Brain Research, 174 (1979) 221-228 © Elsevier/North-Holland Biomedical Press
221
S U B C E L L U L A R LOCALIZATION OF L U T E I N I Z I N G H O R M O N E R E L E A S I N G H O R M O N E D E G R A D I N G ACTIVITY IN THE HYPOTHALAMUS
C.
RICHARD PARKER, Jr.*, MARK
M.
FOREMAN and JOHN C. PORTER
The Cecil H. and Ida Green Center for Reproductive Biology Sciences, The Departments of Obstetrics and Gynecology and Physiology, The University of Texas Southwestern Medical School, Dallas, Texas 75235 (U.S.A.)
(Accepted February 1st, 1979)
SUMMARY The in vitro degradation of endogenous as well as exogenous luteinizing hormone releasing hormone (LHRH) by subcellular fractions of rat hypothalamic tissue was studied. Endogenous LHRH, localized in isolated nerve terminals (synaptosomes), was found to be resistant to enzymatic degradation (60 rain, 37 °C) as long as the synaptosomal membrane remained intact. Endogenous L H R H was rapidly degraded by the 900 × g supernatant fluid and cytosol but not by myelin/microsomes, intact synaptosomes, or mitochondria. Lysed synaptosomes rapidly degraded exogenous LHRH. The L H R H degrading activity of synaptosomes was highly concentrated in the 'synaptosol', i.e., the cytosol of the nerve terminal. These data suggest that the L H R H degrading activity of the rat hypothalamus is a readily solubilized component of neurons, and possibly of non-neuronal cells.
INTRODUCTION The capacity of neurons to synthesize and degrade putative neurotransmitters such as acetylcholine and norepinephrine is well established3,7, 21. It has recently been demonstrated that homogenates of rat brain tissue possess the enzymatic capacity to degrade L H R H with a resultant loss of its biological and immunoreactive properties 4,5,8-1°,13,2°. The L H R H degrading activity of hypothalamic homogenates has been shown to vary with the sex and endocrine status of the animal from which the tissue was obtained6, lo. These findings are suggestive that such enzymatic activity may * To whom requests for reprints and correspondence should be addressed.
222 have a regulatory role in the control of L H R H secretion or in the expression of L H R H activity in the brain. Although L H R H has been shown to be sequestered in dense-cored granules of hypothalamic synaptosomes 18, the question whether L H R H stored within, or released from, nerve terminals is subject to enzymatic degradation has not been addressed. The purpose of the current investigation was to evaluate the extent of L H R H degradation under in vitro conditions by several subcellular fractions of rat hypothalamic tissue. MATERIALS AND METHODS Adult male rats (200-275 g) of the Long-Evans strain were decapitated; the brains were excized, and the hypothalami were dissected and placed in ice-cold 0.32 M sucrose-10 # M CaC12. All sucrose solutions contained 10 # M CaCI2, and subcellular fractionation was carried out at 4 °C. The hypothalamic fragments were rinsed and homogenized in 10 vols. of 0.32 M sucrose and centrifuged at 900 x g for 10 min at 4 '~C as described previously 17. The 900 Y. g supernatant fluid (0.9K S) was fractioned on discontinuous sucrose density gradients consisting of 1.5 ml each of 0.8 and 1.2 M sucrose TM. Subcellular fractions consisting of cytosol, myelin/microsomes, synaptosomes, and mitochondria were recovered from the gradients, in previous studies, electron microscopic examination of the particulate fractions has confirmed the nature of subcellular elements recoverable from such gradients 18. The particulate fractions were diluted te approximately 0.4 M sucrose and were pelleted by centrifugation for 60 min at 100,000 ~< g in a Ti-75 rotor (Beckman Instruments, Inc.). The subcellular particles in the pellets were resuspended intact in 0.32 M sucrose or in 10 ,aM CaCI2. Incubations consisted of 1.0 ml aliquots of the various subcellular fractions (0.9 K S, cytosol, myelin/microsomes, synaptosomes, and mitochondria) mixed with 1.0 ml Hanks balanced salt solution. Incubations were carried out at 37 °C in glass flasks using a Dubnoff metabolic incubator. In experiments designed to follow the degradation of exogenous L H R H , subcellular fractions were pre-incubated at 37 °C for 30 min prior to the addition of 10 ng L H R H dissolved in 100 #1 of 0.01 M phosphate buffer, pH 7.0, containing 0.14 M NaCI. Triplicate aliquots were removed at designated times, extracted in acid ethanol ~ and assayed for L H R H by radioimmunoassay 15 using synthetic L H R H (Beckman Instruments, Inc.) as the reference standard. Protein was quantified by the method of Lowry et al. 11 using bovine serum albumin as the standard. The activity of marker enzymes, cytochrome oxidase (EC 1.9.3.1), N A D P H cytochrome c reductase (EC 1.6.2.4), and acid phosphatase (EC 3,1.3.2), was assayed by established methods 12,14,19. RESULTS When incubated with the 0.9K S or cytosol, exogenous L H R H was rapidly degraded as shown in Fig. 1. The half-life of L H R H in the cytosol or 0:9K S ranged from 5 to 20 min depending on the amount of protein in the incubation vial. No significant disappearance of L H R H was observed in incubations containing medium alone
223 100
80 FZ LIJ I-Z
8 T
60
I
40 LL O
20
0
30
60
INCUBATION TIME (min)
Fig. 1. Time course of the degradation of exogenous L H R H by medium, cytosol, and 0.9K S. Flasks containing medium or the indicated amounts of cytosolic or 0.9K S protein (in mg) were pre-incubated for 30 min at 37 ' C and then incubated with 10 ng LHRH as described in Materials and Methods. Data are presented as a mean percentage of the initial LHRH content remaining at each time period sampled. The initial LHRH content was determined immediately after the addition of LHRH at time 0.
(Fig. 1). Similarly, in incubations of cytosol or 0.9K S which had been heated at 100 °C for 10 min prior to the addition of LHRH, there was no degradation of L H R H (data not shown). Little, if any, degradation of L H R H occurred during a 60-min incubation with intact or hypo-osmotically shocked myelin/microsomes or mitochondria (data not shown). In addition, exogenous L H R H was not degraded by intact synaptosome preparations (Fig. 2). However, significant degradation of L H R H was observed during incubation with hypo-osmotically shocked (or lysed) synaptosomes, and the degree of degradation was related to the amount of synaptosomal protein in the incubation flask (Fig. 2). As with the 0.9K S and cytosol, prior heating for 10 min at 100 °C of lysed synaptosomes prevented the degradation of L H R H (data not shown). Of the several subcellular fractions isolated from the 0.9K S, only synaptosomes contained significant quantities of endogenous LHRH. As shown in Fig. 3, the endogenous L H R H content of the synaptosome fraction was stable for at least 60 rain at 37 °C. Conversely, when synaptosomes were lysed and then incubated at 37 °C, endogenous LHRH was degraded. In order to define the localization of the L H R H degrading activity in synaptosomes, the synaptosomes were lysed and centrifuged at 4 °C for 60 rain at 100,000 × g. The pellet, i.e., synaptosomal membranes and intra-synaptosomal organelles, was
224 • Intact Synaptosomas o Lysed Synoptosomes
._.__..----e
4.0mg
iO07rag 0.6mg
500
2.0 rng 8O I'O,8rng T 60 n," T ..J
O
'~
13rag
i
-~ 4o u_ o
o
20
t,9mg
0
0
Q 50
0
2,5rag
60
INCUBATION TIME (min)
Fig. 2. Time course of the degradation of exogenous LHRH by intact and lysed hypotha!amic synaptosomes. Synaptosome pellets, containing the designated amount of protein (rag), were either suspended intact in 0.32 M sucrose or were lysed by suspension in 10 ~M CaCI~. See Fig. 1 and text for other details.
-lO0
Intoct Synaptosomes
5 60
\~
E
0 0
I 2~O
60
90
INCUBATION TIME (mini
Fig. 3. Time course of the degradation of endogenous LHRH in lysed and intact hypothalamic synaptosome preparations. Intact and lysed synaptosome preparations (1.5 mg protein each) were incubated in the absence of exogenous L H R H as described in Materials and Methods. Data, expressed as percentage of the endogenous L H R H content at time 0, are presented as the mean 7~ S . E .
a
225 TABLE 1 Distribution of L H R H degrading activity and marker enzymes in subee/lular ./~'actions of adult male rat hypothalamus Snbcellular fraction
0.9K S Cytosol Myelin/microsomes Mitochondria Intact synaptosomes Lysed synaptosomes Synaptosol
Enzyme activity* LHRH degrading activity
Cytochrome oxidase
NADPH cytoehrome c reductase
Acid phosphatase
10.0 36.0 0.8 1.3 1.0 17.0 49.0
ND 6 18 894 196 ND ND
ND 10 I0 3 4 ND ND
ND 8 18 39 35 ND ND
* The LHRH degrading activity is expressed as ng LHRH degraded/60 rain/rag protein. Incubations of subcellular fractions -+ 10 ng LHRH were performed as indicated in the text (n 2 6 separate experiments). Other enzyme activities are expressed as nmol product formed/rain/rag protein (n : 3). ND not determined. resuspended in 10 # M CaCI~. The pellet fraction or the supernatant fluid was incubated with 10 ng L H R H . The specific activity of the L H R H degrading enzyme(s) in the synaptosol, the cytosol of the nerve terminals, was 6-8 times greater than that in the pellet fraction. A comparison of specific activities of the L H R H degrading enzyme(s) in the subcellular fractions are shown in Table I. L H R H degrading activity was highest in the cytosol and synaptosol. With the exception of the 0.9K S and lysed synaptosomes, little L H R H degrading activity was expressed in the remaining subcellular fractions. The subcellular localization of the L H R H degrading activity was different from that of the enzyme markers (Table I). The activity of the mitochondrial marker, cytochrome oxidase, was greatest in the mitochondrial fraction and was also present in synaptosomes, which are known to contain mitochondria. The activity of the microsomal enzyme, N A D P H cytochrome c reductase, was greatest in the myelin/microsome fraction and in the cytosol, whereas the activity of the lysosomal enzyme, acid phosphatase, was greatest in the mitochondria and in the synaptosomes. DISCUSSION The functional organization of cholinergic and noradrenergic neurons in the brain is well established. However, the mechanisms controlling the synthesis, release, and degradation of the substances contained in peptidergic neurons, such as L H R H , are poorly understood. Of significance in neural transmission is the capacity of nerve terminals to release neurotransmitters which can be rapidly degraded after eliciting their action at the postsynaptic receptor site. Acetylcholine is inactivated by acetylcholine esterase (EC 3.1.1.7) which is present in plasma membranes and microsomes of
226 neurons a,21. In the brain, norepinephrine is degraded by monoamine oxidase (EC 1.4.3.4), which is present in mitochondria :~, and catechol-o-methyltransferase (EC 2.1.1.6), a soluble enzyme 7. The brain also contains enzymes which can degrade peptides such as thyrotropin releasing hormone (TRH), oxytocin, and LHRH6,8,1~ Variation in the rate of enzymatic degradation of T R H has been observed in the rat brain during postnatal development TM. In addition, the capacity of brain homogenates to degrade L H R H and oxytocin has been shown to be affected by the hormonal status of experimental animals6,10. Thus, it is possible that peptide degrading enzymes may be of importance in peptidergic neurons. However, little is known concerning the cell of origin or subcellular localization of such peptide degrading enzymes. Griffiths and co-workers4, 5 have found L H R H degrading activity in a soluble fraction of hypothalamic homogenates and in a particulate fraction which was pelleted at 25,000 ~ g for 60 min. However, the soluble fraction probably contained microsomal elements, and the particulate fraction undoubtedly contained many elements, including myelin, synaptosomes, and mitochondria. In other studies, hypothalami were homogenized under conditions in which some, if not most, of the subcellular organelles would have been lysed s,~3,e°. In the current study, the L H R H degrading enzyme activity in the 0.9K S was shown to be present largely in the cytosol, and little, if any, L H R H degrading enzyme activity was expressed in the myelin and microsomes, intact synaptosomes, or mitochondria. However, significant L H R H degradation occurred after lysis of the synaptosomes, and the specific activity of the L H R H degrading enzyme(s) in the synaptosol was as high as that of the cytosol. In a prior investigation, T R H degrading activity was found in the 0.9K S of rat hypothalamic homogenates, but little TRH degradation occurred in incubations containing isolated intact synaptosomeslL The L H R H degrading enzyme activity in rat hypothalamic tissue is not associated with microsomes and synaptic plasma membranes as is acetylcholine esterase or with mitochondria as is monoamine oxidase. Instead, L H R H degrading activity is distributed among subcellular fractions of rat hypothalamic homogenates in a manner similar to that of lactate dehydrogenase (EC 1.1.1.27) and catechol-o-methyltransferase7, 21. Although we cannot exclude non-neuronal cells as a source of L H R H degrading enzyme activity in the cytosol, there is strong evidence that the enzyme activity is present in the synaptosol, the cytosol of isolated nerve terminals. However, since the hypothalamic synaptosome fraction is composed of nerve terminals containing L H R H as well as those containing TRH, ct-melanocyte stimulating hormone, dopamine, etc., it would be improper to conclude that the L H R H degrading enzyme activity which was expressed in the lysed synaptosome preparation was derived fi'om L H R H containing neurons. However, it may be possible to address this question in the future since the L H R H containing synaptosomes, after fractionation on continuous sucrose density gradients, exhibit a slightly different banding density than do those containing T R H or catecholamines a. Several other questions remain to be answered concerning peptidergic neurons of the brain and the hypothalamic LHRH-containing neurons in particular. What are the by-products of L H R H degradation and are there differences in
227 either the degree o f L H R H d e g r a d a t i o n or the nature o f L H R H b r e a k d o w n p r o d u c t s themselves which are manifested by different brain cell types? D o the p r o d u c t s o f L H R H d e g r a d a t i o n possess any intrinsic biological activity? Is the d e g r a d a t i o n of L H R H subject to biological a n d p h a r m a c o l o g i c a l m a n i p u l a t i o n ? O f practical i m p o r t a n c e to future studies is our finding that s y n a p t o s o m a l L H R H is stable during i n c u b a t i o n s o f isolated synaptosomes. In addition, it m a y be possible to investigate L H R H actions on n e u r o n s under certain in vitro conditions since exogenous L H R H is n o t d e g r a d e d by intact synaptosomes. It is a p p a r e n t , however, t h a t cytosolic c o n t a m i n a n t s must be r e m o v e d before such studies are p e r f o r m e d a n d that care must be exercised to avoid lysis o f nerve terminals during such studies. ACKNOWLEDGEMENTS The a u t h o r s t h a n k M i l d r e d A r n o l d for excellent editorial assistance and G a y e Burnsed, Sue Sherwin, L i n d a Akers, Jodie Roberts, and D a v i d D o w d for technical assistance. This w o r k was s u p p o r t e d by the following grants: AM01237, HD07062, AG00306, a n d 5-S07-RR-05426-16.
HDllI49,
REFERENCES 1 Barnea, A., Ben-Jonathan, N., Colston, C., Johnston, J. M. and Porter, J. C., Differential subcellular compartmentalization of thyrotropin releasing hormone (TRH) and gonadotropin releasing hormone (LRH) in hypothalamic tissue, Proc. nat. Aead. Sci. (Wash.), (1975) 3153 3157. 2 Barnea, A., Oliver, C. and Porter, J. C., Subcellular localization of a-melanocyte stimulating hormone in the rat hypothalamus, J. Neurochem., 29 (1977) 619-624. 3 De Robertis, E. and Rodriguez de Lores Arnaiz, G., Structural components of the synaptic region. In A. Lajtha (Ed.), Handbook ofNeurochemistry, Vol. H, Plenum Press, New York, 1969, pp. 365392. 4 Griffiths, E. C., Hooper, K. C. and Hopkinson, C. R. N., Evidence for an enzymic component in the rat hypothalamus capable of inactivating luteinizing hormone releasing factor (LRF), Acta endocr., 74 (1973) 49 55. 5 Griffiths, E. C., Hooper, K. C., Jeffcoate, S. L. and Holland, D. T., The presence of peptidases in the rat hypothalamus inactivating luteinizing hormone-releasing hormone (LH-RH), Acta endocr., 77 (1974) 435 442. 6 Griffiths, E. C. and Hooper, K. C., Peptidase activity in different areas of the rat hypothalamus, Acta endocr., 77 (1974) 10-18. 7 Guldberg, H. C. and Marsden, C. A., Catechol-o-methyl transferase: pharmacological aspects and physiological role, Pharmacol. Rev., 27 (1975) 135 206. 8 Koch, Y., Baram, T. and Chobsieng, P., Enzymic degradation of luteinizing hormone-releasing hormone (LH-RH) by hypothalamic tissue, Biochem. biophys. Res. Commun., 6l (1974) 95-103. 9 Kuhl, H. and Taubert, H.-D., Inactivation of luteinizing hormone releasing hormone by rat hypothalamic L-cystine arylamidase, Acta endocr., 78 (1975) 634-648. l0 Kuhl, H. and Taubert, H.-D., Short-loop feedback mechanism of luteinizing hormone: LH stimulates hypothalamic L-cystine arylamidase to inactivate LH-RH in the rat hypothalamus, Acta endocr., 78 (1975) 649-663. l 1 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 12 Masters, B. S. S., Williams, C. H., Jr. and Kamin, H., The preparation and properties ofmicrosomal TPNH-cytochrome c reductase from pig liver. In R. W. Estabrook and M. E. Pullman (Eds.), Methods. in Enzymology, Vol. X, Academic Press, New York, 1967, pp. 565 573.
228 13 McKelvy, J. F., LeBlanc, P., Laudes, C., Perrie, S., Grimm-Jorgensen, Y. and Kordon, C., The use of bacitracin as an inhibitor of the degradation of thyrotropin releasing factor and luteinizing hormone releasing factor, Biochem. biophys. Res. Commun., 73 (1976) 507-515. 14 Michell, R. H., Karnovsky, M. J. and Karnovsky, M. L., The distribution of some granuleassociated enzymes in guinea-pig polymorphonuclear leucocytes, Biochem. J., 116 (1970) 2.07-216. 15 Nett, T. M., Akbar, A. M., Niswender, G. D., Hedlund, M. T. and White, W. F., A radioimmunoassay for gonadotropin-releasing hormone (Gn-RH) in serum, J. olin. Endocr., 36 (1973) 880-885. 16 Oliver, C., Parker, C. R., Jr. and Porter, J. C., Developmental changes in the degradation of thyrotrophin releasing hormone by the serum and brain tissues of the male rat, J. Endocr., 74 (1977) 339-340. 17 Parker, C. R., Jr., Neaves, W. B., Barnea, A. and Porter, J. C., Studies on the uptake of [:3H]thyrotropin-releasing hormone and its metabolites by synaptosome preparations of the rat brain, Endocrinology, 101 (1977) 66-75. 18 Parker, C. R., Jr., Neaves, W. B., Barnea, A. and Porter, J. C., Studies on the subsynaptosomal localization of luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the rat hypothalamus, Endocrinology, 102 (1978) 1167-1175. 19 Smith, L., Spectrophotometric assay of cytochrome c oxidase. In D. Glick (Ed.), Methods. of Biochemical Analysis, Vol. 11, lnterscience Publishers, New York, 1955, pp. 427-434. 20 Sundberg, D. K. and Knigge, K. M., Luteinizing hormone-releasing hormone (LH-RH) production and degradation by rat medial basal hypothalami in vitro, Brain Research, 139 (1978) 89-99. 21 Whittaker, V. P., Michaelson, 1. A. and Kirkland R. J. A., The separation of synaptic vesicles from nerve-ending particles ('Synaptosomes'), Biochem. J., 90 (1964) 293-303.
221
S U B C E L L U L A R LOCALIZATION OF L U T E I N I Z I N G H O R M O N E R E L E A S I N G H O R M O N E D E G R A D I N G ACTIVITY IN THE HYPOTHALAMUS
C.
RICHARD PARKER, Jr.*, MARK
M.
FOREMAN and JOHN C. PORTER
The Cecil H. and Ida Green Center for Reproductive Biology Sciences, The Departments of Obstetrics and Gynecology and Physiology, The University of Texas Southwestern Medical School, Dallas, Texas 75235 (U.S.A.)
(Accepted February 1st, 1979)
SUMMARY The in vitro degradation of endogenous as well as exogenous luteinizing hormone releasing hormone (LHRH) by subcellular fractions of rat hypothalamic tissue was studied. Endogenous LHRH, localized in isolated nerve terminals (synaptosomes), was found to be resistant to enzymatic degradation (60 rain, 37 °C) as long as the synaptosomal membrane remained intact. Endogenous L H R H was rapidly degraded by the 900 × g supernatant fluid and cytosol but not by myelin/microsomes, intact synaptosomes, or mitochondria. Lysed synaptosomes rapidly degraded exogenous LHRH. The L H R H degrading activity of synaptosomes was highly concentrated in the 'synaptosol', i.e., the cytosol of the nerve terminal. These data suggest that the L H R H degrading activity of the rat hypothalamus is a readily solubilized component of neurons, and possibly of non-neuronal cells.
INTRODUCTION The capacity of neurons to synthesize and degrade putative neurotransmitters such as acetylcholine and norepinephrine is well established3,7, 21. It has recently been demonstrated that homogenates of rat brain tissue possess the enzymatic capacity to degrade L H R H with a resultant loss of its biological and immunoreactive properties 4,5,8-1°,13,2°. The L H R H degrading activity of hypothalamic homogenates has been shown to vary with the sex and endocrine status of the animal from which the tissue was obtained6, lo. These findings are suggestive that such enzymatic activity may * To whom requests for reprints and correspondence should be addressed.
222 have a regulatory role in the control of L H R H secretion or in the expression of L H R H activity in the brain. Although L H R H has been shown to be sequestered in dense-cored granules of hypothalamic synaptosomes 18, the question whether L H R H stored within, or released from, nerve terminals is subject to enzymatic degradation has not been addressed. The purpose of the current investigation was to evaluate the extent of L H R H degradation under in vitro conditions by several subcellular fractions of rat hypothalamic tissue. MATERIALS AND METHODS Adult male rats (200-275 g) of the Long-Evans strain were decapitated; the brains were excized, and the hypothalami were dissected and placed in ice-cold 0.32 M sucrose-10 # M CaC12. All sucrose solutions contained 10 # M CaCI2, and subcellular fractionation was carried out at 4 °C. The hypothalamic fragments were rinsed and homogenized in 10 vols. of 0.32 M sucrose and centrifuged at 900 x g for 10 min at 4 '~C as described previously 17. The 900 Y. g supernatant fluid (0.9K S) was fractioned on discontinuous sucrose density gradients consisting of 1.5 ml each of 0.8 and 1.2 M sucrose TM. Subcellular fractions consisting of cytosol, myelin/microsomes, synaptosomes, and mitochondria were recovered from the gradients, in previous studies, electron microscopic examination of the particulate fractions has confirmed the nature of subcellular elements recoverable from such gradients 18. The particulate fractions were diluted te approximately 0.4 M sucrose and were pelleted by centrifugation for 60 min at 100,000 ~< g in a Ti-75 rotor (Beckman Instruments, Inc.). The subcellular particles in the pellets were resuspended intact in 0.32 M sucrose or in 10 ,aM CaCI2. Incubations consisted of 1.0 ml aliquots of the various subcellular fractions (0.9 K S, cytosol, myelin/microsomes, synaptosomes, and mitochondria) mixed with 1.0 ml Hanks balanced salt solution. Incubations were carried out at 37 °C in glass flasks using a Dubnoff metabolic incubator. In experiments designed to follow the degradation of exogenous L H R H , subcellular fractions were pre-incubated at 37 °C for 30 min prior to the addition of 10 ng L H R H dissolved in 100 #1 of 0.01 M phosphate buffer, pH 7.0, containing 0.14 M NaCI. Triplicate aliquots were removed at designated times, extracted in acid ethanol ~ and assayed for L H R H by radioimmunoassay 15 using synthetic L H R H (Beckman Instruments, Inc.) as the reference standard. Protein was quantified by the method of Lowry et al. 11 using bovine serum albumin as the standard. The activity of marker enzymes, cytochrome oxidase (EC 1.9.3.1), N A D P H cytochrome c reductase (EC 1.6.2.4), and acid phosphatase (EC 3,1.3.2), was assayed by established methods 12,14,19. RESULTS When incubated with the 0.9K S or cytosol, exogenous L H R H was rapidly degraded as shown in Fig. 1. The half-life of L H R H in the cytosol or 0:9K S ranged from 5 to 20 min depending on the amount of protein in the incubation vial. No significant disappearance of L H R H was observed in incubations containing medium alone
223 100
80 FZ LIJ I-Z
8 T
60
I
40 LL O
20
0
30
60
INCUBATION TIME (min)
Fig. 1. Time course of the degradation of exogenous L H R H by medium, cytosol, and 0.9K S. Flasks containing medium or the indicated amounts of cytosolic or 0.9K S protein (in mg) were pre-incubated for 30 min at 37 ' C and then incubated with 10 ng LHRH as described in Materials and Methods. Data are presented as a mean percentage of the initial LHRH content remaining at each time period sampled. The initial LHRH content was determined immediately after the addition of LHRH at time 0.
(Fig. 1). Similarly, in incubations of cytosol or 0.9K S which had been heated at 100 °C for 10 min prior to the addition of LHRH, there was no degradation of L H R H (data not shown). Little, if any, degradation of L H R H occurred during a 60-min incubation with intact or hypo-osmotically shocked myelin/microsomes or mitochondria (data not shown). In addition, exogenous L H R H was not degraded by intact synaptosome preparations (Fig. 2). However, significant degradation of L H R H was observed during incubation with hypo-osmotically shocked (or lysed) synaptosomes, and the degree of degradation was related to the amount of synaptosomal protein in the incubation flask (Fig. 2). As with the 0.9K S and cytosol, prior heating for 10 min at 100 °C of lysed synaptosomes prevented the degradation of L H R H (data not shown). Of the several subcellular fractions isolated from the 0.9K S, only synaptosomes contained significant quantities of endogenous LHRH. As shown in Fig. 3, the endogenous L H R H content of the synaptosome fraction was stable for at least 60 rain at 37 °C. Conversely, when synaptosomes were lysed and then incubated at 37 °C, endogenous LHRH was degraded. In order to define the localization of the L H R H degrading activity in synaptosomes, the synaptosomes were lysed and centrifuged at 4 °C for 60 rain at 100,000 × g. The pellet, i.e., synaptosomal membranes and intra-synaptosomal organelles, was
224 • Intact Synaptosomas o Lysed Synoptosomes
._.__..----e
4.0mg
iO07rag 0.6mg
500
2.0 rng 8O I'O,8rng T 60 n," T ..J
O
'~
13rag
i
-~ 4o u_ o
o
20
t,9mg
0
0
Q 50
0
2,5rag
60
INCUBATION TIME (min)
Fig. 2. Time course of the degradation of exogenous LHRH by intact and lysed hypotha!amic synaptosomes. Synaptosome pellets, containing the designated amount of protein (rag), were either suspended intact in 0.32 M sucrose or were lysed by suspension in 10 ~M CaCI~. See Fig. 1 and text for other details.
-lO0
Intoct Synaptosomes
5 60
\~
E
0 0
I 2~O
60
90
INCUBATION TIME (mini
Fig. 3. Time course of the degradation of endogenous LHRH in lysed and intact hypothalamic synaptosome preparations. Intact and lysed synaptosome preparations (1.5 mg protein each) were incubated in the absence of exogenous L H R H as described in Materials and Methods. Data, expressed as percentage of the endogenous L H R H content at time 0, are presented as the mean 7~ S . E .
a
225 TABLE 1 Distribution of L H R H degrading activity and marker enzymes in subee/lular ./~'actions of adult male rat hypothalamus Snbcellular fraction
0.9K S Cytosol Myelin/microsomes Mitochondria Intact synaptosomes Lysed synaptosomes Synaptosol
Enzyme activity* LHRH degrading activity
Cytochrome oxidase
NADPH cytoehrome c reductase
Acid phosphatase
10.0 36.0 0.8 1.3 1.0 17.0 49.0
ND 6 18 894 196 ND ND
ND 10 I0 3 4 ND ND
ND 8 18 39 35 ND ND
* The LHRH degrading activity is expressed as ng LHRH degraded/60 rain/rag protein. Incubations of subcellular fractions -+ 10 ng LHRH were performed as indicated in the text (n 2 6 separate experiments). Other enzyme activities are expressed as nmol product formed/rain/rag protein (n : 3). ND not determined. resuspended in 10 # M CaCI~. The pellet fraction or the supernatant fluid was incubated with 10 ng L H R H . The specific activity of the L H R H degrading enzyme(s) in the synaptosol, the cytosol of the nerve terminals, was 6-8 times greater than that in the pellet fraction. A comparison of specific activities of the L H R H degrading enzyme(s) in the subcellular fractions are shown in Table I. L H R H degrading activity was highest in the cytosol and synaptosol. With the exception of the 0.9K S and lysed synaptosomes, little L H R H degrading activity was expressed in the remaining subcellular fractions. The subcellular localization of the L H R H degrading activity was different from that of the enzyme markers (Table I). The activity of the mitochondrial marker, cytochrome oxidase, was greatest in the mitochondrial fraction and was also present in synaptosomes, which are known to contain mitochondria. The activity of the microsomal enzyme, N A D P H cytochrome c reductase, was greatest in the myelin/microsome fraction and in the cytosol, whereas the activity of the lysosomal enzyme, acid phosphatase, was greatest in the mitochondria and in the synaptosomes. DISCUSSION The functional organization of cholinergic and noradrenergic neurons in the brain is well established. However, the mechanisms controlling the synthesis, release, and degradation of the substances contained in peptidergic neurons, such as L H R H , are poorly understood. Of significance in neural transmission is the capacity of nerve terminals to release neurotransmitters which can be rapidly degraded after eliciting their action at the postsynaptic receptor site. Acetylcholine is inactivated by acetylcholine esterase (EC 3.1.1.7) which is present in plasma membranes and microsomes of
226 neurons a,21. In the brain, norepinephrine is degraded by monoamine oxidase (EC 1.4.3.4), which is present in mitochondria :~, and catechol-o-methyltransferase (EC 2.1.1.6), a soluble enzyme 7. The brain also contains enzymes which can degrade peptides such as thyrotropin releasing hormone (TRH), oxytocin, and LHRH6,8,1~ Variation in the rate of enzymatic degradation of T R H has been observed in the rat brain during postnatal development TM. In addition, the capacity of brain homogenates to degrade L H R H and oxytocin has been shown to be affected by the hormonal status of experimental animals6,10. Thus, it is possible that peptide degrading enzymes may be of importance in peptidergic neurons. However, little is known concerning the cell of origin or subcellular localization of such peptide degrading enzymes. Griffiths and co-workers4, 5 have found L H R H degrading activity in a soluble fraction of hypothalamic homogenates and in a particulate fraction which was pelleted at 25,000 ~ g for 60 min. However, the soluble fraction probably contained microsomal elements, and the particulate fraction undoubtedly contained many elements, including myelin, synaptosomes, and mitochondria. In other studies, hypothalami were homogenized under conditions in which some, if not most, of the subcellular organelles would have been lysed s,~3,e°. In the current study, the L H R H degrading enzyme activity in the 0.9K S was shown to be present largely in the cytosol, and little, if any, L H R H degrading enzyme activity was expressed in the myelin and microsomes, intact synaptosomes, or mitochondria. However, significant L H R H degradation occurred after lysis of the synaptosomes, and the specific activity of the L H R H degrading enzyme(s) in the synaptosol was as high as that of the cytosol. In a prior investigation, T R H degrading activity was found in the 0.9K S of rat hypothalamic homogenates, but little TRH degradation occurred in incubations containing isolated intact synaptosomeslL The L H R H degrading enzyme activity in rat hypothalamic tissue is not associated with microsomes and synaptic plasma membranes as is acetylcholine esterase or with mitochondria as is monoamine oxidase. Instead, L H R H degrading activity is distributed among subcellular fractions of rat hypothalamic homogenates in a manner similar to that of lactate dehydrogenase (EC 1.1.1.27) and catechol-o-methyltransferase7, 21. Although we cannot exclude non-neuronal cells as a source of L H R H degrading enzyme activity in the cytosol, there is strong evidence that the enzyme activity is present in the synaptosol, the cytosol of isolated nerve terminals. However, since the hypothalamic synaptosome fraction is composed of nerve terminals containing L H R H as well as those containing TRH, ct-melanocyte stimulating hormone, dopamine, etc., it would be improper to conclude that the L H R H degrading enzyme activity which was expressed in the lysed synaptosome preparation was derived fi'om L H R H containing neurons. However, it may be possible to address this question in the future since the L H R H containing synaptosomes, after fractionation on continuous sucrose density gradients, exhibit a slightly different banding density than do those containing T R H or catecholamines a. Several other questions remain to be answered concerning peptidergic neurons of the brain and the hypothalamic LHRH-containing neurons in particular. What are the by-products of L H R H degradation and are there differences in
227 either the degree o f L H R H d e g r a d a t i o n or the nature o f L H R H b r e a k d o w n p r o d u c t s themselves which are manifested by different brain cell types? D o the p r o d u c t s o f L H R H d e g r a d a t i o n possess any intrinsic biological activity? Is the d e g r a d a t i o n of L H R H subject to biological a n d p h a r m a c o l o g i c a l m a n i p u l a t i o n ? O f practical i m p o r t a n c e to future studies is our finding that s y n a p t o s o m a l L H R H is stable during i n c u b a t i o n s o f isolated synaptosomes. In addition, it m a y be possible to investigate L H R H actions on n e u r o n s under certain in vitro conditions since exogenous L H R H is n o t d e g r a d e d by intact synaptosomes. It is a p p a r e n t , however, t h a t cytosolic c o n t a m i n a n t s must be r e m o v e d before such studies are p e r f o r m e d a n d that care must be exercised to avoid lysis o f nerve terminals during such studies. ACKNOWLEDGEMENTS The a u t h o r s t h a n k M i l d r e d A r n o l d for excellent editorial assistance and G a y e Burnsed, Sue Sherwin, L i n d a Akers, Jodie Roberts, and D a v i d D o w d for technical assistance. This w o r k was s u p p o r t e d by the following grants: AM01237, HD07062, AG00306, a n d 5-S07-RR-05426-16.
HDllI49,
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