CHARACTERIZATION OF THE PGE(2) RECEPTOR SUBTYPE IN BOVINE CHONDROCYTES IN CULTURE

1 Prostaglandin E(2) (PGE(2)) is an autacoid that decreases proteoglyc an synthesis, increases metalloprotease production by cultured chondro cytes, and can modulate some of the actions of interleukin-l on cartil age. The objective of the present study was to characterize the subtyp e of prostaglandin E(2) receptor present in bovine chondrocytes in cul ture. 2 Primary cultures of articular chondrocytes were prepared from slices of bovine carpal cartilage by sequential digestion with type II I hyaluronidase, trypsin, type II collagenase, followed by overnight i ncubation in Dulbecco's Modified Eagle's Medium (DMEM) with type II co llagenase, washing, and seeding at a density of 2x10(5) cells cm(-2) i n DMEM with 10% foetal bovine serum. 3 PGE(2) and carbaprostacyclin in duced dose-dependent increases in intracellular cyclic AMP in bovine c hondrocytes in culture. The potencies of these compounds were differen t, and maximal doses of PGE, and carbaprostacyclin had an additive eff ect. PGD(2) induced a small increase in intracellular cyclic AMP only at a high concentration (10(-5) M). 4 PGE(2) was more potent that the EP(2) agonist 11-deoxy-PGE(1) at inducing increases in intracellular c yclic AMP. The EP(2) agonist butaprost, however, induced only a small increase at a concentration of 10(-5) M. 17-Phenyl-PGE(2) (EP(1) agoni st), sulprostone and MB 28767 oxy-9-oxo-16-phenoxy-omega-tetranorprost -13E-enoic acid) (EP(3) agonists) did not induce an increase in intrac ellular cyclic AMP at concentrations up to 10(-5) M. 5 The EP(4) antag onist AH 23848B ([1 alpha(Z),2 beta,5 yl-2-(4-morpholinyl)-3-oxocyclop entyl]-5-heptenoic acid) antagonized PGE(2) but not carbaprostacyclin effects on intracellular cyclic AMP. The Schild plot slope was differe nt from 1 but this could be due to an interaction of PGE(2) with IP re ceptors in high doses. The exact nature of the antagonism by compound AH 23848B could not be definitely established in these experimental co nditions. 6 Neither PGE(2) nor any of its analogues inhibited the incr ease in intracellular cyclic AMP induced by forskolin, and pertussis t oxin did not alter the response to PGE(2), suggesting that no Gi-coupl ed PGE(2) receptors are present in these cells. Stimulation with PGE(2 ) did not induce significant increases in intracellular inositol-trisp hosphate levels nor increases in intracellular free calcium as determi ned by confocal microscopy, suggesting the absence of phospholipase-C- coupled or of calcium channel-coupled PGE(2) receptors in bovine chond rocytes in these experimental conditions. 7 These results show for the first time that bovine chondrocytes in culture present a functional P GE(2) receptor that has some pharmacological characteristics of an EP( 4) subtype, as well as an IP receptor.