High affinity vasoactive intestinal peptide receptors on fetal human nonpigniented ciliary epithelial cells

Abstract

The effect of vasoactive intestinal peptide (VIP) on stimulation of adenylyl cyclase in fetal human nonpigmented ciliary epithelial (NPE) and pigmented ciliary epithelial (CPE) cells was studied. 1 microM VIP elicited a 5-10 fold increase in intracellular cAMP in NPE cells from three fetal donors, but caused little or no response in CPE from two fetal donors and other ocular cell types employed as controls. Appearance of cAMP in the extracellular medium was stimulated in NPE but not in CPE in response to VIP. Both NPE and CPE gave similar cAMP responses (8-13 fold) to the beta-adrenergic agonist, isoproterenol. Binding studies of [125I]VIP to intact NPE and CPE revealed that VIP bound to NPE cells at a high affinity site (KD = .33 nM and a low affinity site (KD = 16 nM), whereas VIP bound to CPE cells only at the low affinity site (KD = 18 nM). In NPE cells, VIP stimulated cAMP formation with an EC50 of approximately 0.6-1 nM, similar to the high affinity binding site KD, with maximal stimulation at 10 nM. Four peptides with various degrees of sequence homology to VIP were also studied. Of these, PHM and PHI stimulated cAMP with EC50s of 50 and 300 nM, respectively, while secretin and glucagon stimulated only at concentrations above 0.1 microM. These results suggest that in fetal human ciliary epithelium, as in rabbit ciliary epithelium (Mittag et al., J Pharm Exp Ther 241: 230, [1987]), VIP stimulation of adenylyl cyclase is a characteristic of NPE but not CPE cells.

Catecholaminergic Regulation of Na-K-Cl Cotransport in Pigmented Ciliary Epithelium: Differences Between PE and NPE

“…Adenylate cyclase and the cAMP pathway were suggested in several early studies to be involved in the regulation of aqueous humor formation, although the effect was reported to be inhibitory rather than stimulatory (Sears et al, 1981;Caprioli and Sears, 1984). Isoproterenol and dopamine have previously been reported to stimulate cAMP formation in human PE cells (Polansky et al, 1993;Crook et al, 1994). In the present work, both CPT cAMP, a cell-permeable cAMP analogue, and forskolin, an activator of adenylate cyclase elevated Na-K-Cl cotransport activity.…”

“…On the other hand, b 2 adrenergic agonists have been reported to be effective regardless of route of administration, which is also consistent with our ®ndings (Topper and Brubaker, 1985;Kacere, Dolan and Brubaker, 1992). Crook and Riese (1996) and Riese et al (1998).…”

“…Rb uptake was carried out as previously described (Crook and Riese, 1996) with the following modi®ca-tions. One mM indomethacin was added to the medium for more than 1 hr prior to the experiment to inhibit endogenous prostaglandin production, which could increase intracellular cAMP levels (Yabu and Crook, unpublished data).…”

“…Since isoproterenol increases cAMP levels in PE cells (Crook et al, 1994), we asked whether the cAMP pathway mediated activation of Na-K-Cl cotransport by isoproterenol. CPT-cAMP, a cell-permeable cAMP analogue, caused a 1 .…”

Down-regulation of Na-K-Cl Cotransport by Protein Kinase C is Mediated by Protein Phosphatase 1 in Pigmented Ciliary Epithelial Cells

“…This increase may involve Na-K-Cl cotransport since isoproterenol stimulates both blood-to-aqueous chloride transport and Na-K-Cl cotransport in rabbit ciliary epithelium (Crook et al, 2000). Adrenergic stimulation of cotransport occurs via the cAMP pathway in cultured ciliary epithelial cells (Hochgesand, Dunn and Crook, 2001) and is rapidly reversed when the agonist is removed from the culture medium (Crook and Riese, 1996). In the rabbit, adrenergic agonists appear to evoke a coordinated regulatory response which includes stimulation of both chloride entry into the bilayer via cotransport and chloride exit into the aqueous humor via chloride channels (Crook et al, 2000).…”