zyxwv zyxwvutsrqponmlk zyxwvutsrqponmlkjihg zyxwvutsrqponm zyxwvutsrqponmlk zyxwvutsrqponm zyxwvutsrqpo 00??-1042 78 0901-0751102000 Jourtrol u/Mulrrorhr,,ll,rn. Vol. 31. pp 751 753 Pcrgsmon Press Ltd 1978 Printed in Great Britain 0 International Society lor Neurochcmistry Ltd. SHORT COMMUNICATION Subcellular localization of growth hormone release inhibiting hormone in rat hypothalamus, cerebral cortex, striatum and thalamus (Receiivd 9 January 1978. Accepted 23 March 1978) GROWTHhormone release inhibiting hormone (GH-RIH) is present in high concentration in the hypothalamus, but has a wide extrahypothalamic distribution in the CNS (BROWSTEINet al., 1975). Although immunocytologic studies have revealed GH-RIH immunoreactivity in nerve endings in the external layer of the median eminence as et al., 1975). viswell as in hypothalamic axons (HBKFELT ualisation of GH-RIH immunoreactivity in extrahypothalamic CNS has been unsuccessful using these techniques. We have used subcellular fractionation of CNS homogenates as a more sensitive means of studying localisation in these areas employing a sensitive and specific radioimmunoassay for GH-RIH (KRONHEIM et a\.. 1976). We describe in this paper our findings on the subcellular distribution of GH-RIH immunoreactivity in the hypothalamus, striatum. thalamus and cerebral cortex of the rat, and show the concentration of this immunoreactivity to reside largely in the synaptosome fraction. 30 min whilst controls of identical composition were incubated at 4°C to assess non-specific choline or noradrenaline uptake. Uptake was terminated by rapid cooling in an ice bath following which unincorporated tracer was largely removed by centrifuging the mixture at 10,000g for 5 min at 4°C. The resultant pellet. resuspended by gentle homogenisation in 0.32 M-sucrose. was then applied to a linear continuous density gradient of 0.32 M-1.5 M-SUCrose prepared in 30 ml cellulose centrifuge tubes (Beckman Instruments) and centrifuged at 75,000 g for 90 min in an zyxwvutsrqp MATERIALS AND METHODS Male Long-Evans rats (20G300g) housed under constant conditions were rapidly decapitated between 09.00 and 10.00 h, the brains removed to an ice tray and dissected, as previously described (KRONHEIM er al., 1976),into the striatum, thalamus, hypothalamus and cerebral cortex. In each experiment, the areas from 5 to 10 rats were pooled and a 107, w/v homogenate prepared in 0.32~-sucrose using a Perspex pestle in a glass homogeniser (clearance 0.25 mm) (ALDRIGEe f al., 1960) at 900 rev./min. Homogenisation was performed as described by B ~ N N E T & T EUWARUSON (1975). G H - R I H profile and neurotransrnirter uptake on continuous sucrose density gradient. In initial experiments. the profile of homogenate immunoreactive GH-RIH was compared to that of '"C choline and 'H noradrenaline uptake by tissue fractionated on a continuous sucrose density gradient. The homogenate was allowed to warm slowly to room temperature. One millilitre was then added to 5 ml of Krebs bicarbonate buffer (pregassed with 95:: O,, 5:/, C 0 2 ) containing. in addition, glucose (10 mM). adenosine triphosphate (0.45 mM), sodium pyruvate (10 mM), ascorbic acid (0.03", w,'v) and either 50 mM-physostigmine hydrochloride (Eserine) with 5 pl 14C choline (methyl I4C choF R A C T I O N NUMBER line chloride. 52 mCi/mmol, Amersham) or 100 mM-phenelzine sulphate (Nardil) with 25 pl of 'H noradrenaline (L-7- FIG. 1. Profiles of GH-RIH immunoreactivity in ng,ml 'H noradrenaline, 5.8 Ci/mmol, Amersham). The mixture (+a), I4C choline (-0) and 'H noradrenaline was incubated with agitation in a water bath at 30°C for (@--O) specific uptakes. expressed as percentages of total radioactivity. after continuous sucrose density gradient Abhrei*iation used: GH-RIH, growth hormone release ultracentrifugation of homogenates from different areas of inhibiting hormone. rat brain. 751 Short communication 752 PERCENTAGE OF zyxwvutsrqpo GRADIENT CONCEN? RATION zyxwvutsr zyxwvutsrqp zyxwvutsrq zyxwvutsrqp zyxwvutsrqp FIG. 2. Distribution of protein, succinic cytochrome c reductase activity (SCCR), occluded lactate dehydrogenase activity (LDH) and GH-RIH immunoreactivity on discontinuous sucrose density gradients from rat cortex, hypothalamus, striaturn and thalamus. See text for details. MY represents 0.32 ~ - 0 . 8M interface (myelin), S is 0.8 M-1.2 k interface (synaptosomes) and M represents the pellet (mitochondria). SW 25.1 rotor (Spinco). The tubes were punctured and I ml fractions of the gradient collected. 100 pl was solubilised in 500 pl Soluene (Packard Instruments), suspended in 8oOpI Instagel (Packard Instruments) and counted in a Packard Automatic Liquid Scintillation Counter. The remainder was snap frozen and stored until assayed in dilution for GH-RIH. The same homogenisation and density gradient centrifugation procedure was followed on three occasions per area, without prior incubation, to ensure that the GH-RIH profile was unchanged. Production of a purified synaptosomal fraction. Following homogenisation of brain tissue, as described above, a purified synaptosome fraction was prepared by the method of (1962) as modified by BENNE-IT& GRAY& WHITTAKER EDWARDSON (1975). The purity of the final preparation was assessed for contamination by microsomes using D-ghCOSe6-phosphate phosphohydrolase (glucose-6-phosphatase) et al., 1967). and mitoEC 3.1.3.9, activity (DE LAMIRANDE chondria (using succinic-cytochrome c reductase-complex 11-111 activity) (TISDALE, 1967). Occluded lactate dehydrogenase @-lactate: NAD oxidoreductase) EC 1.1.1.27 activity was used as a marker for intact synaptosomes (MARCHBANKS, 1967). Protein was measured by the method of L o m v et al. (1951). Samples of all fractions were made up to 2M-acetic acid, well mixed, centrifuged and the supernatant freeze dried for GH-RIH assay. G H - R l H radioimmunoassay. The radioimmunoassay for GH-RIH was performed as previously described (KRONHEIM et al., 1976) using rabbit-anti GH-RIH haemocyanin (1 : 125,000 final concentration), L Z 5 1 - T ~ 1 - G H - R Itracer H (750 pCi/pg). synthetic cyclic GH-RIH standards in serial dilution (Ayerst AY-24910) and dextran coated charcoal, in the presence of inactivated horse serum, to separate anti- body bound tracer from free. The assay is sensitive to 7 ppi assay tube and has been demonstrated to be specific for GH-RIH. RESULTS Continuous sucrose density gradient ultracentrifugation of homogenised hypothalamus, thalamus, striatum and cerebral cortex revealed a peak of GH-RIH immunoreactivity, coinciding with the peaks of specific 14C choline and 'H noradrenaline uptake (Fig. I). The GH-RIH peak retained the same sedimentation characteristics when studied without preceding incubation. Another peak of GH-RIH immunoreactivity in high concentration occurred in the 0.32 M area of the gradient coinciding with unincorporated 14C choline or 3H noradrenaline. The purified synaptosomal fraction obtained following differential centrifugation and ultracentrifugation on a discontinuous sucrose gradient contained &60% of the GH-RIH immunoreactivity recovered in all areas studied. This immunoreactivity coincided with the major concentration of occluded lactic dehydrogenase activity and gradient protein as well as mitochondria1 marker enzyme (succinic cytochrome C reductase) activity (Fig. 2). Electron microscopy performed on glutaraldehyde fixed samples of all fractions confirmed the major concentration of intact synaptosomes to coincide with GH-RIH immunoreactivity. DISCUSSION GH-RIH immunoreactivity has been shown (KRONHEIM et al., 1976; KOBAYASHI et al., 1977; PALKOVITS et al., 1976) to have a wide distribution in the CNS with the highest zyxwvut zyxwvutsr zyxwvut zyxwvutsrqponm zyxwvutsrq zyxwvu zyxwvutsrqponm zyxwvutsrqp zyxwvut Short communication 753 concentrations in the hypothalamus and septum and Energy Board, the University of Cape Town Staff Research preoptic areas hut with considerable levels in extrahypo- Fund and Nellie Atkinson Bequest. and the International thalamic CNS. namely thalamus, striatum, cerebral cortex, Atomic Energy Agency (Contract No. 1806/RB). We thank brain stem and spinal cord. Immunohistochemistry has the University of Cape Town Merrin Bequest for support revealed the presence of GH-RIH immunoreactivity in of G.W.B. during his stay at the University of Cape Town. hypothalamus alone, where it is located in nerve endings in the median eminence and axons in the hypothalamic Isotope and Immunoassay Laboratory. M. BtRELOWlTZ A. HUDSON nuclei, although some workers have been successful in Department of Medicine. demonstrating GH-RIH containing perikarya in the peri- University of Cape Town Medical School, B. PIMSTONE'.' S. KRONHEIM ventricular areas of the anterior hypothalamus (HOKFELT Republic of South Africa G. W. BENNETT' et a/.. 1975). The reason for the more diffuse localisation and of immunoreactive GH-RIH as measured by immunoassay Department of Physiology, probably lies in the much greater sensitivity of the tech- St. George's Hospital Medical School, nique: GH-RIH has been demonstrated to have an inhibi- London SW17 OPT. tory effect on nerve activity following microiontophoretic England application (RENAUDet a/., 1975) and has been shown to decrease seizure duration in rats given strychnine, increasREFERENCES ing the LD50 of the toxin (BROWN & VALE,1975). Behavioural effects have been demonstrated following direct ALDRIDGEW. M., EMERYR. C. & STREET B. W. (1960) GH-RIH application to amygdala ( R ~ Z E Ket al., 1977a), Biochem. J. 17, 326-327. neostriatal areas (REZEK et a!., 1977b) or cerebral cortex BENNETT G. W. & EDWARDSON J. A. (1975) J. Endocririol. (REZEKet a/., 1976) of the rat. 65, 33-44. The wide CNS distribution of GH-RIH, together with BENNETT G. W. & EDWARDSONJ. A. (1977) J. Endrocripzoi. its effects on nerve activity and behaviour, place it in the 73, 27-28P. same category as other neuropeptides with similar properBROWN M. & VALE W. (1975) Endocrinology %, ties. Many of these peptides including thyrotrophin-releas1333-1 336. ing hormone, TRH (BENNETT & EDWARDSON, 1977), suhBROWNSTEINM., ARIMURAA,, SATOH.. SCHALLY A. V. stance P (DUFFY e t al., 1975), neurotensin (UHL& SNYDER, & KIZERJ. S. (1975) Endocrinology %, 1456- 1461. 1976, and vasoactive intestinal polypeptide (GIACHETTI et DE LAMIRANDE G., MORAlS R. & BLACKSTEIN M. (1967) al., 1977) have been shown to be concentrated in nerve Archs Biochem. Biophys. 118, 347-351. endings and have been postulated to comprise a new group DUFFYM. J., MULHALL D. & POWELL D. (1975) J . Neuroof peptidergic neurotransmitters. EPELBAUMet al. (1977) chern. 25, 305-307. have previously demonstrated a suhcellular localisation of EPELBAUM J., BRAZEAU P., TSANGD., BRAWER J. & MARGH-RIH in the synaptosome fraction of hypothalamus, TIN J. B. (1977) Brain Res. 126, 309-323. preoptic area and amygdala, areas of importance in regulaGIACHETTI A,, SAID S. I., REYNOLDS R. C. & K O N I GF. ~ SC. tion of pituitary growth hormone secretion (EPELBAUM et (1977) Proc. Natn. Acad. Sci., U.S.A. 74, 3424-3428. al., 1977). We confirm the hypothalamic localisation of GRAYE. G. & WHITTAKER V. P. (1962) J. Anal., Lond. GH-RIH in nerve endings but also find a similar subcellu98, 79-87. lar distribution in striatum, thalamus. and cerebral cortex. HOKFELT T., EFENDIC S., HELLERSTROM C., JOHANSSON 0.. suggesting a more diffuse role for the peptide. LUFTR . & ARIMURAA. (1975) Acta endocririol. 80, Suppl. That not all the GH-RIH recovered from the gradient 200, 5-41. is present in our synaptosome preparation is shown by the KOBAYASHI R. M., BROWNM. & VALEW. (1977) Brain presence of non-sedimented GH-RIH immunoreactivity Res. 126, 548-588. coinciding with free I4C choline and 'H noradrenaline. KRONHEIM S., BERELOWITZ M. & PIMSTONE B. L. (1976) Whether this is a function of synaptosome rupture during Clin. Endocrinol. 5, 619-630. homogenisation or represents GH-RIH in axons or periLOWRY H. O., ROSEBROUGHN. J., FARRA. L. & RANDALL karya released during homogenisation is unclear. R . J. (1951) J. hiol. Chem. 53, 265-275. The localisation of GH-RIH in nerve endings in extraMARCHBANKS R . M. (1967) Biochem. J. 104, 148-157. hypothalamic CNS suggests a neurotransmitter role for the PALKOVITS M., BROWNSTEIN M. J., ARIMURA A,, SATOH.. peptide whilst its presence in synaptosomes of the median SCHALLY A. V. & KlzER J. S. (1976) Brain Res. 109, eminence is consistent with a hypophysiotrophic role on 43w34. growth hormone secretion. Further studies on the mechanRENAUDL. P., MARTIN J. B. & BRAZEAU P. (1975) Nature isms controlling release of synaptosomal GH-RIH in 255, 233-235. various CNS sites may elucidate differential physiologic REZEK M., HAVLICEK V., HUGHES K. R . & FRIESEN H. functions. (1976) Pharmacol. Biochem. Behac. 5, 73-77. REZEK M., HAVLICEK V., HUGHESK. R. & FRIESEN H. Acknowledye~nentsFinancialsupport was obtained from (1977a) Neuropharmacology 16, 157-1 62. the South African Medical Research Council and Atomic REZEKM., HAVLICEK v., LEYBlN L., PINSKY c., KROEGER E. A., HUGHES K. R. & FRIESEN H. (19776) Carl. J. Physiol. Pharmac. 55, 234-242. TISDALE H. D. (1967) in Methods in Enzymology (ESTATo whom correspondence should be sent. BROOK R. W. & PULLMAN M. E., eds.) Vol. X, pp. 'To whom reprint requests should be sent, at Depart213-215. Academic Press, New York. ment of Medicine, University of Capetown Medical UHLG. R . & SNYDERS. H. (1976) L f e Sci. 19, 1827--1832. School. Observatory 7925, Republic of South Africa. '