an antioxidant that inhibits lipoxygenase; and icosatetraynoic acid, an acetylenic ... 5,6-epoxyicosatrienoic acid was the most potent of the compounds tested.
Proc. Natl. Acad. Sci. USA Vol. 80, pp. 3504-3507, June 1983 Medical Sciences
Action of luteinizing hormone-releasing hormone: Involvement of novel arachidonic acid metabolites (adenohypophysis/secretion)
GARY D. SNYDER*, J. CAPDEVILAt, N. CHACOSt, S. MANNAt, AND J. R. FALCKt *Departments of Physiology, tBiochemistry and NMolecular Genetics, University of Texas Health Science Center, Dallas, Texas 75235
Communicated by Ronald W Estabrook, March 7, 1983
MATERIALS AND METHODS Adult Sprague-Dawley male rats (Holtzman, Madison, WI) were killed by decapitation. The anterior pituitary from each animal was dissected from the intermediate and posterior lobes. The anterior pituitaries were minced with a razor blade and enzymatically dispersed to a single-cell suspension in 0.1% trypsin (Difco; 1:250)/0.1% bovine serum albumin (Sigma) in minimal essential medium (GIBCO) without Ca2+. The cells were recovered from the trypsin solution by centrifugation (800 x g, 10 min) and then suspended in medium 199 (GIBCO)/10% horse serum for culture overnight (10). The following morning the cells were incubated in medium 199/0.1% bovine serum albumin for 30 min with or without 5,8,11,14-icosatetraynoic acid (ETYA, a gift of Hoffiman-La Roche), indomethacin (Merck Sharp and Dohme) or nordihydroguaiaretic acid (NDGA, Sigma), all at 50 ,AM. The cells were centrifuged and the medium was discarded. The cell pellet was resuspended in the experimental medium (medium 199/0.1% bovine serum albumin) in the presence or absence of test compounds. LHRH was added to some samples in 10 ,ul of 0.154 M NaCl to final concentrations of 10-100 nM. Arachidonic acid (Nu-Chek Prep, 99.9% by gas chromatographic analysis) was added in some cultures to a final concentration of 1 ,uM. The synthetic epoxyicosatrienoic acids (EETs) were prepared by literature methods (11-13) and stored at -20°C in benzene under argon. The components were purified by HPLC (14) immediately prior to addition to the incubation medium. The chromatographic solvent was evaporated under argon and the EET was dissolved in 10 ,ul of ethanol. Samples were incubated with EETs for either 30 or 60 min. EETs are rapidly hydrated in aqueous solutions; however, in aqueous solution, albumin (0.1%) or membrane phospholipid prevents this hydration for extended periods of time (15). Secretion of LH was determined by radioimmunoassay and is expressed as the LH-S1 reference standard of the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases.
ABSTRACT Anterior pituitary cells were incubated in the presence of luteinizing hormone-releasing hormone and one of three inhibitors of arachidonic acid metabolism: indomethacin, an inhibitor of the cyclooxygenase system; nordihydroguaiaretic acid, an antioxidant that inhibits lipoxygenase; and icosatetraynoic acid, an acetylenic analogue of arachidonic acid that blocks all known pathways of arachidonic acid metabolism. Indomethacin was ineffective in blocking luteinizing hormone-releasing hormone-stimulated luteinizing hormone secretion. Nordihydroguaiaretic acid was only marginally capable of inhibiting luteinizing hormone-releasing hormone-stimulated luteinizing hormone secretion. Icosatetraynoic acid at 10 pM completely inhibited stimulated luteinizing hormone secretion. Addition of several epoxygenated arachidonic acid metabolites to cells in vitro resulted in secretion of luteinizing hormone equal to or greater than that induced by 10 nM luteinizing hormone-releasing hormone. The half-maximal effective dose for these compounds was approximately 50 nM. The 5,6-epoxyicosatrienoic acid was the most potent of the compounds tested. These studies suggest that luteinizing hormone-releasing hormone-stimulated luteinizing hormone release is closely coupled with the production of oxidized arachidonic acid metabolites. Moreover, one or more of the epoxygenated arachidonic acid metabolites might be a component of the cascade of reactions initiated by luteinizing hormone-releasing hormone that ultimately results in secretion of luteinizing hormone.
Calcium ions, cyclic nucleotides, and combinations thereof are postulated to be the "second messenger" of luteinizing hormone-releasing hormone (LHRH) (1, 2). The action of LHRH in stimulating the release of luteinizing hormone (LH) from anterior pituitary gonadotrophs also appears to involve turnover of phosphatidylinositol (3) and release of arachidonic acid from membrane phospholipid stores (4). Previous studies have eliminated a role for prostaglandins in the action of LHRH because LHRH-stimulated LH secretion is insensitive to blockage of prostaglandin formation by indomethacin (5). At least two additional pathways of arachidonate metabolism are currently recognized: a lipoxygenase reaction leading to hydroperoxides and leukotrienes (6) and the recently reported NADPH-supported cytochrome P450-dependent epoxygenase pathway (79). Since arachidonic acid mobilization from cellular pools is apparently required for LHRH-stimulated LH secretion (4), we presumed that either arachidonic acid or one or more of its metabolites of the lipoxygenase or epoxygenase pathways were active in stimulated secretion of LH. The studies reported here suggest that conversion of arachidonic acid to physiologically active metabolites constitutes an essential link in the cascade of intracellular events leading to LHRH-stimulated LH release.
RESULTS Effect of Inhibitors of Arachidonic Acid Metabolism on LH Secretion. As has been reported (5), indomethacin (10 ,uM), a cyclooxygenase inhibitor (5, 16), was ineffective in blocking either basal LH secretion or LHRH-induced LH secretion (Table 1). Likewise, NDGA, a lipoxygenase inhibitor (17, 18), at a concentration (10 ,uM) reported to block leukotriene production in neutrophils (19) had no significant effect on either basal or LHRH-stimulated LH secretion (Table 1). In fact, in both seAbbreviations: LHRH, luteinizing hormone-releasing hormone; LH, luteinizing hormone; ETYA, 5,8,11,14-icosatetraynoic acid; NDGA, nordihydroguaiaretic acid; EET, epoxyicosatrienoic acid; DHET, dihydroxyicosatrienoic acid; HETE, hydroxyicosatetraenoic acid.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3504
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Table 1. Effect of NDGA or indomethacin on LHRH-stimulated LH secretion LH released, ng per 10' cells Without LHRH With LHRH* 9.6 ± 0.7 No inhibitor 3.5 ± 0.3 NDGA 2.8 ± 0.2 13.0 ± 0.4 10.9 ± 0.6 3.4 ± 0.3 Indomethacin Cells were incubated in the presence or absence of 10 ,uM NDGA or 10 uM indomethacin for 30 min prior to and during the 30-min stimulation with 20 pM LHRH. Data represent mean ± SEM LH release in 30 min for five mean replicate samples. *P < 0.01; vs. control, indomethacin, or NDGA by analysis of variance and Neuman-Keul multiple comparison tests.
ries of experiments, the presence of indomethacin or NDGA seemed to slightly potentiate the LH-releasing capability of LHRH (Table 1). However, at concentrations -40 AuM, NDGA decreased LHRH-stimulated LH secretion by 50% (Table 2). Addition of 10 AM ETYA to medium containing anterior pituitary cells had no effect on the unstimulated secretion of LH during incubations lasting up to 1 hr. On the other hand, for cells previously incubated for 30 min with 10 AM ETYA, LHRHinduced LH secretion was almost completely blocked (Fig. 1). The ability of ETYA to inhibit LHRH-stimulated LH secretion was found to be dose related but only over a narrow range of concentrations (Fig. 2). Effect of Epoxygenated Arachidonic Acid Metabolites on LH Secretion. The results of experiments with inhibitors suggested that products of the cyclooxygenase and lipoxygenase systems are less important than products of the epoxygenase pathway. Therefore, synthetically produced products of the epoxygenase pathway of arachidonic acid metabolism were tested in vitro to determine their ability to cause LH secretion. At 1 AtM, all epoxygenated compounds tested stimulated LH secretion during a 1-hr incubation (Table 3). 5,6-EET was the most potent metabolite tested, increasing LH secretion to 4-8 times the control level in several experiments. The other EETs tested were, among themselves, equipotent, increasing LH secretion from the cells to 3-4 times that of controls (Table 3). The release of LH by EETs other than 5,6-EET was of approximately the same magnitude as that produced by 10 nM LHRH, a dose that usually produces maximal secretion of LH in this experimental model (10). At the end of each experiment, the cells were incubated at 37TC for 15 min in medium containing 0.01% trypan blue. More than 95% of the cells in control and all treatment groups excluded the dye, suggesting that exposure to either the EETs at Table 2. Effect of NGDA concentration on LHRH-stimulated LH release LH released, ng per 10' cells Sample NDGA, XM Control 3.0 ± 0.2 NDGA 2.8 ± 0.3 10 16.9 ± 0.6 LHRHI(0.l1bM) 10 14.6 ± 1.3* LHRH/NDGA 40 10.6 ± 0.5* 11.2 ± 0.3* 80 100 9.9 ± 0.8* Cells were incubated in the presence or absence of NDGA at the concentrations indicated for 30 min; then the medium was removed and fresh medium containing LHRH and NDGA (10-100 ^M) was added for an additional 60 min. Data represent mean + SEM of five replicate samples. *P < 0.05; vs. 0.1 pM LHIIR by analysis of variance and Neuman-Keul multiple comparison tests.
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8.0
W
6.0
4.0-
2.0
2
1
3
4
FIG. 1. Effect of 10 ,uM ETYA on LHRH-stimulated LH secretion from anterior pituitary cells. Cells were incubated in the presence or absence of ETYA for 30 min; then, the medium was changed, 10 nM LHRH in 10 ,ul of saline was added, and incubation was continued for 30 min. Bars: 1, control; 2, LHRH only; 3, ETYA only; 4, ETYA/LHRH. *P < 0.01 by Student's t test.
the highest concentration used (1 ,uM) or to the various inhibitors was not damaging to cell viability. Effect of 5,6-EET Concentration on LH Secretion. 5,6-EET was added to cell samples at concentrations of 10 nM to 1 ,uM. The concentration that induced half-maximal LH secretion was calculated in three separate experiments to be 30-80 nM. Minimal stimulation of LH secretion was observed in tests in which 10 nM 5,6-EET was present. At 1 ,uM, 5,6-EET caused maximal LH secretion (Fig. 3). Effect of Arachidonic Acid and of Chemical Modification of 5,6-EET on LH Secretion. Arachidonic acid (1 AM), the immediate precursor of 5,6-EET, when added to cell samples was essentially equipotent with 5,6-EET in eliciting LH secretion (103% of 5,6-EET stimulation in two experiments). Others (4, 20) have also reported that arachidonic acid promotes LH secretion; however, in their experiments, slightly higher concentrations (20 ,uM) were required. Several compounds structurally similar to 5,6-EET were also tested at 1 ,uM to determine their ability to stimulate LH secretion. Two products likely to be produced in the tissue or cells from 5,6-EET-the 6-lactone of 5,6-dihydroxy-8,11,14-icosatrienoic acid (5,6-DHET) and the methyl ester of 5,6-EET-were also equipotent with 5,6-EET in stimulating LH secretion (93% and 96% of 5,6-EET-stimulated LH secretion, respectively). However, the hydration 100 r._
80
-
od
60 a
-
n.
.._4
40-
: 20
-
i
, 1
I
a
100
1,000
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ETYA, nM
FIG. 2. Effect of concentration of ETYA on inhibition of LHRHstimulated LH secretion. For protocol, see Fig. 1.
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Table 3. Effect of selected EET on LH secretion from dispersed anterior pituitary cells Sample LH released, ng per 10' cells Control 2.6 ± 0.7 5,6-EET 16.2 ± 1.6* 8,9-EET 9.9 ± 0.9* 11,12-EET 10.3 ± 0.7* 14,15-EET 8.3 ± 0.8* LIIRH 8.4 ± 0.8* Cells were incubated in 1 ml of medium containing either 1 pM EET or 10 nM LHRH for 60 min. Data represent mean ± SEM of seven replicate samples. *P < 0.001; vs. control by analysis of variance and Neuman-Keul multiple comparison tests.
product of 5,6-EET, 5,6-DHET, proved to be less potent in causing LH secretion than the parent molecule in three separate experiments (i = 82% of 5,6-EET-stimulated LH release). DISCUSSION Recent studies concerning the mechanism of LHRH action suggest that Ca2', rather than cAMP or cGMP, functions as the second messenger of LHRH (1). However, as yet there is no explanation as to how the binding of LHRH to its membrane receptor alters the cell membrane of the gonadotroph to a state that permits greater permeability to Ca2+. One hypothesis is that the calcium channels of the cell membrane are closely linked with a specific class of phospholipid, phosphatidylinositol (21). Although opinions differ as to how the metabolism of phosphatidylinositol contributes to the opening of the calcium channels (20, 21), there is a consensus that increased turnover of phosphatidylinositol occurs in a variety of tissues seemingly coincident with the opening of Ca2' channels (20, 21). It has recently been reported that LHRH stimulates turnover of a specific phospholipid, phosphatidylinositol, in anterior pituitary cells (3). It had previously been suggested that LHRH may cause turnover of phospholipids because stimulation of gonadotrophs by LHRH was shown to release arachidonic acid from the phospholipids of the cell (4). Moreover, inhibition of the release of arachidonic acid from phospholipids by quinicrine or chloroquine (phospholipase A2 inhibitors) blocks LHRH-stimulated LH release (4). 20
°15 54
0
84)
54
1
10 100 5,6-EET, nM
1,000
FIG. 3. Effect of concentration of 5,6-EET on LH secretion. Cells were incubated with 5,6-EET for 60 min. Data are mean ± SEM of five
replicate samples.
The metabolism of arachidonic acid proceeds via one of three enzymatic pathways: (i) cyclooxygenase, which leads to prostaglandins and thromboxane formation; (ii) lipoxygenase, which leads to formation of hydroperoxyicosatetraenoic acids (HPETEs) and subsequently to hydroxyicosatetraenoic acids (HETEs) and leukotrienes, or (iii) a NADPH-supported cytochrome P-450dependent epoxygenase, which leads to the formation of epoxygenated acids (7-9, 14). An inhibitor of arachidonic acid metabolism, ETYA, effectively inhibits the function of both cyclooxygenase and lipoxygenase (17), as well as the NADPHcytochrome P450-dependent epoxygenase (R. W. Estabrook, personal communication). Indomethacin, on the other hand, at low concentrations inhibits only the cyclooxygenase system (S, 16, 17) while NDGA, at the concentrations used in these experiments, is reported to inhibit the lipoxygenase enzyme pathway (18). Since indomethacin and NDGA (at low concentrations) are without effect on LHRH-stimulated LH release, the results of the experiments using these inhibitors suggest that oxidation of arachidonic acid by either the cyclooxygenase or possibly the lipoxygenase pathway is not of primary importance in the initial metabolic step leading to the final icosanoid product(s) required in the releasing action of LHRH. In support of this contention, Sundberg et aL (5) have tested numerous prostaglandins for their ability to stimulate LH secretion. They found no evidence that prostaglandins or other products of cyclooxygenase are involved in LH secretion. Naor et al. (22) have reported that, of numerous lipoxygenase products tested on anterior pituitary cells in vitro, only 5-HETE at high concentrations (e.g., >1 AuM) is capable of stimulating LH secretion. Furthermore, the maximal secretion of LH elicited by 5-HETE was not comparable with maximal LHRH-stimulared LH secretion. However, the data from the present study (e.g., effect of ETYA) as well as the data from that of Naor et aL (22) do indicate that one or more metabolites of arachidonic acid is essential for expression of LHRH-stimulated LH release. Finally, the fact that the antioxidant NDGA, only at concentrations higher than needed to suppress lipoxygenase activity (18, 22), decreases the LHRH-stimulated LH secretion suggests that an oxygenase (e.g., epoxygenase) reaction is, indeed, a critical initial reaction leading to the release process (Table 2). When added to the cells, all of the epoxygenated acids tested were capable of stimulating secretion of LH. 5,6-EET was, however, twice as potent as the other isomers. The ability of 5,6-EET to stimulate LH secretion to values over and above that of 10 nM LHRH cannot yet be explained. However, the cells are exposed to higher levels of 5,6-EET in vitro, in contrast to the amount that might normally be produced by the cell itself. In LHRH-stimulated cells, the endogenous EET may subsequently be rapidly converted to less active metabolites enzymatically. This might then explain why the addition of 5,6EET to the cells causes unexpectedly high levels of LH secretion. The observation that 5,6-EET is the most biologically potent EET (in regard to stimulation of LH secretion) is significant in that, in another study, we have found evidence that anterior pituitary microsomal fractions actively oxidize arachidonic acid to 5,6-EET and its hydration product, 5,6-DHET. We have noted that several of EETs, as well as the derivatives, are capable of stimulating LH secretion. These results point out that, as yet, we cannot identify a specific arachidonate metabolite(s) essential to the mechanism of LHRH-stimulated LH secretion. It is possible that the cell is capable of using any of the EETs applied exogenously as a precursor of the compound(s) naturally produced under a secretion stimulus or, alternatively, by substituting the exogenous compound for the natural product. The data we have at this time do not allow us to favor either possibility. Current data favor 5,6-EET as a pre-
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Proc. Natl. Acad. Sci. USA 80 (1983)
Snyder et al.
cursor or intermediate species of one of these molecules cause it is a primary metabolite of arachidonic acid and is
bethe
most potent naturally occurring stimulatory agent of LH secretion yet reported. Both the 8-lactone and the methyl ester
of 5,6-EET can be enzymatically converted to 5,6-DHET by the cell. Moreover, the greater. biological potency of 5,6-EET may be related to its greater chemical reactivity, which results from the proximity of the epoxide to the carboxylate. The intracellular mechanisms involved in the action of the arachidonate metabolites described here remain to be elucidated. However, there-is some evidence in neutrophils that have been stimulated to release lysosomal enzymes that the production of HPETEs, HETEs, or their metabolites increases the permeability of the cell membrane to Ca2" (23). Whether or not a similar mechanism (e.g., production of EETs) occurs in gonadotrophs that have been stimulated to release LH by LHRH remains to be explored. Such reactions, if indeed they exist, would strengthen the hypothesis regarding Ca2" as an intracellular second messenger of LHRH and also provide a mechanism to explain the increased permeability of the cells to c2+,
Ca2~
We wish to thank Mrs. Michelle Norris for expert technical assistance and Mrs. Diane Doach for typing and editing the manuscript. We wish to express our appreciation to Dr. R. W. Estabrook for critical evaluation of the manuscript. This work was supported by National Institute of Child Health and Human Development Grant HD-154654 (G.S.), National Institute of General Medical Sciences Grant NIGMS-16488 (J.C. and N.C.), and Grant 1-782 from the Robert A. Welch Foundation (J.R.F. and S.M.). 1. Conn, P. M., Marian, J., McMillian, M., Stem, J., Rogers, D., Hamby, M., Penna, A. & Grant, E. (1981) Endocr. Rev. 2, 174185. 2. Labrie, F., Borgeat, P., Drouin, J., Beaulieu, M., Lagace, L., Ferland, L. & Raymond, V. (1979) Annu. Rev. Physiot 41, 555569.
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3. Snyder, G. & Bleasdale, J. E. (1982) Mol, Cell Endocrinol 28, 5563. 4. Naor, Z. & Catt, K. J. (1981)J. Biol Chem. 256, 2226-2229. 5. Sundberg, D. K., Fawcett, C. P., lIner, P. & McCann, S. M. (1975) Proc. Soc. Exp.,BioL Med..148, 54-59. 6. Borgeat, P. & Samuelsson, B. (1979)J. Biol Chem. 254, 7865-7869. 7. Capdevila, J., Marnett, L. J., Chacos, N., Prough, R. A. & Estabrook, R. W (1982) Proc. Natl Acad. Sci. USA 79, 767-770. 8. Oliw, E. H. & Oates, J. A. (1981) Biochim. Biophys. Acta 666, 327340. 9. Morrison, A. R. & Pascoe, N. (1981) Proc. Natl Acad. Sci. USA 78, 7375-7378. 10. Naor, Z., Snyder, G., Fawcett, C. P. & McCann, S. M. (1978)J. Cyclic Nucleotide Res. 4, 475-486. 11. Corey, E. J., Marfat, A., Falck, J. R. & Albright, J. 0. (1980)J. Am. Chem. Soc. 102, 1433-1435. 12. Falck, J. R. & Manna, S. (1982) Tetrahedron Lett. 23, 1755-1756. 13. Corey, E. J., Niwa, H. & Faick, J. R. (1979) J.Am. Chem. Soc. 101, 1586-1587. 14. Chacos, N., Falck, J. R., Wixtrom, C. & Capdevila, J. (1982) Biochem. Biophys. Res. Commun. 104, 916-922. 15. Chacos, N., Capdevila, J., Falck, J. R., Manna, S., Martin-Wixtrom, C., Gill, S. S., Hammock, B. D. & Estabrook, R. W. (1983) Arch. Biochem. Biophys., in press. 16. Ojeda, S. R., Naor, Z. & Negro-Vilar, A. (1979) Prostaglandins Med. 5, 249-275. 17. Hamberg, M. (1976) Biochim. Biophys. Acta 431, 651-654. 18. Tappel, A. L., Lundberg, W O. & Boyer, P. D. (1953) Arch. Biochem. Biophys. 42, 293-304. 19. Smith, R. J. & Bowman, B. J. (1982) Biochem. Biophys. Res. Commun. 104, 1495-1501. 20. Berridge, M. J. (1981) Mol CelL Endocrinol 24, 115-140. 21. Michell, R. H. (1975) Biochim. Biophys. Acta 415, 81-147. 22. Naor, Z., Vanderhoek, J. Y., Linder, H. R. & Catt, K. J. (1982) in Advances in Prostaglandin, Thromboxane and Leukotriene Research, eds. Samuel, B., Paoleti, R. & Ramwell, P. W (Raven, New York), pp. 259-263. 23. Volpi, M., Maccache, P. H. & Shaati, R. I. (1980) Biochem. Biophys. Res. Commun. 94, 1231-1237.
