Functional Pharmacological Evidence for EP₂and EP₄Prostanoid Receptors in Immortalized Human Trabecular Meshwork and Non-Pigmented Ciliary Epithelial Cells

Abstract

The aim of these studies was to characterize the molecular pharmacology of the prostanoid receptors positively coupled to stimulation of adenylyl cyclase activity in immortalized human trabecular meshwork (TM-3) cells and to compare these results with that of the receptors in immortalized human nonpigmented epithelial (NPE) cells. In general, the TM-3 and NPE cells showed a similar profile with respect to their responses to various prostaglandin (PG) receptor agonists. The rank order of potency (EC50; means +/- SEM) for these compounds in the TM-3 cells was: PGE2 (124 +/- 21 nM) > 13,14-dihydro-PGE1 (430 +/- 110 nM) = PGE1 (522 +/- 345 nM) > 11-deoxy-PGE1 (1063 +/- 118 nM) = 16,16-dimethyl-PGE2 (1776 +/- 460 nM) = butaprost (1920 +/- 527 nM) >> PGD2 = PGI2 = PGF2alpha (n = 3 - 12). While the agonist profile indicated the presence of EP2 receptors, the effects of the EP4 receptor antagonists suggested the additional expression of EP4 receptors in both of these cells. Thus, the EP4 receptor antagonist, AH23848B, at a concentration of 30 microM, caused a dextral shift in the PGE2 concentration-response curves in both TM-3 and NPE cells coupled with a 20-28% decrease in the maximal response of PGE2, indicating apparent noncompetitive antagonism profiles. The antagonist potency of AH23848B in these cells was: Kb = 38.4 +/- 14.8 microM and 23.5 +/- 4.5 microM; -log Kb = 4.7. The other EP4 receptor antagonist, AH22921 (-log Kb = 4.1 - 4.7), was weaker than AH23848B. Taken together, these pharmacological studies have shown than TM-3 and NPE cells apparently contain functional EP2 and EP4 prostanoid receptors positively coupled to adenylyl cyclase.

Human Choroidal Melanocyte Signal Transduction Responses to Various Pharmacological Agents: Focus on Endothelin Receptors

“…The concentrations of the compounds for initial exploratory studies and the full concentration-response experiments were chosen based on the known pharmacological properties of each receptor of interest. 6,7,10,15,[22][23][24][25][26][27][28][29][30][31][32][33][34][35] Initial studies utilized maximally effective concentrations of each compound in order to maximally stimulate the receptors under investigation. 6,7,10,15,[22][23][24][25][26][27][28][29][30][31][32][33][34][35] These studies revealed that PGs (100 µM final) known to activate DP-receptors (e.g., PGD 2 ), EP-receptors (e.g., PGE 2 ), FP-receptors (e.g., PGF 2α , cloprostenol, latanoprost acid [PhXA85], latanoprost), and TP-receptors (e.g., U-46619) were essentially inactive in these assays (e.g., Figure 1).…”

“…6,7,10,15,[22][23][24][25][26][27][28][29][30][31][32][33][34][35] While isoproterenol (10 µM), ET-1 (1 µM), and PGE 2 (10 µM) stimulated cAMP production, numerous other PGs (e.g., PGD 2 , PGF 2α , PGI 2 , latanoprost, latanoprost acid, U-46619, and BW245C [all at > 10 µM]) were inactive ( Figure 4). Due to limited supplies of HCOMs, and difficulties in propagating existing cells beyond a few passages without loss of responsiveness, further studies on the cAMP system were not pursued at this time.…”

“…PGs that then stimulate cAMP generation via EP 2 / EP 4 receptors. 35 As demonstrated in other melanocytes isolated from different tissues, [8][9][10]20,31-34 a variety of pharmacologically distinct receptors participate in the melanogenic and/or proliferative activity of HCOMs (Table 2). How and in which sequence such interactions occur to promote the mitogenic and melanization of HCOM cells in vitro and in vivo remains to be determined.…”

“…Concentrations of test compounds were selected based on our prior experience and the literature. 6,7,10,15,[22][23][24][25][26][27][28][29][30][31][32][33][34][35] After this time, the experiments were terminated and all (total) [ 3 H]-inositol phosphates ([ 3 H]-IPs) were isolated as one batch and quantified by anion exchange chromatography and liquid scintillation spectrometry, respectively, as previously described. 10,22,23 When antagonists were tested, they were pre-incubated with the cells for 20 min prior to the addition of the agonists.…”

The Pathophysiologic Role of Cyclooxygenases in the Eye

“…15,16 EP 2 is associated with the corneal epithelium and endothelium, lens epithelium, and ciliary nonpigmented epithelium. [16][17][18][19][20] In pigs, EP 3 is localized to the corneal epithelium and endothelium, the lens epithelium, and the pigmented ciliary epithelium, bipolar cell, horizontal cell, amacrine cell, ganglion cell, and Müller cell. 15 EP 2 is associated with the trabecular meshwork in humans and pigs, 15,21 and EP 3 is localized to all uveal tissues in pigs.…”