Misidentification of prostamides as prostaglandins

Prostaglandins and endogenous cannabinoid metabolites share the same lipid backbone with differing polar head groups at exactly the position through which a large molecule is attached to provide antigenicity and thus raise antisera. Hence, we hypothesized that antisera raised against prostaglandins linked to a large molecule such as BSA at the carboxyl functional group would also recognize endogenous cannabinoid metabolites and lead to highly misleading interpretations of data. We found major cross-reactivity of commercial antisera raised to prostaglandins with endocannabinoid metabolites. Furthermore, in a well-characterized cell line (WISH) or primary amnion tissue explants, endocannabinoid treatment led to increased production of endocannabinoid metabolites as opposed to primary prostaglandins. This was apparent only after separation of products by thin-layer chromatography, because they measured as prostaglandins by radioimmunoassay. These findings have major implications for our interpretation of data in situations in which these prostaglandin-like molecules are formed, and they stress the need for chromatographic or spectrometric confirmation of prostaglandin production detected by antibody-based methods. —Glass, M. , J. Hong, T. A. Sato, and M. D. Mitchell. Misidentification of prostamides as prostaglandins. J. Lipid Res. 2005. 46: 1364–1368. Supplementary key words ethanolamides • prostamides • endocannabinoids • anandamide The arachidonic acid derivatives anandamide (1), 2-arachidonyl-glycerol (2AG) (2, 3), virodhamine (4), and 2-arachidonyl-glyceryl ether (5) are exciting not only for their isolation as putative endogenous cannabinoids but also because they represent a novel class of signaling molecules (6). Although all endocannabinoids can bind to and activate cannabinoid receptors, there is increasing evidence of nonreceptor-mediated actions. Recent studies have demonstrated that anandamide and 2AG can be metabolized by cyclooxygenase-2 (COX-2) into prostaglandin-like molecules (7, 8) that consist of a prostaglandin with a polar head group, ethanolamide or glycerol, respectively. The prostaglandin-ethanolamides have been termed prostamides. The activity of prostaglandin E 2 (PGE 2 )-ethanolamide at prostaglandin receptors has been investigated, and it possesses 100to 1,000-fold lower affinity and potency than PGE 2 itself, making this an unlikely target (9); likewise, this compound has little affinity for the cannabinoid receptors (10) or endocannabinoid-metabolizing enzymes (11). Hence, prostamides and glycerol equivalents may represent a novel class of mediators with separate receptors/transduction pathways. Prostamides are produced in vivo (12) and reduce intraocular pressure (13); indeed, bimatoprost (LumiganTM), one compound in a new class of highly efficacious ocular hypotensive agents, is a close analog of prostaglandin F 2 (PGF 2 )-ethanolamide (13). Moreover, the glyceryl ester of PGE 2 can mobilize calcium and activate signaling pathways (14). Given that it is unlikely that prostamides and glycerol equivalents produce their biological actions through existing prostaglandin receptors, and therefore are likely to generate a novel set of signaling pathways, it is essential that studies differentiate between the prostaglandins and the prostamides. However, many recent studies demonstrating prostaglandin production use a radioimmunoassay to detect and quantify the prostaglandin. The antibodies used in these studies are generally produced by the addition of an antigenic head group, thereby possibly conferring a similar conformation to the prostaglandin as it would adopt with the addition of an ethanolamide or glycerol. Therefore, we investigated the ability of PGE 2 and PGF 2 antibodies to recognize PGE 2 -ethanolamide and PGF 2 -ethanolamide, respectively. To determine the possible magnitude and thus significance of the problem, we investigated the relative production of these two prostamides by stimulated amnion-derived WISH cells and the production of PGE 2 -ethanolamide by primary human amnion explants. MATERIALS AND METHODS