Neuroendocrinology46: 131-136 (1987)
`
`© 1987 S. Karger AG, Basel
`s 2.75/0
`0028-3835/87/0462-0131
`
`Regional Distribution of Calcitonin Gene-Related Peptide and
`Its Specific Binding Sites in Rats with Particular Reference to the
`Nervous System
`
`Sunil J. Wimatawansa», Piers C. Emsons,
`lain Macintyre»
`sEndocrine Unit, Department of Chemical Pathology, Royal Postgraduate Medical School, London;
`bMRC Group, Institute of Animal Physiology, Babraham, Cambridge, UK
`
`Key Words. Calcitonin gene-related
`
`peptide· Nervous
`
`system·
`
`Specific binding
`
`Abstract. The concentration of calcitonin gene-related peptide (CGRP) in rat tissue extracts was determined by a speci-
`fic and sensitive radio-immunoassay,
`and the distribution of its specific binding sites was assessed by radioligand binding
`studies. A high concentration of immunoreactive CORP was found at all levels of the spinal cord, in the trigeminal nu-
`cleus,
`in trigeminal
`and dorsal
`root ganglia,
`in the thyroid gland,
`in blood vessels and in nerves. The highest density of
`specific binding sites was detected in the cerebellum, where the CORP content was minimal. The dorsal portion of the
`spinal cord contained a high concentration of CORP and its specific binding sites. Specific binding of 125I_CORPwas also
`demonstrated in a number of other areas of the brain and in certain peripheral tissues. Among the other tissues examined,
`the spleen, adrenal gland, penis,
`lungs, bladder, heart and blood vessels all contained
`a large number of CGRP binding
`sites, whereas only a negligible number of sites were found in ganglia, nerves, muscle, kidney and liver. The distribution of
`CGRP and its specific binding sites demonstrated
`here suggests that CGRP is a neuropeptide with multiple physiological
`roles.
`
`In addition to calcitonin (CI) and the carboxy terminus-
`adjacent peptide (CAP) in the rat or katacalcin in man, the
`CT gene encodes a 37-amino acid peptide named CT gene-
`related peptide (CGRP)
`[3, 29]. CT is expressed mainly in
`the C cells of the thyroid, whereas CGRP is expressed
`mainly in the nervous system [29J, although the presence of
`CGRP in thyroid gland has been reported in both rats [15,
`25,31,42]
`and in man [40J. Preliminary results of the distri-
`bution and quantitation
`of all three products of the CT gene
`in rats have been reported using specific and sensitive
`radio-immunoassays
`(RIA) [42].
`studies
`Immunocytochemistry
`and RIA quantitation
`have
`shown the presence
`of
`immunoreactive CORP
`(i-CGRP)
`in the nervous system [29, 33, 40, 42], especially in
`the spinal cord [II, 25, 42], in the cardiovascular
`system [24,
`27, 42], in the perivascular nerves often associated with the
`smooth muscles of blood vessels [29] and in a variety of
`other
`tissues [10, 24, 25. 40, 42]. Binding sites for CGRP
`have been demonstrated
`both in the rat [14] and human [39]
`
`Received: July 21,1986
`Accepted after revision: February 5, 1987
`
`in guinea-pig pancreas [30] and in
`central nervous system,
`the rat cardiovascular
`system [32].
`The first evidence for the existence of CORP in human
`was reported by isolation and characterisation of h-CGRP
`(a-hCGRP)
`from mednllary
`thyroid
`carcinoma
`[26].
`Subsequent
`investigations have revealed the presence of
`another gene which encodes
`for a second (P) CGRP in rats
`[2, 28] and in man [I, 35]. This peptide
`differs
`from the
`a-CGRP by only I amino acid in the rat [2] and by 3 amino
`acids in the human [35]. The effects of CGRP on vasodila-
`tion of rabbit skin (bio-assay) has shown that
`the three dif-
`ferent
`forms of synthetically
`available CGRP (rat a-CGRP,
`human a-CGRP
`and human p-CGRP)
`are equipotent
`[5].
`However,
`in an assay based on stimulation of cyclic AMP
`in aortic smooth muscle preparations,
`p-CG RP has been
`shown
`to be 5 times more
`potent
`than
`the a-CG RP
`[McEwan et al., unpubl. data]. Our present RIA and the lig-
`and binding assay are unable to differentiate between a-
`and p-CGRP, which are equipotent
`in these assays
`(data
`not shown). Here, we report detailed regional distribution
`of i-CGRP in the rat and its binding sites, studied by RIA
`and radio ligand binding studies, respectively.
`.
`
`to.
`
`1
`
`EX2248
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01426
`
`

`

`132
`
`Materials
`
`and Methods
`
`the tyrosinated C-terminal deca-
`Synthetic rat CGRP (rCGRP),
`peptide (Tyr"-rCGRP28_37)and human ~-CGRP (~-hCGRP) were
`purchased from Peninsula Laboratories, Belmont, Calif., USA.
`cx.-hCGRP was supplied
`by Dr J. Pless (Sandoz, Basel, Switzer-
`land). Cellulose-coated sheep anti-rabbit gamma globulin was ob-
`tained from IRE, Fleurus, Belgium. Male Wistar rats weighing
`180-210 g were obtained from A. Tuck & Son Ltd. (Laboratory An-
`imal Breeding Station, Essex, UK). J25I-CGRP (spec. act. 2,000 Ci/
`mmol) was obtained from Amersham International, UK. Aprotinin
`(Trasylol) was obtained from Bayer (FRG) and Bacitracin was
`from Sigma (Poole, UK).
`
`Tissue Extraction
`To minimise blood contamination of tissues, animals were ex-
`sanguinated through the dorsal aorta following ether anaesthesia.
`Brain dissections were carried out as previously described [13, 19]
`and tissues collecred on dry ice. Wet weights were recorded and
`tissues homogenised in an acid mixture [4](I MHCl, 15%TFA, 5%
`formic-acid (v/v) and 1 M NaCI) on ice. The homogenateswere
`centrifuged at 7,000 g for 15 min at 4 0 C and the pellets rehomoge-
`nised in fresh acid mixture and recentrifuged. Pooled supernatants
`were applied to C·18 Sep-Pak disposable cartridges (Waters Asso-
`ciates) primed with methanol and eluted with amixture of metha-
`nol, water and trifluroacetic acid (TFA), (90:9:1; v/v). The extracts
`were vacuum-dried and stored at -20 "C. The recoveries of synthe-
`tic rCGRP and mI-rCGRP added to [he control tissues (n= 8) were
`85±0.6 and 87 ±0.8% (mean ± SEM), respectively.
`
`Preparation
`Membrane
`Membranes were prepared by a modification of a previously
`described method [34].Fresh rat tissues were dissected and placed
`in 15 vol of ice-cold 50 roM Tris/HCl buffer (pH 7.5) containing
`0.32 M sucrose, 1 mM dithiothreitol and 5 mM EDTA. They were
`homogenised with a Dounce homogeniser at 4 °C and the homo-
`genate was centrifuged at 1,000 g for 10 min. The supernatant was
`then centrifuged for 30 min at 30,000 g and the pellet resuspended
`in 10vol of 50 mMTris/HCl
`buffer. Following further centrifuga-
`tion at 30,000 g for 30 min, the pellet was finally resuspended in
`fresh 50 mMTris/HCl
`buffer and the protein concentrations were
`determined by the method of Lowry et at. [23].The final protein
`concentration was adjusted to 2 mg protein/ml with assay buffer
`(50 mMTris/HCl,
`10 mMKCI, 5 mMMgS04, bacitracin O.l%, ap-
`rotinin 100 KIU/ml and 30 mMsodium azide). Aliquots (1-2 rot)
`were immediately frozen on dry ice and stored at -70 °C and used
`within 6 weeks of preparation.
`
`Studies
`Receptor Binding
`The labelled peptide (2.5 fmol of [12SI]His-hCGRP)was incu-
`bated with rat tissue membranes (400 ug of protein) in the presence
`of 1%(w/v) heat-inactivated bovine serum albumin, in a total vol-
`ume of 300 ul for 50 min at 23 °C (equilibrium conditions) in poly-
`propylene micro-centrifuge
`tubes
`(Sarstedt). After
`incubation,
`700 ~l of assay buffer containing 1%BSA was added to each tube
`and immediately centrifuged at 11,000gfor 2 min. The supernatant
`was discarded and the pellet
`layered with another 750 ~l of the
`same buffer, recentrifuged and the supernatant discarded. The ra-
`dioactivity present
`in the pellets was measured using an NE·1600
`
`Wimalawansa/Emson/MacIntyre
`
`gamma counter (Nuclear Enterprises) with a counting efficiency of
`60%. Specific binding was calculated from the total amount
`of
`12sI_CGRP bound minus the amount bound in the presence
`of
`0.5 ~M unlabelled peptide and expressed as 12'iI_CGRP bound
`(fmol/g membrane protein). For rat cerebellar
`tissues,
`lC50 for
`I2SI-CGRP was 6·4fmol/tube and the Ka 0.24 nM(data not shown).
`
`Radio-Immunoassay
`Radio-iodination ofthe Cvterminal decapeptide ofrCGRP was
`carried out by a modification of the chloramine T method [16] and
`the products were purified by high-performance liquid chromatog-
`raphy (HPLC). Antiserum (CG·39) was raised in rabbit against
`the
`same decapaptide coupled to ovalbumin [26]. Prior to RIA, sam-
`ples were dissolved in 20 ul of 0.01 Mformic acid, neutralised with
`500 JlI of the assay buffer and centrifuged. RIA was performed as
`previously described [43]using a non-equilibrium assay with addi-
`tion of label after 3 days. The antiserum-bound peptide was preci-
`pitated by sheep anti-rabbit gamma globulin after a further 2 days
`of incubation. Extracts were assayed in at least two dilutions
`in
`duplicate and results were expressed as picomoles per gram wet
`weight of tissues. 50% displacement of the tracer required 20 fmol
`ofrCGRP, with a detection limit ofO.5 fmol/assay tube. Intra- and
`inter-assay variations were 5 and 9%, respectively.
`
`Specificity
`None of the following peptides at a concentration of I nmol/
`tube resulted in displacement of tracer in either RIA or radioligand
`binding assay: corticotrophin, ACIH 1-39, LHRH, Pro-opio-mela-
`nocortin (National
`Institute of Health, Bethesda, Md., USA), so-
`matostatin, glucagon, substance P, bombesin, human and porcine
`insulin, corticotrophin-releasing
`factor, growth 1 hormone, oxyto-
`cin, vasopressin, prolactin,
`leu- and met-enkephalins,
`secretin, gao
`stric inhibitory peptide (Sigma Ltd., Poole, UK) katacalcin and
`CAP (Peninsula). Salmon CT (Sandoz) with 30% homology
`to
`CG RP and human CT (Ciba-Geigy) with 19% homology [25] were
`both able to displace receptor-bound CGRP, but only at 104 and
`105 molar excess, respectively.
`
`Results
`
`Its Binding
`
`Regional Distribution ofCGRPand
`Sites in the Rat Nervous Tissues
`Immunoreactive CGRP. The highest concentrations
`of
`i-CORP were found in the trigeminal nucleus (635 pmollg)
`followed by the dorsal root ganglia, dorsal half of the spinal
`cord and trigeminal ganglia (table I), Levels were intermed-
`iate in the trigeminal and sciatic nerves, medulla and pons,
`pituitary
`and substantia
`nigra. However,
`concentrations
`were found to be low in the hypothalamus, midbrain
`and
`striatum, and minimal
`in the hippocampus,
`cerebral cortex
`and cerebellum, The i-CORP content decreased
`steadily
`between the cervical and the lumbar
`regions of the spinal
`cord, but in contrast a markedly high level was found in the
`sacral
`region.
`
`I
`
`2
`
`

`

`133
`
`300
`150
`
`100
`150
`
`100
`50
`
`0
`
`-cc"r»
`
`~E~
`
`•
`il
`
`-o
`U
`
`a 600
`
`ct 500
`
`J2'400
`
`~::: •••••i100
`
`Ii
`
`o
`
`b
`l'
`tn
`
`co8 10 _I
`
`Distribution ofCGRP and Its Receptors
`
`Table I. The tissue concentration
`of immunoreactive CGRP
`and its specific binding sites .in the nervous system of the rat
`
`Tissue
`
`Cerebellum
`Grey matter
`White matter
`Spinal cord (cervical)
`Dorsal portion
`Ventral portion
`Upper cervical
`Lower cervical
`Thoracic
`Lumbar
`Sacral
`Substantia nigra
`Medulla and pons
`Olfactory tubercle
`Striatum
`Hypothalamus
`Retina
`Olfactory bulb
`Midbrain
`Hippocampus
`Cerebral cortex
`Pituitary
`Trigeminal nucleus
`Trigeminal ganglion
`Dorsal
`root ganglion
`Trigeminal nerve
`Sciatic nerve
`
`125I_CGRPbound
`fmoll g protein
`(n~4)
`
`i-CGRP
`pmollg wet weight
`(n e B)
`
`2,385 ± 67
`2,968 ± 51
`1,381 ± 33
`1,603 ± 61
`2,011 ± 55
`1,235 ± 73
`1,625 ± 52
`1,732 ± 60
`1,130 ± 29
`1,566 ± 41
`1,711 ± 51 ,
`690 ± 31
`635 ± 34
`512 ± 25
`458 ± 27
`415 ± 32
`370 ± 31
`355 ± 22
`341 ± 24
`252 ± 22
`195 ± 19
`174 ± 13
`76 ± 5
`14 ± 2
`II ± 2
`5 ± I
`5 ± I
`
`1.0 ± 0,1
`1.6 ± 0.2
`0,6 ± 0,1
`265,0 ± 6,9
`370,0 ± 10,2
`35,S ± 3,1
`311.0±11.1
`237,0 ± 93
`172,0 ±
`8,1
`86,8 ± 3.5
`270.5 ± 7,8
`20.1 ±
`1.0
`72,6 ± 2.6
`9.7 ±
`1.0
`5.2 ± 0.4
`10.0 ± 0,9
`10.3 ±
`0.8
`6.1 ± 03
`10,5 ± 0.9
`12.0 ± 03
`1.0 ± 0,1
`31.5 ±
`1.6
`635.0 ± 17.5
`182.0 ± 9,6
`411.0 ± 13,1
`91.6 ± 5.0
`85.0 ± 2.9
`
`Values are presented as means ± SEM.
`
`70
`60
`'0•
`50l
`"" 40
`-g
`30
`c,
`0.- 10
`"
`
`0
`
`Illll_
`~
`-c
`c
`ro
`c;
`.~
`c ~ ~
`• c •
`~ D
`" U
`C ~
`<f
`S}
`<t
`
`t
`ro
`I
`
`g,
`3 •
`tissues
`Fig. 1. Specific binding sites and i-CORP in non-neural
`(mean±SEM).
`I25I-CGRP bound (n=4)
`(a) and i-CORP concen-
`tration (n - 8)(b),
`
`Specific Binding Sites. The highest concentration
`of
`specific
`binding
`was
`found
`the
`cerebellum
`In
`(2,385 ± 67 fmol/g protein) where the content of i-CORP
`was minimal (I pmol/ g) (table I). High concentrations of
`specific binding sites were also found in the spinal cord
`(1,603 ± 61 fmol/g protein) where i-CORP levels were also
`high (265 pmol/g), Markedly different numbers of specific
`binding sites were found in the grey and white matter of the
`cerebellum, and in the dorsal and ventral halfs of the spinal
`cord. Higher numbers of specific binding sites were also
`found in the hypothalamus,
`substantia nigra, medulla and
`pons, olfactory tract, striatum, mid brain and hippocampus
`in comparison to the cerebral cortex, pituitary and the trige-
`minal nucleus. Minimal or no specific binding was detected
`in the sensory ganglia and on nerve trunks. Non-specific
`binding Wasrelatively constant at 4-6% of the total radioac-
`tivity added to the membranes.
`
`Other Tissues
`The highest concentration of i-CORP outside the ner-
`vous system was found to be in_the the thyroid gland
`(fig, I), No significant binding could
`(287± 18,8 pmol/g)
`however be demonstrated in this tissue. A high concentra-
`tion of i-CORP and also a high number of specific binding
`sites were found in the adrenal gland. However, the spleen
`contained the highest concentration of specific binding sites
`fmol/g protein) outside the nervous system, de-
`(475±41
`spite the presence of minimal amounts of CORP (0.7 pmol!
`g). In addition,
`i-CORP was measured in the pancreas,
`penis, bladder and intestine. Specific binding was also found
`in several other tissues including penis, lungs, bladder and
`pancreas, but was negligible in muscles, kidney and liver.
`The heart contained only a small quantity (I pmol/g) of
`CORP, but large amounts of its binding sites (95 ± 6 fmol/g
`protein), particularly in the right atrium (not shown).
`
`3
`
`

`

`134
`
`Discussion
`
`The main purpose of this study was to determine the dis-
`tribution of CGRP and its specific binding sites in rat tis-
`sues with special
`reference ot the nervous system. The dis-
`tribution
`of CGRP-producing
`cells and pathways
`in the
`central and peripheral
`nervous
`system and in endocrine
`and other systems suggests potential
`functions
`in nocicep-
`tion, ingestive behaviour
`and modulation
`of the autonomic
`and endocrine
`systems
`[29], cardiovascular
`homeostasis
`and mineral metabolism [28]. The high concentration
`of
`i-CGRP in the neural ganglia and dorsal half of the spinal
`cord with lower levels in the ventral half of the spinal cord
`described
`here
`reinforces
`the possible
`involvement
`of
`CGRP in the modulation
`of sensory activity. The high lev-
`els of i-CORP found in cervical and sacral areas of the spi-
`nal cord could be due to the abundance
`of autonomic
`outflow in these
`areas
`[18J. However,
`this needs
`to be
`proven either by immunocytochemistry
`or by further quan-
`titation of i-CORP in microdissected
`areas in the autono-
`mic outflow. There are only low levels of i-CORP in the
`mid brain, hypothalamus,
`cerebral cortex and cerebellum,
`despite the high number of specific binding sites in the last
`region. The presence of CORP and its specific binding sites
`in the olfactory area of the brain is consistent with the pos-
`sible role of CORP as a neuromodulator/neurotransmitter
`in this sensory pathway.
`The present study has shown the presence of a high con-
`centration of i-CORP as well as a high number of specific
`binding sites in the dorsal spinal cord when compared with
`the ventral spinal cord. No significant
`specific binding was
`found in the trigeminal or dorsal root ganglia and on nerve
`trunks. The relative concentrations
`of specific binding sites
`demonstrated
`in the present
`study are not
`in agreement
`with the ratios previously reported [14, 39J. The highest con-
`centration
`of specific binding sites were detected in the
`cerebellum followed by the spinal cord. Goltzman and Mit-
`chell [14], found specifig binding of CGRP in rat pituitary
`membranes
`comparable
`to that
`in spinal cord and several
`fold greater
`than in the cerebellum. Tschopp
`et a!. [39],
`however, could not find any CGRP binding sites on human
`pituitary membranes. The present
`study shows
`that
`the
`number of specific binding sites for CORP on the pituitary
`membranes was some 14-fold less than that of cerebellum.
`Similarly, binding in the brain stem was found to be 4-fold
`less than in the cerebellum. Marked differences
`in CGRP-
`specific binding sites exist in different areas of the cerebel-
`lum and spinal cord, and this is consistent with the autora-
`diographic
`localization
`of CGRP binding sites [17]. This
`distribution
`of specific binding sites and i-CORP suggests
`that CORP may playa
`part
`in sensory neurotransmission
`and in cerebellar
`function.
`CGRP has been shown to inhibit basal and stimulated
`gastric
`acid and pepsin
`secretion
`[22J together with a
`
`Wimalawansa/Emson/Maclntyre
`
`a
`
`and neuroten-
`lowering of plasma gastrin, enteroglucagon
`sin [22]. Furthermore, CGRP has been shown to cause
`dose-related
`contraction
`of intestine in rats [7J and contrac-
`tion of the guinea-pig ileum which could be blocked by ei-
`ther antihistamine
`or anticholinergic
`drugs [38]. The inte-
`raction of CGRP with its receptors has been shown to re-
`lease amylase from exocrine pancreas
`in a dose-dependent
`manner
`[30], perhaps
`to assist
`the digestion
`of
`food.
`Furthermore,
`the
`finding
`of a high concentration
`of
`i-CORP and its specific binding sites in mesenteric
`arteries
`(not shown) supplying the viscera could reflect a role in the
`diversion of the blood supply following a meal and perhaps
`enhancing absorption.
`ef-
`CORP has been shown to have potent cardiovascular
`fects including positive chronotropic
`and inotropic
`actions
`on the heart [9,24,32,38]
`and to cause vasodilation
`in man
`[6, 12, 36] and in the rabbit [5,6], hamster
`[6] and rat [8]. The
`positive inotropic action of CORP, however, may be due to
`the reflex sympathetic
`stimulation
`as shown by a rise of
`plasma noradrenaline
`levels after administration
`of CG RP
`[8,9,36]. Nevertheless, direct action on the heart cannot be
`excluded,
`in view of the presence of the CGRP-specific
`bin-
`ding sites.
`of i-CORP and its specific bin-
`The high concentrations
`ding sites in peripheral
`arteries may be important
`in in-
`creasing blood supply during exercise.
`It
`is possible
`that
`some of the specific binding sites demonstrated
`in highly
`vascular
`tissues, such as penis and lungs, could be due to
`the binding sites present on the blood vessels. The spleen
`contained
`the highest
`concentration
`of specific binding
`sites outside the nervous system (fig. I), and this may reflect
`the known ability of the spleen to pool blood in certain situ-
`ations. Whether
`this high specific binding reflects
`splenic
`vessels or some other component
`in the spleen remains
`to
`be elucidated.
`The finding
`of a high concentration
`of
`CORP and its specific binding sites in the peripheral vascu-
`lature together with the proven potent vasodilatory
`activity
`ofCGRP [5, 6,12,36] makes it likely that the peptide has an
`important
`role as a neuromodulator
`of peripheral vascular
`tone.
`in plasma from normal man
`CGRP has been measured
`[12] and in rat [43] plasma. Furthermore,
`high concentra-
`tions of i-CGRP have been found in both the thyroid and
`plasma [26] of patients with medullary thyroid carcinoma.
`In the rat thyroid, CG RP has been shown to coexists with
`CT in the C cells and with substance P in the perivascular
`nerve
`fibers
`[15]. On HPLC of Sep-pak
`(C-18)-purified
`thyroid extracts,
`the predominant
`i-CGRP peak co eluted
`with the synthetic rCGRP(l_37) [31]. In addition, gel-perme-
`ation studies of rat thyroid extracts revealed that 70% of the
`total
`yield
`of
`i-CGRP
`co eluted with
`the
`synthetic
`rCGRP(l-37), suggesting the presence of monomeric CORP
`in the thyroid gland [Wimalawansa,
`unpub!. data]. CGRP
`receptors
`associated with smooth muscle cells have been
`
`i
`
`4
`
`

`

`Distribution
`
`of CGRP and Its Receptors
`
`135
`
`shown to be linked to adenylate cyclase [20, 32], but no sim-
`ilar interaction
`has been demonstrated
`in the CNS [14]. Re-
`cently,
`it has also been reported that CORP may act on vas-
`cular smooth muscle indirectly via an endothelial
`cell- de-
`studies are needed
`pendent mechanism [6, 20], but further
`to elucidate
`the nature and in particular
`the cellular
`locali-
`sation of specific binding sites in blood vessels by autora-
`diography or by immunocytochemical
`techniques.
`in
`Although CORP has been shown to be present
`role has been estab-
`plasma [12, 43], no definitive humoral
`lished. However,
`the distribution
`of i-CORP and its specific
`binding sites strengthen the postulated
`actions of CORP as
`a neuromodulator
`involving more than one function. No
`conclusions
`can be drawn about
`the differential
`expression
`of the two calcitonin genes or the possibility of subclasses
`of receptors.
`It has been shown that CGRP coexists at a
`higher
`concentration
`with other neuropeptides
`such as
`substance P in the trigeminal
`and dorsal
`root ganglia [21,
`41] and also in sensory nerve fibres [15, 24]. It has also been
`shown to coexist with acetylcholine
`in neurones
`in the hy-
`poglossal,
`facial and ambiguous nuclei
`[37] of rat brain.
`The widespread
`distribution
`of CORP and its specific
`binding sites suggests that CORP (whether a, ~ or both)
`may have a physiological
`role.
`In view of the close struc-
`tural homology between the a- and ~-CORP,
`the differen-
`tiation of the a- and ~-gene expression may be difficult
`using conventional
`immunological methods,
`and more
`easily approached
`using gene-specific probes.
`
`Acknowledgements
`
`and
`We would like to thank Drs S.L Girgis, D.W.R. Macdonald
`S.D. Brain for helpful criticism of the manuscript. This study was
`supported in part by a grant from British Heart Foundation.
`
`References
`
`Alevizaki, M.; Shiraishi, A.; Rassool, F.V.; Ferrier, G.J.M.;
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`for GABA receptor
`
`_____
`
`6
`
`

`

`Editor-in-Chief
`S.M. McCann, Dallas, Tex.
`
`Associate Editors
`C.P. Fawcett, Dallas, Tex.
`L. Krulich, Dallas, Tex.
`S.R. Ojeda, Dallas, Tex.
`J.e. Porter, Dallas, Tex.
`
`Neuroendocrinology
`
`for Basic and Clinical Studies on Neuroendocrine Relationships
`Journal
`International
`Official Journal of the International
`Society of Neuroendocrinology
`Founded 1965 by E. Bayusz
`Continued
`by K.M. Knigge (1973-1978), W.E Ganong (1979-1984)
`
`Ind.
`
`Editorial Board
`Indianapolis,
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`L.W. Swanson, La Jolla, Calif.
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`Portland, Oreg.
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`
`is
`'Neuroendocrinology'
`Publication data:
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