Influence of bilirubin and other antioxidants on nitrergic relaxation in the pig gastric fundus

Abstract

1. The influence of several antioxidants (bilirubin, urate, ascorbate, alpha-tocopherol, glutathione (GSH), Cu/Zn superoxide dismutase (SOD) and the manganese SOD mimic EUK-8) on nitrergic relaxations induced by either exogenous nitric oxide (NO; 10(-5) M) or electrical field stimulation (4 Hz; 10 s and 3 min) was studied in the pig gastric fundus. 2. Ascorbate (5x10(-4) M), alpha-tocopherol (4x10(-4) M), SOD (300 - 1000 u ml(-1)) and EUK-8 (3x10(-4) M) did not influence the relaxations to exogenous NO. In the presence of GSH (5x10(-4) M), the short-lasting relaxation to NO became biphasic, potentiated and prolonged. Urate (4x10(-4) M) and bilirubin (2x10(-4) M) also potentiated the relaxant effect of NO. None of the antioxidants influenced the electrically evoked relaxations. 3. 6-Anilino-5,8-quinolinedione (LY83583; 10(-5) M) had no influence on nitrergic nerve stimulation but nearly abolished the relaxant response to exogenous NO. Urate and GSH completely prevented this inhibitory effect, while it was partially reversed by SOD and bilirubin. Ascorbate, alpha-tocopherol and EUK-8 were without effect. 4. Hydroquinone (10(-4) M) did not affect the electrically induced nitrergic relaxations, but markedly reduced NO-induced relaxations. The inhibition of exogenous NO by hydroquinone was completely prevented by urate and GSH. SOD and ascorbate afforded partial protection, while bilirubin, EUK-8 and alpha-tocopherol were ineffective. 5. Hydroxocobalamin (10(-4) M) inhibited relaxations to NO by 50%, but not the electrically induced responses. Full protection versus this inhibitory effect was obtained with urate, GSH and alpha-tocopherol. 6. These results strengthen the hypothesis that several endogenous antioxidant defense mechanisms, enzymatic as well as non-enzymatic, might play a role in the nitrergic neurotransmission process.

Figure 1

Representative traces showing the responses to electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s and 3 min) and to a bolus of exogenous NO (10⁻⁵ M) in a time control (a), before and in the presence of 5×10⁻⁴ M glutathione (GSH, b) or 4×10⁻⁴ M urate (UA, c). During intervals (//—–//), the paper speed was reduced 5 fold.

Figure 2

Relaxant responses to electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s and 3 min) and exogenous NO (10⁻⁵ M) in the presence of antioxidants or aqua, the solvent of most antioxidants. The relaxations are expressed as a percentage of the response to the same stimulus before administration of the antioxidants. All results are the means±s.e.mean of six to eight strips. ^**P<0.01: significantly different from the response before administration of the antioxidant (paired t-test). SOD=superoxide dismutase; ASC=ascorbate; α-TOC=α-tocopherol; BILI=bilirubin; UA=uric acid; GSH=glutathione.

Figure 3

Representative traces showing the effect of 2×10⁻⁴ M bilirubin (BILI) on the relaxations induced by electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s and 3 min) and exogenous NO (10⁻⁵ M) in the absence (a) and in the presence (b) of 10⁻⁵ M LY83583. During intervals (//—–//), the paper speed was reduced 5 fold.

Figure 4

Representative traces showing the influence of 10⁻⁵ M LY83583 on the relaxations induced by electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s and 3 min) and exogenous NO (10⁻⁵ M) (a), and the effect of 5×10⁻⁴ M glutathione (GSH, b), 4×10⁻⁴ M urate (UA, c) and 1000 u ml⁻¹ SOD (d) on the inhibitory action of LY83583. During intervals (//—–//), the paper speed was reduced 5 fold.

Figure 5

Relaxations induced by exogenous NO infused for 10 min before and in the presence of 10⁻⁵ M LY83583 (b) and its solvent ethanol (a), 10⁻⁴ M hydroxocobalamin (HC, c) and 10⁻⁴ M hydroquinone (HQ, d). The response measured at 1, 2, 3, 5, 8 and 10 min of infusion was expressed as a percentage of the maximum relaxation obtained during the NO-infusion before administration of the drugs. Means±s.e.mean of n=6 are shown. ^*P<0.05; ^**P<0.01; ^***P<0.001: significantly different versus before.

Figure 6

Representative traces showing the influence of 10⁻⁵ M LY83583 (a), 10⁻⁴ M hydroxocobalamin (HC, b) and 10⁻⁴ M hydroquinone (HQ, c) when added 5 min after starting a continuous NO-infusion. The NO-infusion was maintained for 15 min.

Figure 7

Relaxant responses to electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s and 3 min) and exogenous NO (10⁻⁵ M), in the presence of 10⁻⁵ M LY83583 (a), 10⁻⁴ M hydroxocobalamin (b) or 10⁻⁴ M hydroquinone (c) alone or plus one of the antioxidants indicated. 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 six to eight strips. ^*P<0.05; ^**P<0.01; ^***P<0.001: significantly different from the response in the presence of 10⁻⁵ M LY83583, 10⁻⁴ M HC or 10⁻⁴ M HQ alone (unpaired t-test). HC=hydroxocobalamin; HQ=hydroquinone; SOD=superoxide dismutase; ASC=ascorbate; α-TOC=α-tocopherol; BILI=bilirubin; UA=uric acid; GSH=glutathione.