Different Fatty Acids Compete with Arachidonic Acid for Binding to the Allosteric or Catalytic Subunits of Cyclooxygenases to Regulate Prostanoid Synthesis*

Prostaglandin endoperoxide H synthases (PGHSs), also called cyclooxygenases (COXs), convert arachidonic acid (AA) to PGH2. PGHS-1 and PGHS-2 are conformational heterodimers, each composed of an (Eallo) and a catalytic (Ecat) monomer. Previous studies suggested that the binding to Eallo of saturated or monounsaturated fatty acids (FAs) that are not COX substrates differentially regulate PGHS-1 versus PGHS-2. Here, we substantiate and expand this concept to include polyunsaturated FAs known to modulate COX activities. Non-substrate FAs like palmitic acid bind Eallo of PGHSs stimulating human (hu) PGHS-2 but inhibiting huPGHS-1. We find the maximal effects of non-substrate FAs on both huPGHSs occurring at the same physiologically relevant FA/AA ratio of ∼20. This inverse allosteric regulation likely underlies the ability of PGHS-2 to operate at low AA concentrations, when PGHS-1 is effectively latent. Unlike FAs tested previously, we observe that C-22 FAs, including ω-3 fish oil FAs, have higher affinities for Ecat than Eallo subunits of PGHSs. Curiously, C-20 ω-3 eicosapentaenoate preferentially binds Ecat of huPGHS-1 but Eallo of huPGHS-2. PGE2 production decreases 50% when fish oil consumption produces tissue EPA/AA ratios of ≥0.2. However, 50% inhibition of huPGHS-1 itself is only seen with ω-3 FA/AA ratios of ≥5.0. This suggests that fish oil-enriched diets disfavor AA oxygenation by altering the composition of the FA pool in which PGHS-1 functions. The distinctive binding specificities of PGHS subunits permit different combinations of non-esterified FAs, which can be manipulated dietarily, to regulate AA binding to Eallo and/or Ecat thereby controlling COX activities.

that are COX substrates and others that are not substrates. Additionally, huPGHS-1 appears to be allosterically inhibited by celecoxib (10), while huPGHS-2 is inhibited allosterically by some NSAIDs, including naproxen and flurbiprofen (7).
As noted above, agents that bind E allo regulate not only COX activity but interactions of E cat with NSAIDs and coxibs. For example, palmitic acid potentiates and celecoxib attenuates the response of huPGHS-1 to aspirin (8). Because of the functional interplay between FAs that bind E allo and the substrates and COX inhibitors that bind E cat , there are likely to be dietary effects on both total COX activity and the responses of PGHSs to NSAIDs. Some of these interactions may underlie adverse drug responses.
In the study reported here, we have documented details of the interactions of FAs that are not COX substrates, nsFAs, with E allo . Additionally, we have determined the E allo versus E cat specificities of several polyunsaturated FAs that interact with PGHSs.
nsFAs act allosterically on PGHSs by binding to E allo (7)(8)(9)(11)(12)(13)(14). Interestingly, the binding of saturated and monounsaturated FAs (i.e. nsFAs) to E allo of huPGHS-1 causes enzyme inhibition, whereas binding of several of these same FAs, notably palmitic acid (PA), to E allo of huPGHS-2 markedly increases enzyme activity (7,8). One goal of this study was to determine the relative concentrations of AA and nsFAs that elicit a maximal difference between PGHS-1 versus PGHS-2 activities. The results of these experiments lead us to a plausible explanation for how PGHS-2 can function at low AA concentrations, when PGHS-1 is effectively latent in cells co-expressing both isoforms (15).
One surprising finding from our studies of polyunsaturated FAs is that C-22 FAs bind more tightly to E cat than E allo . Additionally, -3 fish oil FAs, when tested alone, are poorer inhibitors of purified huPGHSs than anticipated based on the magnitude of the effects of dietary fish oil on PG formation in vivo (17). The in vivo effects of fish oil may result from a combination of nsFAs and fish oil FAs working in tandem to inhibit PGHS-1.
Expression, Purification, and Assay of huPGHS-1 and huPGHS-2 Variants-Procedures for the expression and purification of native huPGHS-1 and native huPGHS-2 from insect cells were essentially as described previously (7)(8)(9). COX activities were typically determined using measurements of O 2 consumption with an O 2 electrode essentially as detailed previously (7). One unit of COX activity is defined as 1 mol of O 2 consumed per min at 37°C in the standard assay mixture. The purity of the recombinant huPGHSs was determined by SDS-PAGE and Western blot analysis (8,12). The average specific activities with 100 M AA were 20 units/mg for purified huPGHS-1 and 40 units/mg for purified huPGHS-2. Radio-thin layer chromatography assays were performed as described previously using [1-14 C]AA (12).
Measurements of FA Binding to E allo -COX assays were performed at high enzyme/substrate ratios to quantify AA or EPA binding to E allo of huPGHSs and FA-induced displacement of AA or EPA from E allo . To measure AA or EPA binding, reaction mixtures (100 l final volume) containing 1 M [1-14 C]AA or 1 M [ 3 H]EPA, 0.10 -2.0 M huPGHS-1 or huPGHS-2, 5 M hematin, and 1 mM phenol in 0.1 M Tris-HCl, pH 8.0, were incubated at 37°C for 1-8 min, and the products were separated and quantified by radio-reverse phase-HPLC as detailed previously (7,8). The principle underlying this method is described in detail in earlier references (7,9). Briefly, when [1-14 C]AA and huPGHS-2 were incubated at high enzyme to substrate ratios, significant amounts of unreacted AA remain after the oxygenation reaction is largely complete. The amounts of unreacted AA remaining are directly proportional to the amount of added PGHS because much of the unreacted AA becomes bound to E allo . This occurs because E allo , although catalytically inert, has a 30-fold higher affinity (K d ϳ0.25 M) than E cat (K d ϳ7.5 M) for AA. Non-substrate FAs (e.g. PA) bind E allo (K d ϳ 7.5 M) more tightly than E cat (K d Ն50 M), and when added to the reaction mixture they displace the small amounts of unreacted [1][2][3][4][5][6][7][8][9][10][11][12][13][14]

Results
Optimal Non-substrate FA/AA Ratio for Regulating huPGHSs-Most common saturated and monounsaturated FAs that are not COX substrates allosterically inhibit huPGHS-1 (8,12), and several of these nsFAs, notably palmitate and oleate, allosterically stimulate huPGHS-2 (7,9,12). With both huPGHS isoforms, nsFAs function by binding E allo and displacing AA from E allo (7,8). The K d values for AA binding to E allo of huPGHS-1 and huPGHS-2 are similar (ϳ 0.25 M), whereas the K d values for the binding of various nsFA to E allo are all about 30-fold higher (ϳ 7.5 M).
We performed a series of measurements to determine what combination of nsFA concentrations and nsFA/AA ratios would cause the biggest difference between the rates of AA oxygenation by huPGHS-1 versus huPGHS-2. We first tested the effect of PA on AA oxygenation (Fig. 1). Both maximal relative inhibition of huPGHS-1 and maximal relative activation of huPGHS-2 occurred under very similar conditions, at a molar ratio of PA/AA of ϳ20 and at relatively low AA concentrations (ϳ0.5-2.0 M AA). With 20 M PA and 1 M AA, we observed a 45% inhibition of huPGHS-1 and 215% of the starting huPGHS-2 activity, a 4.8 ratio of huPGHS-2 to huPGHS-1 activities. The fact that maximal effects of PA on PGHS-1 and PGHS-2 occur under the same conditions in vitro implies that the enzymes can be coordinately regulated in cells.
When oleic acid and stearic acid were tested at a ratio of nsFA/AA of 20, oleate and stearate showed patterns similar to those seen with PA, although the magnitudes of the differences between huPGHS-1 and huPGHS-2 were less (Fig. 2). The ratios of huPGHS-2/huPGHS-1 activities with palmitic, oleic, and stearic acid were 4.9, 3.7, and 2.2, respectively. The differences are a consequence of oleate and stearate being poorer activators than PA of huPGHS-2.
Interactions of EPA with huPGHS-1 Versus huPGHS-2-EPA is poor substrate and a weak inhibitor of AA oxygenation by both recombinant huPGHS-1 (  Table 2 is 0.28 M, which is essentially identical to the K d for AA (7). Also like AA (9), EPA fails to bind to E allo of Y385F R120A/Native huPGHS-2 (data not shown).
In marked contrast to what is observed with huPGHS-2, insignificant amounts of [ 3 H]EPA remain after 1 M [ 3 H]EPA is incubated for 4 min with 5 M huPGHS-1 (Fig. 3). The results indicate that EPA fails to efficiently bind E allo of huPGHS-1. However, EPA can bind to E cat as a substrate, albeit a poor substrate, of huPGHS-1. According to our model for the functioning of PGHS conformational heterodimers (7-9), the K d value for substrate binding to E cat -, EPA in this case, is the same as the K m value (i.e. 11 M). Control values (i.e. without PA, oleic, or stearic acids) for huPGHS-1 and huPGHS-2 were normalized to 100%. Significant differences from the control value were determined using the Student's t test (p Ͻ 0.001) and are denoted by asterisks. FEBRUARY 19, 2016 • VOLUME 291 • NUMBER 8

Allosteric Regulation of Cyclooxygenases by Fatty Acids
Consistent with the concept that EPA binds poorly to E allo of huPGHS-1, we found that C-15 to C-19 saturated FAs as well as oleic acid at a relatively low concentration of 10 M inhibit EPA oxygenation by huPGHS-1. Inhibition occurs even when EPA is present at high concentrations (100 M) under conditions that optimize EPA oxygenation ( Fig. 4A) (20,21). nsFAs can exert an inhibitory effect on AA oxygenation by binding E allo of huPGHS-1, but this occurs only at high nsFA/AA ratios (i.e. Ն5) (8). In a related experiment (Fig. 4B), EPA alone caused 12% inhibition of AA oxygenation by huPGHS-1, whereas a mixture of nsFAs caused 16% inhibition. An additive response would yield 28% inhibition, a level only less than the 35% inhibition caused by the combination of EPA plus nsFAs. Although this is not a large change, it suggests that nsFAs bound to E allo enhance the binding of EPA versus AA to E cat when EPA and AA are present at low concentrations, 1 and 2 M, respectively. as tested in Fig. 4B. Overall, our results indicate that the most abundant nsFAs inhibit EPA oxygenation by huPGHS-1, that EPA preferentially binds E cat versus E allo of huPGHS-1, and that nsFAs bound to E allo of huPGHS-1 function in combination with EPA bound to E cat to inhibit PGHS-1.
Although the inhibitory effect of EPA is augmented by nsFAs in the case of huPGHS-1, the stimulatory effects of nsFAs on AA oxygenation by huPGHS-2 are attenuated in the presence of EPA (Fig. 5). For example, PA causes a 62% increase in the rate of AA oxygenation by huPGHS-2, but the stimulatory effect of PA is reduced to 26% in the presence of EPA. Similarly, the stimulatory effect of OA plus EPA is 16% versus a 30% stimulatory effect of OA on AA alone (i.e. a net 53% decrease in stimulation). EPA and AA bind about 30 times more tightly than nsFAs to E allo (Table 2) (7). Thus, increasing the concentration of EPA has the effect of displacing nsFAs from E allo of huPGHS-2. Because nsFAs are better activators of AA oxygenation than EPA, the net effect of added EPA is a decreased rate of AA oxygenation by huPGHS-2 in the presence of PA and oleic acid. Conversely, increasing the concentration of nsFAs reduces EPA and AA binding to E allo of huPGHS-2 and stimulates COX activity and thus can enhance PGE 2 formation.
Inhibition of huPGHS-2-mediated AA Oxygenation by DHA and DPA-We previously reported that DHA is a poor substrate for huPGHS-1 and huPGHS-2 and that DHA is converted only to monohydroxy docosahexaenoic acids by huPGHS-2 (7,12,13). Under optimal conditions in the presence of 100 M H 2 O 2 , we determined a V max ϭ 4.1 units/mg and K m ϭ 27 M for DHA oxygenation (data not shown). huPGHS-2 undergoes a suicide inactivation when oxygenating polyunsaturated FA substrates (2,22). We determined that 1 nmol (ϳ150 g) of huPGHS-2 can convert 100 nmol of DHA to product under these conditions.
The rates of oxygenation of 100 M DPA by both huPGHS-1 and huPGHS-2 in the presence of 100 M H 2 O 2 were 70 -80% that of DHA (Table 1) (12). DHA and DPA are also about equally effective inhibitors of AA oxygenation by both isoforms. For example, in the case of DHA and huPGHS-2, an ϳ50% reduction in O 2 consumption occurred at a 5:1 ratio of DHA (100 M)/AA (20 M), and a similar 50% inhibition of the conversion of [1-14 C]AA to products occurs at this DHA/AA ratio (     As noted above, AA and EPA bind equally well to E allo of huPGHS-2 with K d values of ϳ0.25 M (Table 2) (7). We assumed the same would be true for DHA. To test this, we examined the ability of DHA to displace [1-14 C]AA from E allo of huPGHS-2. As shown in Table 3, even with 10 M DHA, which constitutes an ϳ100-fold excess over the ϳ0.12 M bound AA (i.e. 0.135-0.015 M [1-14 C]AA remaining for control conditions minus 5 M PA conditions) was unable to displace unreacted [1-14 C]AA from E allo of huPGHS-2. Under these conditions, 5 M PA, which has a relatively high K d value for E allo (ϳ7.5 M) (7), displaced most if not all of the bound AA. There is sufficient enzyme available under the conditions used in the experiment with 10 M DHA to convert all of the available DHA to product so the fact that AA levels are not decreased by DHA is because AA is not displaced from E allo and not because DHA prevents the binding of AA to E cat . DPA, like DHA, was also ineffective in displacing [1-14 C]AA from E allo (Table 3). Likewise, the C-22 6 FA adrenic acid had surprisingly little effect on AA binding to E allo . These results suggest that 3and 6-polyunsaturated FAs having 22 carbon atoms fail to bind tightly to E allo . We also examined linoleic acid, which contains 18 carbons. Linoleate displaced [1-14 C]AA from E allo of huPGHS-2 (Table 3) but was no more potent on a molar basis than PA (Tables 1 and 3).
Effects of nsFAs on the Interactions of DHA and EPA with huPGHS-2-As shown in Table 1, DHA and DPA when tested alone with either huPGHS-1 or huPGHS-2 are comparably effective in inhibiting both isoforms. DHA inhibits huPGHS-1 with approximately the same potency of EPA. However, DHA is a more potent inhibitor than EPA of huPGHS-2 when each -3 FA is tested alone with AA as the primary substrate.
In earlier studies, we observed that PA had no effect on the rate of DHA oxygenation by huPGHS-2 (7, 13) as can also be seen in Fig. 6. However, we observed that the modest inhibitory effect of DHA on AA oxygenation by PGHS-2 is slightly augmented in the presence of common nsFAs (Fig. 6). Specifically, a mixture of the most common nsFAs stimulated AA oxygenation by 27% in the absence of DHA compared with 21% in the presence of DHA. Thus, DHA modestly interfered with the stimulation of AA oxygenation by nsFAs that function by binding E allo of huPGHS-2. Because DHA does not appear to bind to E allo efficiently, we interpret the results in Fig. 6 as indicating that nsFAs promote the binding of DHA versus AA to E cat of huPGHS-2. The small decrease in AA oxygenation observed with the concentrations of DHA plus nsFA tested in Fig. 6 may not be biologically significant.

Discussion
Each PGHS isoform is encoded by a single gene (23,24). Ovine PGHS-1 was originally found to be a homodimer (25), and it is now clear that PGHS-1 and PGHS-2 are each sequence homodimers composed of subunits having identical primary structures (2,4). There is some heterogeneity involving differences in levels of N-glycosylation that leads to subunits with molecular masses that differ by about 2 kDa and are distinguishable by SDS-PAGE (4, 19, 26 -28). However, N-glycosylation of PGHSs is involved in folding and trafficking (28,29) and    [1-14 C]AA (1 M) was incubated with huPGHS-2 (1 M) at 37°C for 4 min and then the indicated amounts of an unlabeled FA was added, and the incubation was continued for another 4 min. The reactions were stopped by the addition of ethyl acetate/acetic acid (20:1), and an aliquot of the organic phase was subjected to radio-reverse phase-HPLC to separate the radioactive products and unreacted AA as described under "Experimental Procedures." The results are shown as the percentage of total 14 C label that remained in the RP-HPLC fraction co-eluting with unreacted AA. deglycosylation of native oPGHS-1 does not alter enzyme activity (29). Furthermore, even removal of the near C-terminal targeting domain of PGHS-2 does not significantly affect enzyme activity (28,30). X-ray crystal structures of PGHSs suggest that the enzymes are structural homodimers and that each monomer has a bound heme. When crystallized with ligands that bind the COX active sites, both COX sites are typically occupied. When crystallized with sub-stoichiometric amounts of flurbiprofen, only molecules of oPGHS-1 having flurbiprofen in both monomers are observed in the structure (31). Thus, PGHSs tend to crystallize only in symmetric forms in which both of the heme-binding sites and both COX active sites are fully occupied. There are a few exceptions. AA and EPA bind in different orientations in the two monomers (32,33), and PA is bound in only one of the two monomers (7) of muPGHS-2; additionally, celecoxib and certain other non-steroidal anti-inflammatory drugs are partially bound in only one of the two monomers of oPGHS-1 (10,31).
There is now considerable evidence dating back to studies by Kulmacz and co-workers (6 -14, 31, 34 -38) in the mid-1980s that PGHSs function in solution as conformational heterodimers composed of E cat and E allo monomers. The work reported here serves to identify the E allo versus E cat specificities of several physiologically important FAs that had not previously been examined in detail. Fig. 7 is a diagram that summarizes the subunit preferences for a large number of nsFAs and COX substrates and inhibitors that are able to interact with the E allo and/or E cat COX sites of huPGHS-1 and huPGHS-2. The physio-logical and pharmacological significance of these many potential interactions have yet to be studied. However, these biochemical interactions are likely to have important biological consequences. This contention is based on findings that have indicated that even relatively subtle differences in COX activities and responses to NSAIDs are important clinically (5,39). For example, incomplete (50 -75%) suppression of PG formation correlates with analgesic and anti-inflammatory responses (40 -42).
Differential Coordinate Regulation of PGHSs by nsFAs-Previous studies had shown that huPGHS-1 and huPGHS-2 are allosterically regulated, but in different directions, by nsFAs (7)(8)(9). We determined that the biggest differential between the two activities, about 5-fold, occurs at concentrations of AA of 0.5-2 M and a PA/AA ratio of about 20.
Although often described as "low," the effective concentration of AA, which is the "free" AA available to COXs at their site(s) action, is unknown (43). Addition of exogenous AA at concentrations of 0.5-1 M to murine 3T3 cells expressing muPGHS-1 versus muPGHS-2 are sufficient to elicit PG formation, and the maximal ratio of PGHS-2 versus PGHS-1 activity occurs with 1-2.5 M exogenous AA (44).
Although there is limited information on the topic (see LIPID MAPS Lipidomics Gateway), in RAW264.7 cells treated with endotoxin to stimulate COX-2 formation, the most abundant non-esterified FAs in the cells are palmitate, oleate, and stearate, and the relative ratios of non-esterified PA to AA range from 5 to 60. Maximal ratios of nsFA to AA occur during a period 4 -8 h after endotoxin treatment when PGHS-2 expression peaks and maximal PGHS-2-derived PGD 2 synthesis occurs.
Our experiments indicate that at normal nsFA/AA levels and low AA concentrations (1-2.5 M), huPGHS-1 has, on a molar basis, only a fifth of the activity as huPGHS-2 toward AA. We propose that it is this reciprocal allosteric regulation of the PGHS isoforms by nsFAs that permits PGHS-2 to function, although PGHS-1 is essentially latent when PGHS-1 and PGHS-2 are co-expressed in cells (15).  EPA Interactions with E allo and E cat of huPGHSs-huPGHS-1 and huPGHS-2 interact differently with EPA. EPA binds E cat but fails to efficiently bind E allo of huPGHS-1. The affinities of AA and EPA for E cat of huPGHS-1 are similar, but the V max for EPA is less than 20% that for AA. Consequently, EPA is a modest competitive inhibitor of AA oxygenation by huPGHS-1. The inhibitory potency of EPA toward huPGHS-1 is similar to those observed for DHA and DPA. Common nsFAs, including palmitate, oleate, and stearate, that allosterically inhibit huPGHS-1 by binding E allo have effects additive with EPA, which binds E cat . Interestingly, the ability of EPA to inhibit huPGHS-1 is augmented somewhat in the presence of nsFAs perhaps because nsFAs potentiate the binding of EPA versus AA to E cat of huPGHS-1.
The degree of inhibition of PGHS-1-mediated PGE 2 formation by normal colonic tissue and of urinary PGE 2 levels in animals fed fish oil supplements is much greater than predicted based simply on the EPA/AA ratio in tissue phospholipids (17). Thus, 50% inhibition of PGE 2 formation occurs with a tissue EPA/AA ratio of 0.2, whereas 50% inhibition of PGH 2 synthesis by huPGHS-1 occurs at an EPA/AA ratio of five (Table 1). This apparent augmentation of EPA-mediated inhibition of PGE 2 formation could reflect a high level of nsFAs in the milieu in which PGHS-1 operates in vivo and/or could be attributable to a PGHS-1 substrate pool having a high EPA/AA ratio. AA and EPA appear to be handled similarly during cellular uptake and metabolism (45)(46)(47), and a relative excess of dietary EPA could lead to relatively high levels of EPA versus AA in substrate pools available to PGHSs. Dietary fish oil also raises the levels of other -3 FAs, including DPA and DHA, that may further augment PGHS-1 inhibition.
AA and EPA each bind with similar affinities to E cat (K m ϭ K d ϭ 5-10 M) of huPGHS-2, and we have shown here that EPA binds to E allo with the same affinity as AA (K d ϳ0.25 M (7)). Paradoxically, huPGHS-2 uses AA in preference to EPA when examined under Michaelis-Menten conditions (i.e. [E] Ͼ Ͼ [S]) at concentrations of EPA that are up to five times that of AA (9,11,12). The simplest explanation is that EPA bound to E allo of huPGHS-2 behaves in the same way as AA itself, both EPA and AA promote the use of AA by E cat . It is only when EPA concentrations are much greater than AA that EPA is oxygenated by huPGHS-2. The products are PGH 3 and monohydroxy acids and are formed in approximately equal amounts (11,13).
EPA interferes with the binding of nsFAs to E allo of huPGHS-2 and thus interferes with the activation of huPGHS-2 by nsFAs. This process, along with displacing AA from phospholipid precursors, is an indirect way that EPA can interfere with AA oxygenation by huPGHS-2.
Interactions of C-18 and C-22 Polyunsaturated FAs with huPGHSs-We found that DHA is oxygenated with about 5% of the efficiency of AA by huPGHS-2. Similar results were obtained by Malkowski and co-workers (33). However, studies by Kulmacz and co-workers (48) have indicated that DHA has V max and K m values comparable with those of AA; this latter study takes into consideration a higher rate of substrate turnover-dependent inactivation with DHA versus AA of huPGHS-2. DPA has not previously been compared as a substrate or inhibitor of purified huPGHS-1 and huPGHS-2. We find that this -3 fish oil FA behaves like its homolog DHA with both huPGHS isoforms.
A surprising observation of our studies of DPA and DHA is that these C-22 polyunsaturated FAs bind preferentially to E cat rather than E allo of huPGHS-2. Thus, C-22 polyunsaturated FAs such as DPA and DHA probably interfere with AA oxygenation mainly by competing with AA for binding to E cat and not E allo . This also appears to be the case with C-22 adrenic acid, an -6 FA. Although linoleic acid, a C-18 FA, does displace [1-14 C]AA from E allo of huPGHS-2 (Table 2), it is no more effective than PA on a molar basis, and thus, like C-22, FAs appears to bind relatively more tightly to E cat than E allo . We conclude that only certain C-20 -6 FAs (i.e. AA and dihomo-␥-linolenic acid) bind to E allo of huPGHS-1, whereas both -3 and -6 C-20 FAs (i.e. AA, dihomo-␥-linolenic acid and EPA) bind to E allo of the huPGHS-2 isoform. It is unclear why DHA fails to efficiently bind to E allo , although it binds to E cat , albeit with a lower affinity than its 20 carbon homolog EPA. We speculate that in solution E allo is constricted in such a way that the carboxyl group of DHA cannot interact well with Arg-120 of E allo (33).
Based on studies of huPGHS-2 inhibition by DHA and DPA, we conclude that in the absence of other interfering FAs (or other ligands), DHA and DPA would be expected to cause 50% inhibition of PGHS-1 and PGHS-2 only when present at a 5-fold molar excess of AA. This is consistent with a 5-fold difference in K m (i.e. the K d for E cat ) between AA and DHA. A 5-fold higher concentration of free DHA (or DPA) does not occur in cells, except in special cases like retina or testes that have significantly higher levels of DHA than AA (49) or in cells cultured with high concentrations of -3 FAs (16). Accordingly, our results would suggest that the biologic effects of DHA and DPA are not mediated primarily via effects on PGHSs but rather through other mechanisms.
In earlier studies, we had found that PA, linoleate, DHA, and 2-arachidonoylglycerol were ineffective in preventing crosslinking between monomers of huPGHS-2, whereas EPA and AA and most COX inhibitors did prevent cross-linking (12). These experiments were performed with huPGHS-2 variants having sulfhydryl groups juxtaposed in adjoining subunits of huPGHS-2 (e.g. at positions 127 and 541). We can now interpret these cross-linking data to imply that ligands only interfere with cross-linking if simultaneously bound to E cat and E allo and that this only occurs with FAs and COX inhibitors that bind with submicromolar affinities to E allo (e.g. AA and EPA). A corollary to this is that ligands having a preference for binding E cat versus E allo are those that do not interfere with cross-linking. DHA, PA and oleate all fail to interfere with cross-linking (12). The FAs that do interfere with crosslinking between huPGHS-2 monomers include all C-20 carbon FAs with three or more double bonds and 18:36 and 18:43. We speculate that these latter FAs all bind E allo of huPGHS-2 with high affinities.