Journal of Neurochemisrry, 1977. Vol. 29, pp. 619-624. Pergamon Press. Printed in Great Britain.

SUBCELLULAR LOCALIZATION OF a-MELANOCYTESTIMULATING HORMONE IN THE RAT HYPOTHALAMUS’ AYALLABARNEA, CHARLES OLIVER’ and JOHN C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Physiology, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, TX 75235 USA. (Received 17 January 1977. Accepted 7 M a y 1977)

Abstract-Homogenates of male rat hypothalami were fractionated by means of differential centrifugation, and cx-melanocyte-stimulating hormone (a-MSH) in the various fractions was quantified by radioimmunoassay. Of the total quantity of a-MSH in the homogenate, 36% was recovered in the 11,500g pellet and 31% sedimented between 11,500 and 105,OOOg. a-MSH was not detected in the 105,000 g supernatant fluid. When the 9009 supernatant fluid was fractionated on continuous sucrose density gradients at non-equilibrium conditions, two populations of particles containing a-MSH were observed. When fractionated at equilibrium conditions, the two populations were recovered in a single band. These sedimentation characteristics indicate that the particles that contain a-MSH differ in size but are similar in density. After hypo-osmotic shock, the large particles containing a-MSH were not demonstrable, whereas the small particles appeared to be resistant to such treatment. In their sedimentation, the particles containing a-MSH were indistinguishable from particles containing thyrotropin releasing hormone (TRH) but were separable from those that contained luteinizing hormone releasing hormone (LHRH). It is suggested that the large particles containing a-MSH are synaptosomes.

SEVERAL ACTH-like peptides, some of which are of Since the mode of action of ACTH-like peptides and pituitary origin (SHAPIROet al., 1972; RUDMANet al., their distribution in the brain resemble those of classi1973; LOWRY& SCOTT,1975; USTEGUIet al., 1976), cal neurotransmitters, it can be envisioned that have direct effects on such brain functions as behav- ACTH-like peptides and classical neurotransmitters ior, learning, memory (FERRARI et al., 1963; GISPEN are localized in similar subcellular sites in the brain. It has been well established that neurotransmitters & SCHOTMAN, 1973; GREVEN & DE WIED, 1973; KASTIN et al., 1973; LANDEet al., 1973; STRATTON & KAS- in the brain, such as ACh and biogenic amines, are TIN, 1974), protein synthesis (GISPEN et al., 1971; highly concentrated at presynaptic axonal terminals DE LORESARNAIZ,1969; GISPEN& SCHOTMAN, 1973; RUDMANet al., 1974a), (DE ROBERTIS& RODR~GUES and the levels of cyclic AMP in the CSF (RUDMAN WHITTAKER,1969). Upon homogenization, these ter& ISAACS,1975). Some of these peptides also have minals are shorn from their axons and form particles melanocyte stimulating activity (GUILLEMINet al., which have been called synaptosomes. In hypothala1962; SCHALLY et al., 1962; LEE et at., 1963; BAKER, mic homogenates, two peptides, TRH-a tripeptide1973). Melanocyte stimulating activity has been and LHRH-a decapeptide-are sequestered in pardemonstrated in bovine, simian, human and rodent ticles having several physical properties resembling brain tissue (RUDMANet al., 1973; 1974b; 1975). synaptosomes (RAMIREZ& KORDON,1975; BARNEA Moreover, the rat brain contains appreciable amounts et al., 1975, 1976, 1977; BENNETTet al., 1975; WARof a-MSH (OLIVER& PORTER,submitted). a-MSH is BERG et a/., 1977). In the present study, the following a potent melanocyte stimulating peptide which con- questions were addressed: is GI-MSH stored in presists of the first thirteen amino acid residues of synaptic axonal terminals in the rat hypothalamus? ACTH, with acetylation of the N-terminal serine and If so, d o these same axonal terminals contain other amidation of the C-terminal valine (LEE et al., 1963). peptides such as TRH or LHRH? To answer these questions, the subcellular compartmentalization of Supported by grants from the National Institute of immunoreactive a-MSH in the rat hypothalamus was Arthritis, Metabolism, and Digestive Diseases (AM01237) investigated using differential centrifugation as well and from the National Institute of Child Health and as non-equilibrium and equilibrium density gradient Human Development (HD08672). centrifugation. Present address: Laboratoire de Medecine Experimentale, U.E.R. MCdecine Nord, Boulevard Pierre Dramard, Marseille, France 13326. MATERIALS AND METHODS Abbreuiations used: ACTH, adrenocorticotropic horAnimals. Adult male rats of the Long-Evans strain were mone; a-MSH, a-melanocyte stimulating hormone; LHRH, luteinizing hormone releasing hormone; TRH, used. The animals were decapitated, and their brains were removed rapidly and chilled in ice-cold 0.15 M-NaCl. The thyrotropin releasing hormone. 619



NC

29/4--~

AYALLABARNEA, CHARLES OLIVER and JOHNC. PORTER

620

TABLE I. RECOVERIESOF a-MSH, TRH, AND LHRH .FROM VARIOUS Heated* TRH

z-MSH

LHRH

Quantity in the homogenate (ng)

12.7 & 0.7t

7.6

k 0.5

6.8 & 0.5

Quantity in the 9009 SFS (ng)

10.5 & 0.5

6.0 & 0.2

4.8 & 0.4

Recovery in the 9009 SF (%)$

82.5 k 3.1

79.8 $- 4.5

71.8

Recovery lrom the gradient (%)I1

62.3 & 3.9

74.0

75.0 & 4.57

* Heated in a boiling water bath t Mean k s.E.M.; N = 10. 1SF = Supernatant

5.9'

3.9

SUBCELLULAR FRACTIONS

a-MSH 10.1 & 0.7

Acetic acid ethanolTRH 6.8

LHRH

0.4

5.8 & 0.6

7.8

k 0.8

5.6 & 0.4

3.9 ?c 0.4

76.8

k 5.6

X3.9 & 4.4

66.8 & 2.6

-

for 10 min.

fluid.

8 % = (Quantity in the 9009 SF + Quantity in the homogenate) I] "/, = (Quantity recovered from gradient q:

N

=

x 100. the 9009 SF) x 100

+ Quantity in

7. homogenates were diluted with 1 vol of 0.32 M-sucrose and centrifuged at 900 g for 10 min. The recoveries of a-MSH, TRH, and LHRH in the 900 g supernatant fluid are shown in Table 1. The 9009 supernatant fluid was layered on continuous sucrose density gradients which were prepared et a / . (1975). The gradients were as described by BARNEA centrifuged for 30 min (non-equilibrium fractionation) or for 300 min (equilibrium fractionation) (BARNEAet a/., 1976) at 29,000 rev./min (100,000gdv)at 4°C in a Beckman Model L2-65 Ultracentrifuge using an SW 40 rotor (Beckman Instruments, Inc., Palo Alto, CA). At the end of each run, 0.4 ml fractions were collected using an ISCO Model 640 Density Gradient Fractionator (Instrument Specialties

nypothalami were then excised. The boundaries of the hypothalamic fragments (about 7 mm long and 4 mm wide; 25 mg wet weight) extended from the anterior margin of the preoptic area to the posterior border of the mammillary bodies. Subcellular.fruc.1ionations.All operations were performed at W" C . and all sucrose solutions contained 10 pM-CaCI2. The hypothalami were homogenized in 10 vol of 0 . 3 2 ~ sucrose with a Dual1 tissue grinder and Teflon pestle (K-885480, Kontes Glass Co., Vineland, NJ) using ten upand-down strokes. Differential centrifugation was carried et a/., 1975). For fracout as described previously (BARNEA tionation by means of density gradient centrifugation, the

SYNTHETIC a - ~ s ~ DUNBOILED OBOILED

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l UNBOILED

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SYNTHETIC a-MSH

A ACID ETHANOL EXTRACT o SALINE EXTRACT - BOILED

29

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8 I v)

2

q-J 3c n a N

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5

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,*I

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,,,I

10 60 100 SYNTHETIC a-MSH (p9)

I

50C

I

I 1 1 1 1

I

I I

5 I0 50 100 500 SYNTHETIC a-MSH (p9) or HYPOTHALAMIC EXTRACT (pL)

FIG. 1. Displacement of [' 2si]a-MSH binding to anti-x-MSH by synthetic a-MSH or hypothalamic extracts. Displacement of the binding of ['251]a-MSH to anti-a-MSH was achieved by the addition of increasing amounts of synthetic a-MSH, synthetic a-MSH heated for 10 min, hypothalamic tissue homogenized in saline and then heated for IOmin, and acetic acid ethanol extract of hypothalamic tissue. Heating, extraction, and radioimmunoassay were carried out as described in Materials and Methods.

.

Hypothalamic a-melanocyte stimulating hormone Co., Lincoln, NE); and aliquots were analyzed for a-MSH, LHRH, and TRH. Assays. Subcellular fractions were prepared for radioimmunoassay by one of the following methods: (1) Ten vol of ice-cold acetic acid-ethanol (1M acetic acid-ethanolwater, 10:75:15) were added to each subcellular fraction. Extracts were centrifuged, the supernatant fluid decanted and lyophilized, and the dry residue was dissolved in 0.01 M-sodium phosphat4.14 M-NaCl containing 1% egg albumin, pH 7.6 (PBS-EA). (2) The tubes containing the subcellular fractions were covered with marbles, heated for 10min in a boiling water bath, cooled on ice, mixed, and stored at -20°C. The fact that heating could be used as an alternative to acetic acid-ethanol extraction is illustrated by the finding that hypothalamic tissues treated by either procedure gave parallel displacement curves in the radioimmunoassay of a-MSH (Fig. I), TRH, or LHRH (BARNEAet al., 1977). The recoveries of a-MSH, TRH, and LHRH were slightly higher in the heated samples than in those extracted with acetic acid ethanol (Table 1). a-MSH was quantified by radioimmunoassay according to the procedure of USATECUI et al. (1976), modified as foler lows: ["SI]a-MSH, prepared according to GREENWWO af. (1963), was purified by gel filtration using Sephadex (3-25. The incubation was conducted at 2°C for 5 days. On the first day, a-MSH antiserum (antiserum provided by C.O.), ['251]or-MSH, and synthetic a-MSH or the test sample in PBS-EA were added to 10 x 75 mm glass tubes. On the third day, 200 p1 of sheep antiserum against rabbit gamma globulin and loop1 of 1% normal rabbit serum in PBS4.05 M-EDTA,pH 7.6, were added simultaneously. On the fifth day, the reaction tubes were centrifuged; and the radioactivity in the precipitate was counted. Synthetic a-MSH (Ciba-Geigy Ltd., Basel, Switzerland) served as the reference preparation (Oliver & Porter, unpublished data). The lowest level of detectability of a-MSH was 2G30pg. TRH (ESKAYet a/., 1976), and LHRH (NETTet al., 1973) were quantified by radioimmunoassay using synthetic TRH and LHRH (Beckman Instruments, Inc., Palo Alto, CA) as reference standards.

62 I

matter in the homogenate during the fractionation procedure. To test this possibility, the 900 g supernatant fluid, prepared from hypothalamic tissue homogenized in 0.32 M-sucrose containing 251]a-MSH, was fractionated on continuous sucrose density gradients. Essentially all of the radioactivity was recovered at the top of the gradients in a volume equal to that of the sample (Fig. 3). This result indicates that the particle-bound a-MSH observed in this investigation represents endogenous compartmentalization of the peptide, and negates the possibility that it represents adsorption of soluble peptide to particulate matter in the homogenate. When the 900g supernatant fluid was fractionated at non-equilibrium conditions, a-MSH distributed as two peaks (Fig. 4). However, when fractionated at equilibrium conditions, the a-MSH was recovered in a single peak (Fig. 5). These results and those obtained by differential centrifugation indicate that the particles containing a-MSH differ in size but are similar in density. i t has been shown previously (BARNEAet a!., 1976) that other small peptides in the hypothalamus, i.e. ,TRH and LHRH, are each sequestered in two populations of particles. Therefore, it is of interest to know whether the particles containing a-MSH are distinguishable from those containing TRH or LHRH. The gradient profiles of TRH and LHRH, shown in Figs. 5 and 6, demonstrate that the particles containing a-MSH are separable from those containing LHRH but are not separable from those containing TRH.

['

PROTEIN (I-MSH

RESULTS Distribution ofa-MSH in subcellular fractions obtained by differential centrijiugation Hypothalamic homogenates were fractionated by means of differential centrifugation. Of the total a-MSH in the homogenate, 6.5% was recovered in the 900g pellet; 35.8% in the 11,500g pellet; 15.5% in the 20,000 g pellet; and 15.4% in the 105,000 g pellet (Fig. 2). a-MSH was not demonstrable in the 105,000 g supernatant fluid. Although the recovery of protein was 90%, that of a-MSH was only 73%. Since a-MSH was found to be sequestered in a variety of particles sedimenting at centrifugal forces higher than 900g, the 9009 supernatant fluid was further fractionated on continuous sucrose density gradients. Fractionation of a-MSH-containing particles by means of continuous density gradient centrijiugation It could be argued that particle-bound a-MSH, demonstrated in Fig. 2, represents extracellular or soluble a-MSH which had been adsorbed to particulate

PELLET

PELLET

20,000X~ 105,000xq PELLET PELLET

SUPERNATANT

FIG.2. Distribution of a-MSH and protein in subcellular fractions obtained by differential centrifugation of hypothalamic homogenates. A 10% homogenate was centrifuged for 10min at 900g. The pellet was washed twice, and the supernatant fluid was centrifuged 20 min at 11,500g. The t 1,500g pellet was washed once, and the supernatant fluid was centrifuged for 30 min at 20,0000 g. The 20,000 g supernatant fluid was centrifuged for 60min at l05,OOOg. a-MSH was extracted with acetic acid-ethanol. Bars represent the mean ~ s . E . M .of 5 determinations.

AYALLA BARNEA,CHARLES OLIVER and JOHN C. PORTER

622

'ooo GRADIENT FRACTION

I0.4

4

SUCROSE CONCENTRATION (MI

1:4

FIG. 3. Lack of adsorption of ['251]a-MSH to hypothalamic particles. c(-MSH was iodinated in the procedure decf al. (1963). Two hypothalami scribed by GRI.ENWOOD were homogenized in a solution of 0.32 M-sucrose-1Op~CaCl, containing ['2sI]a-MSH (0.5 ml). The 900 g supernatant fluid was prepared and fractionated on continuous sucrose density gradients centrifuged for 30 min at 29,000 1.o

SUCROSE CONCENTRATION (M)

0.6

'.6

FIG.5. Equilibrium density fractionation of hypothalamic particles containing (r-MSH. TRH, and LHRH. Two hypothalami were homogenized in 0.5 ml of 0.32 M-SUCrose-10 pM-CaCI,. The 900 g supernatant fluid was prepared and fractionated on sucrose density gradients centrifuged for 300 min at 29,000 rev./min (100,000gJ,,). The gradient distributions of a-MSH, TRH, and LHRH were similar after centrifugation for 180 or 300 rnin, indicating that density equilibrium was achieved by 180 min.

i ._0

t 0.6

e "-

L

al

a 0

I

cn

P

r)

0.2

C

I

0

I

I I I I 10 20 GRADIENT FRACTION

I

30

I

7dr I

0'4 SUCROSE CONCENTRATION (M) 1'4

FIG.4. Non-equilibrium density fractionation of hypothalamic particles containing a-MSH. Two hypothalami were homogenized in 0.32 M-SUCrOSe-lO pM-CaCI, (0.5 ml). The 900 g supernatant fluid was prepared and fractionated on continuous sucrose density gradients centrifuged for 30 min at 29,000 re;.jmin (100,000gJr

I

I

10 20 GRADIENT 'FRACTION

0.4

I

I

1

30 1.4

(M) FIG. 6. Comparison of the gradient profiles of a-MSH, TRH, and LHRH fractionated at non-equilibrium conditions. For experimental details, see Fig. 4. SUCROSE CONCENTRATION

Hypothalamic a-melanocyte stimulating hormone

623

Thus, it can be inferred that the subcellular compartmentalization of the immunoreactive a-MSH de0 ISO-OSMOTIC scribed in this investigation represents that of a-MSH. 0 HYPO-OSMOTIC 40c In this study, we have demonstrated that in rat hypothalamic homogenates, a-MSH is sequestered in two populations of particles differing in size but simiE .lar in density. These particles are clearly distinguishc e able from hypothalamic subcellular particles containing LHRH but are indistinguishable from those cona & taining TRH in their sedimentation properties. P 0 Although the specific particle containing a-MSH has f 320 not been identified, circumstantial evidence indicates that the large particles containing a-MSH are synapa tosomes. Firstly, their sedimentation properties are identical to those of synaptosomes containing TRH and similar to those of synaptosomes containing dopamine, norepinephrine, 5-HT, and histamine (GREENet al., 1969; KUHARet al., 1971a, b ; BARNEA 160 et al., 1975, 1976); secondly, in a previous study which 0 10 20 30 was carried out under conditions identical to those GRADIENT FRACTION , used in the present investigation, we observed that 0‘4 SUCROSE CONCENTRATION (MI ‘.I4 synaptosomes predominated the gradient fractions FIG. 7. Differential effect of hypo-osmotic shock on which were enriched with the large particles containet al., 1977); thirdly, the large parhypothalamic particles containing a-MSH. Two hypotha- ing TRH (BARNEA lami were homogenized in iso-osmotic solution (0.32 M-SUC- ticles containing a-MSH are susceptible to hyporose-10 pM-CaC12; 0.5 ml) or hypo-osmotic solution osmotic shock, a feature characteristic of synapto(10 pM-CaC1,; 0.5 ml). The 900y supernatant fluid was pre- somes (DE ROBERTIS& RODR~GUEZ DE LOREZ pared and fractionated on continuous sucrose density gra- ARNAIZ,1969; WHITTAKER, 1969). dients centrifuged for 30 min at 29,000 rev./min The small a-MSH-containing particles were indis(100,000 9.”). tinguishable from the small TRH-containing particles in their sedimentation properties. However, TRH asDifferential susceptibility t o hypo-osmotic shock of the sociated with the small particles was released by two populations of particles containing u-MSH hypo-osmotic shock (BARNEAet al., 1975, 1976), Hypothalami were homogenized in iso-osmotic whereas a-MSH associated with the small particles solution (0.32 M-sucrose10 pM-CaCl,) or in hypo- appeared to be resistant to such treatment. The possiosmotic solution (10 pM-CaCl,), and the 900 g super- bility should be considered that the small particles natant fluids prepared from the homogenates were containing a-MSH are not synaptosomes. We have fractionated on sucrose density gradients at non-equi- reported earlier that small membrane-bound particles librium conditions (Fig. 7). Hypo-osmotic conditions and dense granules predominate the gradient fracresulted in the disappearance of the peak of a-MSH tions which were enriched with the small particles et al., 1977). In the present associated with the large particles, whereas that as- containing TRH (BARNEA sociated with the small particles was only slightly study, when fractionated as described by BARNEAet al. (1977), the small particles containing a-MSH and affected. the small particles containing TRH were recovered in the same gradient fractions. Therefore, the small DISCUSSION particles containing a-MSH could represent memSubstances which possess melanocyte stimulating brane-bound peptide. On the other hand, in the activity have been extracted from brain tissues of neural lobe of the hypophysis, vasopressin was found various species (GUILLEMIN et al., 1962; RUDMAN et to be localized in small dense granules banding at a/., 1973, 1974b, 1975). This biological activity could 1.61.7 M-sucrose (WEINSTEINet al., 1961; SACHS, be attributed to a number of different peptides (GUIL- 1963). Moreover, in non-neuronal tissues, polypepet al., LEMIN et a/., 1962; SCHALLY et al., 1962; LEE et a/., tides viz., parathyroid hormone (MACGREGOR 1963; RUDMAN et al., 1973). Using a specific antiserum 1973), insulin (KEMMLER et al., 1973), and gastrin et al., 1976), are sequestered in small dense against a-MSH (USATEGUI et al., 1976), OLIVER& (TROTMAN PORTER (submitted) have shown that a-MSH is granules having banding densities around 1 M-sucrose. present in the rat brain. This immunoreactive a-MSH These granules differ appreciably from synaptic vesiwas indistinguishable from synthetic a-MSH in its cles containing neurotransmitters in the brain which chromatographic, electrophoretic, immunologic, and have a banding density of approx 0.4~-sucrose 1973). Thus, some of the small particles biologic properties. In the present study, we used the (WHITTAKER, same antiserum that was used by OLIVER& PORTER. containing a-MSH, which have a banding density of

P

624

CHARLES OLIVER and JOHNC. PORTER AYALLA BARNEA,

about 1.1 M-sucrose, bear a closer resemblance in their density t o the vasopressin and other polypeptide hormone-containing granules. Since a-MSH exhibits neurotransmitter-like actions on the brain (FERRARI et al., 1963; GISPEN & SCHOTMAN, 1973; GREVEN & DE WED, 1973; KASTINet d., 1973; STRATTON & KASTIN, 1974) and since the large particles containing a-MSH resemble synaptosomes, we propose that some of the a-MSH in the hypothalamus is localized at presynaptic axonal terminals. These axonal terminals may contain other peptides, 1.e. TRH. O n e can envision that in addition t o a neurotransmitter-like action on the brain, a-MSH may also function in a manner similar t o hormones, i.e. at a site remote from its storage site. A possible route of transport of a-MSH t o its target site in the brain may be the CSF which possesses melanotropic activity (RUDMANet al., 1973).

GUILLEMIN R., SCHALLY A. V., LIPSCOMB H. S., ANDERSEN R. N . & LONGJ. M. (1962) Endocrinology 70, 471-477. KASTINA. J., MILLERL. H., NOCKTONR., SANDMAN C. A., SCHALLY A. V. & STRATTON L. 0. (1973) Prog. Brain Res. 39, 461470. KUHARM. J., SHASKANE. G. & SNYDERS. H. (1971a) J. Neurochem. 18, 333-343. KUHARM. J., TAYLOR K. M. & SNYDERS. H. (1971b) J . Neurochem. 18, 1515-1527. KEMMLERW., STEINERD. F. & BORG J. (1973) J . b i d . Chem. 248, 45444551. LANDES., DE WED D. & WITTERA. (1973) P r y . Brain Res. 39, 421427. V. (1963) LEET. H., LERNERA. B. & BUETTNER-JANUSCH Ann. N . Y Acad. Sci. 100, 658-668. LOWRYP. J. & SCOTTA. P. (1975) Gen. comp. Endocrinol. 26, 1 6 2 3 . MACGREGORR. R., CHU L. L. H., HAMILTON J. W. & COHND. V. (1973) Endocrinology 93, 1387-1397. NETT T. M., AKBARA. M., NISWENDER G. D., HEDLUNII M. T. & WHITE W. F. (1973) J. clin. Endocr. Metah. 36, 88&885. Acknowledgements-The authors thank JUDYWAGERSand V. D. & KORDONC. (1975) in Hypothalamic HorBRENDAROWLANDfor their generous assistance in the RAMIREZ P. G . & MARTINIL., preparation of this manuscript. We thank SUE SHERWIN, mones (MOTTA M., CROSIGNANI eds.) pp. 57-74. Academic Press, New York. MARGUERITE GUNDER,LINDAAKERS,GLORIACHO,and ROBERT LIPSEYfor valuable technical assistance. The anti- RUDMAND., CHAWLAR. K. & KHATRAB. S. (1975) Am. J : Physiol. 228, 1245-1248. serum used in the assay for LHRH was a gift from Dr. D., DEL Rio A. E., HOLLINSB. M., HOUSERD. G. D. NISWENDER, the a-MSH antiserum was provided RUDMAN H., KEELINGM. E., SUTINJ., SCOTTJ. W., SEARSK. by Dr. C. OLIVER, and synthetic a-MSH was a gift from A. & ROSENBERGM. Z. (1973) Endocrinology 92, Drs. W. RITTELand P. DESAULLES, Ciba-Geigy, Ltd., Basel, 372-379. Switzerland. RUDMAND. & ISAACS J. W. (1975) Endocrinology 97, 1476-1480. REFERENCES RUDMAN D., SCOTTJ. W., DEL RIO A. E., HOUSERD. H. BAKERB. 1. (1973) J. Endocrinol. 57, 393-404. & SHEENS. (1974a) Am. J . Physiol. 226, 687-692. BARNEA A,, BEN-JONATHAN N. & PORTERJ. C. (1976) J. RUDMAND., SCOTTJ. W., DEL RIO A. E., HOUSERD. H. & SHEENS. (1974b) Am. J . Physiol. 226, 682-686. Neurochern. 27, 477484. BARNEA A,, BEN-JONATHAN N., COLSTON C., JOHNSTONJ. SACHSH. (1963) J . Neurochem. 10, 289-297. M. & PORTERJ. C. (1975) Proc. nutn. Acad. Sci., U.S.A. SCHALLYA. V., LIPSCOMBH. S., LONG J. M., DEAR W. E. & GUILLEMIN R. (1962) Endocrinology 70, 478-480. 72, 3153-3157. BARNEAA,, NEAVES W. B. & PORTERJ. C. (1977) Endo- SHAPIRON., NICHOLSONW. E., ORTHD. N., MITCHELL W. M., ISLANDD. P. & LIDDLEG. W. (1972) Endocrinocrinology 100, 1068-1079. J. A., HOLLAND D., JEFFCOATE logy 90, 249-256. BENNETT G. N., EDWARDSON S. L. & WHITEN. (1975) Nature, Lond. 257, 323-325. STRATTONL. 0. & KASTINA. J. (1974) Horm. Behau. 5, DE ROBERTISE. & RODR~GUEZ DE LORESARNAIZG . (1969) 149-155. in Handbook of Neurochemistry (LAJTHAA,, ed.) Vol. 11, TROTMAN C. N. A., FIDDES I. J. S., GRUND E. R., MCHANpp. 365-392. Plenum Press, New York. WELL S., SANDERS D. J., SANDERSON C. & SHAWB. (1976) J. EndocrinoL 68, 5-12. ESKAYR. L., OLIVER C., WARBERG J. & PORTERJ. C. (1976) USATEGUIR., OLIVERC., VAUDRYH., LOMBARDIG.. Endocrinology 98, 269-277. ROZENBERG I. & MOURREA. M. (1976) Endocrinology FERRARI W., GESSA G. L. & VARGIUL. (1963) Ann. N.Y. 98, 189-196. Acad. Sci. 104, 330-345. R. C. GISPENW. H., DE WIED D., SCHOTMAN P. & JANSZH. WARBERGJ., ESKAVR. L., BARNEAA,, REYNOLDS & PORTER J. C. (1977) Endocrinology 100, 814-825. S. (1971) Brain Res. 31, 341-351. H., MALAMED A. & SACHSH. (1961) Biochim. CISPEN W. H. & SCHOTMAN P. (1973) Prog. Brain Res. WEINSTEIN 39, 441b459. biophys. Acta 50, 386389. GREENA. I., SNYDER S. H. & IVERSEN L. L. (1969) J . Phar- WHITTAKERV. P. (1969) in Handbook of Neurochemistry (LAJTHAA., ed.) Vol. 11, pp. 327-364. Plenum Press, New mac. exp. Thrr. 168, 264271. York. GREENWOOD F. C., HUNTERW. H. & GLOVERJ. S. (1963) WHITTAKER V. P. (1973) in Proreins of the Nervous System Biochem. J . 89, 114-123. H. M. & DE WIED D. (1973) Prog. Brain Res. (SCHNEIDER D. J., ed.) pp. 155-169. Raven Press, New GREVEN 39, 429--442. York.

Subcellular localization of alpha-melanocyte-stimulating hormone in the rat hypothalamus.

Journal of Neurochemisrry, 1977. Vol. 29, pp. 619-624. Pergamon Press. Printed in Great Britain. SUBCELLULAR LOCALIZATION OF a-MELANOCYTESTIMULATING...
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