PROSTAGLANDIN E(2) ALTERS HUMAN ORBITAL FIBROBLAST SHAPE THROUGH A MECHANISM INVOLVING THE GENERATION OF CYCLIC ADENOSINE-MONOPHOSPHATE

Orbital fibroblasts from patients with Graves' ophthalmopathy, when tr eated with prostaglandin E(2) (PGE(2)), become stellate and develop pr ominent cellular processes. In this paper, we describe results of stud ies designed to characterize the action of PGE(2) on orbital fibroblas t shape changes in vitro. Orbital and dermal fibroblasts were incubate d with PGE(2), one of several prostanoid analogues, 8-br-cAMP or forsk olin and were then visualized by phase-contrast microscopy. Other stud ies involved seeding cells in special chambers equipped with electrode s for cell sensing using electric cell-substrate impedance sensing (EC IS) to detect changes in shape. PGE(2) (10(-7) mol/L) elicited a rapid and dramatic alteration in the shape of orbital fibroblasts but not t hose derived from the skin. Cells became stellate and developed promin ent cytoplasmic processes that extended out from the central area cont aining the cell nucleus. The effects were stereoselective in that a nu mber of structurally related compounds, including Sulprostone, PGI(2), PGF(2 alpha), thromboxane A(2), thromboxane B-2, and 11 deoxy,16,16 d imethyl PGE(2) failed to elicit a similar shape change. Butaprost (10( -5) mol/L), a specific EP(2) agonist, elicited a similar shape-change as that observed with PGE(2). 16,16-dimethyl PGE(2), nonselective agon ist, could mimic the action of PGE(2). The effect of PGE(2) was appare nt at 10(-8) mol/L, maximal at a concentration of 10(-7) mol/L and too k 4-8 hr to evolve completely. Cycloheximide (10 mu g/mL) and actinomy cin D (1 mu g/mL) failed to block the shape change. The morphologic ch ange could be reproduced by addition of 8-br-cAMP (3 mmol/L) and by fo rskolin (5 mu mol/L). Moreover, PGE(2) and Butaprost treatment elicite d in orbital cultures a massive increase in endogenous cAMP production while analogues not affecting cell shape failed to influence cyclic n ucleotide generation. Three strains of orbital fibroblasts from patien ts with Graves' ophthalmopathy and three from normal orbits were teste d and all responded to PGE(2) (10(-7) mol/L). Four strains of dermal f ibroblasts failed to respond to PGE(2). The changes in orbital fibrobl ast morphology were accompanied by a marked decrease in monolayer impe dance as assessed by electric cell-substrate impedance sensing. The ea rliest effects were apparent within 30 min using this sensitive techni que. The widely recognized roles of PGE(2) and related compounds in th e mediation of the inflammatory response make our current findings in orbital fibroblasts of potential importance to the pathogenesis of Gra ves' ophthalmopathy.