Pre- and postjunctional protective effect of neocuproine on the nitrergic neurotransmitter in the mouse gastric fundus

Abstract

1. Electrical field stimulation (EFS) of non-adrenergic non-cholinergic nerves of the mouse gastric fundus induced frequency-dependent transient relaxations which were mimicked by nitric oxide (NO), added as acidified NaNO(2). The NO donors S-nitrosocysteine, S-nitrosoglutathione, SIN-1 and hydroxylamine induced sustained concentration-dependent relaxations. The NO synthase blocker L-nitro arginine (L-NOARG; 300 microM) abolished the relaxations to EFS without affecting the relaxations to NO. 2. The copper(I) chelator neocuproine (10 microM) enhanced the relaxations to EFS and NO but inhibited those to S-nitrosocysteine and S-nitrosoglutathione. Neocuproine potentiated the relaxations to SIN-1, which releases NO extracellularly, without affecting the relaxations to hydroxylamine, which releases NO intracellularly. 3. The potentiating effect of neocuproine on the relaxations to EFS was more pronounced after inhibition of catalase with 3-amino-1,2,4-triazole (1 mM) but not after inhibition of Cu/Zn superoxide dismutase (SOD) with diethyl dithiocarbamic acid (DETCA, 1 mM). The potentiating effect of neocuproine on relaxations to NO was not altered by 3-amino-1,2,4-triazole or DETCA treatment. 4. The relaxations to EFS were significantly inhibited by the oxidants hydrogen peroxide (70 microM) and duroquinone (10 microM) but only after inhibition of catalase with 3-amino-1,2,4-triazole or after inhibition of Cu/ZnSOD with DETCA respectively. 5. Our results suggest that neocuproine can act as an antioxidant in the mouse gastric fundus and that both catalase and Cu/ZnSOD protect the nitrergic neurotransmitter from oxidative breakdown. Since inhibition of catalase but not inhibition of Cu/ZnSOD potentiated the effect of neocuproine on relaxations to EFS without affecting the relaxations to NO, catalase may protect the nitrergic neurotransmitter mainly at a prejunctional site whereas Cu/ZnSOD protects at a postjunctional site.

Figure 1

Typical tracings of the mouse gastric fundus contracted with prostaglandin F_2α (PGF, 0.3 μM) showing (A) the relaxations induced by electrical field stimulation (EFS, 0.5 – 4 Hz, pulses of 1 ms during 10 s and 8 Hz, pulses of 1 ms during 2 min) and 1 μM NO, added as acidified NaNO₂, and the effect of L-NOARG (300 μM) on the relaxations to EFS and NO. (B) shows the effect of L-NOARG for n=6 experiments. Results are expressed as percentage decrease of the prostaglandin F_2α-induced contraction and shown as mean±s.e.mean. ^*P<0.05 is considered as significantly different from control, Student's t-test for paired values.

Figure 2

Typical tracings obtained from two different muscle strips of the mouse gastric fundus contracted with prostaglandin F_2α (PGF, 0.3 μM). (A) shows the nitrergic relaxations to repetitive electrical field stimulation at 1 Hz (•) and the potentiating effect of 10 μM neocuproine on these relaxations. (B) shows the frequency- and concentration-dependent relaxations to electrical field stimulation (EFS, 0.5 – 4 Hz) and NO (0.3 – 30 μM, added as acidified NaNO₂) in control conditions (left panel) and in the presence of 10 μM neocuproine (right panel).

Figure 3

Frequency-response curves to electrical field stimulation (EFS, 0.5 – 4 Hz) and concentration-response curves to NO (0.3 – 3 μM, added as acidified NaNO₂) in the mouse gastric fundus in control conditions and in the presence of 10 μM neocuproine. Results are expressed as percentage decrease of the prostaglandin F_2α-induced contraction and shown as mean±s.e.mean for n=5 – 7 experiments. ^*P<0.05 is considered as significantly different from control, Student's t-test for paired values.

Figure 4

Concentration-response curves to the NO donors SIN-1 (A, 0.03 – 3 μM), hydroxylamine (B, 0.01 – 1 μM), S-nitrosocysteine (C, 1 – 100 nM) and S-nitrosoglutathione (D, 1 – 1000 nM) in the mouse gastric fundus in control conditions and in the presence of 10 μM neocuproine. Results are expressed as percentage decrease of the prostaglandin F_2α-induced contraction and shown as mean±s.e.mean for n=4 – 6 experiments. ^*P<0.05 is considered as significantly different from control, Student's t-test for paired values.

Figure 5

Relaxations induced by electrical field stimulation (EFS) at 1 Hz in control conditions and in the presence of (A) superoxide dismutase (SOD, 1000 u ml⁻¹), (B) D-mannitol (0.5 mM) and (C) ascorbic acid (30 μM). Results are expressed as percentage decrease of the prostaglandin F_2α -induced contraction and shown as mean±s.e.mean for n=4 – 6 experiments. Student's t-test for paired values did not reveal any significant differences.

Figure 6

Effect of 10 μM neocuproine on the relaxations induced by (A) 1 Hz electrical field stimulation (EFS) and (B) 1 μM NO (added as acidified NaNO₂) in control conditions and after treatment of the muscle strips with DETCA (0.5 mM) or 3-amino-1,2,4-triazole (Atr, 1 mM). Results are expressed as percentage enhancement induced by neocuproine of the control relaxation, which was taken as 100%, in respective conditions (saline, DETCA or 3-amino-1,2,4-triazole). Results are shown as mean±s.e.mean for n=8 – 12 experiments. ^*P<0.05 is considered as significantly different from respective controls (paired Student's t-test) and #P<0.05 is considered as significantly different from the effect of neocuproine in untreated (saline) muscle strips (one way ANOVA followed by post hoc Dunnett's test, NS=not significant).

Figure 7

Effect of duroquinone (10 μM, A,B) and hydrogen peroxide (H₂O₂, 70 μM, C,D) on the relaxations to electrical field stimulation (EFS, 0.5 – 4 Hz) and NO (0.3 – 3 μM, added as acidified NaNO₂) before and after treatment of the muscle strips with DETCA (A,B) or 3-amino-1,2,4 triazole (C,D). Results are expressed as percentage decrease of the prostaglandin F_2α -induced contraction and shown as mean±s.e.mean for n=4 – 5 experiments. ^*P<0.05 is considered as significantly different from control, one way ANOVA followed by post hoc Dunnett's test.