We previously reported that KOR agonists reduce IOP, in part, by reducing aqueous humor formation
3,4 and increasing outflow facility,
5 but the precise site and cellular mechanism of action of these agonists—and the localization and distribution of KORs that modulate aqueous humor dynamics—has not been clearly established. In this study, KORs were found localized to cell membranes of both NPCE and HTM-3 cells (
Fig. 1). The presence of KORs on cultured cells of the inflow and outflow pathways has not been previously reported, and these data suggest that these NPCE and HTM-3 cells may be used to elucidate the mechanisms by which KOR agonists modulate aqueous humor dynamics. KOR proteins were also found localized to the ICB of the New Zealand white rabbit. Specifically, the immunoreactive staining on ciliary processes (CPs) suggests that these receptors could play a role in the regulatory mechanisms that modulate aqueous humor dynamics because CPs are responsible for the active production of aqueous humor. Overall, these immunofluorescence data provide evidence that the nonpigmented ciliary epithelium of the ciliary body, and the trabecular meshwork, could be direct targets for KOR agonists, and supports our previous work that these agonists influence the rate of aqueous humor formation and egress from the anterior chamber in the regulation of IOP.
NO appears to be an important physiologic regulator of IOP, and it might also be directly involved in the increase in IOP observed in POAG as a result of low NO production.
14,15 The underproduction of NO could be corrected by providing NOS substrates or NO donors to lower IOP, increase ocular blood flow, and relax ciliary muscle.
16 The control of NO levels in the eye may therefore be a therapeutic target in glaucoma. The effects of various drugs acting through NO in aqueous humor modulation suggest a role for this cellular mediator in the regulation of IOP. Endothelial NOS (eNOS) and neuronal NOS (nNOS) were found to be present in most ocular tissues, including those responsible for aqueous dynamics—that is, the CPs, ciliary muscle, and trabecular meshwork
17 –19 —further confirming the involvement of these NO producing enzymes in IOP regulation.
A number of inconsistencies currently exist in the literature regarding the effects of NO on IOP. A study performed by Kiel et al.
20 indicated that the inhibition of NOS with L-NAME caused a large rapid decrease in IOP in rabbits. They concluded that this reduction could be related to ciliary vasoconstriction and a reduction in aqueous humor production. Other investigators also indicated the inhibition of IOP by L-NAME in laser irradiated
21 and α-chymotrypsin
22 ocular hypertensive rabbits. No effect of this agent was seen in normotensive rabbits. Another group revealed that the topical application of various NOS inhibitors, including L-NAME, did not prevent the IOP increase induced by water intake in rabbits.
23 Several studies however, have shown IOP-lowering effects of NO. For example, it was shown that by increasing the L-arginine/NO/cGMP pathway, it was possible to lower IOP in rabbits equally to antiglaucoma agents currently being used.
24 A NO donor was also found to enhance the IOP-lowering effects of latanoprost, a commonly used ocular hypotensive agent.
25 In a clinical study, L-arginine, applied intravenously to human subjects, was shown to lower IOP.
26 In addition, a number of other studies indicate that the mechanisms involved in the reduction of aqueous humor flow
27 –30 and increased outflow facility
31,32 are likely to involve the NO/cGMP pathway. Others have shown that the release of NO is implicated in avoiding ocular hypertension in a rabbit glaucoma model.
33 Taken together, these results suggest that the NO/GC system plays a pivotal role in aqueous humor dynamics and, therefore, in the regulation of IOP.
Previous studies in our laboratory have shown that the adenylyl cyclase/cAMP and natriuretic peptide (NP) pathways are involved in KOR-mediated regulation of aqueous humor dynamics in the reduction of IOP. The present study supports our hypothesis that NO could also play a role in KOR-mediated changes in aqueous humor dynamics. Whether or not the NP and NO pathways interact with each other in the reduction of IOP remains to be determined. Here, we focused on NO released by NPCE cells that secrete aqueous humor, and HTM-3 cells that are involved in aqueous humor egress from the anterior chamber. IOP is maintained as a result of a balance between the secretion of aqueous humor by the CPs and its outflow through the trabecular and uveoscleral outflow pathways. We showed that activation of KORs in these cells of the anterior chamber, which participate in aqueous humor regulation, caused a dose-dependent increase in NO levels. The selectivity of SPR for KORs is 84 times that for mu opioid receptors and 100 times that for delta opioid receptors,
34 so the relatively selective, competitive KOR antagonist norBNI was used to confirm that the response to SPR was KOR mediated. The observed increases in NO generated by 100 μM SPR were abolished in the presence of norBNI (100 μM), whereas that produced by the 1000-μM dose of SPR was not significantly affected. At the equivalent molar concentration however, norBNI (1000 μM) significantly inhibited the observed KOR-induced (SPR 1000 μM) increases in NO. These effects of norBNI suggest that in these cells, high levels of KOR activation require an equivalent high molar concentration of antagonist for inhibition.
We have shown in previous studies
5 that activation of KORs in the anterior chamber increases outflow facility, and that this increase, is caused in part, by KOR-activated paracrine effects of natriuretic peptides on tissues within ocular outflow tracts. Based on in vitro evidence presented here, this increase in outflow facility may also be related to the release of NO from trabecular meshwork cells. This NO release could cause in vivo relaxation of trabecular meshwork cells, thereby increasing trabecular outflow facility and lowering IOP. Others have shown that NO induces relaxation of the trabecular meshwork and ciliary muscle cells and therefore may be involved in the regulation of aqueous humor dynamics.
35 Their experiments on relaxation/contraction in trabecular meshwork and ciliary muscle of the bovine eye indicated that cGMP is the final common effector molecule of vasodilators, which activate the NO system, thereby influencing the outflow of aqueous humor. Studies by Ellis et al.
36,37 have revealed that NO plays a direct role in aqueous humor outflow facility in bovine trabecular meshwork. They provided evidence that NO decreases trabecular meshwork cell volume by the activation of the sGC/cGMP/PKG pathway,
36 and that observed changes in trabecular meshwork cell volume are correlated with changes in outflow facility. This group's anterior segment perfusion studies also revealed that the activation of sGC is necessary for NO-induced increases in outflow facility.
37 Taken together, these data suggest that the NO/cGMP pathway plays a role in increasing outflow facility, thereby reducing IOP.
The CPs, which contain the cells responsible for aqueous humor formation, are enriched with nitric oxide synthases, the enzymes involved in the synthesis of NO. Several studies indicate that the NO/cGMP pathway is involved in the secretion of aqueous humor. Sodium azide, a vasodilator drug that acts through the generation of NO, was shown to lower IOP by reducing aqueous humor formation.
38 In a bovine model, this effect of NO donors on aqueous humor secretion was shown to involve cGMP.
28 NO donors were also shown to elevate cGMP in bovine CPs.
39,40 Based on these studies and the data obtained here, we propose that KOR-mediated NO release from NPCE and trabecular meshwork cells could be associated with the mechanistic changes in aqueous humor inflow and outflow that result in reduced IOP.
To our knowledge, we are the first to show that KORs are expressed in CPs and on NPCE and HTM-3 cells, and that these receptors are coupled to NO production in the anterior segment of the eye. However, whether or not the receptor is linked to a specific NOS isoform is yet to be determined. KOR agonists have been shown to elicit their nociceptive and other actions, in part, by activating the L-arginine/NO/cGMP pathway, and there is evidence to suggest that some of these actions may be related to effects on neuronal NOS
41,42 (nNOS; NOS1). In conclusion, results from this study show that KOR receptors are present in the ICB and on HTM-3 and NPCEs. Also, activation of KORs in human NPCE and trabecular meshwork cells increase NO production. These findings provide evidence that KOR-mediated reduction in IOP could be caused, in part, by NO production in both the ciliary body and the trabecular meshwork.
Supported by the National Centers for Research Resources at the National Institutes of Health, Research Centers in Minority Institutions Grant G12-RR03034.
We thank Dr. Peter MacLeish for the SV2 antibody and for allowing use of the facilities in the Neuroscience Institute, Morehouse School of Medicine, Mr. Sidney Pitts of the Department of Neurobiology for his invaluable assistance with sectioning and microscopy, and Dr. Jeffrey Boatright for helpful comments on the manuscript.