Search

Prostaglandins are locally produced mediators of physiologic function which act through specific receptors to influence cellular response pathways. We have conducted a series of experiments designed to obtain a preliminary pharmacologic characterization of the prostaglandin E2 receptor subtype mediating increased synthesis of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in an SV40 transformed line of human non-pigmented ciliary epithelium (ODMC2). Human non-pigmented epithelial (NPE) cells were grown to confluency, preincubated with indomethacin (to prevent endogenous PGE2 synthesis) and with isobutyl-methyl-xanthine (to prevent breakdown of cAMP), challenged with PGE2 or receptor-selective prostanoids and extracted. Prostanoid-stimulated cAMP synthesis was determined using a protein binding assay and normalized to cellular protein. Prostanoid agonists known to be relatively selective for the EP2 receptor subtype stimulated cAMP synthesis by human NPE cells. EP1 and EP3, IP, FP and DP selective prostanoids had negligible effects on stimulation of cAMP synthesis. The results of these studies lead us to conclude that human NPE cells express receptors of the EP2 subtype which mediate PGE2 stimulated synthesis of cyclic AMP.

Corneal endothelial cells synthesize prostaglandin E2 (PGE2), and this synthesis is necessary for the maintenance of the normal polygonal shape of these cells. A series of experiments was done to examine the receptor-effector mechanism responsible for PGE2-mediated effects on cultured rabbit corneal endothelium. When challenged with exogenous PGE2, endothelial cells synthesized cyclic adenosine monophosphate (AMP) in a dose-dependent manner, and this synthesis was not antagonized by AH6809. The synthetic agonist 11-deoxy-PGE1, but not sulprostone, stimulated increased cyclic AMP synthesis. The pharmacologic profile of the endothelial PGE2 receptor is therefore consistent with that of an EP2 receptor linked to activation of adenylate cyclase. The prostaglandin agonists were also tested for their ability to prevent cellular elongation in response to indomethacin. The PGE2, 11-deoxy-PGE1, and 16,16-dimethyl PGE1 prevented elongation, but sulprostone and PGF2 alpha did not. The authors conclude that rabbit corneal endothelium in culture expresses a specific PG receptor of the EP2 subtype which is coupled to cyclic AMP synthesis and is involved in the regulation of cell shape.

Rabbit corneas were treated in vitro and in vivo with cholera toxin (CTX), a specific and irreversible activator of adenylate cyclase. Tissue pieces incubated in vitro in the presence of 10 mug/ml CTX for 15 min continuously synthesized adenosine 3',5'-monophosphate (cyclic AMP) at an increased rate for 3 hr in the absence of CTX in the medium. Corneas exposed for 10 min to CTX topically in vivo and after various time intervals incubated in vitro had an increased ability to synthesize cyclic AMP for at least 30 hr after topical treatment. Epithelial wounds, 6 mm in diameter, were made by brief exposure of corneas in vivo to filter disks soaked in heptanol. Wounds in corneas pretreated with CTX closed at a faster rate and earlier than wounds in corneas pretreated with inactivated CTX. We postulate that cyclic AMP mediates the initial events governing the rate of closure of an epithelial defect.

Serotonin increases the level of cyclic AMP in incubated rabbit corneas; the concentration of agonist producing half-maximal stimulation is approximately 1.5 microM. Nialamide, an inhibitor of monoamine oxidase, potentiates the response to serotonin but not to epinephrine. Amitriptyline, an inhibitor of neuronal uptake of serotonin, does not potentiate the stimulation of cyclic AMP synthesis. Lysergic acid diethylamide, but not timolol, antagonizes the response to serotonin; the half-maximal inhibitory concentration is approximately 6 nM lysergic acid diethylamide. A comparison of the time course of the increase in cyclic AMP synthesis after addition of serotonin or epinephrine to the incubation media indicates that serotonin, but not epinephrine, must penetrate a barrier to its free diffusion. We conclude that the corneal epithelium contains specific serotonergic receptors that, upon activation, cause the synthesis of cyclic AMP, which mediates the stimulation of chloride transport (c.f. companion article, Klyce et al.). The serotonergic receptors must be at a location posterior to the beta-adrenergic receptors, which are on the anterior-surface of the apical cells.