Morphine-stimulated nitric oxide release in rabbit aqueous humor

Abstract

Recent studies in our laboratory have demonstrated a role of nitric oxide (NO) in morphine-induced reduction of intraocular pressure (IOP) and pupil diameter (PD) in the New Zealand white (NZW) rabbit. The present study was designed to determine the effect of morphine on NO release in the aqueous humor of NZW rabbits, as this effect could be associated with morphine-mediated changes in aqueous humor dynamics and iris function. Dark-adapted NZW rabbits were treated as follows: (1) treatment with morphine (10, 33 or 100 μg, 5 min); (2) treatment with morphine or endomorphin-1 for 5, 15 or 30 min; (3) pretreatment with naloxone (100 μg), l-NAME (125 μg) or reduced glutathione (GSH, 100 μg) for 30 min, followed by treatment with morphine (100 μg, 5 min). After the various treatment regimens, aqueous humor samples were obtained by paracenthesis and immediately assayed for nitrates and nitrites (an index of NO production), using a microplate assay kit. Morphine caused a dose-dependent increase in the levels of NO in aqueous humor after 5 min of treatment with each dose. Rabbits treated with endomorphin-1 (100 μg) had no significant change in NO levels in aqueous at any point in the course of time. Aqueous samples from rabbits treated with morphine (100 μg) for 5 min increased from 29.84 ± 2.39 μM (control) to 183.94 ± 23.48 μM (treated). The increase in NO levels by morphine (100 μg, 5 min) was completely inhibited in the presence of naloxone (100 μg), l-NAME (125 μg) or GSH (100 μg). These results indicate that morphine-induced increase in NO production in aqueous humor is a transient response that is linked to the activation of mu opioid receptors. Data obtained suggest that morphine-stimulated changes in ocular hydrodynamics and iris function are due, in part, to increased release of NO in aqueous humor. In addition, the sensitivity of the response to l-NAME and GSH suggests that morphine-induced release of nitric oxide into aqueous humor is mediated by activation of mu-3 opioid receptors found in the anterior segment of the eye.

Introduction

Morphine is a prototypical mu opioid receptor agonist that is used clinically as an analgesic agent. At present, the mu opioid receptor has three recognized subtypes, mu-1, mu-2, and the most recently ascribed mu-3 receptor. The mu-3 subtype is an opiate alkaloid selective receptor that is insensitive to opiate peptides like endomorphin-1 and -2. Correlated with its insensitivity to opiate peptides, the mu-3 receptor is also coupled to constitutive NO synthase (cNOS) derived NO release (Stefano et al., 1993). Morphine-induced NO release is naloxone sensitive and is antagonized by the NOS inhibitors N-nitro-l-arginine and Nω-nitro-l-arginine methyl ester (l-NAME). Furthermore, the mu-3 receptor appears to be more sensitive to inactivation by reduced glutathione (GSH) than are classical mu, delta and kappa opioid receptors.

Although morphine-induced reduction of intraocular pressure (IOP) and pupil diameter (PD) has been previously reported, the exact mechanism(s) involved in these actions has not been completely elucidated. Initial studies by Miyashita (1913) demonstrated that morphine raised the IOP, but subsequent reports revealed morphine's ability to lower IOP (Leopold and Comroe, 1948, Fanciulacci et al., 1980a, Drago et al., 1985, Dortch-Carnes and Russell, 2006, Bonfiglio et al., 2006). The ocular effects of morphine (particularly the miosis) are generally thought to occur primarily through centrally-mediated signaling mechanisms (Lee and Wang, 1975, Murray et al., 1983). To date, however, a direct intraocular component has not been ruled out.

Aqueous humor is produced by the ciliary epithelial cells located in the ciliary body (Krupin et al., 1986). IOP is determined primarily by the dynamic equilibrium between the production of aqueous humor in the ciliary body and its efflux mainly through the trabecular meshwork and Schlemm's canal (Wiederholt et al., 1991). Because recent studies in our laboratory (Dortch-Carnes and Russell, 2006) and others (Bonfiglio et al., 2006) have generated evidence of a role of NO in morphine-mediated reduction of IOP and PD, the present study is designed to determine the effect of morphine on NO formation in aqueous humor, because this effect could play a role in morphine-mediated reduction of IOP and PD. With the use of an inhibitor of NO synthesis and the sulfhydryl agent, GSH, additional evidence that morphine-mediated changes in NO levels in aqueous humor are linked to activation of mu-3 opioid receptors has also been established.