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-l 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 105 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-' M).4 PGE2 was more potent that the EP2 agonist 11-deoxy-PGE, at inducing increases in intracellular cyclic AMP. The EP2 agonist butaprost, however, induced only a small increase at a concentration of l0-5 M. 17-Phenyl-PGE2 (EPI agonist), sulprostone and MB 28767 (15S-hydroxy-9-oxo-16-phenoxy-wotetranorprost-13E-enoic acid) (EP3 agonists) did not induce an increase in intracellular cyclic AMP at concentrations up to l0-' M. The EP4 antagonist AH 23848B ([1a(Z),2fl,5a]-(±)-7-[5-[[(l,1'-biphenyl)-4-yl]methoxy]-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.

cAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21Cip1, and PCNA

“…For instance, it is known that lymphocytes and macrophages are attracted to sites of neoplasia (Witz, 2001). At these sites, tumors stimulate macrophages to secrete a number of regulatory molecules including PGE 2 (Alleva et al, 1993;Brum-Fernandes et al, 1996;Elgert et al, 1998). Most effects of PGE 2 are mediated by the PGE 2 receptors whose activation leads to elevation of intracellular cAMP (Brum-Fernandes et al, 1996;Kiriyama et al, 1997).…”

“…At these sites, tumors stimulate macrophages to secrete a number of regulatory molecules including PGE 2 (Alleva et al, 1993;Brum-Fernandes et al, 1996;Elgert et al, 1998). Most effects of PGE 2 are mediated by the PGE 2 receptors whose activation leads to elevation of intracellular cAMP (Brum-Fernandes et al, 1996;Kiriyama et al, 1997). Thus, it can be postulated that tumor-induced secretion of PGE 2 by macrophages might elevate cAMP levels inside tumor cells and render them resistant to S phase-specific antitumor drugs.…”

Differential effects of nonsteroidal antiinflammatory drugs on the IL-1 altered expression of plasminogen activators and plasminogen activator inhibitor-1 by articular chondrocytes

“…Furthermore, all tested NSAIDs significantly decreased IL-1 induced transcript levels of tPA, albeit at varying degrees, indicating that a common property of NSAIDs is responsible for their inhibition of tPA expression. It has been shown that cultured bovine chondrocytes express a functional prostaglandin E 2 (PGE 2 ) receptor, which possibly shares pharmacological characteristics with the EP4 prostanoid receptor subtype [26]. PGE 2 synthesis is induced by IL-1 and the subsequent binding of PGE 2 to its receptor could increase intracellular cAMP levels.…”

Functional Pharmacological Evidence for EP2and EP4Prostanoid Receptors in Immortalized Human Trabecular Meshwork and Non-Pigmented Ciliary Epithelial Cells

“…Similar antagonist observations for AH23848B have been previously reported for other cell types expressing EP 4 receptors. Thus, a rightward shift in the PGE 2 response curve was observed with AH23848B in bovine chondrocytes that expressed the EP 4 receptor subtype (41). Similarly, Coleman et al (40) showed AH23848B to behave as a weak but selective EP 4 receptor antagonist in the piglet saphenous vein.…”