The Presence of Peptidases in the Rabbit Hypothalamus Capable of Inactivating Luteinizing Hormone-Releasing Hormone*

Vol. 26, No. 8, August 1975 Printed in U.SA.

FERTILITY AND STERILITY Copyright " 1975 The American Fertility Society

THE PRESENCE OF PEPTIDASES IN THE RABBIT HYPOTHALAMUS CAPABLE OF INACTIVATING LUTEINIZING HORMONE-RELEASING HORMONE* EWAN C. GRIFFITHS, B.Sc., PH.D., KENNETH C. HOOPER, B.Sc., PH.D., D. HUTSON, S. L. JEFFCOATE, B.A., M.B.B.CHIR., PH.D., AND D. T. HOLLAND Department of Physiology, University of Sheffield, Sheffield Sl 0 2TN, and Department of Chemical Pathology, St. Thomas's Hospital, London SEl 7EH, England

Since the isolation and characterization of luteinizing hormone-releasing hormone (LH-RH) as the decapeptide pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-ArgPro-Gly NH2 , 1• 2 there have been reports on its enzymic inactivation by various tissues, including liver, kidney, 3 • 4 and brain. 3 • 5 • 7 From studies on oxytocin activation, 8• 9 it has been suggested that the hypothalamus, which is known to be the site of LH-RH synthesis and release, to,u contains peptidases capable of inactivating the releasing hormone. Recently, this has been confirmed in the rat hypothalamus, where peptidases were found in a supernatant (soluble, cytoplasmic) fraction and a particulate (microsomal and mitochondrial) fraction. 6 • 7 As the rabbit hypothalamus has already been shown to contain enzymes acting on other polypeptide hormones 12 • 13 and the enzymes' activities have been closely linked with luteinizing hormone (LH) release, 12 • 14 the presence of peptidases inactivating LH-RH was investigated in this brain region, using a sensitive and specific radioimmunoassay for the releasing hormone, 15 to provide further information on the function of thse enzymes.

been maintained under standard conditions and had had free access to water and pellet diet, were used. Female rabbits were kept for approximately 3 weeks before use, to ensure that none was pregnant or pseudopregnant.

Preparation of the Hypothalamus. Each animal was killed at approximately the same time of morning (9:30 to 9:45 A.M.) by a blow on the neck and exsanguination. The brain was quickly excised and the hypothalamus was dissected out after removal of the optic chiasma and corpora mamillaria. Each hypothalamus was weighed, and those from three animals were bulked together; they were homogenized in 9 volumes of 0.25 M sucrose in a hand-operated homogenizer (all previously cooled to 4° C). The homogenate was centrifuged in an MSE 6L ultracentrifuge at 2° C for 15 minutes at 600 x g, to give a nuclear cell debris fraction and a supernatant fraction. The former was discarded and the latter was centrifuged again, this time for 1 hour at 25,000 x g, giving a particulate fraction containing microsomes and mitochondria and a supernatant fraction. The particulate fraction was resuspended in approximately 1 ml of distilled water, and MATERIALS AND METHODS both this fraction and the supernatant Animals. Adult male and female rab- fraction were dialyzed overnight against bits of the New Zealand White strain distilled water at 4o C. The nondiffusible (average body weight, 2.5 kg), which had nitrogen content of the two fractions was determined by a micro-Kjeldahl method. 16 Incubation Procedure. Incubations Received August 14, 1974. *Supported in part by The Wellcome Trust. were carried out at a total volume of 1 ml, 802

-~

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for various times detailed later, at 37° C and pH 7.3 (0.2 volume of0.4 M Na2HP0 4NaH2P04 buffer). The concentration of synthetic LH-RH in all incubations was 500 ng/ml, as used previously. 6 • 7 A constant amount of supernatant fraction (128 IJ-g N) was employed in the timecourse studies, although varying amounts of both fractions were later incubated with LH-RH to determine their peptidase activity. Enzyme activity was terminated by placing the incubated samples in boiling water for 10 minutes. The following series of incubations was performed for each experiment: (1) buffer plus 500 ng of LH-RH; (2) buffer plus either supernatant or particulate fraction; (3) buffer plus supernatant or particulate fraction preboiled for 10 minutes, then incubated with 500 ng ofLH-RH; (4) buffer plus supernatant or particulate fraction plus 500 ng of LH-RH. Incubated samples were then stored at -20° C until required for assay. Assay of Residual LH-RH by Radioimmunoassay. Residual LH-RH after incubation was determined by radioimmunoassay as described in detail elsewhere.15· 17 The antiserum (DD/14) was raised in a rabbit against LH-RH conjugated to bovine serum albumin and was used at final dilution of 1:150,000. Its specificity was directed toward the COOH terminus of the decapeptide, as with our previous antisera 18 : complete cross-reaction was found with the 3-10 octapeptide, but none (less than 0.01%) was found with the 1-9 nonapeptide. Synthetic LHRH was labeled with 1251 by the method of Greenwood et- al. 19 and purified as previously detailed by J effcoate et al. 15 All dilutions of reagents were made in 0.04 M phosphate buffer (pH 7.4) containing 0.1% bovine serum albumin and 0.6% NaCl; free and antibody-bound radioactivity were separated by ethanol precipitation. Expression of Enzyme Activity in the Hypothalamic Fractions. The recovery of

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LH-RH from the medium after incubation was expressed in terms of substrate recovered from the control experiment (that cont~ining buffer, preboiled hypothalamic fraction, and 500 ng of LH-RH), i.e., as log Ao/A, where Ao and A are the LH-RH concentrations (nanograms per milliliter) in the control (incubation 3) and test (in incubation 4) experiments, respectively. Log Ao!A was plotted against concentration of hypothalamus homogenate used, and fiducial limits at a probability of 0.95 were calculated. 20 Materials. Synthetic LH-RH was kindly provided by Dr. A. L. Walpole and Dr. H. Gregory, of Imperial Chemical Industries, Pharmaceuticals Division, Alderley Park, Cheshire, England. RESULTS

Confirmation of LH-RH Inactivation by Hypothalamic Peptidases. Table 1 illustrates the effects of incubating LH-RH with a constant amount of supernatant and particulate fractions (128 /J-g N) from male and female rabbits for 15 minutes: when incubations 4a and 4b, 5a and 5b are compared with incubation 1, it can be seen that there was a significant decrease in the amount of releasing hormone present in these incubations. Recoveries of approximately 100% of the LH-RH initially added in incubations 1, 3a, and 3b suggest that LH-RH was unaltered by the incubation procedure alone, but comparison with incubations 4a and 4b, 5a and 5b indicates the presence of a heat-labile component, which was obviously enzymic in nature, in these solutions. Some small amount of releasing hormone was found in both fractions on their own (incubations 2a and 2b, combined results from male and female rabbits), although this was relatively insignificant in comparison with the levels found in the other incubations. Time-Course Studies. It can be seen in Figure 1 that, after incubation of 500 ng

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TABLE 1. Residual LH-RH as Measured by Radioimmunoassay in the Various Samples after 15-Minute Incubation No. of determinations

Incubation

Residual LH-RH ± SEM

Statistical significancEf

+:

nglml

1. 2a. 2b. 3a. 3b. 4a. 4b. 5a. 5b.

Buffer + 500 ng of LH-RH Buffer + particulate fraction (male and female) Buffer + suoornatant fraction (male and female) Buffer + preboiled particulate fraction + 500 ng of LH-RH (male and female) Buffer+ preboiled supernatant fraction+ 500 ng ofLH-RH (male and female) Buffer + male particulate fraction + 500 ng of LH-RH Buffer + female particulate fraction + 500 ng of LH-RH Buffer + male supernatant fraction + 500 ng of LH-RH Buffer + female supernatant fraction + 500 ng of LH-RH

5 6 6 6

533.4 1.35 0.34 545.0

6

492.1 ± 25.2

Not significant

4 4 4 4

310.0 250.0 7.0 2.5

p p p p

± ± ± ±

± ± ± ±

22.1 0.29 0.08 19.7

3.7 12.2 0.09 0.07

p < 0.001 p < 0.001

Not significant

< < < <

lr

0.001 0.001 0.001 0.001

"Compared with incubation 1, calculated by Student's t-test.

of LH-RH with a constant amount of incubation period should be used in future hypothalamic supernatant fraction (128 experiments, since, in each sample incup,g N) for various times, residual re- bated, sufficient residual releasing horleasing hormone levels, as detected by mone remained after this time for several radioimmunoassay, decreased very rapid- determinations by radioimmunoassay. Peptidase Activity in the Hypothalami ly and were extremely low after 180 minutes. When considered with the evi- of Male and Female Rabbits. Hypodence from Table 1, this suggests the thalamic peptidase activity in both male presence of very active peptidases in this and female rabbits is shown in Figure 2. hypothalamic fraction, since inactivation Activity was found in both particulate would appear to be virtually complete 25 within the 1st hour of incubation. Similar results were obtained with supernatant log~ A fractions from both male and female 20 rabbits. Working from the results obtained, it was decided that a 15-minute 1.5

E 5"'

400

10

J: 0:

'

J:

300

~

0

~

"'~

200

OS

0:

100 50

100

150

200

Hypothalamus homogonat• mcubalod().Jg N)

0 Time(min)

FIG. 1. Residual LH-RH (nanograms per milliliter) after incubation of an initial LH-RH concentration of 500 ng/ml with a constant amount (128 p.,g N) of supernatant fraction from male and female rabbit hypothalami for 5, 10, 20, 30, 60, 120, and 180 minutes. e, Male supernatant fraction; o, female supernatant fraction.

FIG. 2. Levels of peptidase activity in the hypothalamic fractions from male and female rabbits when incubated with 500 ng of LH-RH. Two or three estimations were made from each group of three rabbits, ± fiducial limits; e, male supernatant enzyme; ;., female supernatant enzyme; o, male particulate-bound enzyme; a, female particulatebound enzyme.

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HYPOTHALAMIC PEPTIDASE INACTIVATION OF LH-RH

and supernatant fractions, although it was about 5 times greater in the supernatant fraction than in the particulate fraction. Supernatant peptidase activity was significantly higher in female than in male animals (P = 0.95); however, particulate activity was approximately the same for both sexes. These results agree closely with those from the rat hypothalamus, 7 except that, in the rat, the male supernatant fraction has a greater activity than the same fraction from female animals. DISCUSSION

Previous work on hypothalamic oxytocin inactivation has suggested that there are enzymes present in this brain region capable of inactivating LH-RH. 12 - 14 The studies described here have confirmed the presence of these peptidases and have extended the studies of the enzymes, from those already performed in the rat, 6 • 7 to the rabbit, where loss of the biologic6 as well as the immunologic activity 7 of LH-RH has been demonstrated. LH-RH inactivation was found to be more rapid in the rabbit than in the rat/ with 80 to 90% inactivation in the first 5 minutes of incubation, whereas less than 80% was found in the rat. However, a similar intracellular distribution of peptidase activity was found in both animals, with the majority of activity in the supernatant (soluble, cytoplasmic) fraction and a small amount of activity in the particulate (microsomal-mitochondrial) fraction. This distribution is in accord with that already shown by Hooper, 12 who used oxytocin as substrate. Determination of enzyme activity in the two fractions from male and female rabbits required a shorter incubation time than that previously used (15 minutes instead of 30 minutes)/ but produced evidence of a sexual difference in peptidase activity. In the supernatant fraction, activity was significantly greater in female than in male animals, the

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reverse of that found with oxytocin as substrate. 21 In the particulate fraction, activity was slightly higher, although not significantly so, in female rabbits. This sexual difference in LH-RH inactivation may indicate that the male and female rabbit hypothalami degrade the releasing hormone at different rates. Inasmuch as the enzymes can rapidly inactivate the releasing hormone, they may be responsible for its metabolism in the hypothalamus and so could control the amount of LH-RH available for release. A fine balance between LH-RH biosynthesis,22 • 23 degradation, and release would thus regulate the amount of releasing hormone produced, with the peptidases providing a part of the control mechanism. Knights et al.2 4 have suggested that polypeptide hormone levels are controlled by enzyme degradation; the enzymes studied here may provide an example of this type of physiologic mechanism at the site of hormone synthesis and release. The radioimmunoassay for LH-RH has provided a suitable means for investigating the releasing homone's inactivation by the hypothalamic peptidases. It has provided a more accurate and direct method for measuring LH-RH than had been possible in other studies 3 • 6 and should form a useful method for further investigation of the peptidases. These enzymes could have a role in the control ofLH-RH production in the hypothalamus through their action on the releasing hormone; consequently, this method could give useful information on the ways in which the central nervous system regulates reproductive function. SUMMARY

The presence of peptidases in the rabbit hypothalamus capable of inactivating luteinizing hormone-releasing hormone (LH-RH) was investigated with the use of a sensitive and specific radioimmunoassay for the releasing hormone. En-

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zymes acting on LH-RH were found in two fractions from rabbit hypothalami, a particulate fraction and a supernatant fraction, which very rapidly inactivated the decapeptide. Peptidase activity in the particulate fraction from male and female rabbits was approximately the same, but the supernatant fraction from female animals had significantly greater peptidase activity than that from male animals. These findings confirm the hypothesis that rabbit hypothalamus contains peptidases which inactivate LH-RH, and give some indication that the enzymes may be involved in the control by the central nervous system of reproductive function. Acknowledgments. We wish to thank Dr. A. L. Walpole and Dr. H. Gregory, of I. C. I. Pharmaceuticals, for the generous gift of synthetic LH-RH, and Mrs. B. Lenihan for her assistance in preparing the manuscript. REFERENCES 1. Schally AV, Arimura A, Kastin AJ: Hypothalamic regulatory hormones. Science 179:341, 1973 2. Blackwell RE, Guillernin R: Hypothalamic control of adenohypophysial secretions. Annu Rev Physiol 35:357, 1973 3. Sandow J, Heptner W, Neimann E: Kinetic studies with LH-RH. Naunyn-Schmeidebergs Arch Pathol Pharmacol[Suppl]274:R64, 1973 4. Redding TW, Schally AV: The distribution, half-life and excretion of tritiated LH-RH in rats. Life Sci 12:23, 1973 5. Griffiths EC, Hooper KC, Jeffcoate SL, Holland DT: Peptidases in different areas of the rat brain inactivating luteinizing hormone-releasing hormone (LH-RH). Brain Res 85:161, 1975 6. Griffiths EC, Hooper KC, Hopkinson CRN: Evidence for an enzymic component in the rat hypothalamus capable of inactivating luteinizing hormone releasing factor (LRF). Acta Endocrinol (Kbh) 74:49, 1973 7. Griffiths EC, Hooper KC, Jeffcoate SL, Holland DT: The presence of peptidases in the rat hypothalamus inactivating luteinizing hormonereleasing hormone (LH-RH). Acta Endocrinol (Kbh) 77:435, 1974 8. Griffiths EC, Hooper KC: The stimulatory influence of gonadal steroids and methallibure (ICI 33,828) on peptidase activity in the female rat hypothalamus. Fertil Steril 24:269, 1973 9. Griffiths EC, Hooper KC: Competitive inhibition between oxytocin and luteinizing hormone releasing factor (LRF) for the same enzyme system

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in the rat hypothalamus. Acta Endocrinol (Kbh) 75:435, 1974 10. Mess B, Zanisi M, Tima L: Site of production of releasing and inhibiting factors. In The Hypothalamus, Edited by L Martini, M Motta, F Fraschini. New York, Academic Press, 1970, p 259 11. Crighton DB, Schneider HPG, McCann SM: Localization of LH-releasing factor in the hypothalamus and neurohypophysis as determined by an in vitro method. Endocrinology 87:323, 1970 12. Hooper KC: The effects of ovariectomy and injected oestradiol monobenzoate on polypeptide metabolism in the hypothalamus. Biochem J 110:151, 1968 13. Hopkinson CRN, Hooper KC: Peptidase activity in the hypothalamus. Biochem J 117:36p, 1970 14. Frith DA, Hooper KC: The effect of coitus on the activity of certain enzymes in the female rabbit hypothalamus. Acta Endocrinol (Kbh) 65:213, 1971 15. Jeffcoate SL, Fraser HM, Holland DT, Gunn A: Radioimmunoassay of luteinizing hormonereleasing hormone in serum from man, sheep and rat. Acta Endocrinol (Kbh) 75:625, 1974 16. Fleck A, Munro HN: The determination of organic nitrogen in biological materials-a review. Clin Chim Acta 11:2, 1965 17. Jeffcoate SL, Fraser HM, Gunn A, Holland DT: Radioimmunoassay of luteinizing hormone releasing factor. J Endocrinol 57:189, 1973 18. Jeffcoate SL, Holland DT, Fraser HM, Gunn A: Preparation and specificity of antibodies to the decapeptide, luteinizing hormone-releasing hormone (LH-RH). Immunochemistry 11:75, 1974 19. Greenwood FC, Hunter WM, Glover TS: The preparation of 131 I-labelled human growth hormone of high specific radioactivity. Biochem J 89:114, 1963 20. Burn JA: Biological Standardization, Second Edition. Oxford, Oxford University Press, 1952, p37 21. Frith DA: Ph.D. thesis, University of Sheffield, 1971, p 103 22. Johansson HG, Hooper F, Sievertsson H, Currie BL, Folkers K, Bowers CY: Biosynthesis in vitro of luteinizing releasing hormone by hypothalamic tissue. Biochem Biophys Res Commun 49:656, 1972 23. Johansson KNG, Currie BL, Folkers K, Bowers CY: Biosynthesis of the luteinizing hormonereleasing hormone in mitochondrial preparations and by a possible pantetheine-template mechanism. Biochem Biophys Res Commun 53:502, 1973 24. Knights EB, Baylin SB, Foster GV: Control of polypeptide hormones by enzymatic degradation. Lancet 2:719, 1973