Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3153-3157, August 1975
Cell Biology
Differential sub-cellular compartmentalization of thyrotropin releasing hormone (TRH) and gonadotropin releasing hormone (LRH) in hypothalamic tissue (brain/synaptosomes/neurohormones/neurotransmitters/catecholamines)
A. BARNEA, N. BEN-JONATHAN, C. COLSTON, J. M. JOHNSTON, AND J. C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology, Biochemistry, and Physiology, University of Texas Southwestern Medical School, Dallas, Texas 75235
Communicated by Roger Guillemin, May 19,1975
ABSTRACT Homogenates of male rat hypothalami were fractionated by differential centrifugation. Of the total quantity of TRH and LRH in the homogenate, about 50% was in particles sedimenting at 11,500 X g, and 15-20% was in particles sedimenting between 20,000 and 105,000 X g. No LRH or TRH was recovered in the 105,000 X g supernatant fluid. When the 900 X g supernatant fluid was subjected to continuous sucrose density gradient centrifugation, TRH-containing particles distributed as two peaks located at 0.9 and 1.1 M sucrose. On the other hand, LRH-containing particles distributed as only one peak located at 1.2 M sucrose. The 11,500 X gpellet contained those particles comprising the 1.2 M peak of LRH and the 1.1 M peak of TRH. Acid phosphatase activity was found in the gradient fractions containing TRH and LRH, whereas NADPH-cytochrome c reductase and cytochrome c oxidase activities were separated from the peaks of TRH and LRH. Norepinephrine, dopamine, and TRH distributed identically on the gradient. H smotic treatment changed the gradient profile of TRH btot that of LRH. Most of the TRH was found near the top of the gradient, but a small amount of TRH was associated with particles which were lighter than those previously noted. The peak of LRH was reduced but its location on the gradient was unchanged. It is concluded that in hypothalamic homogenates TRH and LRH are localized in synaptosome-like particles which have different physical properties.
Evidence that LRH and TRH serve as neurotransmitters in the brain includes observations that these peptides have effects on brain function other than regulation of the pituitary gland (1-5). TRH is found throughout the brain (6-8), but LRH appears mostly in the hypothalamus (8, 9). If LRH and TRH are neurotransmitters, it is reasonable to assume that their sub-cellular localization is similar to that of known neurotransmitters, e.g., acetylcholine, norepinephrine, and dopamine. These substances are concentrated in synaptic vesicles located at presynaptic nerve endings. Upon homogenization of brain tissue in iso-osmotic sucrose, nerve terminals are shorn from their axons but seal spontaneously, forming particles which are called synaptosomes. As much as half of the neurotransmitter content of the brain is present in synaptosomes (10, 11). In the present investigation, the subcellular localization of TRH and LRH in hypothalamic homogenates was studied. These peptides were found to be compartmentalized in heterogeneous populations of particles. On the basis of sedimentation characteristics and other properties of these particles, we suggest that TRH and perhaps some of the LRH are contained in synaptosomes. MATERIALS AND METHODS Male rats of the Long-Evans strain (180-220 g) were killed by decapitation, and the brains were removed rapidly and Abbreviations: TRH, thyrotropin releasing hormone; LRH, gonadotropin releasing hormone. 3153
placed in cold 0.15 M NaCl. All operations were performed at 0-4°. Three to five hypothalami (20-25 mg per hypothalamus) were gently homogenized in 10 volumes of 0.32 M sucrose-10 ,M CaCG2 with a Duall tissue grinder and a Teflon pestle. For hypo-osmotic shock, hypothalami were homogenized in 10 volumes of 10 ,uM CaCl2 and chilled on ice for 5 min. Sub-Cellular Fractionations. All sucrose solutions contained 10 ,mol of CaCl2 per liter. Differential -centrifugation of hypothalamic homogenates was performed according to Kataoka and De Robertis (12). The pellets were prepared for analytical assays by resuspension in a solution of 0.01 M sodium phosphate buffer containing 0.14 M NaCl and 0.01% merthiolate, pH 7.0 (phosphate-buffered saline). Continuous sucrose density gradients were prepared using a two-chamber mixing device. One chamber was filled with 6.4 ml of a solution of low sucrose concentration and the other with 5.8 ml of a solution of high sucrose concentration. The gradients were allowed to stand at 20 for 20 hr before use. Pellets obtained by centrifuging the 900 X g supernatant fluid (S) at 11,500 X g for 20 min (P) or 40,000 X g for 30 min were suspended in a volume of 0.32 M sucrose-10 ,M CaCl2 equal to the homogenization volume. Aliquots (0.71.2 ml) of the suspension or of S were layered on the gradients. The tubes were centrifuged at designated speeds and times at 40 in a Beckman model L2-65 Ultracentrifuge using an SW 40 rotor. At the end of the run, fractions (0.1-0.4 ml) were collected using an ISCO model 640 Density Gradient Fractionator. In order to facilitate comparison of the locations of the various substances, it was assumed that the concentration of sucrose changed linearly on the gradients. Assays. TRH and LRH in hypothalamic homogenates or in fractions obtained by differential centrifugation were extracted with 9 volumes of methanol as previously described (6). Fractions collected from sucrose density gradients were diluted with phosphate-buffered saline, sonicated for 1 hr in an ice-water bath using an Ultrasonicator (125 W, Heat Systems Ultrasonics, Inc.). These two methods of preparing samples for assay gave similar results. TRH and LRH were quantified by radioimmunoassay procedures described by Oliver et al. (13) and Nett et al. (14). Synthetic TRH and LRH (Beckman Instruments, Inc.) served as reference preparations. Enzyme activities were determined according to the following procedures: NADPH-cytochrome c reductase (EC 1.6.2.4) according to Masters et al. (15), cytochrome c oxidase (EC 1.9.3.1) by the method of Smith (16), and acid phosphatase (EC 3.1.3.2) by the procedure of Michell et al. (17). Norepinephrine and dopamine were measured by
Cell Biology: Barnea et al.
3154
Proc. Nat. Acad. Sci. USA 72 (1975)
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FIG. 1. Distribution of TRH and LRH in sub-cellular fractions obtained by differential centrifugation of hypothalamic homogenates. A 10% homogenate was centrifuged 10 min at 900 X g. The pellet was washed twice, and the supernatant fluid (S) was centrifuged 20 min at 11,500 X g. The 11,500 X g pellet (P) was washed, and the supernatant fluid was centrifuged 30 min at 20,000 X g. The 20,000 X g supernatant fluid was centrifuged 60 min at 105,000 X g. The height of the bars is the average of two fractionations, indicated by dots.
means of a radiometric-enzyme assay as described by Coyle and Henry (18). Protein was determined according to Lowry et al. (19) using crystalline bovine serum albumin as the standard. RESULTS
Sub-cellular fractionation by differential centrifugation Of the total amount of TRH or LRH in the hypothalamic homogenate, 5-10% was recovered in the 900 X g pellet; 40-50% in the 11,500 X g pellet; 10% in the 20,000 X g pellet; and 17% of the TRH and 7% of the LRH in the 105,000 X g pellet. TRH and LRH were not demonstrable in the 105,000 X g supernatant fluid (Fig. 1). The recovery of TRH or LRH after fractionation was 75%. The recovery of protein was 95%, and its sub-cellular distribution was similar to that reported by others (12).
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FIG. 2. Comparison of gradient profiles of TRH obtained from S and P. These two sub-cellular fractions, prepared as described in Fig. 1, were layered on sucrose gradients and centrifuged 1.5 hr at 29,000 rpm. The arrows in Figs. 2-5 denote the lower limit of sensitivity of the assays.
ing P on a gradient ranging from 0.9 to 1.6 M sucrose (Fig. 4). The recovery of TRH and LRH from the gradients was 75 1 13.2% and 52 A 9.2% (mean and SD of eight gradients), respectively. 200 r
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Sub-cellular fractionation by density gradient centrifugation When S, which contained 95% of the TRH in the homogenate, was fractionated on sucrose (0.6-1.6 M) gradients, two populations of TRH-containing particles were found. The maximal concentrations were in 0.90 and 1.11 M sucrose (Fig. 2). When P. which contained 40% of the TRH in the homogenate, was fractionated, only those particles comprising the 1.11 M peak were recovered (Fig. 2). However, both populations of TRH-containing particles were sedimented by centrifuging S 30 min at 40,000 X g. When S. which contained 90% of the LRH in the homogenate, was fractionated, only one population of LRH-containing particles was recovered (Fig. 3). The peak concentration of these particles was in 1.22 M sucrose, indicating a slight difference between the heavy particles containing TRH and the LRH-containing particles. Better separation of these two populations of particles was effected by fractionat-
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FIG. 3. Distribution of TRH and LRH on a sucrose gradient. S was layered on sucrose gradients and centrifuged 3 hr at 20,000 rpm.
Cell Biology: Bamea et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
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and LRH S was fractionated on a sucrose (0.6-1.6 M) gradient, and the distributions of several biochemical markers for sub-cellular organelles were determined. The gradient profiles of norepinephrine and dopamine, synaptosomal markers (10), were identical; and both distributed in two peaks coinciding j 56 3.6 c 0
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FIG. 6. Distribution of enzyme markers, TRH, and LRH on a sucrose gradient. (See Fig. 3 for details.) The activity (E.U.) of acid phosphatase, cytochrome c oxidase, and NADPH-cytochrome c reductase is expressed as nmol/min of p-nitrophenol produced, cytochrome c oxidized, and cytochrome c reduced, respectively. The bars denote the positions of the TRH and LRH peaks at half their
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with those of TRH (Fig. 5). The distribution of the heavier particles containing norepinephrine and dopamine was similar to that of particles containing LRH. Unlike TRH and LRH, 40% of the norepinephrine or dopamine recovered was located near the top of the gradient. Acid phosphatase activity, a lysosomal marker (20), was distributed throughout the gradient; however, peak activities were present near the top of the gradient and at 1.16 M sucrose (Fig. 6). Peak activities of cytochrome c oxidase, a mitochondrial marker, and NADPH-cytochrome c reductase, a microsomal marker (20), were recovered in 1.40 M and 0.67 M sucrose, respectively (Fig. 6). Effect of centrifugal (g) force on particle size We noted that some properties of the LRH-containing particles could be altered, depending on the g force used for preparing sub-cellular fractions. For example, S contained only one population of LRH-containing particles (Fig. 3). LRHcontaining particles in the pellet prepared by centrifuging S at 11,500 X g distributed on the gradient as a large peak in 1.16-1.36 M sucrose and as a small peak in 1.40-1.47 M sucrose* (Fig. 4). LRH-containing particles in the pellet prepared by centrifuging S at 40,000 X g distributed on the gradient as a broad peak in the region of 1.08-1.53 M sucrose. We interpret these findings as indicating that LRHcontaining particles have a tendency to aggregate. TRHcontaining particles do not aggregate as easily; however, some aggregation was observed in the 40,000 X g pellet. *
The boundaries of the peaks were measured at half their heights.
3156
Cell Biology: Barnea et al.
Proc. Nat. Acad. Sci. USA 72 (1975) 125
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FIG. 7. Effect of hypo-osmotic treatment on the distribution of TRH and LRH on sucrose gradients. S prepared in 0.32 M sucrose-10
MM CaC12 (iso-osmotic) or 10MM CaCl2 (hypo-osmotic) was layered on sucrose gradients and centrifuged 2 hr at 20,000 rpm.
Effect of hypo-osmotic treatment on particle-bound TRH and LRH S obtained from iso- or hypo-osmotically treated hypothalami was analyzed on gradients ranging from 0.4 to 1.4 M sucrose. Hypo-osmotic treatment changed the distribution of TRH markedly (Fig. 7). Eighty percent of the TRH recovered was present near the top of the gradient in 0.32-0.46 M sucrose, and 10% appeared as a peak located at 0.60 M sucrose. In the iso-osmotically treated samples, the TRH-containing particles were recovered in 0.70 and 1.05 M sucrose. After hypo-osmotic treatment, the LRH peak was reduced but its location on the gradient was not altered. A small amount of LRH was recovered near the top of the gradient (Fig. 7). The recovery of TRH and LRH from the gradients was 90% and 60%, respectively, for the iso-osmotically treated samples and 50% and 30%, respectively, for the hypo-
osmotically treated samples. DISCUSSION The sub-cellular location of TRH in the brain has not been studied heretofore. However, a particulate site of storage for gonadatropin releasing activity, determined by bioassay, has been proposed (21-24). In the present study, we found that TRH as well as LRH in hypothalamic homogenates are associated with particles having sedimentation characteristics similar to those of synaptosomes containing norepinephrine and dopamine. The fact that two populations of TRH-containing particles and one population of LRH-containing particles were found does not exclude the identity of these particles with synaptosomes, since it has been demonstrated that synaptosomes vary widely in size (10). These TRH- and LRH-containing particles are not mitochondria or microsomes since the sedimentation characteristics of these sub-
cellular organelles, as indicated by enzyme markers (20), are clearly different from those of the particles containing TRH and LRH. Although a peak of acid phosphatase activity, a lysosomal marker (20), was found in the gradient fraction containing LRH and one of the fractions containing TRH, it seems unlikely that these peptides are bound to lysosomes; however, such a possibility cannot be totally excluded. Despite the similarities of the sedimentation characteristics of the LRH- and some of the TRH-containing particles, they do differ appreciably in other physical properties. When sedimented at high g forces, the LRH-containing particles were found to aggregate more readily than did those containing TRH. In addition, the two sets of particles differ in their susceptibility to hypo-osmotic shock. After hypoosmotic treatment, most of the TRH recovered was near the top of the gradient, presumably in non-particulate form. The small fraction of TRH that was associated with particles had a sedimentation characteristic similar to that of synaptic vesicles containing acetylcholine (11). The sedimentation characteristics of most of the particles containing LRH were not altered by hypo-osmotic shock. Thus, the question arises, why did hypo-osmotic treatment fail to release LRH? Since all synaptosomes described so far are ruptured by hypo-osmotic shock (10, 11), thus releasing their neurotransmitter content, the possibility arises that some of the LRH-containing particles are not synaptosomes. The presence of TRH in synaptosome-like particles and the wide distribution of TRH in brain tissue (6-8) provide additional evidence for a neurotransmitter role for TRH. We thank Robert Lipsey, Bob Athey, and Mary Bob Wylie for excellent technical assistance. This research was supported by Research Grants, AM01237 and SPOl HD08672-01, from the National Institutes of Health.
Cell Biology: Barnea et al. 1. Pfaff, D. W. (1973) Science 182, 1148-1149. 2. Moss, R. L., & McCann, S. M. (1973) Science 181, 177-179. 3. Kastin, A. J., Ehrensing, R. H., Schalch, D. S. & Anderson, M. S. (1972) Lancet ii, 740-742. 4. Prange, A. J., Jr., Wilson, I. C., Lara, P. P., Alltop, L. B. & Breese, G. R. (1972) Lancet ii, 999-1002. 5. Wilson, I. C., Prange, A. J., Jr., Lara, P. P., AlItop, L. B., Stikeleather, R. A. & Lipton, M. A. (1973) Arch. Gen. Psychiat. 29, 15-21. 6. Oliver, C., Eskay, R. L., Ben-Jonathan, N. & Porter, J. C.
(1974) Endocrinology 95,540-546. 7. Brownstein, M. J., Palkovits, M., Saaredra, J. M., Bassiri, R. M. & Utiger, R. D. (1974) Science 185,267-269. 8. Winters, A. J., Eskay, R. L. & Porter, J. C. (1974) J. Clin. Endocrinol. Metab. 39,960-963. 9. Araki, S., Ferin, M., Zimmerman, E. A. & Vande Wiele, R. L. (1975) Endocrinology 96,644-650. 10. De Robertis, E. & Rodriguez de Lores Arnaiz, G. (1969) in Handbook of Neurochemistry, ed. Lajtha, A. (Plenum Press, New York), pp. 365-392. 11. Whittaker, V. P. (1969) in Handbook of Neurochemistry, ed. Lajtha, A. (Plenum Press, New York), pp. 327-364. 12. Kataoka, K. & De Robertis, E. (1967) J. Pharmacol. Exp. Ther. 156, 114-125. 13. Oliver, C., Eskay, R. L., Mical, R. S. & Porter, J. C. (1973)
Proc. Nat. Acad. Sci. USA 72 (1975)
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49th Meeting Amer. Thyroid Assoc., Inc., Seattle, Washington, p. T-5 (abstr.). 14. Nett, T. M., Akbar, A. M., Niswender, G. D., Hedlund, M. T. & White, W. F. (1973) J. Clin. Endocrinol. Metab. 36, 880885. 15. Masters, B. S. S., Williams, C: H., Jr. & Kamin, H. (1967) in Methods in Enzymology, eds. Estabrook, R. W. & Pullman, M. E. (Academic Press, New York), Vol. 10, pp. 565-573. 16. Smith, L. (1955) in Methods of Biochemical Analysis, ed. Click, D. (Interscience Publishers Inc., New York), pp. 427434. 17. Michell, R. H., Karnovsky, M. J. & Karnovsky, M. L. (1970) Biochem. J. 116, 207-216. 18. Coyle, J. T. & Henry, D. (1973) J. Neurochem. 21, 61-67. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Blol. Chem. 193,265-275. 20. De Duve, C. (1971) J. Cell Biol. 50, 20D-55D. 21. Clementi, F., Ceccarelli, B., Cerati, E., Demonte, M. L., Felici, M., Motta, M. & Pecile, A. (1970) J. Endocrinol. 48, 205213. 22. Ishii, S. (1970) Endocrinology 86,207-216. 23. Johansson, K. N. G., Currie, B. L. & Folkers, K. (1973) Biochem. Biophys. Res. Commun. 52,967-973. 24. Taber, C. A. & Karavolas, H. J. (1975) Endocrinology 96, 446-452.
Cell Biology
Differential sub-cellular compartmentalization of thyrotropin releasing hormone (TRH) and gonadotropin releasing hormone (LRH) in hypothalamic tissue (brain/synaptosomes/neurohormones/neurotransmitters/catecholamines)
A. BARNEA, N. BEN-JONATHAN, C. COLSTON, J. M. JOHNSTON, AND J. C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology, Biochemistry, and Physiology, University of Texas Southwestern Medical School, Dallas, Texas 75235
Communicated by Roger Guillemin, May 19,1975
ABSTRACT Homogenates of male rat hypothalami were fractionated by differential centrifugation. Of the total quantity of TRH and LRH in the homogenate, about 50% was in particles sedimenting at 11,500 X g, and 15-20% was in particles sedimenting between 20,000 and 105,000 X g. No LRH or TRH was recovered in the 105,000 X g supernatant fluid. When the 900 X g supernatant fluid was subjected to continuous sucrose density gradient centrifugation, TRH-containing particles distributed as two peaks located at 0.9 and 1.1 M sucrose. On the other hand, LRH-containing particles distributed as only one peak located at 1.2 M sucrose. The 11,500 X gpellet contained those particles comprising the 1.2 M peak of LRH and the 1.1 M peak of TRH. Acid phosphatase activity was found in the gradient fractions containing TRH and LRH, whereas NADPH-cytochrome c reductase and cytochrome c oxidase activities were separated from the peaks of TRH and LRH. Norepinephrine, dopamine, and TRH distributed identically on the gradient. H smotic treatment changed the gradient profile of TRH btot that of LRH. Most of the TRH was found near the top of the gradient, but a small amount of TRH was associated with particles which were lighter than those previously noted. The peak of LRH was reduced but its location on the gradient was unchanged. It is concluded that in hypothalamic homogenates TRH and LRH are localized in synaptosome-like particles which have different physical properties.
Evidence that LRH and TRH serve as neurotransmitters in the brain includes observations that these peptides have effects on brain function other than regulation of the pituitary gland (1-5). TRH is found throughout the brain (6-8), but LRH appears mostly in the hypothalamus (8, 9). If LRH and TRH are neurotransmitters, it is reasonable to assume that their sub-cellular localization is similar to that of known neurotransmitters, e.g., acetylcholine, norepinephrine, and dopamine. These substances are concentrated in synaptic vesicles located at presynaptic nerve endings. Upon homogenization of brain tissue in iso-osmotic sucrose, nerve terminals are shorn from their axons but seal spontaneously, forming particles which are called synaptosomes. As much as half of the neurotransmitter content of the brain is present in synaptosomes (10, 11). In the present investigation, the subcellular localization of TRH and LRH in hypothalamic homogenates was studied. These peptides were found to be compartmentalized in heterogeneous populations of particles. On the basis of sedimentation characteristics and other properties of these particles, we suggest that TRH and perhaps some of the LRH are contained in synaptosomes. MATERIALS AND METHODS Male rats of the Long-Evans strain (180-220 g) were killed by decapitation, and the brains were removed rapidly and Abbreviations: TRH, thyrotropin releasing hormone; LRH, gonadotropin releasing hormone. 3153
placed in cold 0.15 M NaCl. All operations were performed at 0-4°. Three to five hypothalami (20-25 mg per hypothalamus) were gently homogenized in 10 volumes of 0.32 M sucrose-10 ,M CaCG2 with a Duall tissue grinder and a Teflon pestle. For hypo-osmotic shock, hypothalami were homogenized in 10 volumes of 10 ,uM CaCl2 and chilled on ice for 5 min. Sub-Cellular Fractionations. All sucrose solutions contained 10 ,mol of CaCl2 per liter. Differential -centrifugation of hypothalamic homogenates was performed according to Kataoka and De Robertis (12). The pellets were prepared for analytical assays by resuspension in a solution of 0.01 M sodium phosphate buffer containing 0.14 M NaCl and 0.01% merthiolate, pH 7.0 (phosphate-buffered saline). Continuous sucrose density gradients were prepared using a two-chamber mixing device. One chamber was filled with 6.4 ml of a solution of low sucrose concentration and the other with 5.8 ml of a solution of high sucrose concentration. The gradients were allowed to stand at 20 for 20 hr before use. Pellets obtained by centrifuging the 900 X g supernatant fluid (S) at 11,500 X g for 20 min (P) or 40,000 X g for 30 min were suspended in a volume of 0.32 M sucrose-10 ,M CaCl2 equal to the homogenization volume. Aliquots (0.71.2 ml) of the suspension or of S were layered on the gradients. The tubes were centrifuged at designated speeds and times at 40 in a Beckman model L2-65 Ultracentrifuge using an SW 40 rotor. At the end of the run, fractions (0.1-0.4 ml) were collected using an ISCO model 640 Density Gradient Fractionator. In order to facilitate comparison of the locations of the various substances, it was assumed that the concentration of sucrose changed linearly on the gradients. Assays. TRH and LRH in hypothalamic homogenates or in fractions obtained by differential centrifugation were extracted with 9 volumes of methanol as previously described (6). Fractions collected from sucrose density gradients were diluted with phosphate-buffered saline, sonicated for 1 hr in an ice-water bath using an Ultrasonicator (125 W, Heat Systems Ultrasonics, Inc.). These two methods of preparing samples for assay gave similar results. TRH and LRH were quantified by radioimmunoassay procedures described by Oliver et al. (13) and Nett et al. (14). Synthetic TRH and LRH (Beckman Instruments, Inc.) served as reference preparations. Enzyme activities were determined according to the following procedures: NADPH-cytochrome c reductase (EC 1.6.2.4) according to Masters et al. (15), cytochrome c oxidase (EC 1.9.3.1) by the method of Smith (16), and acid phosphatase (EC 3.1.3.2) by the procedure of Michell et al. (17). Norepinephrine and dopamine were measured by
Cell Biology: Barnea et al.
3154
Proc. Nat. Acad. Sci. USA 72 (1975)
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SUB-CELLULAR FRACTION 1
FIG. 1. Distribution of TRH and LRH in sub-cellular fractions obtained by differential centrifugation of hypothalamic homogenates. A 10% homogenate was centrifuged 10 min at 900 X g. The pellet was washed twice, and the supernatant fluid (S) was centrifuged 20 min at 11,500 X g. The 11,500 X g pellet (P) was washed, and the supernatant fluid was centrifuged 30 min at 20,000 X g. The 20,000 X g supernatant fluid was centrifuged 60 min at 105,000 X g. The height of the bars is the average of two fractionations, indicated by dots.
means of a radiometric-enzyme assay as described by Coyle and Henry (18). Protein was determined according to Lowry et al. (19) using crystalline bovine serum albumin as the standard. RESULTS
Sub-cellular fractionation by differential centrifugation Of the total amount of TRH or LRH in the hypothalamic homogenate, 5-10% was recovered in the 900 X g pellet; 40-50% in the 11,500 X g pellet; 10% in the 20,000 X g pellet; and 17% of the TRH and 7% of the LRH in the 105,000 X g pellet. TRH and LRH were not demonstrable in the 105,000 X g supernatant fluid (Fig. 1). The recovery of TRH or LRH after fractionation was 75%. The recovery of protein was 95%, and its sub-cellular distribution was similar to that reported by others (12).
GRADIENT VOLUME (ml) -
1.6
0.32 0.6
SUCROSE CONCENTRATION (M)
FIG. 2. Comparison of gradient profiles of TRH obtained from S and P. These two sub-cellular fractions, prepared as described in Fig. 1, were layered on sucrose gradients and centrifuged 1.5 hr at 29,000 rpm. The arrows in Figs. 2-5 denote the lower limit of sensitivity of the assays.
ing P on a gradient ranging from 0.9 to 1.6 M sucrose (Fig. 4). The recovery of TRH and LRH from the gradients was 75 1 13.2% and 52 A 9.2% (mean and SD of eight gradients), respectively. 200 r
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Sub-cellular fractionation by density gradient centrifugation When S, which contained 95% of the TRH in the homogenate, was fractionated on sucrose (0.6-1.6 M) gradients, two populations of TRH-containing particles were found. The maximal concentrations were in 0.90 and 1.11 M sucrose (Fig. 2). When P. which contained 40% of the TRH in the homogenate, was fractionated, only those particles comprising the 1.11 M peak were recovered (Fig. 2). However, both populations of TRH-containing particles were sedimented by centrifuging S 30 min at 40,000 X g. When S. which contained 90% of the LRH in the homogenate, was fractionated, only one population of LRH-containing particles was recovered (Fig. 3). The peak concentration of these particles was in 1.22 M sucrose, indicating a slight difference between the heavy particles containing TRH and the LRH-containing particles. Better separation of these two populations of particles was effected by fractionat-
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FIG. 3. Distribution of TRH and LRH on a sucrose gradient. S was layered on sucrose gradients and centrifuged 3 hr at 20,000 rpm.
Cell Biology: Bamea et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
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and LRH S was fractionated on a sucrose (0.6-1.6 M) gradient, and the distributions of several biochemical markers for sub-cellular organelles were determined. The gradient profiles of norepinephrine and dopamine, synaptosomal markers (10), were identical; and both distributed in two peaks coinciding j 56 3.6 c 0
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FIG. 6. Distribution of enzyme markers, TRH, and LRH on a sucrose gradient. (See Fig. 3 for details.) The activity (E.U.) of acid phosphatase, cytochrome c oxidase, and NADPH-cytochrome c reductase is expressed as nmol/min of p-nitrophenol produced, cytochrome c oxidized, and cytochrome c reduced, respectively. The bars denote the positions of the TRH and LRH peaks at half their
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with those of TRH (Fig. 5). The distribution of the heavier particles containing norepinephrine and dopamine was similar to that of particles containing LRH. Unlike TRH and LRH, 40% of the norepinephrine or dopamine recovered was located near the top of the gradient. Acid phosphatase activity, a lysosomal marker (20), was distributed throughout the gradient; however, peak activities were present near the top of the gradient and at 1.16 M sucrose (Fig. 6). Peak activities of cytochrome c oxidase, a mitochondrial marker, and NADPH-cytochrome c reductase, a microsomal marker (20), were recovered in 1.40 M and 0.67 M sucrose, respectively (Fig. 6). Effect of centrifugal (g) force on particle size We noted that some properties of the LRH-containing particles could be altered, depending on the g force used for preparing sub-cellular fractions. For example, S contained only one population of LRH-containing particles (Fig. 3). LRHcontaining particles in the pellet prepared by centrifuging S at 11,500 X g distributed on the gradient as a large peak in 1.16-1.36 M sucrose and as a small peak in 1.40-1.47 M sucrose* (Fig. 4). LRH-containing particles in the pellet prepared by centrifuging S at 40,000 X g distributed on the gradient as a broad peak in the region of 1.08-1.53 M sucrose. We interpret these findings as indicating that LRHcontaining particles have a tendency to aggregate. TRHcontaining particles do not aggregate as easily; however, some aggregation was observed in the 40,000 X g pellet. *
The boundaries of the peaks were measured at half their heights.
3156
Cell Biology: Barnea et al.
Proc. Nat. Acad. Sci. USA 72 (1975) 125
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FIG. 7. Effect of hypo-osmotic treatment on the distribution of TRH and LRH on sucrose gradients. S prepared in 0.32 M sucrose-10
MM CaC12 (iso-osmotic) or 10MM CaCl2 (hypo-osmotic) was layered on sucrose gradients and centrifuged 2 hr at 20,000 rpm.
Effect of hypo-osmotic treatment on particle-bound TRH and LRH S obtained from iso- or hypo-osmotically treated hypothalami was analyzed on gradients ranging from 0.4 to 1.4 M sucrose. Hypo-osmotic treatment changed the distribution of TRH markedly (Fig. 7). Eighty percent of the TRH recovered was present near the top of the gradient in 0.32-0.46 M sucrose, and 10% appeared as a peak located at 0.60 M sucrose. In the iso-osmotically treated samples, the TRH-containing particles were recovered in 0.70 and 1.05 M sucrose. After hypo-osmotic treatment, the LRH peak was reduced but its location on the gradient was not altered. A small amount of LRH was recovered near the top of the gradient (Fig. 7). The recovery of TRH and LRH from the gradients was 90% and 60%, respectively, for the iso-osmotically treated samples and 50% and 30%, respectively, for the hypo-
osmotically treated samples. DISCUSSION The sub-cellular location of TRH in the brain has not been studied heretofore. However, a particulate site of storage for gonadatropin releasing activity, determined by bioassay, has been proposed (21-24). In the present study, we found that TRH as well as LRH in hypothalamic homogenates are associated with particles having sedimentation characteristics similar to those of synaptosomes containing norepinephrine and dopamine. The fact that two populations of TRH-containing particles and one population of LRH-containing particles were found does not exclude the identity of these particles with synaptosomes, since it has been demonstrated that synaptosomes vary widely in size (10). These TRH- and LRH-containing particles are not mitochondria or microsomes since the sedimentation characteristics of these sub-
cellular organelles, as indicated by enzyme markers (20), are clearly different from those of the particles containing TRH and LRH. Although a peak of acid phosphatase activity, a lysosomal marker (20), was found in the gradient fraction containing LRH and one of the fractions containing TRH, it seems unlikely that these peptides are bound to lysosomes; however, such a possibility cannot be totally excluded. Despite the similarities of the sedimentation characteristics of the LRH- and some of the TRH-containing particles, they do differ appreciably in other physical properties. When sedimented at high g forces, the LRH-containing particles were found to aggregate more readily than did those containing TRH. In addition, the two sets of particles differ in their susceptibility to hypo-osmotic shock. After hypoosmotic treatment, most of the TRH recovered was near the top of the gradient, presumably in non-particulate form. The small fraction of TRH that was associated with particles had a sedimentation characteristic similar to that of synaptic vesicles containing acetylcholine (11). The sedimentation characteristics of most of the particles containing LRH were not altered by hypo-osmotic shock. Thus, the question arises, why did hypo-osmotic treatment fail to release LRH? Since all synaptosomes described so far are ruptured by hypo-osmotic shock (10, 11), thus releasing their neurotransmitter content, the possibility arises that some of the LRH-containing particles are not synaptosomes. The presence of TRH in synaptosome-like particles and the wide distribution of TRH in brain tissue (6-8) provide additional evidence for a neurotransmitter role for TRH. We thank Robert Lipsey, Bob Athey, and Mary Bob Wylie for excellent technical assistance. This research was supported by Research Grants, AM01237 and SPOl HD08672-01, from the National Institutes of Health.
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49th Meeting Amer. Thyroid Assoc., Inc., Seattle, Washington, p. T-5 (abstr.). 14. Nett, T. M., Akbar, A. M., Niswender, G. D., Hedlund, M. T. & White, W. F. (1973) J. Clin. Endocrinol. Metab. 36, 880885. 15. Masters, B. S. S., Williams, C: H., Jr. & Kamin, H. (1967) in Methods in Enzymology, eds. Estabrook, R. W. & Pullman, M. E. (Academic Press, New York), Vol. 10, pp. 565-573. 16. Smith, L. (1955) in Methods of Biochemical Analysis, ed. Click, D. (Interscience Publishers Inc., New York), pp. 427434. 17. Michell, R. H., Karnovsky, M. J. & Karnovsky, M. L. (1970) Biochem. J. 116, 207-216. 18. Coyle, J. T. & Henry, D. (1973) J. Neurochem. 21, 61-67. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Blol. Chem. 193,265-275. 20. De Duve, C. (1971) J. Cell Biol. 50, 20D-55D. 21. Clementi, F., Ceccarelli, B., Cerati, E., Demonte, M. L., Felici, M., Motta, M. & Pecile, A. (1970) J. Endocrinol. 48, 205213. 22. Ishii, S. (1970) Endocrinology 86,207-216. 23. Johansson, K. N. G., Currie, B. L. & Folkers, K. (1973) Biochem. Biophys. Res. Commun. 52,967-973. 24. Taber, C. A. & Karavolas, H. J. (1975) Endocrinology 96, 446-452.
