long-term EFS elicited a complete, stable reversal of relaxation starting after a delay. During continuous NO-infusion, HX/XO induced an immediate, complete but transient reversal. The antioxidants bilirubin, ascorbate, alpha-tocopherol, urate, glutathione and Cu/Zn SOD, the hydrogen peroxide degrading enzyme catalase, the hydroxyl radical scavengers dimethylsulphoxide and mannitol, and the cofactor flavin adenine dinucleotide did not influence the reversal induced by HX/XO during either EFS or NO-infusion. The cell-permeable manganese SOD mimetic EUK-8 modified the stable reversal during long-term EFS into a transient one. The results suggest that a nitrated uric acid derivative is responsible for the prolonged second phase in the relaxation to a bolus of exogenous NO in the presence of HX/XO. The exact underlying mechanism of the reversal induced by HX/XO during sustained relaxation remains unclear.

Figure 1

Effect of HX (3×10⁻⁴ M)/XO (64 mu ml⁻¹) on the relaxant responses to 2 NO-boli (10⁻⁵ M) consecutively given with an interval of 8 min. On the left, the control situation is depicted where the strips receive the solvent of either HX or XO. In the presence of HX/XO (on the right), the relaxant response to NO became biphasic. Both phases are shown (1 and 2). The relaxations are expressed as a percentage of the response to the same stimulus before addition of interfering drugs. ^***P<0.001: significantly different from the response before (paired t-test). HX=hypoxanthine; XO=xanthine oxidase.

Figure 2

Representative traces showing the relaxant responses to 5 NO-boli (10⁻⁵ M) in the presence of XO (64 mu ml⁻¹) alone (a), and when the substrate HX (3×10⁻⁴ M) is added before the first and fifth bolus (b) or before each bolus (c). HX=hypoxanthine; XO=xanthine oxidase.

Figure 3

Typical tracings illustrating the influence of HX (3×10⁻⁴ M) / XO (64 mu ml⁻¹) when added 5 min after starting a NO-infusion (b) or electrical field stimulation (EFS, 40 V, 0.1 ms, 4 Hz) (d). Time controls, which receive only the enzyme (a) or the substrate (c), are also shown. Both the NO-infusion and EFS were sustained for 20 min. HX=hypoxanthine; XO=xanthine oxidase.

Figure 4

Influence of uricase (100 mu ml⁻¹) on the relaxant responses to 2 NO-boli (10⁻⁵ M), administered with an interval of 8 min, in the presence of HX (3×10⁻⁴ M) / XO (64 mu ml⁻¹). In the presence of HX/XO, the relaxant response to NO was biphasic and both phases (1 and 2) are shown. Results are expressed as a percentage of the same stimulus before addition of the interfering drugs. ^*P<0.05: significantly different from the response in the presence of HX/XO alone (unpaired t-test). HX=hypoxanthine; XO=xanthine oxidase.

Figure 5

Representative traces demonstrating the effect of 4×10⁻⁴ M uric acid (UA) on relaxations induced by boli of exogenous NO (10⁻⁵ M) (a,b), and the influence of 100 mu ml⁻¹ (c) and 200 mu ml⁻¹ (d) of uricase on exogenous NO in the absence and presence of 4×10⁻⁴ M UA. During intervals (//- - - - - -//), the paper speed was reduced 5 fold.

Figure 6

Relaxant responses to two boli of exogenous NO (10⁻⁵ M), applied with an interval of 8 min, in the presence of HX (3×10⁻⁴ M) / XO (64 mu ml⁻¹) alone or plus one of the antioxidants indicated. The first (1) and second (2) phase of the relaxant response to NO in the presence of HX/XO is shown. The relaxations are expressed as a percentage of the response to the same stimulus before administration of the drugs under study. All results are the means±s.e.mean of 6–7 strips from different animals. ^*P<0.05; ^**P<0.01; ^***P<0.001: significantly different from the response in the presence of HX/XO alone (unpaired t-test). ASC=ascorbic acid; GSH=glutathione; α-TOC=α-tocopherol; UA=uric acid; SOD=superoxide dismutase; BILI=bilirubin; HX=hypoxanthine; XO=xanthine oxidase.