title: Characterization of the PGE2 receptor subtype in bovine chondrocytes in culture.
abstract: 1. Prostaglandin E2 (PGE2) 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 E2 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 x 10(5) cells cm-2 in DMEM with 10% foetal bovine serum. 3. PGE2 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 PGE2 and carbaprostacyclin had an additive effect. PGD2 induced a small increase in intracellular cyclic AMP only at a high concentration (10(-5) M). 4. PGE2 was more potent that the EP2 agonist 11-deoxy-PGE1 at inducing increases in intracellular cyclic AMP. The EP2 agonist butaprost, however, induced only a small increase at a concentration of 10(-5)M. 17-Phenyl-PGE2 (EP1 agonist), sulprostone and MB 28767 (15S-hydroxy-9-oxo-16-phenoxy-omega-tetranorprost-13E-enoic acid) (EP3 agonists) did not induce an increase in intracellular cyclic AMP at concentrations up to 10(-5)M. 5. The EP4 antagonist AH 23848B ([1 alpha(Z),2 beta, 5 alpha]-(+/-) -7-[5-[[(1,1'-biphenyl)-4-yl]methoxyl-2-(4-morpholinyl) -3-oxocyclopentyl]-5-heptenoic acid) antagonized PGE2 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 PGE2 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 PGE2 nor any of its analogues inhibited the increase in intracellular cyclic AMP induced by forskolin, and pertussis toxin did not alter the response to PGE2, suggesting that no Gi-coupled PGE2 receptors are present in these cells. Stimulation with PGE2 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 PGE2 receptors in bovine chondrocytes in these experimental conditions. 7. These results show for the first time that bovine chondrocytes in culture present a functional PGE2 receptor that has some pharmacological characteristics of an EP4 subtype, as well as an IP receptor.
authors.affiliation: Department of Medicine, Faculty of Medicine, Université de Sherbrooke, Fleurimont, Quebec, Canada.
authors.forename: A J
authors.initials: AJ
authors.lastname: de Brum-Fernandes
authors.forename: S
authors.initials: S
authors.lastname: Morisset
authors.forename: G
authors.initials: G
authors.lastname: Bkaily
authors.forename: C
authors.initials: C
authors.lastname: Patry
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism* / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D007293:Inosine Triphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D011459:Prostaglandins E, Synthetic / Q000494:pharmacology; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D007295:Inositol Phosphates; D008024:Ligands; D011459:Prostaglandins E, Synthetic; D018078:Receptors, Prostaglandin E; D007293:Inosine Triphosphate; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D015232:Dinoprostone; D002118:Calcium