Kappa Opioid Receptor Expression and Nitric Oxide Production in Nonpigmented Ciliary Epithelial and Trabecular Meshwork Cells

Abstract

Purpose: : Considerable evidence suggests that the nitric oxide (NO)/cGMP signaling pathway plays an integral role in opioid receptor-mediated responses. Data from our lab and others demonstrate that kappa opioid receptor activation causes IOP reduction but the specific cellular mechanisms are still unknown. The present study was designed to determine if kappa opioid receptors and nitric oxide synthase (NOS) isoforms are present in cells found in the inflow and outflow pathways of the eye, and if activation of these opioid receptors has an effect on NO production in the specific cell types, as these effects could play a role in kappa opioid agonist-induced ocular hypotension.

Methods: : Transformed non-pigmented ciliary epithelial (NPCE) and human trabecular meshwork (HTM-3) cells used in these studies were grown and maintained in Dulbecco’s Modified Eagle Medium (DMEM) at 37^oC. For immunofluorescence experiments, cells were seeded onto poly-l-lysine coated glass coverslips placed in 6-well culture dishes and grown for 24 hr. Cells were stained by direct immunofluorescence using antibodies specific to kappa opioid receptor and NOS. Immunohistochemical detection of kappa opioid receptors and NOS isozymes was done using fluorescence and confocal microscopy. For the NOassays, cells were grown to 70 - 80% confluency in 6 well plates. Experimental protocols were carried out on cells incubated at 37^oC in dye-free DMEM containing protease inhibitor cocktail and L-arginine. Cells were treated with either spiradoline (10, 100 and 1000 µM) or estradiol (100 µM) for 24 hr. In other experiments, cells were pretreated with the selective kappa opioid receptor antagonist nor-binaltorphimine (norBNI) for 30 min, followed by addition of spiradoline (100 or 1000 µM). NO levels were measured in the incubation medium using a Greiss assay.

Results: : Preliminary immunohistochemical data demonstrated the localization of kappa receptors to the cell membranes of NPCE and HTM-3 cells. The NOS isoforms (nNOS and eNOS) were shown to be primarily localized to the cytoplasm. Spiradoline caused a concentration-dependent increase in nitric oxide release from both NPCE and HTM-3 cells. Spiradoline (100 µM and 1000 µM)-induced release of NO was partially inhibited by norBNI (10 and 100 µM).

Keywords: nitric oxide • trabecular meshwork • signal transduction: pharmacology/physiology