Characterization of the PGE 2 receptor subtype in bovine chondrocytes in culture

1 . Prostaglandin E 2 (PGE 2 ) is an autacoid that decreases proteoglycan synthesis, increases metalloprotease production by cultured chondrocytes, and can modulate some of the actions of interleukin‐1 on cartilage. The objective of the present study was to characterize the subtype of prostaglandin E 2 receptor present in bovine chondrocytes in culture. 2 . Primary cultures of articular chondrocytes were prepared from slices of bovine carpal cartilage by sequential digestion with type III hyaluronidase, trypsin, type II collagenase, followed by overnight incubation in Dulbecco's Modified Eagle's Medium (DMEM) with type II collagenase, washing, and seeding at a density of 2 × 10 5 cells cm −2 in DMEM with 10% foetal bovine serum. 3 . PGE 2 and carbaprostacyclin induced dose‐dependent increases in intracellular cyclic AMP in bovine chondrocytes in culture. The potencies of these compounds were different, and maximal doses of PGE 2 and carbaprostacyclin had an additive effect. 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 cyclic 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 agonist), sulprostone and MB 28767 (15S‐hydroxy‐9‐oxo‐16‐phenoxy‐γ‐tetranorprost‐13E‐enoic acid) (EP 3 agonists) did not induce an increase in intracellular cyclic AMP at concentrations up to 10 −5 m . 5 . The EP 4 antagonist AH 23848B ([1α(Z),2β,5α]‐(±)‐7‐[5‐[[(1,1′‐biphenyl)‐4‐yl]methoxy]‐2‐(4‐morpholinyl)‐3‐oxocyclopentyl]‐5‐heptenoic acid) antagonized PGE 2 but not carbaprostacyclin effects on intracellular cyclic AMP. The Schild plot slope was different from 1 but this could be due to an interaction of PGE 2 with IP receptors in high doses. The exact nature of the antagonism by compound AH 23848B could not be definitely established in these experimental conditions. 6 . Neither PGE 2 nor any of its analogues inhibited the increase in intracellular cyclic AMP induced by forskolin, and pertussis toxin did not alter the response to PGE 2 , suggesting that no Gi‐coupled PGE 2 receptors are present in these cells. Stimulation with PGE 2 did not induce significant increases in intracellular inositol‐trisphosphate levels nor increases in intracellular free calcium as determined by confocal microscopy, suggesting the absence of phospholipase‐C‐coupled or of calcium channel‐coupled PGE 2 receptors in bovine chondrocytes in these experimental conditions. 7 . These results show for the first time that bovine chondrocytes in culture present a functional PGE 2 receptor that has some pharmacological characteristics of an EP 4 subtype, as well as an IP receptor.