ABSTRACT. This study demonstrates that leukotriene C4, at concentrations in the picomolar range, released luteinizing hormone (LH) but not growth hormone ...
Proc. Nati. Acad. Sci. USA Vol. 82, pp. 3834-3838, June 1985 Medical Sciences
Leukotriene C4 as a mediator of luteinizing hormone release from rat anterior pituitary cells (luteinizing hormone-releasing hormone/growth hormone/lipoxygenase products/hypothalamus)
ANNA-LENA HULTING*, JAN AKE LINDGRENt, TOMAS HOKFELTt, PETER ENEROTH§, SIGBRITT WERNER*, CARLO PATRONO¶, AND BENGT SAMUELSSONt *Department of Endocrinology and §The Hormone Laboratory, The Karolinska Hospital, and Departments of tPhysiological Chemistry and *Histology, Karolinska Institutet, S-104 01 Stockholm, Sweden; and ¶Department of Pharmacology, Catholic University, 1-00168 Rome, Italy
Contributed by Tomas Hokfelt, September 5, 1984
eases (Bethesda, MD). LTC4 antiserum was raised against
LTC4 conjugated with bovine serum albumin (6). LHRH antiserum was purchased from RIA (Tyne and Wear, England). Aprotinin (Trasylol) was obtained from Bayer (Leverkusen, F.R.G.). Pituitary Cell Dispersion and Culture Procedure. Adenohypophyses from male Sprague-Dawley rats (200 g) were enzymatically dispersed (7). The cells were plated on multiwell plates (1-2 x 105 cells per ml per well), whereby 24 adenohypophyses typically yielded 144 incubation wells. The culture medium consisted of Dulbecco's modified Eagle's medium supplemented with fresh sera. Cells were counted in a Buerker's chamber; the trypan blue dye exclusion technique was applied to assure >95% viability of the dispersed cells used. On the third day, fresh Krebs-Ringer bicarbonate medium (containing 1% bovine serum albumin), test substances, and Trasylol (2 x 10 M) were added in a total volume of 1 ml per well. Control media and various solutions of peptides and LTs were kept at pH 7.4. Controls and test substances were incubated in 10 equivalent wells for 0.5 and 3 hr. One-hundred microliters was aspirated from each well after 0.5 and 3 hr, dissolved in 900 gl of buffer, and stored at -200C. RIA. LH was determined by using a RIA kit (kindly supplied by the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, and used as described in ref. 8). GH was determined by using a double-antibody technique (9) with monkey anti-rat GH serum supplied by the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. Highly purified rat GH was used as a reference standard in the RIA. Rat GH was iodinated with "25I by the lactoperoxidase method of Thorell and Johansson (10). Immunohistochemisty. Male rats (body weight, 150-200 g) were anesthetized and perfused via the ascending aorta with Ca2'-free Tyrode's solution followed by ice-cold formalin (11). The brains were dissected out, rinsed for at least 24 hr in 0.1 M phosphate buffer containing 5% sucrose, and cut on a cryostat (section thickness, 8 or 14 ,um). The sections were processed for indirect immunofluorescence (12). Briefly, adjacent sections taken from various levels of the hypothalamus were incubated in a humid atmosphere for 1824 hr at 4°C with LTC4 antiserum (1:400) or LHRH antiserum (1:100; both antisera contained 0.3% Triton X-100), rinsed in phosphate-buffered saline, incubated with fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit antibodies (1:10) (Dakopatts, Copenhagen), rinsed in phosphate-buffered saline, mounted in a mixture of glycerol and phosphate-buffered saline (3:1) containing p-phenylene-diamine (13, 14), and examined in a Zeiss fluorescence microscope equipped with proper filter combinations.
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.
Abbreviations: GH, growth hormone; LT, leukotriene; LH, luteinizing hormone; LHRH, LH-releasing hormone; FITC, fluorescein isothiocyanate.
ABSTRACT This study demonstrates that leukotriene C4, at concentrations in the picomolar range, released luteinizing hormone (LH) but not growth hormone (GH) from dispersed rat anterior pituitary cells. Leukotriene B4, another lipoxygenase pathway product of arachidonic acid, had no effect on LH or GH release. The stimulatory effect of leukotriene C4 could be seen after 0.5 but not after 3 hr of incubation. This was in contrast to the dose-dependent LH-releasing hormone (LHRH)-induced LH release that was not measurable after 0.5 hr but was fully established after incubation for 3 hr. Furthermore, the LH-releasing ability of leukotriene C4 was blocked in the presence of high doses of LHRH. The immunohistochemical analysis revealed leukotriene C4-immunoreactive fibers at all levels of the median eminence, mainly in the lateral parts. These fibers exhibited a marked overlap distribution with LHRH-immunoreactive fibers and elution-restaining experiments revealed identity of at least a large proportion of the leukotriene C4- and LHRH-immunoreactive fibers. Furthermore, cell bodies in the preoptic area contained both leukotriene C4- and LHRH-like immunoreactivities, suggesng localization of these two compounds in the same neurons. The leukotrienes (LTs) are compounds with biological effects related to anaphylaxis and inflammation. LT formation is initiated by 5-lipoxygenation of arachidonic acid to 5 (S)hydroperoxyicosatetraenoic acid, which is further transformed into an unstable epoxide, LTA4. Enzymatic addition of glutathione to this intermediate leads to formation of LTC4, which can be further metabolized to LTD4 and LTE4. Alternatively, LTA4 is enzymatically hydrolyzed to the dihydroxy acid LTB4 (see ref. 1 for review). Formation of LTC4, LTD4, and LTE4 in the rat brain has been described recently (2, 27, 28). The largest amounts of LTC4 were produced in the hypothalamus and the median eminence. This is of interest since a role for lipoxygenase products in anterior pituitary function has been suggested (3, 4). The present report demonstrates that LTC4 released luteinizing hormone (LH) but not growth hormone (GH) from dispersed rat anterior pituitary cells after 0.5 hr in culture.
MATERIALS AND METHODS Materials. Synthetic LTC4 was a kind gift from J. Pike (Upjohn, Kalamazoo, MI). LTB4 was biosynthetically prepared as described (5). Synthetic LH-releasing hormone (LHRH) was obtained from Hoechst (Basel, Switzerland). Highly purified rat GH was obtained from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Dis-
3834
Medical Sciences:
Hulting et aL
Proc. NatL Acad Sci USA 82 (1985)
3835
1600 .5
e 200
0 '4-
0 4) 4)
1200
4) C
= 100
ll800
oJ
lo-15
1o-7 lo-11 1o-9 LT, M FIG. 1. Effects of LTB4 (m) and LTC4 (0) on LH secretion from dispersed rat anterior pituitary cells after 0.5 hr in culture. LH values are expressed as % of basal release in control cultures. The curves show the mean ± SEM from four different experiments with 10 identical cultures in each experiment. *P < 0.02; **P < 0.01; ***P < 0.001 (Student's t test). 0 1o-17
lo-i3
To analyze the possible occurrence of LTC4- and LHRHlike immunoreactivity, the elution-restaining technique of Tramu et al. (15) was used. Briefly, after photography of the LTC4 staining patterns in the median eminence, the sections were eluted with acid potassium permanganate, rinsed in phosphate-buffered saline, incubated with FITC-conjugated antibodies (as above), and analyzed in the fluorescence microscope. If no staining was observed, the sections were processed as above-i.e., incubated with LHRH antiserum, rinsed, incubated with FITC-conjugated antibodies, rinsed, mounted, examined, and photographed in the fluorescence microscope. The LTC4 and LHRH staining patterns were then compared on the photographs.
RESULTS Hormone Release Experiments. LTC4 at concentrations of 0.01 pM to 1.0 nM significantly stimulated basal LH release 400 .
** 300 -
400
l
0
Control LHRH LTB4 LTB4 LTC4 LTC4 +
+
LHRH
LHRH
FIG. 3. Effects of LTB4 and LTC4 (10-14 M) and LTs combined with LHRH (10-6 M) on LH release (ng/ml) from rat anterior pituitary cells after 3 hr in culture. Bars represent the mean t SEM from two experiments; n = 10 in each experiment. ***P < 0.001 (Student's t test).
from dispersed rat anterior pituitary cells after 0.5 hr in culture (Figs. 1 and 2). At 0.01 pM, LTC4 caused a 90% increase of LH levels. Maximal effects were seen at concentrations of 0.1-1.0 pM (123-129%o stimulation). At higher concentrations the effect decreased (Fig. 1). When incubations were kept for 3 hr, the ability of LTC4 to release LH could no longer be observed (Fig. 3). LTB4 at concentrations of 0.01 fM to 0.1 juM had no significant effect on basal LH release (Figs. 1-3). Neither LTB4 nor LTC4 exerted any significant effect on basal GH release at concentrations ranging from 1 fM to 0.1 gM (Fig. 4). LHRH at concentrations of 1.0 nM and 1.0 ,LM had no stimulatory effect on LH release after 0.5 hr (Table 1). However, after 3 hr, LHRH stimulated LH release in a dose-dependent manner (Table 1). 200 1 -62
,: 200'
'4-a
C.)
0
I-j
e 100
I------
4)
100 4) 0
0 I
0
Control LHRH LTB4 LTB4 LTC4 LTC4 +
+
LHRH LHRH FIG. 2. Effects of LTB4 and LTC4 (10-14 M) and LTs combined with LHRH (10-6 M) on LH release (ng/ml) from rat anterior pituitary cells after 0.5 hr in culture. Bars represent the mean ± SEM from two experiments; n = 10 in each experiment. **P < 0.01 (Student's t test).
0
10-15
lo- 13
I
lo-11
10-9
10-7
LT, M FIG. 4. Effects of LTB4 (-) and LTC4 (-) on GH secretion from dispersed anterior pituitary cells after 0.5 hr in culture. GH values are expressed as % of basal release in control cultures. The curves show the mean ± SEM from four different experiments with 10 identical cultures in each experiment. No statistically significant changes were observed.
3836
Medical Sciences: Hulting et aL
Proc. NatL Acad Scd USA 82 (1985)
FIG. 5. Immunofluorescence micrographs of the median eminence at anterior (a), posterior (b and c), and middle (d-h) levels after incubation with antiserum to LTC4 (a, b, d, and g) or LHRH (c, e, f, and h). (e) The same section as d, which, after elution, has been restained with LHRH antiserum. (g and h) Higher magnifications of parts of d and e, respectively, as indicated by rectangles. LTC4-like immunoreactivity is present in fibers in the midline at anterior levels (a) and at middle and posterior levels mainly in the lateral part of the external layer (b and d). At all levels there is a close overlap with the LHRH-positive fibers (compare b with c and d with f). Restaining experiments revealed that most LTC4 fibers are identical to LHRH-positive ones (compare d with e and g with h). Arrows in d, e, g, and h indicate some examples of identical fibers. Asterisks indicate third ventricle. (Bars = 50 Eum.)
Proc. NatL Acad ScL USA 82 (1985)
Medical Sciences: Hulting et aL Table 1. LH release from cultures of rat anterior pituitary cells in the presence of LTC4 and LHRH LXI release, ng/ml Condition 3 hr 0.5 hr 97 ± 11 Control 238 + 12 183 ± 21* 259 ± 18 LTC4, 10 fM LHRH 1 nM 97 ± 16 407 ± 38* 1 AM 913 + 117t 129 ± 40 LH release was measured after 0.5-hr and 3-hr incubations. Values represent the mean ± SEM from two experiments; n = 10 in each experiment. Student's t test was used for statistical comparisons. *P < 0.01.
tp < o.ooi.
The stimulatory effect of LTC4 on LH release after 0.5 hr was inhibited in the presence of 1.0 FM LHRH (Fig. 2). LTB4 or LTC4 (at concentrations of 0.01 pM) did not influence the stimulated LH release induced by LHRH (1.0 .M) after 3 hr (Fig. 3). Immunohistochenistry. The immunohistochemical analysis revealed moderately dense LTC4- and LHRH-immunoreactive fiber networks in the external layer of the median eminence (Fig. 5). At anterior levels the fibers covered the entire superficial zone (Fig. Sa) and in the caudal direction they assumed a more lateral location (Fig. 5 b and d), extending into the proximal parts of the pituitary stalk. The intensity of the LTC4-positive fibers was weaker than that of the LHRHpositive ones and their number often seemed to be somewhat lower. However, the two distribution patterns exhibited a high degree of similarity. Furthermore, the elution-restaining experiments revealed that most LTC4-immunoreactive fibers also contained LHRH-like immunoreactivity. Analysis of adjacent sections of the rostral hypothalamus revealed cell bodies that were both LTC4- and LHRH-positive.
DISCUSSION Arachidonic acid metabolites have been indicated to participate in the control of secretion of different hypothalamic and pituitary hormones. Thus, prostaglandin E2 stimulates release of LHRH from the hypothalamus (16). Prostaglandins seem, however, not to be involved in LH secretion from the pituitary gland (17). Epoxygenated fatty acids formed from arachidonic acid by a NADPH-dependent cytochrome P450-linked monooxygenase (18-20) have recently been shown to stimulate release of hypothalamic and pituitary hormones. Thus, 5,6-epoxyicosatrienoic acid causes release of somatostatin and LHRH from rat median eminence (21) and also stimulates (at concentrations above 0.1 ,uM) release of LH from rat anterior pituitary cells (22). Formation of 5,6epoxyicosatrienoic acid in rat hypothalamus has been re-
ported (21). Recently, a role for lipoxygenase products in pituitary function has been suggested (3, 4). Such a role was further indicated by our finding of LTC4 production in the hypothal-
amus and the median eminence from rat in vitro (2). The present report shows that LTC4 but not LTB4 stimulated LH release in vitro, whereas no effects were observed on GH release. LTC4 seems to stimulate LH secretion through a mechanism with rapid onset and short duration. In contrast, an effect of LHRH on LH release was not obvious after incubation for 0.5 hr in our system but was seen after 3 hr. At this time interval a marked effect was observed with high concentrations of LHRH (1.0 j&M), but, at 1.0 nM, the increase of LH levels was only about 709o--i.e., less than the effect
3837
observed at 0.5 hr with LTC4 at a concentration of 10 fM. These findings suggest that LTC4 is a potent stimulator of LH release. The fast action of LTC4 is in contrast to the apparently slow action of LHRH as seen in our model. This may indicate that LTC4 and LHRH stimulate LH release at least partly through different mechanisms. A possible hypothesis is that LTC4 and LHRH cooperate in the control of LH release, whereby LTC4 may be preferentially responsible for initial release and LHRH for a more long-lasting effect. The LH-releasing ability of LTC4 seen at 30 min was inhibited in the presence of high concentrations of LHRH. The mechanisms behind this interaction of the two compounds are at present not understood. Similarly, the lack of effect of LTC4 on LH release after a 3-hr incubation period needs further analysis. In this context, it is of interest that immunohistochemical studies using antibodies raised against LTC4 (conjugated to bovine serum albumin) revealed a network of LTC4-immunoreactive nerve endings in the lateral part of the external layer of the median eminence (2), and this was confirmed in the present study. The identity of the LTC4-like immunoreactivity is still unclear. It was earlier shown that it could be absorbed out with an LTC4-bovine serum albumin conjugate and with glutathione in about lOx higher concentrations, but not with LTC4 alone (2). These nerve endings had a distribution that was similar to those containing LHRH-like immunoreactivity throughout the median eminence, as shown in earlier immunohistochemical studies (23). Since elution-re-
staining experiments according to Tramu et al. (15) revealed that at least a large proportion of the LTC4- and LHRH-positive fibers were identical and since LHRH- and LTC4-like immunoreactivities in addition were observed in the same cell bodies in the preoptic area, it seems likely that the two are compounds are present in the same neurons and perhapsemireleased from the same nerve endings in the median nence.
In conclusion, the present results suggest a close interaction between LTC4 and LHRH in the regulation of LH release that is supported by immunohistochemical findings of a possible coexistence of the two compounds in the same neurons. These findings may be related to the recent demonstration that many central and peripheral neurons release two (or more) messengers at their synapses, whereby in several cases one compound seems to exert a fast effect and the other factor a more sustained response (24-26). We thank Agneta Hilding, Gaby Astrom, Kerstin Englund, Margareta Maxe, and Anne Peters for skillful technical assistance and Elisabet Bjorklund for expert secretarial help. This work was supported by grants from The Swedish Medical Research Council (03X06805, 04X-02887, 19X-06865, and 03X-00217), Alice och Knut Wallenbergs Stiftelse, The Karolinska Institut's Research Funds, and the Soderberg Foundation.
1. Samuelsson, B. (1983) Science 220, 568-575. 2. Lindgren, J. A., Hokfelt, T., Dahldn, S.-E., Patrono, C. & Samuelsson, B. (1984) Proc. Nati. Acad. Sci. USA 81, 62126216. 3. Canonico, P. L., Schettini, G., Valdenegro, C. A. & MacLeod, R. M. (1983) Neuroendocrinology 37, 212-217. 4. Naar, Z., Vanderhoek, J. Y., Lindner, H. R. & Call, K. J. (1983) in Prostaglandin, Thromboxane and Leukotriene Research, eds. Samuelsson, B., Paoletti, R. & Ramwell, P. (Raven, New York), Vol. 12, pp. 259-263. 5. Lindgren, J. A., Hansson, G. & Samuelsson, B. (1981) FEBS Lett. 128, 329-335. 6. Aehringhaus, U., Wolbling, R. H., Konig, W., Patrono, C., Peskow, B. M. & Peskar, B. A. (1982) FEBS Lett. 146, 111114. 7. Nakano, H., Fawcett, C. P. & McCann, S. M. (1976) Endocrinology 98, 278-288.
3838
Medical Sciences: Hulting et al.
8. Andersson, K., Nielsen, 0. G., Toftgard, R., Eneroth, P., Gustavsson, J.-A., Battistini, N. & Agnati, L. S. (1983) Neurotoxicology 4, 43-56. 9. Eden, S., Albertsson-Wiklund, K. & Isaksson, 0. (1978) Acta Endocrinol. 88, 676-690. 10. Thorell, J. L. & Johansson, B. G. (1971) Biochim. Biophys. Acta. 251, 363-369. 11. Zamboni, L. & Martini, C. (1967) J. Cell Biol. 148, 35 (abstr.). 12. Coons, A. H. (1958) in General Cytochemical Methods, ed. Danielli, J. F. (Academic, New York), pp. 399-422. 13. Johnson, D. G. & de C Nogueira Aranjo, G. M. (1981) J. Immunol. Methods 43, 349. 14. Platt, J. L. & Michael, A. F. (1983) J. Histochem. Cytochem. 31, 840-842. 15. Tramu, G., Pillez, A. & Leonardelli, J. (1978) J. Histochem. Cytochem. 26, 322-324. 16. Ojeda, S. R., Naar, Z. & Negro-Vilar, A. (1979) Prostaglandins Med. 2, 249-275. 17. Sundberg, D. K., Fawcett, C. P., Iluier, P. & McCann, S. M. (1975) Proc. Soc. Exp. Biol. Med. 148, 54-59. 18. Capdevila, J., Marnett, L. J., Chacos, N., Prough, R. A. & Estabrook, R. W. (1980) Proc. Natl. Acad. Sci. USA 79, 757770.
Proc. Natl AcadJ Scd USA 82 (1985) 19. Oliw, E. H., Lawson, J. A., Brash, A. R. & Oates, J. A. (1982) J. Biol. Chem. 257, 3771-3779. 20. Chacos, N., Falck, J. R., Wixtrom, C. & Capdevila, J. (1982) Biochem. Biophys. Res. Commun. 104, 916-922. 21. Capdevila, J., Chacos, N., Falck, J. R., Manna, S., Negro-Vilar, A. & Ojeda, S. R. (1983) Endocrinology 113, 421-423. 22. Snyder, G. D., Capdevila, J., Chacos, N., Manna, S. & Falck, J. R. (1983) Proc. Natl. Acad. Sci. USA 80, 3504-3507. 23. Barry, J. (1979) Int. Rev. Cytol. 60, 179-221. 24. Lundberg, J. M., Hedlund, B., Anggard, A., Fahrenkrug, J., Hbkfelt, T., Tatemoto, K. & Bartfai, T. (1982) in Systemic Role ofRegulatory Peptides, eds. Bloom, S. R., Polak, J. M. & Lindenlaub, E. (Schattauer, Stuttgart, F.R.G.), pp. 93-119. 25. Lundberg, J. M. & Hokfelt, T. (1983) Trends Neuro. Sci. 6, 325-333. 26. Hokfelt, T., Johansson, 0. & Goldstein, M. (1984) Science 225, 1326-1334. 27. Dembinska-Kied, A., Simmet, T. & Peskar, B. A. (1984) Eur. J. Pharmacol. 99, 57-62. 28. Moskowitz, M. A., Kiwak, K. J., HIekimian, K. & Levine, L. (1984) Science 224, 886-889.
