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0013-7227/78/1024-1167$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 4
Printed in U.S.A.
• Studies on the Subsynaptosomal Localization of Luteinizing Hormone-Releasing Hormone and Thyrotropin-Releasing •4 Hormone in the Rat Hypothalamus* C. RICHARD PARKER, jR.,t4 WILLIAM B. NEAVES, AYALLA BARNEA, AND JOHN C. PORTER i
Cecil H. and Ida Green Center for Reproductive Biology Sciences, The Departments of Obstetrics and Gynecology, Physiology, and Cell Biology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 ABSTRACT. In the current investigation, subcellular particles (synaptosomes) of hypothalamic homogenates were isolated by differential centrifugation and discontinuous sucrose density gradient fractionation and found to be rich in LHRH, TRH, and the neuronal marker, norepinephrine (NE). Of the total quantity of LHRH, TRH, or NE in the nuclei-free homogenate, 52-65% was recovered in synaptosomes, whereas the cytosol, myelin/microsomes, and mitochondria contained only 1-12%. To determine the subsynaptosomal localization of LHRH and TRH, purified synaptosomes were lysed and the resulting suspensions were fractionated on discontinuous sucrose density gradients. LHRH (30-40%) was found to be localized primarily in subsynaptosomal
T
HE PEPTIDES, LHRH and TRH, are highly concentrated in the median eminence of the rat (1, 2). Although LHRH and TRH dramatically affect the secretory function of the anterior pituitary, both peptides also possess neuromodulatory activities in the central nervous system (CNS) (cf. 3). More than 50% of the total brain content of TRH is localized in extrahypothalamic tissues (2). These findings suggest that these two peptides may have a role in neural function. It has been shown that when brain tissues are homogenized in iso-osmotic sucrose, axonal terminals are sheared off, forming subcellular particles which have been called synReceived July 29, 1977. * This work was supported by grants from the NIAMDD (AM01237), the NICHHD (HD08672), and the NIA (AG00306). fNIH Postdoctoral Training Fellow, Grant 5 T01 HD00256. $ To whom all correspondence and requests for reprints should be addressed: Department of Obstetrics and Gynecology, Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
particles which banded at sucrose densities between 0.6-1.0 M. Electron microscopic analysis of these particles revealed the presence of dense-cored granules (70-80 nm diameter) and synaptosomal membrane remnants. Norepinephrine was found in two pools within the isolated nerve endings: 15-25% of synaptosomal NE was associated with the synaptic vesicles (45-55-nm diameter); about 40% was in the cytosol. TRH was present primarily as a soluble component of the nerve ending. No apparent association of TRH with densecored granules was demonstrable in this study; however, there may be some TRH in synaptic vesicles. (Endocrinology 102: 1167, 1978)
aptosomes (4, 5). By means of both differential centrifugation and density gradient sedimentation procedures, it has been demonstrated that several putative as well as proven CNS neurotransmitters are concentrated within synaptosomes (4, 5). By utilizing such techniques, several investigators have shown that LHRH and TRH in homogenates of hypothalamic tissue are associated with subcellular particles which resemble synaptosomes (6-11). Immunohistochemical studies also support the view that LHRH and TRH are present within neural elements of the hypothalamus (12-16). Although such CNS neurotransmitters as acetylcholine and norepinephrine (NE) are stored in synaptic vesicles which are localized in axonal terminals (17-19), the mode of storage of LHRH and TRH is not established. It is possible that these peptides are sequestered in synaptic vesicles like other neurotransmitters, or they may be present in larger secretory granules as are peptide and protein hormones of other endocrine tissues (20-23). Barnea et
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PARKER, JR. ET AL.
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at. (9) have shown that homogenization of hypothalami in hypo-osmotic medium resulted in a significant decline in particle-bound TRH and LHRH. It is, therefore, possible that either or both peptides are stored in a readily solubilized pool in the nerve ending. The purpose of this study was to investigate the subneuronal localization of hypothalamic LHRH and TRH. Subsynaptosomal elements were released by lysis of purified synaptosomes and fractionated by means of discontinuous sucrose density gradient centrifugation. The content of NE in the subsynaptosomal fractions was used as a biochemical marker for synaptic vesicles. In addition, an ultrastructural analysis of each subsynaptosomal fraction was performed. Materials and Methods Subcellular fractionation of hypothalmic tissue Adult male rats (Long-Evans strain) weighing 200-300 g were decapitated; the brains were excised, and the hypothalami were dissected and placed in ice-cold 0.15 M NaCl. Hypothalamic fragments were then rinsed, homogenized in 10 vol 0.32 M sucrose10 JUM CaCh, and centrifuged at 900 X g for 10 min at 4 C as described previously (24). All sucrose solutions contained 10 JUM CaCh, and each preparative procedure was carried out at 1-4 C. The 900 X g supernatant fluid was fractionated on a discontinuous sucrose density gradient, identified as gradient A, consisting of 1.5 ml each 0.8 and 1.2 M sucrose (4, 24). Subcellular fractions consisting of cytosol, myelin/microsomes, synaptosomes, and mitochondria were recovered by aspiration from the gradient as shown in Fig. 1.
(gradient B), and were centrifuged at 100,000 X g for 60 min in a Beckman L5-75 ultracentrifuge by using an SW 60 rotor, and fractions were collected by aspiration. Each fraction was identified according to the position on the gradient from which it was recovered, as illustrated in Fig. 1. Distribution of radiolabeled LHRH and TRH on gradient B To test the possibility that soluble LHRH and/or TRH were adsorbed by subsynaptosomal organelles during lysis and subsequent fractionation, the following experiment was conducted. The pelleted synaptosomes were lysed in 10 JUM CaCb containing [3H]LHRH and [3H]TRH (New England Nuclear), and the suspension was fractionated on gradient B as described above. Gradient fractions were collected, mixed with Aquasol (New England Nuclear), and counted in a Packard Tri-Carb liquid scintillation spectrophotometer. Determination of protein, LHRH, TRH, and NE Aliquots of the 900 X g supernatant fluid and subcellular fractions were removed for analysis. GRADIENT A
GRADIENT B
Cytosol 0.32M
0-Cytosol
s
Myelin/ Microsomes
0.4M D-Synaptic Vesicles 0.8M 0.6M ~
Synaptosomes
I.2M
F -Granules & Membranes 0.8M
1.2M } Mitochondria
E - Vesicles & Granules
a
1.0M
Subsynaptosomal fractionation The purified synaptosome fraction, which was concentrated at the interface of the 0.8 and 1.2 M layers of gradient A, was diluted to approximately 0.3 M sucrose and pelleted by centrifugation at 40,000 X g for 20 min. The pelleted synaptosomes (equivalent to four to eight hypothalami) were suspended in 0.32 M sucrose (iso-osmotic) or lysed by suspension in 10 /AM CaCb. The resulting suspensions of intact or lysed synaptosomes were fractionated by means of discontinuous sucrose density gradient centrifugation by using a modification of the method used by Whittaker et al. (18). Discontinuous sucrose density gradients consisted of 0.6ml layers of 0.4, 0.6, 0.8, 1.0, and 1.2 M sucrose
Kndo i > 1978 Vol 102 t No 4
G - Synaptosome Ghosts & Granules H-Partially Lysed Synaptosomes I - Synaptosomal Mitochondria
FIG. 1. Fractions from discontinuous sucrose density gradients A and B. Each fraction was analyzed for protein, LHRH, TRH, and NE. Gradient A, utilized for subcellular fractionation of the 900 X g supernatant fluid, was centrifuged at 100,000 x g for 30 min at 4 C. Gradient B was utilized for fractionation of purified synaptosomes after suspension in 0.32 M sucrose or 10 /HM CaCk. Tubes containing gradient B were marked to indicate the position of each gradient layer before centrifugation at 100,000 X g for 60 min at 4 C. The predominant subsynaptosomal particle in each fraction, as shown by electron microscopic analysis, is indicated in the figure. Each fraction, excepting the pellets, was removed by aspiration. The pellets were resuspended in 0.5 ml 0.32 M sucrose.
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SUBSYNAPTOSOMAL POOLS OF LHRH AND TRH Protein was measured by the method of Lowry et al. (25) by using bovine serum albumin as the reference standard. NE was determined by a ra^ dioenzymatic assay (26). Samples taken for LHRH and TRH measurements were extracted with acid«< ified ethanol (27) and analyzed by RIA (28, 29) by using synthetic LHRH and TRH (Beckman Instruments, Inc.) as standards. ** Electron microscopy *-
Subcellular fractions derived from the 900 X g supernatant fluid as well as the subsynaptosomal * fractions were diluted to 0.3 M sucrose and pelleted by centrifugation at 100,000 X g for 60 min. The pellets were fixed in 0.1 M cacodylate buffer (pH * 7.3) containing 2% glutaraldehyde and analyzed by transmission electron microscopy as described previously (24). H
Results
Electron microscopic analysis of subcellular •t and subsynaptosomal fractions -i.
When the 900 X g supernatant fluid was fractionated on gradient A, three particulate
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fractions were obtained. It was found by electron microscopy that the particles banding at the 0.32-0.8 M interphase consisted primarily of myelin and microsomes, whereas the pellet contained mostly mitochondria (data not shown). The fraction which banded at the 0.8-1.2 M interphase consisted mainly of synaptosomes (Fig. 2a). When the purified synaptosomes were diluted, pelleted, resuspended in 0.32 M sucrose, and fractionated on gradient B, 85-90% of the protein was recovered in gradient fractions G, H, and I. Although each of these three gradient fractions contained intact synaptosomes, fraction H contained the most. No specific morphological differences among the synaptosomes in fractions G, H, and I were apparent. A representative electron photomicrograph of particles in fraction H is displayed in Fig. 2b. However, when purified synaptosomes were lysed in a hypo-osmotic solution (10 /XM CaCh) and fractionated on gradient B, a different morphological profile was obtained for
FIG. 2. Electron transmission micrographs of purified synaptosome fractions, a, Gradient fractions were diluted to 0.3 M sucrose and pelleted by centrifugation at 100,000 X g for 60 min. Pellets were processed as described in Materials and Methods. Profiles of particles in the 900 x g supernatant fluid which banded at the 0.8-1.2 M interphase of gradient A are shown. Many synaptosomes (S) are present. Within the synaptosomes are seen synaptic vesicles, mitochondria, and some dense-cored granules (arrow). A few free mitochondria are also present in this fraction (X 17,500). b, Profiles of intact purified synaptosomes (S) which banded in fraction H of gradient B are shown. A dense-cored granule within a synaptosome is indicated (arrow), (x 17,500).
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PARKER, JR. ET AL.
the gradient fractions. Fraction 0 was devoid of participate material as judged by transmission electron microscopy. Fraction D from gradient B consisted almost exclusively of synaptic vesicles (45-55-nm diameter) (Fig. 3a). Fraction E was composed of synaptic vesicles, dense-cored granules, various large vesicles, and membranes (Fig. 3b). Fraction F contained both dense-cored granules (70-80-nm diameter) and large vesicles (190-200-nm diameter) as well as membrane elements (Fig. 4a). Fraction G contained synaptosome ghosts and some dense granules (Fig. 4b). In addition to synaptosome ghosts, fraction H contained partially lysed synaptosomes (Fig. 5a), whereas fraction I contained mitochondria released, apparently, from synaptosomes during lysis (Fig. 5b). The particles in these gradient fractions obtained from lysed hypothalamic synaptosomes resemble those described for lysed synaptosomes of cerebral origin (18). Subcellular localization of NE, LHRH, and TRH When the 900 X g supernatant fluid was
Endo • 1978 Vol 102 • No 4
fractionated on gradient A, about 50% of the total quantity of NE, LHRH, and TRH placed on the gradient was recovered in the synaptosome fraction (Table 1). Minimal amounts of LHRH were recovered in the cytosol, myelin/microsome, and mitochondrial fractions. About 12% of the total TRH and NE was recovered in the myelin/microsome fraction. The distribution of protein throughout the gradient fractions is shown in Table 1; the greatest amount (25%) was recovered in the synaptosome band. The total recoveries of protein, LHRH, TRH, and NE ranged from 56-86%. Failure to recover 100% of the material placed on gradient A was due largely, if not entirely, to the fact that, other than the entire cytosol fraction, only those particles banding at each gradient interphase and the mitochondrial pellet were recovered from the gradient (Fig. 1). Substances between the interfaces were discarded. When the synaptosome fraction from gradient A was diluted to 0.32 M sucrose, pelleted, and then resuspended in 0.32 M sucrose or 10 jiiM CaCk, approximately 70% of the protein,
FIG. 3. Electron transmission micrographs of subsynaptosomal particles from lysed synaptosomes which banded in fractions D and E of gradient B. a, Particles which banded in fraction D are shown. The predominant particles are synaptic vesicles (45-55 nm diameter) (X 17,500). b, Profiles of particles which banded in fraction E are shown. Synaptic vesicles, dense-cored granules (70-80 nm diameter; arrow), and at least two populations of electron-lucent vesicles having diameters of 90-110 and 150-200 nm are discernible (x 17,500). The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 19:01 For personal use only. No other uses without permission. . All rights reserved.
SUBSYNAPTOSOMAL POOLS OF LHRH AND TRH
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FIG. 4. Electron transmission micrographs of subsynaptosomal particles from lysed synaptosomes in fractions F and G from gradient B. a, Numerous dense-cored granules (70-80 nm diameter; arrows) as well as synaptosome ghosts are seen in fraction F (x 17,500). b, Synaptosome ghosts (G) and occasional electron-dense granules (arrows) appear in fraction G. There are some dumbbell-shaped, dense particles that resemble isolated postsynaptic membranes (30) (x 17,500).
FIG. 5. Electron transmission micrographs of subsynaptosomal particles from lysed synaptosomes in fractions H and I from gradient B. a, Partially disrupted synaptosomes (arrow) in which cytoplasm, granules, and vesicles are still contained in varying degrees within a limiting membrane are characteristic of fraction H (x 17,500). b, Free mitochondria (arrows) released from synaptosomes during lysis are seen frequently in fraction I. Granules and vesicles of varying size and density as well as membranes also occur here (x 17,500).
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LHRH, TRH, and NE was recovered. Inasmuch as the losses of LHRH, TRH, and NE paralleled the overall loss of protein, there was no evidence for the presence or expression of peptide- and catecholamine-degrading enzymes in this preparation. Subsynaptosomal TRH, and NE
localization
TRH, and NE on gradient B (in four to seven separate experiments) were 80, 79, 99, and 116%, respectively. When the recoveries of protein, LHRH, TRH, and NE were analyzed by using a two-way analysis of variance, no E3
of LHRH,
When the intact synaptosomes (resuspended in 0.32 M sucrose) were fractionated on gradient B, 50-75% of the total LHRH, TRH, NE, and protein placed on the gradient was recovered in fraction H, whereas fractions G and I contained 10-20% (Fig. 6). Fractions O, D, E, and F contained less than 15% of the total. Overall recoveries of protein, LHRH, TRH, and NE (in three to seven separate experiments) were 87, 92, 121, and 107%, respectively. However, when purified synaptosomes were lysed by suspension in 10 JUM CaCk, the distribution of protein, NE, LHRH, and TRH on gradient B was altered (Fig. 6). TRH was released from the lysed synaptosomes (fractions H and I) and appeared primarily in the cytosol (fraction 0); 16% was recovered in synaptic vesicles (fraction D). About 25% of the NE was recovered in synaptic vesicles and 40% was recovered in the cytosol. After lysis of the synaptosomes, some LHRH (15%) appeared in the cytosol; and the remainder of the released LHRH (30-40%) was localized in the granule/membrane fractions (F and G). From 15-25% of the LHRH, TRH, NE, and protein was recovered in fractions containing partially disrupted synaptosomes (fraction H and I). Total recoveries of protein, LHRH,
Kndo • 1978 Vol 102 • No 4
O.32M SUCROSE
10 nM CaCI2
PROTEIN • n=7 • n=7
60
NE ED n = 3
• n=4
40
20
I
I I u 60
ill
LHRH Q n=7
TRH 0 n=6
• n=6
• n=7
k
40
20
hi I O
D
E
F
G
H
I
O
GRADIENT
FRACTION
D
E
F
G
H
I
FIG. 6. Recovery of protein, NE, LHRH, and TRH in various fractions from gradient B (Fig. 1). Intact synaptosomes (in 0.32 M sucrose-10 /XM CaCb) and lysed synaptosomes (in 10 JUM CaCb) were fractionated on gradient B as described in Materials and Methods. Results are expressed as a percentage of the total material placed on the gradient. The height of each bar denotes the mean from three to seven separate experiments. The vertical lines represent the magnitude of the SE.
TABLE 1. Recovery of protein, LHRH, TRH, and NE in subcellular fractions of the hypothalamic 900 X g supernatant fluid % recovery" Subcellular fraction Protein Cytosol Myelin/microsomes Synaptosomes Mitochondria
14.3 ± 4.4 11.2 ± 3.4 25.6 ± 4.0 5.0 ± 1.2
LHRH 1.0 ± 0.5 1.5 ± 0.8 53.4 ± 9.4 5.1 ± 1.7
TRH 3.4 ± 0.7 11.9 ± 1.7 52.2 ± 8.2 2.5 ± 0.6
NE 9.1 10.3 64.7 3.7
" Recovery was calculated as the percentage of applied material in the 900 X g supernatant fluid which was detected in each gradient fraction, n = four separate experiments except in the case of NE which was determined in two experiments. Data are presented as the mean ± SE.
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SUBSYNAPTOSOMAL POOLS OF LHRH AND TRH statistically significant difference ( P > 0.1) was found in the recoveries from lysed synaptosome fractions compared to that from •*• intact synaptosome fractions. Distribution TRH
of radiolabeled
LHRH
and
When [ 3 H]LHRH or [ 3 H]TRH was added to synaptosomes which were then lysed and fractionated on gradient B, 55-72% of the i added radioactivity was recovered in fraction 0; 15-18% was recovered in fraction D. Minimal amounts ( the endogenous T R H recovered in fraction D (synaptic vesicles) could be due to diffusion of soluble material into the gradient during centrifugation. However, this does not negate the possibility that some T R H may be associated with synaptic vesicles.
Discussion The work of several groups of investigators demonstrates that LHRH and TRH are associated with neural elements of the hypothalamus. After homogenization of the hypo* thalamus in an iso-osmotic medium and fracs, tionation by density gradient or differential centrifugation, LHRH and TRH have been found to be associated with synaptosome-like particles (6-11). Such results obtained by biophysical fractionation methods are complementary to immunocytochemical evidence k which implies that TRH (12) and LHRH (13-16) are contained in neural elements of the hypothalamus. In the current investigation, we found that LHRH, TRH, and NE are concentrated primarily in subcellular fractions * of the hypothalamus which are rich in synap^ tosomes. The subneuronal localization of these pep-
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tides has not, however, been clearly established. It has been demonstrated by means of immunohistochemical methods that certain electron-dense granules (having diameters of 75-95 nm) in axons and nerve endings of the hypothalamus contain LHRH (14-16). Somatostatin (16) and vasopressin (21) have also been found in granules of hypothalamic neurons. Recently, Winokur et al. (11) presented evidence which indicated that extrahypothalamic TRH is localized in synaptosomes and exists as a soluble substance in addition to being associated with subcellular fractions containing synaptic vesicles. Although Winokur et al. found hypothalamic TRH in synaptosomes, they did not report on the subsynaptosomal storage sites of TRH in the hypothalamus. There is, to date, no immunohistochemical evidence to demonstrate the association of TRH with synaptic vesicles, dense granules, or other subsynaptosomal organelles. An established property of synaptosomes is their susceptibility to hypo-osmotic shock (4, 5). The subneuronal localization of NE and acetylcoline was established by utilization of this property (17, 18). By lysing and fractionating hypothalamic synaptosomes on density gradients, we have found a divergence in the subsynaptosomal localization of LHRH, TRH, and NE. In agreement with the findings of De Robertis et al. (17), we found that hypothalamic NE was present in a soluble form and in association with synaptic vesicles. After lysis of synaptosomes, 15-20% of the LHRH was solubilized and 30-40% was associated with subsynaptosomal particles banding between 0.6 and 1.0 M sucrose (fractions F and G). Electron microscopic analysis of these fractions revealed the presence of dense-cored granules having diameters of 70-80 nm (Fig. 5). It is tempting to speculate that the densecored granules in fractions F and G are analogous to those in which LHRH has been detected by electron microscopic immunohistochemistry (14-16). However, since other structures are also present in these fractions, the specific organelle with which LHRH is associated is impossible to identify conclusively. Furthermore, the exclusive association of
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PARKER, JR. ET
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LHRH with secretory granules, as indicated by immunohistochemical methods, is unproven because a large fraction of the LHRH is lost during the processing of the tissue for immunohistochemical analysis (15). TRH, on the other hand, was almost totally solubilized after synaptosome lysis. Barnea et al. (9) reported similar findings after direct homogenization of hypothalami in hypo-osmotic solutions. In addition, however, we found that a portion (16%) of synaptosomal TRH fractionated in a manner similar to synaptic vesicles. The significance of this finding is difficult to interpret; a portion or all of the TRH in the synaptic vesicle fraction may have arisen through diffusion of soluble peptide into the gradient during centrifugation. Our failure to find significant quantities of this peptide in some subsynaptosomal particle (s) implies that TRH is a soluble component of hypothalamic nerve endings. However, it is conceivable that some synaptosomal TRH is stored within, or bound to particles which are extremely labile in hypoosmotic solutions. It is known that cholinergic and adrenergic synaptic vesicles of the CNS are sensitive to osmotic shock (19). Winokur et al. (11) also found that the TRH in subsynaptosomal particles isolated from the cerebrum could be partially solubilized by suspending them in water. Therefore, additional studies are necessary to determine whether TRH is a completely soluble component of hypothalamic nerve endings or whether some of the TRH is sequestered in particles within neurons of the hypothalamus. Acknowledgments The authors thank Judy Wagers and Miriam Long for editorial assistance and Linda Akers, Marguerite Gunder, Sue Sherwin, and Bob' Lipsey for excellent technical assistance.
References 1. Wheaton, J. E., L. Krulich, and S. M. McCann, Localization of luteinizing hormone-releasing hormone in the preoptic and hypothalamus of the rat using radioimmunoassay, Endocrinology 97: 30, 1975. 2. Oliver, C, R. L. Eskay, N. Ben-Jonathan, and J. C. Porter, Distribution and concentration of TRH in the rat brain, Endocrinology 95: 540, 1974.
AL.
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3. Wilber, J. F., E. Montoya, N. P. Plotnikoff, W. F. White, R. Gendrich, L. Renaud, and J. B. Martin, Gonadotropin-releasing hormone and thyrotropin-releasing hormone: distribution and effects in the central nervous system, Recent Prog Horm Res 32: 117, 1976. 4. Whittaker, V. P., The synaptosome, In Lajtha, A. (ed.), Handbook of Neurochemistry, vol. II, Plenum Press, N. Y., 1969, p. 327. 5. De Robertis, E., and G. Rodriguez de Lores Arnaiz, Structural components of the synaptic region, In Lajtha, A. (ed.), Handbook of Neurochemistry, vol. II, Plenum Press, New York, 1969, p. 365. 6. Clementi, F., B. Ceccarelli, E. Cerati, M. L. Demonte, M. Felici, M. Motta, and A. Pecile, Subcellular localization of neurotransmitters and releasing factors in the rat median eminence, J Endocrinol 48: 205,1970. 7. Shin, S. H., A. Morris, J. Snyder, W. C. Hymer, and J. V. Milligan, Subcellular localization of LH releasing hormone in the rat hypothalamus, Neuroendocrinology 16: 191, 1974. 8. Barnea, A., N. Ben-Jonathan, C. Colston, J. M. Johnston, and J. C. Porter, Differential sub-cellular compartmentalization of thyrotropin releasing hormone (TRH) and gonadotropin releasing hormone (LRH) in hypothalamic tissue, Proc NatlAcad Sci USA 72: 3153, 1975. 9. Barnea, A., N. Ben-Jonathan, and J. C. Porter, Characterization of hypothalamic subcellular particles containing luteinizing hormone releasing hormone and thyrotropin releasing hormone, J Neurochem 27: 477, 1976. 10. Ishii, S., Association of luteinizing hormone-releasing factor with granules separated from equine hypophysial stalk, Endocrinology 86: 207, 1970. 11. Winokur, A., R. Davis, and R. D. Utiger, Subcellular distribution of thyrotropin-releasing hormone (TRH) in rat brain and hypothalamus, Brain Res 120: 423, 1977. 12. Hokfelt, T., K. Fuxe, O. Johansson, S. Jeffcoate, and N. White, Distribution of thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, Eur J Pharmacol 34: 389, 1975. 13. Kordon, C, B. Kerdelhue, E. Pattou, and M. Jutisz, Immunocytochemical localization of LHRH in axons and nerve terminals of the rat median eminence, Proc Soc Exp Biol Med 147: 122,1974. 14. Pelletier, G., F. Labrie, R. Puviani, A. Arimura, and A. V. Schally, Immunohistochemical localization of luteinizing hormone-releasing hormone in the rat median eminence, Endocrinology 95: 314, 1974. 15. Goldsmith, P. C, and W. F. Ganong, Ultrastructural localization of luteinizing hormone-releasing hormone in the median eminence of the rat, Brain Res 97: 181, 1975. 16. Styne, D. M., P. C. Goldsmith, S. R. Burstein, S. L. Kaplan, and M. M. Grumbach, Immunoreactive somatostatin and luteinizing hormone releasing hormone (LHRH) in median eminence synaptosomes of
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SUBSYNAPTOSOMAL POOLS OF LHRH AND TRH
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the rat: detection of immunohistochemistry and quantification by radioimmunoassay, Endocrinology 101: 1099, 1977. De Robertis, E., A. Pellegrino de Iraldi, G. Rodriguez de Lores Arnaiz, and L. M. Zieher, Synaptic vesicles from the rat hypothalamus. Isolation and norepinephrine content, Life Sci 4: 193, 1965. Whittaker, V. P., I. A. Michaelson, and R. J. A. Kirkland, The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'), Biochem J 90: 293, 1964. Marchbanks, R. M., Isolation and study of synaptic vesicles, In Marks, N., and R. Rodnight (eds.), Research Methods in Neurochemistry, vol. II, Plenum Press, New York, 1974, p. 79. MacGregor, R. R., L. L. H. Chu, J. W. Hamilton, and D. V. Cohn, Studies on the subcellular localization of proparathyroid hormone and parathyroid hormone in the bovine parathyroid gland: separation of newly synthesized from mature forms, Endocrinology 93: 1387, 1973. Sachs, H., Studies on the intracellular distribution of vasopressin, J Neurochem 10: 289, 1963. Kemmler, W., D. F. Steiner, and J. Borg, Studies on the conversion of proinsulin to insulin, J Biol Chem 248: 4544, 1973. Costoff, A., Ultrastructure of the Rat Adenohypophysis: Correlation with Function, Academic Press,
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N. Y., 1973. 24. Parker, C. R., Jr., W. B. Neaves, A. Barnea, and J. C. Porter, Studies on the uptake of [3H]thyrotropin-releasing hormone and its metabolites by synaptosome preparations of the rat brain, Endocrinology 101: 66, 1977. 25. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193: 265, 1951. 26. Ben-Jonathan, N., and J. C. Porter, A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue, Endocrinology 98: 1497, 1976. 27. Barnea, A., C. Oliver, and J. C. Porter, Subcellular localization of a-melanocyte stimulating hormone in the rat hypothalamus, J Neurochem 29: 619, 1977. 28. Nett, T. M., A. M. Akbar, G. D. Niswender, M. T. Hedlund, and W. F. White, A radioimmunoassay for gonadotropin-releasing hormone (Gn-RH) in serum, J Clin Endocrinol Metab 36: 880, 1973. 29. Eskay, R. L., C. Oliver, J. Warberg, and J. C. Porter, Inhibition of degradation and measurement of immunoreactive thyrotropin-releasing hormone in rat blood and plasma, Endocrinology 98: 269, 1976. 30. Jones, D. G., Synapses and Synaptosomes: Morphological Aspects, Chapman and Hall, London, 1975, p. 98.
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0013-7227/78/1024-1167$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 4
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• Studies on the Subsynaptosomal Localization of Luteinizing Hormone-Releasing Hormone and Thyrotropin-Releasing •4 Hormone in the Rat Hypothalamus* C. RICHARD PARKER, jR.,t4 WILLIAM B. NEAVES, AYALLA BARNEA, AND JOHN C. PORTER i
Cecil H. and Ida Green Center for Reproductive Biology Sciences, The Departments of Obstetrics and Gynecology, Physiology, and Cell Biology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 ABSTRACT. In the current investigation, subcellular particles (synaptosomes) of hypothalamic homogenates were isolated by differential centrifugation and discontinuous sucrose density gradient fractionation and found to be rich in LHRH, TRH, and the neuronal marker, norepinephrine (NE). Of the total quantity of LHRH, TRH, or NE in the nuclei-free homogenate, 52-65% was recovered in synaptosomes, whereas the cytosol, myelin/microsomes, and mitochondria contained only 1-12%. To determine the subsynaptosomal localization of LHRH and TRH, purified synaptosomes were lysed and the resulting suspensions were fractionated on discontinuous sucrose density gradients. LHRH (30-40%) was found to be localized primarily in subsynaptosomal
T
HE PEPTIDES, LHRH and TRH, are highly concentrated in the median eminence of the rat (1, 2). Although LHRH and TRH dramatically affect the secretory function of the anterior pituitary, both peptides also possess neuromodulatory activities in the central nervous system (CNS) (cf. 3). More than 50% of the total brain content of TRH is localized in extrahypothalamic tissues (2). These findings suggest that these two peptides may have a role in neural function. It has been shown that when brain tissues are homogenized in iso-osmotic sucrose, axonal terminals are sheared off, forming subcellular particles which have been called synReceived July 29, 1977. * This work was supported by grants from the NIAMDD (AM01237), the NICHHD (HD08672), and the NIA (AG00306). fNIH Postdoctoral Training Fellow, Grant 5 T01 HD00256. $ To whom all correspondence and requests for reprints should be addressed: Department of Obstetrics and Gynecology, Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
particles which banded at sucrose densities between 0.6-1.0 M. Electron microscopic analysis of these particles revealed the presence of dense-cored granules (70-80 nm diameter) and synaptosomal membrane remnants. Norepinephrine was found in two pools within the isolated nerve endings: 15-25% of synaptosomal NE was associated with the synaptic vesicles (45-55-nm diameter); about 40% was in the cytosol. TRH was present primarily as a soluble component of the nerve ending. No apparent association of TRH with densecored granules was demonstrable in this study; however, there may be some TRH in synaptic vesicles. (Endocrinology 102: 1167, 1978)
aptosomes (4, 5). By means of both differential centrifugation and density gradient sedimentation procedures, it has been demonstrated that several putative as well as proven CNS neurotransmitters are concentrated within synaptosomes (4, 5). By utilizing such techniques, several investigators have shown that LHRH and TRH in homogenates of hypothalamic tissue are associated with subcellular particles which resemble synaptosomes (6-11). Immunohistochemical studies also support the view that LHRH and TRH are present within neural elements of the hypothalamus (12-16). Although such CNS neurotransmitters as acetylcholine and norepinephrine (NE) are stored in synaptic vesicles which are localized in axonal terminals (17-19), the mode of storage of LHRH and TRH is not established. It is possible that these peptides are sequestered in synaptic vesicles like other neurotransmitters, or they may be present in larger secretory granules as are peptide and protein hormones of other endocrine tissues (20-23). Barnea et
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PARKER, JR. ET AL.
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at. (9) have shown that homogenization of hypothalami in hypo-osmotic medium resulted in a significant decline in particle-bound TRH and LHRH. It is, therefore, possible that either or both peptides are stored in a readily solubilized pool in the nerve ending. The purpose of this study was to investigate the subneuronal localization of hypothalamic LHRH and TRH. Subsynaptosomal elements were released by lysis of purified synaptosomes and fractionated by means of discontinuous sucrose density gradient centrifugation. The content of NE in the subsynaptosomal fractions was used as a biochemical marker for synaptic vesicles. In addition, an ultrastructural analysis of each subsynaptosomal fraction was performed. Materials and Methods Subcellular fractionation of hypothalmic tissue Adult male rats (Long-Evans strain) weighing 200-300 g were decapitated; the brains were excised, and the hypothalami were dissected and placed in ice-cold 0.15 M NaCl. Hypothalamic fragments were then rinsed, homogenized in 10 vol 0.32 M sucrose10 JUM CaCh, and centrifuged at 900 X g for 10 min at 4 C as described previously (24). All sucrose solutions contained 10 JUM CaCh, and each preparative procedure was carried out at 1-4 C. The 900 X g supernatant fluid was fractionated on a discontinuous sucrose density gradient, identified as gradient A, consisting of 1.5 ml each 0.8 and 1.2 M sucrose (4, 24). Subcellular fractions consisting of cytosol, myelin/microsomes, synaptosomes, and mitochondria were recovered by aspiration from the gradient as shown in Fig. 1.
(gradient B), and were centrifuged at 100,000 X g for 60 min in a Beckman L5-75 ultracentrifuge by using an SW 60 rotor, and fractions were collected by aspiration. Each fraction was identified according to the position on the gradient from which it was recovered, as illustrated in Fig. 1. Distribution of radiolabeled LHRH and TRH on gradient B To test the possibility that soluble LHRH and/or TRH were adsorbed by subsynaptosomal organelles during lysis and subsequent fractionation, the following experiment was conducted. The pelleted synaptosomes were lysed in 10 JUM CaCb containing [3H]LHRH and [3H]TRH (New England Nuclear), and the suspension was fractionated on gradient B as described above. Gradient fractions were collected, mixed with Aquasol (New England Nuclear), and counted in a Packard Tri-Carb liquid scintillation spectrophotometer. Determination of protein, LHRH, TRH, and NE Aliquots of the 900 X g supernatant fluid and subcellular fractions were removed for analysis. GRADIENT A
GRADIENT B
Cytosol 0.32M
0-Cytosol
s
Myelin/ Microsomes
0.4M D-Synaptic Vesicles 0.8M 0.6M ~
Synaptosomes
I.2M
F -Granules & Membranes 0.8M
1.2M } Mitochondria
E - Vesicles & Granules
a
1.0M
Subsynaptosomal fractionation The purified synaptosome fraction, which was concentrated at the interface of the 0.8 and 1.2 M layers of gradient A, was diluted to approximately 0.3 M sucrose and pelleted by centrifugation at 40,000 X g for 20 min. The pelleted synaptosomes (equivalent to four to eight hypothalami) were suspended in 0.32 M sucrose (iso-osmotic) or lysed by suspension in 10 /AM CaCb. The resulting suspensions of intact or lysed synaptosomes were fractionated by means of discontinuous sucrose density gradient centrifugation by using a modification of the method used by Whittaker et al. (18). Discontinuous sucrose density gradients consisted of 0.6ml layers of 0.4, 0.6, 0.8, 1.0, and 1.2 M sucrose
Kndo i > 1978 Vol 102 t No 4
G - Synaptosome Ghosts & Granules H-Partially Lysed Synaptosomes I - Synaptosomal Mitochondria
FIG. 1. Fractions from discontinuous sucrose density gradients A and B. Each fraction was analyzed for protein, LHRH, TRH, and NE. Gradient A, utilized for subcellular fractionation of the 900 X g supernatant fluid, was centrifuged at 100,000 x g for 30 min at 4 C. Gradient B was utilized for fractionation of purified synaptosomes after suspension in 0.32 M sucrose or 10 /HM CaCk. Tubes containing gradient B were marked to indicate the position of each gradient layer before centrifugation at 100,000 X g for 60 min at 4 C. The predominant subsynaptosomal particle in each fraction, as shown by electron microscopic analysis, is indicated in the figure. Each fraction, excepting the pellets, was removed by aspiration. The pellets were resuspended in 0.5 ml 0.32 M sucrose.
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SUBSYNAPTOSOMAL POOLS OF LHRH AND TRH Protein was measured by the method of Lowry et al. (25) by using bovine serum albumin as the reference standard. NE was determined by a ra^ dioenzymatic assay (26). Samples taken for LHRH and TRH measurements were extracted with acid«< ified ethanol (27) and analyzed by RIA (28, 29) by using synthetic LHRH and TRH (Beckman Instruments, Inc.) as standards. ** Electron microscopy *-
Subcellular fractions derived from the 900 X g supernatant fluid as well as the subsynaptosomal * fractions were diluted to 0.3 M sucrose and pelleted by centrifugation at 100,000 X g for 60 min. The pellets were fixed in 0.1 M cacodylate buffer (pH * 7.3) containing 2% glutaraldehyde and analyzed by transmission electron microscopy as described previously (24). H
Results
Electron microscopic analysis of subcellular •t and subsynaptosomal fractions -i.
When the 900 X g supernatant fluid was fractionated on gradient A, three particulate
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fractions were obtained. It was found by electron microscopy that the particles banding at the 0.32-0.8 M interphase consisted primarily of myelin and microsomes, whereas the pellet contained mostly mitochondria (data not shown). The fraction which banded at the 0.8-1.2 M interphase consisted mainly of synaptosomes (Fig. 2a). When the purified synaptosomes were diluted, pelleted, resuspended in 0.32 M sucrose, and fractionated on gradient B, 85-90% of the protein was recovered in gradient fractions G, H, and I. Although each of these three gradient fractions contained intact synaptosomes, fraction H contained the most. No specific morphological differences among the synaptosomes in fractions G, H, and I were apparent. A representative electron photomicrograph of particles in fraction H is displayed in Fig. 2b. However, when purified synaptosomes were lysed in a hypo-osmotic solution (10 /XM CaCh) and fractionated on gradient B, a different morphological profile was obtained for
FIG. 2. Electron transmission micrographs of purified synaptosome fractions, a, Gradient fractions were diluted to 0.3 M sucrose and pelleted by centrifugation at 100,000 X g for 60 min. Pellets were processed as described in Materials and Methods. Profiles of particles in the 900 x g supernatant fluid which banded at the 0.8-1.2 M interphase of gradient A are shown. Many synaptosomes (S) are present. Within the synaptosomes are seen synaptic vesicles, mitochondria, and some dense-cored granules (arrow). A few free mitochondria are also present in this fraction (X 17,500). b, Profiles of intact purified synaptosomes (S) which banded in fraction H of gradient B are shown. A dense-cored granule within a synaptosome is indicated (arrow), (x 17,500).
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PARKER, JR. ET AL.
the gradient fractions. Fraction 0 was devoid of participate material as judged by transmission electron microscopy. Fraction D from gradient B consisted almost exclusively of synaptic vesicles (45-55-nm diameter) (Fig. 3a). Fraction E was composed of synaptic vesicles, dense-cored granules, various large vesicles, and membranes (Fig. 3b). Fraction F contained both dense-cored granules (70-80-nm diameter) and large vesicles (190-200-nm diameter) as well as membrane elements (Fig. 4a). Fraction G contained synaptosome ghosts and some dense granules (Fig. 4b). In addition to synaptosome ghosts, fraction H contained partially lysed synaptosomes (Fig. 5a), whereas fraction I contained mitochondria released, apparently, from synaptosomes during lysis (Fig. 5b). The particles in these gradient fractions obtained from lysed hypothalamic synaptosomes resemble those described for lysed synaptosomes of cerebral origin (18). Subcellular localization of NE, LHRH, and TRH When the 900 X g supernatant fluid was
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fractionated on gradient A, about 50% of the total quantity of NE, LHRH, and TRH placed on the gradient was recovered in the synaptosome fraction (Table 1). Minimal amounts of LHRH were recovered in the cytosol, myelin/microsome, and mitochondrial fractions. About 12% of the total TRH and NE was recovered in the myelin/microsome fraction. The distribution of protein throughout the gradient fractions is shown in Table 1; the greatest amount (25%) was recovered in the synaptosome band. The total recoveries of protein, LHRH, TRH, and NE ranged from 56-86%. Failure to recover 100% of the material placed on gradient A was due largely, if not entirely, to the fact that, other than the entire cytosol fraction, only those particles banding at each gradient interphase and the mitochondrial pellet were recovered from the gradient (Fig. 1). Substances between the interfaces were discarded. When the synaptosome fraction from gradient A was diluted to 0.32 M sucrose, pelleted, and then resuspended in 0.32 M sucrose or 10 jiiM CaCk, approximately 70% of the protein,
FIG. 3. Electron transmission micrographs of subsynaptosomal particles from lysed synaptosomes which banded in fractions D and E of gradient B. a, Particles which banded in fraction D are shown. The predominant particles are synaptic vesicles (45-55 nm diameter) (X 17,500). b, Profiles of particles which banded in fraction E are shown. Synaptic vesicles, dense-cored granules (70-80 nm diameter; arrow), and at least two populations of electron-lucent vesicles having diameters of 90-110 and 150-200 nm are discernible (x 17,500). The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 19:01 For personal use only. No other uses without permission. . All rights reserved.
SUBSYNAPTOSOMAL POOLS OF LHRH AND TRH
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FIG. 4. Electron transmission micrographs of subsynaptosomal particles from lysed synaptosomes in fractions F and G from gradient B. a, Numerous dense-cored granules (70-80 nm diameter; arrows) as well as synaptosome ghosts are seen in fraction F (x 17,500). b, Synaptosome ghosts (G) and occasional electron-dense granules (arrows) appear in fraction G. There are some dumbbell-shaped, dense particles that resemble isolated postsynaptic membranes (30) (x 17,500).
FIG. 5. Electron transmission micrographs of subsynaptosomal particles from lysed synaptosomes in fractions H and I from gradient B. a, Partially disrupted synaptosomes (arrow) in which cytoplasm, granules, and vesicles are still contained in varying degrees within a limiting membrane are characteristic of fraction H (x 17,500). b, Free mitochondria (arrows) released from synaptosomes during lysis are seen frequently in fraction I. Granules and vesicles of varying size and density as well as membranes also occur here (x 17,500).
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LHRH, TRH, and NE was recovered. Inasmuch as the losses of LHRH, TRH, and NE paralleled the overall loss of protein, there was no evidence for the presence or expression of peptide- and catecholamine-degrading enzymes in this preparation. Subsynaptosomal TRH, and NE
localization
TRH, and NE on gradient B (in four to seven separate experiments) were 80, 79, 99, and 116%, respectively. When the recoveries of protein, LHRH, TRH, and NE were analyzed by using a two-way analysis of variance, no E3
of LHRH,
When the intact synaptosomes (resuspended in 0.32 M sucrose) were fractionated on gradient B, 50-75% of the total LHRH, TRH, NE, and protein placed on the gradient was recovered in fraction H, whereas fractions G and I contained 10-20% (Fig. 6). Fractions O, D, E, and F contained less than 15% of the total. Overall recoveries of protein, LHRH, TRH, and NE (in three to seven separate experiments) were 87, 92, 121, and 107%, respectively. However, when purified synaptosomes were lysed by suspension in 10 JUM CaCk, the distribution of protein, NE, LHRH, and TRH on gradient B was altered (Fig. 6). TRH was released from the lysed synaptosomes (fractions H and I) and appeared primarily in the cytosol (fraction 0); 16% was recovered in synaptic vesicles (fraction D). About 25% of the NE was recovered in synaptic vesicles and 40% was recovered in the cytosol. After lysis of the synaptosomes, some LHRH (15%) appeared in the cytosol; and the remainder of the released LHRH (30-40%) was localized in the granule/membrane fractions (F and G). From 15-25% of the LHRH, TRH, NE, and protein was recovered in fractions containing partially disrupted synaptosomes (fraction H and I). Total recoveries of protein, LHRH,
Kndo • 1978 Vol 102 • No 4
O.32M SUCROSE
10 nM CaCI2
PROTEIN • n=7 • n=7
60
NE ED n = 3
• n=4
40
20
I
I I u 60
ill
LHRH Q n=7
TRH 0 n=6
• n=6
• n=7
k
40
20
hi I O
D
E
F
G
H
I
O
GRADIENT
FRACTION
D
E
F
G
H
I
FIG. 6. Recovery of protein, NE, LHRH, and TRH in various fractions from gradient B (Fig. 1). Intact synaptosomes (in 0.32 M sucrose-10 /XM CaCb) and lysed synaptosomes (in 10 JUM CaCb) were fractionated on gradient B as described in Materials and Methods. Results are expressed as a percentage of the total material placed on the gradient. The height of each bar denotes the mean from three to seven separate experiments. The vertical lines represent the magnitude of the SE.
TABLE 1. Recovery of protein, LHRH, TRH, and NE in subcellular fractions of the hypothalamic 900 X g supernatant fluid % recovery" Subcellular fraction Protein Cytosol Myelin/microsomes Synaptosomes Mitochondria
14.3 ± 4.4 11.2 ± 3.4 25.6 ± 4.0 5.0 ± 1.2
LHRH 1.0 ± 0.5 1.5 ± 0.8 53.4 ± 9.4 5.1 ± 1.7
TRH 3.4 ± 0.7 11.9 ± 1.7 52.2 ± 8.2 2.5 ± 0.6
NE 9.1 10.3 64.7 3.7
" Recovery was calculated as the percentage of applied material in the 900 X g supernatant fluid which was detected in each gradient fraction, n = four separate experiments except in the case of NE which was determined in two experiments. Data are presented as the mean ± SE.
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SUBSYNAPTOSOMAL POOLS OF LHRH AND TRH statistically significant difference ( P > 0.1) was found in the recoveries from lysed synaptosome fractions compared to that from •*• intact synaptosome fractions. Distribution TRH
of radiolabeled
LHRH
and
When [ 3 H]LHRH or [ 3 H]TRH was added to synaptosomes which were then lysed and fractionated on gradient B, 55-72% of the i added radioactivity was recovered in fraction 0; 15-18% was recovered in fraction D. Minimal amounts ( the endogenous T R H recovered in fraction D (synaptic vesicles) could be due to diffusion of soluble material into the gradient during centrifugation. However, this does not negate the possibility that some T R H may be associated with synaptic vesicles.
Discussion The work of several groups of investigators demonstrates that LHRH and TRH are associated with neural elements of the hypothalamus. After homogenization of the hypo* thalamus in an iso-osmotic medium and fracs, tionation by density gradient or differential centrifugation, LHRH and TRH have been found to be associated with synaptosome-like particles (6-11). Such results obtained by biophysical fractionation methods are complementary to immunocytochemical evidence k which implies that TRH (12) and LHRH (13-16) are contained in neural elements of the hypothalamus. In the current investigation, we found that LHRH, TRH, and NE are concentrated primarily in subcellular fractions * of the hypothalamus which are rich in synap^ tosomes. The subneuronal localization of these pep-
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tides has not, however, been clearly established. It has been demonstrated by means of immunohistochemical methods that certain electron-dense granules (having diameters of 75-95 nm) in axons and nerve endings of the hypothalamus contain LHRH (14-16). Somatostatin (16) and vasopressin (21) have also been found in granules of hypothalamic neurons. Recently, Winokur et al. (11) presented evidence which indicated that extrahypothalamic TRH is localized in synaptosomes and exists as a soluble substance in addition to being associated with subcellular fractions containing synaptic vesicles. Although Winokur et al. found hypothalamic TRH in synaptosomes, they did not report on the subsynaptosomal storage sites of TRH in the hypothalamus. There is, to date, no immunohistochemical evidence to demonstrate the association of TRH with synaptic vesicles, dense granules, or other subsynaptosomal organelles. An established property of synaptosomes is their susceptibility to hypo-osmotic shock (4, 5). The subneuronal localization of NE and acetylcoline was established by utilization of this property (17, 18). By lysing and fractionating hypothalamic synaptosomes on density gradients, we have found a divergence in the subsynaptosomal localization of LHRH, TRH, and NE. In agreement with the findings of De Robertis et al. (17), we found that hypothalamic NE was present in a soluble form and in association with synaptic vesicles. After lysis of synaptosomes, 15-20% of the LHRH was solubilized and 30-40% was associated with subsynaptosomal particles banding between 0.6 and 1.0 M sucrose (fractions F and G). Electron microscopic analysis of these fractions revealed the presence of dense-cored granules having diameters of 70-80 nm (Fig. 5). It is tempting to speculate that the densecored granules in fractions F and G are analogous to those in which LHRH has been detected by electron microscopic immunohistochemistry (14-16). However, since other structures are also present in these fractions, the specific organelle with which LHRH is associated is impossible to identify conclusively. Furthermore, the exclusive association of
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PARKER, JR. ET
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LHRH with secretory granules, as indicated by immunohistochemical methods, is unproven because a large fraction of the LHRH is lost during the processing of the tissue for immunohistochemical analysis (15). TRH, on the other hand, was almost totally solubilized after synaptosome lysis. Barnea et al. (9) reported similar findings after direct homogenization of hypothalami in hypo-osmotic solutions. In addition, however, we found that a portion (16%) of synaptosomal TRH fractionated in a manner similar to synaptic vesicles. The significance of this finding is difficult to interpret; a portion or all of the TRH in the synaptic vesicle fraction may have arisen through diffusion of soluble peptide into the gradient during centrifugation. Our failure to find significant quantities of this peptide in some subsynaptosomal particle (s) implies that TRH is a soluble component of hypothalamic nerve endings. However, it is conceivable that some synaptosomal TRH is stored within, or bound to particles which are extremely labile in hypoosmotic solutions. It is known that cholinergic and adrenergic synaptic vesicles of the CNS are sensitive to osmotic shock (19). Winokur et al. (11) also found that the TRH in subsynaptosomal particles isolated from the cerebrum could be partially solubilized by suspending them in water. Therefore, additional studies are necessary to determine whether TRH is a completely soluble component of hypothalamic nerve endings or whether some of the TRH is sequestered in particles within neurons of the hypothalamus. Acknowledgments The authors thank Judy Wagers and Miriam Long for editorial assistance and Linda Akers, Marguerite Gunder, Sue Sherwin, and Bob' Lipsey for excellent technical assistance.
References 1. Wheaton, J. E., L. Krulich, and S. M. McCann, Localization of luteinizing hormone-releasing hormone in the preoptic and hypothalamus of the rat using radioimmunoassay, Endocrinology 97: 30, 1975. 2. Oliver, C, R. L. Eskay, N. Ben-Jonathan, and J. C. Porter, Distribution and concentration of TRH in the rat brain, Endocrinology 95: 540, 1974.
AL.
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3. Wilber, J. F., E. Montoya, N. P. Plotnikoff, W. F. White, R. Gendrich, L. Renaud, and J. B. Martin, Gonadotropin-releasing hormone and thyrotropin-releasing hormone: distribution and effects in the central nervous system, Recent Prog Horm Res 32: 117, 1976. 4. Whittaker, V. P., The synaptosome, In Lajtha, A. (ed.), Handbook of Neurochemistry, vol. II, Plenum Press, N. Y., 1969, p. 327. 5. De Robertis, E., and G. Rodriguez de Lores Arnaiz, Structural components of the synaptic region, In Lajtha, A. (ed.), Handbook of Neurochemistry, vol. II, Plenum Press, New York, 1969, p. 365. 6. Clementi, F., B. Ceccarelli, E. Cerati, M. L. Demonte, M. Felici, M. Motta, and A. Pecile, Subcellular localization of neurotransmitters and releasing factors in the rat median eminence, J Endocrinol 48: 205,1970. 7. Shin, S. H., A. Morris, J. Snyder, W. C. Hymer, and J. V. Milligan, Subcellular localization of LH releasing hormone in the rat hypothalamus, Neuroendocrinology 16: 191, 1974. 8. Barnea, A., N. Ben-Jonathan, C. Colston, J. M. Johnston, and J. C. Porter, Differential sub-cellular compartmentalization of thyrotropin releasing hormone (TRH) and gonadotropin releasing hormone (LRH) in hypothalamic tissue, Proc NatlAcad Sci USA 72: 3153, 1975. 9. Barnea, A., N. Ben-Jonathan, and J. C. Porter, Characterization of hypothalamic subcellular particles containing luteinizing hormone releasing hormone and thyrotropin releasing hormone, J Neurochem 27: 477, 1976. 10. Ishii, S., Association of luteinizing hormone-releasing factor with granules separated from equine hypophysial stalk, Endocrinology 86: 207, 1970. 11. Winokur, A., R. Davis, and R. D. Utiger, Subcellular distribution of thyrotropin-releasing hormone (TRH) in rat brain and hypothalamus, Brain Res 120: 423, 1977. 12. Hokfelt, T., K. Fuxe, O. Johansson, S. Jeffcoate, and N. White, Distribution of thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, Eur J Pharmacol 34: 389, 1975. 13. Kordon, C, B. Kerdelhue, E. Pattou, and M. Jutisz, Immunocytochemical localization of LHRH in axons and nerve terminals of the rat median eminence, Proc Soc Exp Biol Med 147: 122,1974. 14. Pelletier, G., F. Labrie, R. Puviani, A. Arimura, and A. V. Schally, Immunohistochemical localization of luteinizing hormone-releasing hormone in the rat median eminence, Endocrinology 95: 314, 1974. 15. Goldsmith, P. C, and W. F. Ganong, Ultrastructural localization of luteinizing hormone-releasing hormone in the median eminence of the rat, Brain Res 97: 181, 1975. 16. Styne, D. M., P. C. Goldsmith, S. R. Burstein, S. L. Kaplan, and M. M. Grumbach, Immunoreactive somatostatin and luteinizing hormone releasing hormone (LHRH) in median eminence synaptosomes of
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SUBSYNAPTOSOMAL POOLS OF LHRH AND TRH
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the rat: detection of immunohistochemistry and quantification by radioimmunoassay, Endocrinology 101: 1099, 1977. De Robertis, E., A. Pellegrino de Iraldi, G. Rodriguez de Lores Arnaiz, and L. M. Zieher, Synaptic vesicles from the rat hypothalamus. Isolation and norepinephrine content, Life Sci 4: 193, 1965. Whittaker, V. P., I. A. Michaelson, and R. J. A. Kirkland, The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'), Biochem J 90: 293, 1964. Marchbanks, R. M., Isolation and study of synaptic vesicles, In Marks, N., and R. Rodnight (eds.), Research Methods in Neurochemistry, vol. II, Plenum Press, New York, 1974, p. 79. MacGregor, R. R., L. L. H. Chu, J. W. Hamilton, and D. V. Cohn, Studies on the subcellular localization of proparathyroid hormone and parathyroid hormone in the bovine parathyroid gland: separation of newly synthesized from mature forms, Endocrinology 93: 1387, 1973. Sachs, H., Studies on the intracellular distribution of vasopressin, J Neurochem 10: 289, 1963. Kemmler, W., D. F. Steiner, and J. Borg, Studies on the conversion of proinsulin to insulin, J Biol Chem 248: 4544, 1973. Costoff, A., Ultrastructure of the Rat Adenohypophysis: Correlation with Function, Academic Press,
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N. Y., 1973. 24. Parker, C. R., Jr., W. B. Neaves, A. Barnea, and J. C. Porter, Studies on the uptake of [3H]thyrotropin-releasing hormone and its metabolites by synaptosome preparations of the rat brain, Endocrinology 101: 66, 1977. 25. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193: 265, 1951. 26. Ben-Jonathan, N., and J. C. Porter, A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue, Endocrinology 98: 1497, 1976. 27. Barnea, A., C. Oliver, and J. C. Porter, Subcellular localization of a-melanocyte stimulating hormone in the rat hypothalamus, J Neurochem 29: 619, 1977. 28. Nett, T. M., A. M. Akbar, G. D. Niswender, M. T. Hedlund, and W. F. White, A radioimmunoassay for gonadotropin-releasing hormone (Gn-RH) in serum, J Clin Endocrinol Metab 36: 880, 1973. 29. Eskay, R. L., C. Oliver, J. Warberg, and J. C. Porter, Inhibition of degradation and measurement of immunoreactive thyrotropin-releasing hormone in rat blood and plasma, Endocrinology 98: 269, 1976. 30. Jones, D. G., Synapses and Synaptosomes: Morphological Aspects, Chapman and Hall, London, 1975, p. 98.
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