Inhibition of cAMP Accumulation by -Receptor Activation in Isolated Iris-Ciliary Bodies: Role of Phosphodiesterase and Protein Kinase C

ABSTRACT The present study was designed to examine the roles of protein kinase C (PKC) and phosphodiesterase (PDE) in modulating the action of receptor stimulation on cAMP accumulation in isolated iris-ciliary bodies (ICBs) of New Zealand White rabbits. The receptor agonist, (Ϯ)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine (BRL-52537) (BRL), and the PKC activator, phorbol 12,13-dibutyrate (PDBu), both caused a concentration-dependent inhibition of forskolin-stimulated cAMP production. The inhibitory effect of BRL on cAMP levels was significantly reduced in the presence of the selective receptor antagonist, norbinaltorphimine (10 Ϫ6 M), but the effect of PDBu was not, thus supporting the involvement of -opioid receptors in the response to BRL. In the presence of 3-isobutyl-1-methylxanthine or rolipram (10 Ϫ5 M), the inhibitory effect of BRL or PDBu (10 Ϫ6 M) on cyclic AMP accumulation was abolished. In the presence of the selective PKC antagonist, chelerythrine (10 Ϫ6 M), the inhibitory effect of PDBu or BRL (10 Ϫ6 M) was significantly reduced. Direct measurement of PDE activity demonstrated the ability of BRL and PDBu (10 Ϫ6 M) to augment the activity of these enzymes. Preincubation of ICBs with rolipram (10 Ϫ5 M) or chelerythrine (10 Ϫ6 M) caused significant reversal of both BRL-and PDBu-induced increases in PDE activity. These results indicate that stimulation of PKC and PDE4 activity is part of the complex mechanism whereby -opioid receptor agonists reduce levels of cAMP in the rabbit ICB. This mechanism of action could contribute to the ability of -opioid agonists to suppress aqueous flow rate and to lower intraocular pressure

Kappa Opioid Receptor Localization and Coupling to Nitric Oxide Production in Cells of the Anterior Chamber

Kappa opioid receptor (KOR) agonists lower IOP, but the specific cellular mechanisms involved have not been fully elucidated. This study demonstrates the presence of KORs in cells of the anterior chamber and also that nitric oxide may play a role in KOR-mediated IOP reduction

Regulation of Nitric Oxide Production by δ-Opioid Receptors during Glaucomatous Injury

To determine the roles of nitric oxide in glaucomatous injury and its regulation by δ-opioid-receptor activation, animals were treated with: 1) a selective inducible nitric oxide synthase (iNOS) inhibitor (aminoguanidine; AG; 25 mg/kg, i.p.); 2) δ-opioid-receptor agonist (SNC-121; 1 mg/kg, i.p.); or 3) with both drugs simultaneously for 7 days, once daily. The loss in retinal ganglion cell (RGC) numbers and their function in glaucomatous eyes were significantly improved in the presence of AG or SNC-121; however, we did not see any significant additive or synergistic effects when animals were treated with both drugs simultaneously. The levels of nitrate-nitrite were significantly increased in the glaucomatous retina when compared with normal retina (normal retina 86±9 vs. glaucomatous retina 174±10 mM/mg protein), which was reduced significantly when animals were treated either with SNC-121 (121±7 mM/mg protein; P<0.05) or AG (128±10 mM/mg protein; P<0.05). Additionally, SNC-121-mediated reduction in nitrate-nitrite levels was not only blocked by naltrindole (a δ-opioid-receptor antagonist), but naltrindole treatment potentiated the nitrate-nitrite production in glaucomatous retina (235±4 mM/mg protein; P<0.001). As expected, naltrindole treatment also fully-blocked SNC-121-mediated retina neuroprotection. The nitrotyrosine level in the glaucomatous retina was also increased, which was significantly reduced in the SNC-121-treated animals. Additionally, the expression level of iNOS was clearly increased over the control levels in the glaucomatous retina and optic nerves, which was also reduced by SNC-121 treatment. In conclusion, our data support the notion that nitric oxide plays a detrimental role during glaucomatous injury and inhibition of nitric oxide production provided RGC neuroprotection. Furthermore, δ-opioid receptor activation regulates the production of nitric oxide via inhibiting the activity of iNOS in the retina and optic nerve