Effect of neocuproine, a selective Cu(I) chelator, on nitrergic relaxations in the mouse corpus cavernosum

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Abstract

The effects of neocuproine and bathocuproine, Cu(I) and Cu(II) chelators, respectively, were studied on relaxations in response to electrical field stimulation, acetylcholine, S-nitrosoglutathione, acidified sodium nitrite and sodium nitroprusside in the mouse corpus cavernosum precontracted with phenylephrine. Neocuproine significantly inhibited relaxations induced by electrical field stimulation, acetylcholine and S-nitrosoglutathione, but not by acidified sodium nitrite and sodium nitroprusside. The pre-prepared neocuproine–Cu(I) complex was ineffective on the responses. The discrepancy between the shape of relaxations in response to electrical field stimulation or to acetylcholine and S-nitrosoglutathione was abolished by adding CuCl2 into the bathing medium. The copper action was blocked by neocuproine but not by bathocuproine. However, the pre-prepared bathocuproine–Cu(II) complex did not accelerate the relaxations affected by CuCl2. These findings suggest that a Cu(I)-dependent mechanism may play a role in the relaxation induced by the endogenous relaxant factor as well as by S-nitrosoglutathione in mouse cavernosal tissue.

Introduction

The role of nitric oxide in non-adrenergic non-cholinergic neurotransmission is now well established, but the exact nature of the nitrergic transmitter has remained unclear. It is suggested that, in some tissues, such as gastric fundus Hobbs et al., 1991, Kitamura et al., 1993, Barbier and Lefebvre, 1994, Rand and Li, 1995, mouse anococcygeus Hobbs et al., 1991, Gibson et al., 1992 and bovine retractor penis Gillespie and Sheng, 1990, Liu et al., 1994 muscles, the actual nitrergic neurotransmitter might not be free nitric oxide but a superoxide-resistant, nitric oxide-carrying molecule, such as S-nitrosothiols. However, results of subsequent studies performed on the same tissues and on rat anococcygeus muscle, and the canine ileocolonic junction revealed that the nitrergic neurotransmitter may be free nitric oxide but not an S-nitrosothiol Liu and Szurszewski, 1994, Martin et al., 1994, Lilley and Gibson, 1995, De Man et al., 1995, De Man et al., 1996, De Man et al., 1998. On the other hand, we had suggested that the relaxant factor released from non-adrenergic non-cholinergic nerves (tetrodotoxin-sensitive) or endothelial cells in mouse cavernosal tissue displays an S-nitrosoglutathione-like character Göçmen et al., 1997, Göçmen et al., 1998. Recently, results of a study on sheep urethra suggest that the urethral nitrergic transmitter may not be free nitric oxide (Garcia-Pascual et al., 2000). In addition, it has been suggested that a cell-mediated mechanism for the biotransformation of S-nitrosoglutathione is present in some cell types (Gordge et al., 1998). Some studies showed a Cu(I)-dependent mechanism to be responsible for the release of nitric oxide from endogenous nitrosothiols Gordge et al., 1996, Al-Sa'doni et al., 1997. These results prompted us to investigate whether there is an actual contribution of a similar Cu(I)-dependent mechanism for the relaxant factor released from non-adrenergic non-cholinergic nerves or endothelial cells in mouse cavernosal tissue. We thus studied the effect of a selective Cu(I) chelator, neocuproine (2,9-dimethyl-1,10-phenanthroline), on relaxations induced by electrical field stimulation, acetylcholine, S-nitrosoglutathione, acidified sodium nitrite and sodium nitroprusside compared with the effect of bathocuproine, a Cu(II) chelator (De Man et al., 1996) on the relaxations in response to all these stimuli.

Section snippets

Organ bath experiments

Male albino mice weighing 30–35 g were killed by cervical dislocation. Penises were removed and placed in a Petri dish containing Krebs solution (composition in mM: NaCl 119, KCl 4.6, CaCl2 1.5, MgCl2 1.2, NaHCO3 15, NaHPO4 1.2, glucose 11). Corpus cavernosum was prepared according to a previously described method (Göçmen et al., 1997). The preparations were mounted under 0.2-g tension in 5-ml organ baths maintained at 37°C and containing Krebs solution aerated with 95% O2 and 5% CO2. The

Relaxant effects of electrical field stimulation, acetylcholine, acidified sodium nitrite, sodium nitroprusside and S-nitrosoglutathione in the mouse corpus cavernosum

Electrical field stimulation (2–8 Hz), acetylcholine (0.1–1 μM), sodium nitroprusside (0.05–1 μM) and exogenous nitric oxide, applied as acidified sodium nitrite (50–500 μM) all relaxed the tissues in a frequency- or concentration-dependent manner Fig. 1, Fig. 2. The S-nitrosoglutathione (2–100 μM)-induced reproducible relaxations were relatively slow to develop when compared to electrically induced relaxation (Fig. 3). However, the start of the relaxation induced by S-nitrosoglutathione became

Discussion

The present results suggest that a Cu(I)-dependent mechanism may play a role in the relaxation induced by especially small amounts of the nitrergic relaxant factor released from non-adrenergic non-cholinergic nerves or endothelial cells, and that, in the mouse corpus cavernosum, this relaxant factor may have some properties resembling those of S-nitrosoglutathione. Neocuproine, a Cu(I) chelator, significantly inhibited electrical field stimulation- and acetylcholine-induced relaxation. The

Acknowledgements

This work was supported by the Çukurova University Research Foundation (TF-99-8 We are indebted to Çukurova University Experimental Research Centre (TIPDAM) for the supply of mice.

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