Fatty acid substrate specificities of human prostaglandin-endoperoxide H synthase-1 and -2. Formation of 12-hydroxy-(9Z, 13E/Z, 15Z)- octadecatrienoic acids from alpha-linolenic acid.

Human prostaglandin-endoperoxide H synthase-1 and -2 (hPGHS-1 and hPGHS-2) were expressed by transient transfection of COS-1 cells. Microsomes prepared from the transfected cells were used to measure the rates of oxygenation of several 18- and 20-carbon polyunsaturated fatty acid substrates including eicosapentaenoic, arachidonic, dihomo-gamma-linolenic > alpha-linolenic (delta 9, 12, 15), gamma-linolenic, and linoleic acids. Comparisons of kcat/Km values indicate that the order of efficiency of oxygenation is arachidonate > dihomo-gamma-linolenate > linoleate > alpha-linolenate for both isozymes; while the order of efficiency was the same for hPGHS-1 and hPGHS-2, alpha-linolenate was a particularly poor substrate for hPGHS-1. Gamma-Linolenate and eicosapentaenoate were poor substrates for both isozymes, but in each case, these two fatty acids were better substrates for hPGHS-2 than hPGHS-1. These studies of substrate specificities are consistent with previous studies of the interactions of PGHS isozymes with nonsteroidal anti-inflammatory drugs that have indicated that the cyclooxygenase active site of PGHS-2 is somewhat larger and more accommodating than that of PGHS-1. The major products formed from linoleate and alpha-linolenate were characterized. 13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to be the main product formed from alpha-linoleate by both isozymes. The major products of oxygenation of alpha-linolenate were determined by mass spectrometry to be 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids. This result suggests that alpha-linolenate is positioned in the cyclooxygenase active site with a kink in the carbon chain such that hydrogen abstraction occurs from the omega 5-position in contrast to abstraction of the omega 8-hydrogen from other substrates.

Differential inhibition of human prostaglandin endoperoxide H synthases-1 and -2 by nonsteroidal anti-inflammatory drugs.

We developed an in vitro expression system for accurate kinetic analyses of the inhibition of the human prostaglandin H synthase isozymes (hPGHS-1 and -2) by nonsteroidal anti-inflammatory drugs (NSAIDs). Assays of instantaneous inhibition in which enzyme, 10 microM arachidonate, and an NSAID were mixed simultaneously were used to determine apparent affinities of 14 common NSAIDs for hPGHS-1 and hPGHS-2. All NSAIDs except salicylate had appreciable apparent affinities (IC50 < or = 100 microM) for hPGHS-1. Most NSAIDs also exhibited appreciable affinities toward hPGHS-2, but three prominent exceptions were indomethacin, piroxicam and phenylbutazone. We subsequently performed measurements of time-dependent inhibition in which either (a) enzyme and an NSAID were preincubated before substrate was added to initiate the reactions or (b) recovery of activity after time-dependent inhibition was measured using intact cells preincubated with various NSAIDs. Indomethacin, flurbiprofen, meclofenamate and diclofenac, but not ibuprofen, piroxicam or phenylbutazone, caused time-dependent inhibition of both hPGHS-1 and -2 in vitro. For cells pretreated with flurbiprofen or meclofenamate, hPGHS-2 activities, but not hPGHS-1 activities, were recovered relatively rapidly; with indomethacin, recoveries of hPGHS-1 and hPGHS-2 activities were both slow. hPGHS-2 is thought to be the target of NSAIDs acting as anti-inflammatory agents. However, our results indicate that neither measurements of affinities of NSAIDs for hPGHS-2 conducted in vitro with 10 microM arachidonate nor measurements of time-dependent inhibition of hPGHS-2 always predict whether a compound (e.g., piroxicam or phenylbutazone) has anti-inflammatory activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)