Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra

Abstract

1 The eects of superoxide anion generators, the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoine-1-oxyl 3-oxide (carboxy-PTIO), the speci®c guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. 2 Methylene blue (MB, 10 mM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml 71) inhibited NO-mediated relaxations, without aecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. 3 Carboxy-PTIO (0.1 ± 0.5 mM) inhibited NO-mediated relaxations but had no eect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml 71 ). 4 Relaxations to NO were reduced, but not abolished, by ODQ (10 mM), while nitrergic responses were completely blocked. 5 The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB, 0.5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no eect on responses to nitrergic stimulation or NO. In contrast, N-ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. 6 L-cysteine (L-cys, 0.1 mM) had no eect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD-independent manner. 7 Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open.

Effect of Neocuproine, a Copper(I) Chelator, on Rat Bladder Function

“…Previous studies indicated that NO was involved in the actions of other ions (nickel and manganese) on neurally evoked bladder contractions Lin-Shiau, 1997, 1998;Sahin et al, 2000). We considered the possibility that NO released in the bladder is not free NO but a sulfhydryl-containing compound, such as an S-nitrosothiol (Thornbury et al, 1992;Garcia-Pascual et al, 2000) and that neocuproine could affect the responses of S-nitrosothiols by its ability to chelate copper (De Man et al, 1999;Göcmen et al, 2000). However, this mechanism seems unlikely because the NOS inhibitor, L-NOARG, did not alter the effect of neocuproine on contractile responses to EFS.…”

“…For example, copper ions can alter the function of P2X 4 purinergic receptors expressed in Xenopus oocytes (Xiong et al, 1999;Acuna-Castillo et al, 2000), raising the possibility that other types of P2X receptors that are expressed in the bladder may also be sensitive to copper ions. Some of the effects of NO in the bladder may also be dependent on copper ions as suggested for nitrergic transmission at other sites in the urogenital system such as the mouse corpus cavernosum (Göçmen et al, 1997(Göçmen et al, , 1998(Göçmen et al, , 2000 and sheep urethra (Garcia-Pascual et al, 2000). In these tissues, it has been speculated that the nitrergic transmitter may not be free NO but rather a superoxide-resistant, nitric oxide-carrying molecule, such as an S-nitrosothiol.…”

Neocuproine, a Copper (I) Chelator, Potentiates Purinergic Component of Vas Deferens Contractions Elicited by Electrical Field Stimulation

“…This fi nding indicates that an NO pathway does not play a role in the potentiating effect of neocuproine on the purinergic responses of rat vas deferens. We also considered the possibility that NO released in the vas deferens is not free NO but a sulfhydryl-containing compounds, such as S-nitrosothiols [26,27] , and that neocuproine could affect the responses of S-nitrosothiols by its ability to chelate copper [10,11] . However, this mechanism seems unlikely because the NOS inhibitor, L -NOARG, did not alter the effect of neocuproine on contractile responses to EFS.…”

Nitrergic relaxation in urethral smooth muscle: involvement of potassium channels and alternative redox forms of NO

“…These observations suggest strongly the involvement of the L-arginine-NO pathway in the urethral NANC relaxation that mediates the decrease in resistance accompanying micturition. However, whether NO itself or a NO-containing compound is the ultimate transmitter mediating nitrergic relaxation in the urethra remains controversial (García-Pascual and Triguero 1994;García-Pascual et al 2000). In this sense, several relatively stable NO adducts, specially S-nitrosothiols, have been investigated as possible nitrergic carriers (Kerr et al 1992;García-Pascual et al 1999), but definitive data supporting such a role are still lacking.…”