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Distribution of corticotropin-releasing factor in ovine brain determined by radioimmunoassay
Regulatory Peptides, 5 (1983) 189-195
189
Elsevier BiomedicalPress
Distribution of corticotropin-releasing factor in ovine brain determined by radioimmunoassay J. C6t6, G. Lef~vre, F. Labrie and N. Barden * MRC Group in Molecular Endocrinology, Le Centre Hospitalier de I'Universit~ Laval, Qu£,bec GI V 4G2, Canada
(Received 12 July 1982; revisedmanuscriptreceived 18 October 1982; acceptedfor publication 5 November 1982)
Summary We have developed and used a sensitive and specific radioimmunoassay to demonstrate the presence of CRF-like immunoreactivity in extra-hypothalamic areas of ovine brain. Synthetic CRF displaced antibody bound tracer at an EDs0 value of 200 pg and there was no cross-reactivity with LHRH, TRH, ACTH, fl-endorphin and several other peptides. Displacement of bound 125I-CRF by brain extracts exhibited curves parallel to synthetic CRF standards. Highest concentrations (1 n g / m g tissue) of CRF-like immunoreactivity were found in the median eminence but surprisingly, high concentrations of CRF-like immunoreactivity were found in frontal, parietal, occipital and particularly temporal areas of cerebral cortex. Much lower concentrations were found in other brain areas including the basal ganglia, limbic system and brain stem. corticotropin releasing factor; radioimmunoassay; ovine brain
InWoduction Although the importance of hypothalamic corticotropin-releasing factor (CRF) in the regulation of the pituitary-adrenal axis has long been recognized, it is but recently that this substance has been isolated from ovine tissue and characterized as a peptide containing 41 amino acids [1,2]. This peptide has been demonstrated to stimulate the secretion of adrenocorticotropin (ACTH) and fl-endorphin from
* To whomall correspondenceshould be addressed.Tel (418) 656-8253. 0167-0115/83/0000-0000/$03.00 © 1983 ElsevierBiomedicalPress
190 pituitary gland both in vivo [3] and in vitro [4] as well as the formation of cyclic AMP [5]. In addition to effects on the pituitary-adrenal axis, CRF also activates the sympathetic nervous system following intracerebroventricular injection leading to increased arterial pressure and heart rate [6] and suggesting that the peptide may exert effects outside the hypothalamus. We have raised an antiserum against synthetic ovine CRF and developed a sensitive and specific radioimmunoassay for this peptide. Using this assay, we present here results to demonstrate the widespread occurrence of CRF-like immunoreactivity ,(CRF-LI) in ovine brain regions.
Materials and Methods
Antibody production Synthetic ovine CRF, prepared in our laboratory according to a solid-phase method and purified by HPLC [7] was coupled to bovine serum albumin as follows. 10 mg of CRF were dissolved in 1.0 ml of 1 mM acetic acid with 4 mg purified cristalline bovine serum albumin (Sigma). After the addition of 100000 cpm of freshly iodinated CRF [7], 10.5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HC1 (Mann Research Laboratories Inc.) was incorporated and the mixture gently agitated and incubated at room temperature overnight. After the incubation, the opalescent reaction mixture was diluted with 10 ml of 0.4% saline and dialyzed against the same solution for 48 h to remove unreacted carbodiimide. Overall yield of the coupling reaction was estimated to be 70% according to the radioactivity remaining in the dialysis bag.
Production of the antibody 10 New Zealand rabbits were immunized every 2 weeks with 100 /~g of the conjugated CRF diluted in saline and emulsified with:an equal amount of Freund's complete adjuvant (Difco). Each animal was injected intradermally on the back according to the technique of Vaitukaitis et al. [8]. Rabbits were bled by cardiac puncture 1 month after the first injection. Before the beginning of immunization, each animal received 1 ml of DCT vaccine subcutaneously. At the first bleeding, antibodies against CRF were detectable in the serum of every rabbit.
Radioimmunoassay of CRF Ovine CRF was iodinated by the chloramine-T technique according to the method of Hunter and Greenwood [9] and purified by reversed-phase HPLC using acetonitrile and trifluoroacetic acid as elution buffers [7]. 90% of radioiodinated synthetic ovine C R F could b e precipitated by an excess of antibody. This iodinated peptide was kept at 4°C and remained immunoreactive for at least 10 days.
Incubation procedure Radioimmunoassay of ovine CRF was performed in 12 x 75 mm borosilicate or polystyrene tubes. All reagents were dissolved in a buffer containing 25 mM Tris-HC1 (Trizma Base Sigma), 0.1% bovine Serum albumin (Sigma), 1% Trasylol ®
191
(Boehringer), 0.1% sodium azide (Fisher Scientific) and 0.1% Tween 40 (Mann Research Laboratories) at a pH of 7.5. To each tube, we added in the following order 100 /~1 of buffer containing 20000 cpm of radioiodinated CRF, 100 /~1 of unknown or standard in a dose ranging from 1 to 10000 p g / t u b e and I00 #1 of anti-CRF No. 800-3 diluted 1 : 100000 in buffer containing 0.25% normal rabbit serum. The tubes were gently vortexed and incubated at 4°C for a period of 18-24 h when equilibrium was reached. Each tube then received 100/~1 of ice-cold second antibody (diluted in phosphate-buffered saline to assure maximal precipitation of the first antibody) and 500/d of an aqueous solution containing 10% polyethylene glycol and 0.1% Tween 40. Tubes were vortexed and incubation was continued for another hour at 4°C. They were then centrifuged at 3000 × g for 15 rain, decanted and the radioactive content measured.
Peptide extraction Ovine brains were obtained from 5 animals and dissected immediately after killing. The brains were cut into coronal slices approximately 1 cm thick and the areas dissected were weighed and immediately homogenized in a minimum of 20 volumes of 2 M acetic acid. Following centrifugation, the supernatant was lyophilized prior to radioimmunoassay of CRF content whilst the protein content of the pellet was measured according to Lowry et al. [10]
Results
Radioimmunoassay characterization The antibody raised against conjugated CRF we used (No. 800-3) bound approximately 35% of the added radiolabelled CRF at a dilution of 1 : 100000. Under these conditions, a standard curve was generated (Fig. 1) with an EDso of 130 p g / t u b e and a minimum detectable dose of 30 pg/tube. This sensitivity is sufficient to detect small amounts of CRF-like immunoreactivity in various parts of ovine brain. The specificity of the antibody was measured against different peptides. Sauvagine, a peptide isolated from amphibia and sharing some amino acid sequences with C R F showed a cross-reactivity of 0.5%. Other peptides which can be found in various parts of the brain, namely TRH, substance P and neurotensin showed respective cross-reactivities of 0.02, 0.01 and 0.005%. Vasopressin, VIP and oxytocin showed cross-reactivities measureable but smaller than 0.005% whilst other peptides including LHRH, somatostatin, ACTH, a-MSH, fl-endorphin, fl-LPH and y-LPH showed absolutely no cross-reactivity. Extracts from ovine or bovine median eminence and several extrahypothalamic areas of ovine brain exhibited 100% cross-reactivity and complete parallelism with the displacement curve obtained with synthetic CRF. This parallelism, between curves suggests that CRF from ovine and bovine median eminence as well as extrahypothalamic ovine CRF-LI are very similar (Fig. 2). Inter- and intraassay variability were measured as 16 and 11%, respectively on a small number of assays.
192 800-3 1/100,000 ~1 ~ . . . . . . •e...
Bo/T = 35% T C.=20000 S-43%
4~-
N•N
C5
LOGIT Y = 4.132-1.949LOgl0x DE50-132 pg/tube
-
~8-
1
10 1 ~ 0 o
O~j'"
"
~
_
10
100 1000 100004.
3-
O
PG/TUBE Fig. 1. Radioimmunoassay characterization. The anti-CRF (No, 800-3) bound 35% of radiolabelled CRF at a dilution of 1:100000. A standard curve was generated with an EDs0 value of 132 pg/tube and minimum detectable dose of 30 pg/tube.
Too-
100i H
75
MEDIAN EMINENCE TEMPORALCORTEX CEREBELLUM CAUDATENUCLEUS AMYGDALA PREOPTtCAREA
CRF
SAUVAGINE H [D'--C] TRR SUBSTP H NEUROTENSIN VASOPRESSIN VIP OXYTOCIN LHRH SOMATOSTATIN ACTH tr-MSH #-END y-LPH
i
50
25
25
o VOLUME EXTRACT
0 o lo~lo2lo3 lo' ~:~ PEPTIDE (PG/TUBE)
(#L/TUBE)
Fig. 2. Specificity of anti-CRF (No. 800-3). Left: Extracts of ovine median eminence, amygdala, caudate nucleus, preoptic area, temporal cortex or cerebellum exhibited 100% cross-reactivity and complete parallelism with the displacement curve obtained with synthetic CRF. Right: Cross-reactivities of sauvagine, TRH, substance P, neurotensin, vasopressin, VIP, oxytocin, LHRH, somatostatin, ACTH, a-MSH, fl-endorphin and y-LPH with anti-CRF (No. 800-3).
193 TABLE I Distribution of CRF-like immunoreactivity in ovine brain CRF (pg/mg protein) Median eminence Frontal cortex Parietal cortex Temporal cortex Occipital cortex Nucleus accumbens Septum Nucleus caudatus Putamen Globus pallidus Area preoptica Anterior and ventromedian hypothalamus Thalamus dorsalis Thalamus lateralis Thalamus ventralis Corpus amygdaloideum Corpus mamillare Hippocampus Corpus pineale Substantia nigra Colliculus superior Substantia grisea centralis Tegmentum Medulla oblongata Cerebellar vermis Cerebellar hemisphere Ventral spinal cord Dorsal spinal cord
167 500 + 18400 193 + 10 227 + 33 341 + 69 229 + 16 185 + 24 149 + 29 136 + 24 71 + 9 97 + 31 495 + 63 422 + 34 222 + 43 180 + 47 185 + 52 303 + 21 393 + 71 76 + 15 47 + 19 77 + 13 133 + 8 143 + 17 133 + 17 126 + 14 92 + 30 54 + 7 12 ± 1 38 ± 3
Results are the mean ± S.E.M. of values determined individually from 5 animals.
Localization of CRF-like immunoreactivity T h e c o n c e n t r a t i o n s of C R F - l i k e i m m u n o r e a c t i v i t y were d e t e r m i n e d in 28 discrete areas of o v i n e b r a i n as shown in T a b l e I. By far the highest c o n c e n t r a t i o n s of C R F - l i k e i m m u n o r e a c t i v i t y were l o c a t ed in the m e d i a n e m i n e n c e an d c o n c e n t r a t i o n s in o t h er b r a i n areas were at least two orders of m a g n i t u d e lower than in m e d i a n e m i n e n c e a n d did n o t differ so d r a m a t i c a l l y f r o m o n e region to another. Thus, relatively high c o n c e n t r a t i o n s were n o t e d in h y p o t h a l a m i c an d p r e o p t i c areas as well as amygdala, m a m i l l a r y bodies a n d cerebral cortex. S o m e w h a t l o w er c o n c e n t r a t i o n s were seen in t h a l a m u s a n d r h o m b e n c e p h a l i c structures whilst lowest, yet significant, a m o u n t s o c c u r r e d in the basal ganglia, h i p p o c a m p u s , substantia nigra an d cerebellum.
194
Discussion The results document and validate a specific and sensitive radioimmunoassay for CRF. Of numerous peptides tested, only sauvagine, which has a structure similar to that of ovine CRF, showed any cross-reactivity with the antiserum. However, since partial amino acid sequences of CRF were not available, we do not as yet have information on the exact antigenic determinant of our antiserum. Although we cannot be certain of the molecular nature of extracted immunoreactivity, extracts of both ovine and bovine median eminence/hypotha!amus and the CRF-LI of temporal cortex, caudate nucleus, cerebellum and amygdala displaced antibody-bound 125I-labelled CRF parallel to that of authentic CRF standard. Extracts of rat hypothalamus, however, did not cross-react to any appreciable extent, suggesting possible species differences in CRF structure. CRF activity, determined by stimulatory effects on A C T H release in vitro, has been previously localized in hypothalamus and extra-hypothalamic brain [ 11-13], whilst immunohistochemical techniques have shown CRF to be present in rat hypothalamus [14,15]. In agreement with these previous findings, we also noted highest CRF-like immunoreactivity in median eminence, with much lower concentrations elsewhere. Extrahypothalamic concentrations of CRF-like immunoreactivity are comparable to those of other hypothalamic hypophysiotropic hormones [16-19], however, its distribution, with important concentrations found in cerebral cortex, is reminiscent to that of somatostatin but not that of T R H or LHRH. In contrast to other brain peptides, small yet significant concentrations of CRF-like immunoreactivity are found in cerebellum. These results illustrate the wide distribution of CRF-containing neurons in ovine brain and suggest that, in addition to its regulation of corticotropin release, CRF may play a wider role as central nervous system neurotransmitter.
References 1 Spiess, J., Rivier, J., Rivier, C. and Vale, W., Primary structure of corticotropin-releasing factor from ovine hypothalamus, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 6517-6521. 2 Vale, W., Spiess, J., Rivier, C. and Rivier, J., Characterization of a 41 residue ovine hypothalamus peptide that stimulates the secretion of corticotropin and fl-endorphin, Science, 213 (1981) 1394-1397. 3 Rivier, C., Brownstein, M., Spiess, J., Rivier, J. and Vale, W., In vivo corticotropin-releasing factor-induced secretion of adrenocorticotropin, fl-endorphin and corticosterone, Endocrinology, 110 (1982) 272-278. 4 Gigu~re, V., Labile, F., Crtr, J., Coy, D.H., Sueras-Diaz, J. and Schally, A.V., Stimulation of cyclic AMP accumulation and adrenocorticotropin release by synthetic ovine corticotropin-releasing factor in rat anterior pituitary cells: site of glucocorticoid action, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 3466-3469. 5 Labile, F., Veilleux, R., Lef~vre, G., Coy, D.H., Sueiras-Diaz, J. and Schally, A.J., Corticotropin-releasing factor stimulates accumulation of adenosine 3',5'-monophosphate in rat pituitary corticotrophs, Science, 216 (1982) 1007-1008. 6 Fisher, L.A., Rivier, J., Rivier, C., Spiess, J., Vale, W. and Brown, M., Corticotropin-releasing factor (CRF): central effects on mean anterior pressure and heart rate in cats, Endocrinology, 110 (1982) 2222-2224.
195 7 Lefevre, G., Veilleux, R. and Lavoie, M., Solid-phase synthesis and some biological properties of ovine corticotropin-releasing factor, Int. J. Peptides Proteins, (1982) in press. 8 Vaitukaitis, J., Robbins, J.B., Nieschlag, E. and Ross, G.T., A method for producing specific antisera with small doses of immunogen, J. Clin. Endocrinol. Metab., 33 (1971) 988-991. 9 Hunter, W.M. and Greenwood, F.C., Preparation of 131I-labelled human growth hormone of high specific radioactivity, Biochem. J., 89 (1963) 114-123. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 11 Krieger, D.T., Liotta, A. and Brownstein, M.J., Corticotropin releasing factor distribution in normal and Brattleboro rat brain and effect of deafferentation, hypophysectomy and steroid treatment in normal animals, Endocrinology, I00 (1977) 227-237. 12 Lang, R.E., Voigt, K.H., Fehm, H.L. and Pfeiffer, E.F., Localization of corticotropin-releasing activity in the rat hypothalamus, Neurosci. Lett., 2 (1976) 19-22. 13 Yasuda, N. and Greer, M.A., Distribution of corticotropin-releasing factor(s) activity in neural and extra-neural tissues of the rat, Endocrinology, 99 91970) 944-948. 14 Bloom, F.E., Battenberg, E.L.F., Rivier, J. and Vale, W., Corticotropin-releasing factor (CRF): immunoreaetive neurones and fibers in rat hypothalamus, Regul. Peptides, 4 (1982) 43-48. 15 Bugnon, C., Fellmann, D., Gouget, A. and Cardot, J., Ontogeny of the corticoliberin neuroglandular system in rat brain, Nature, 298 (1982) 159-161. 16 Barden, N., M6rand, Y., Rouleau, D., Moore, S., Dockray, G.J. and Dupont, A., Regional distributions of somatostatin and cholecystokinin-like immunoreactivities in rat and bovine brain, Peptides, 2 (1981) 299-302.. 17 Brownstein, M.J., Palkovits, M., Saavedra, J.M., Bassiri, R.M. and Utiger, R.D., Thyrotropin-releasing hormone in specific nuclei of rat brain, Science, 185 (1974) 267-269. 18 Dupont, A., Dussault, J.H., Rouleau, D., Di Paolo, T., Coulombe, P., Gagn6, B., M6rand, Y., Moore, S. and Barden, N., Effect of neonatal thyroid deficiency on the catecholamine, substance P and thyrotropin-releasing hormone contents of discrete brain nuclei, Endocrinology, 108 ( 1981) 2039-2045. 19 Wheaton, J.E., Krulich, L. and McCann, S.M., Localization of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay, Endocrinology, 97 91975) 30-38.
189
Elsevier BiomedicalPress
Distribution of corticotropin-releasing factor in ovine brain determined by radioimmunoassay J. C6t6, G. Lef~vre, F. Labrie and N. Barden * MRC Group in Molecular Endocrinology, Le Centre Hospitalier de I'Universit~ Laval, Qu£,bec GI V 4G2, Canada
(Received 12 July 1982; revisedmanuscriptreceived 18 October 1982; acceptedfor publication 5 November 1982)
Summary We have developed and used a sensitive and specific radioimmunoassay to demonstrate the presence of CRF-like immunoreactivity in extra-hypothalamic areas of ovine brain. Synthetic CRF displaced antibody bound tracer at an EDs0 value of 200 pg and there was no cross-reactivity with LHRH, TRH, ACTH, fl-endorphin and several other peptides. Displacement of bound 125I-CRF by brain extracts exhibited curves parallel to synthetic CRF standards. Highest concentrations (1 n g / m g tissue) of CRF-like immunoreactivity were found in the median eminence but surprisingly, high concentrations of CRF-like immunoreactivity were found in frontal, parietal, occipital and particularly temporal areas of cerebral cortex. Much lower concentrations were found in other brain areas including the basal ganglia, limbic system and brain stem. corticotropin releasing factor; radioimmunoassay; ovine brain
InWoduction Although the importance of hypothalamic corticotropin-releasing factor (CRF) in the regulation of the pituitary-adrenal axis has long been recognized, it is but recently that this substance has been isolated from ovine tissue and characterized as a peptide containing 41 amino acids [1,2]. This peptide has been demonstrated to stimulate the secretion of adrenocorticotropin (ACTH) and fl-endorphin from
* To whomall correspondenceshould be addressed.Tel (418) 656-8253. 0167-0115/83/0000-0000/$03.00 © 1983 ElsevierBiomedicalPress
190 pituitary gland both in vivo [3] and in vitro [4] as well as the formation of cyclic AMP [5]. In addition to effects on the pituitary-adrenal axis, CRF also activates the sympathetic nervous system following intracerebroventricular injection leading to increased arterial pressure and heart rate [6] and suggesting that the peptide may exert effects outside the hypothalamus. We have raised an antiserum against synthetic ovine CRF and developed a sensitive and specific radioimmunoassay for this peptide. Using this assay, we present here results to demonstrate the widespread occurrence of CRF-like immunoreactivity ,(CRF-LI) in ovine brain regions.
Materials and Methods
Antibody production Synthetic ovine CRF, prepared in our laboratory according to a solid-phase method and purified by HPLC [7] was coupled to bovine serum albumin as follows. 10 mg of CRF were dissolved in 1.0 ml of 1 mM acetic acid with 4 mg purified cristalline bovine serum albumin (Sigma). After the addition of 100000 cpm of freshly iodinated CRF [7], 10.5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HC1 (Mann Research Laboratories Inc.) was incorporated and the mixture gently agitated and incubated at room temperature overnight. After the incubation, the opalescent reaction mixture was diluted with 10 ml of 0.4% saline and dialyzed against the same solution for 48 h to remove unreacted carbodiimide. Overall yield of the coupling reaction was estimated to be 70% according to the radioactivity remaining in the dialysis bag.
Production of the antibody 10 New Zealand rabbits were immunized every 2 weeks with 100 /~g of the conjugated CRF diluted in saline and emulsified with:an equal amount of Freund's complete adjuvant (Difco). Each animal was injected intradermally on the back according to the technique of Vaitukaitis et al. [8]. Rabbits were bled by cardiac puncture 1 month after the first injection. Before the beginning of immunization, each animal received 1 ml of DCT vaccine subcutaneously. At the first bleeding, antibodies against CRF were detectable in the serum of every rabbit.
Radioimmunoassay of CRF Ovine CRF was iodinated by the chloramine-T technique according to the method of Hunter and Greenwood [9] and purified by reversed-phase HPLC using acetonitrile and trifluoroacetic acid as elution buffers [7]. 90% of radioiodinated synthetic ovine C R F could b e precipitated by an excess of antibody. This iodinated peptide was kept at 4°C and remained immunoreactive for at least 10 days.
Incubation procedure Radioimmunoassay of ovine CRF was performed in 12 x 75 mm borosilicate or polystyrene tubes. All reagents were dissolved in a buffer containing 25 mM Tris-HC1 (Trizma Base Sigma), 0.1% bovine Serum albumin (Sigma), 1% Trasylol ®
191
(Boehringer), 0.1% sodium azide (Fisher Scientific) and 0.1% Tween 40 (Mann Research Laboratories) at a pH of 7.5. To each tube, we added in the following order 100 /~1 of buffer containing 20000 cpm of radioiodinated CRF, 100 /~1 of unknown or standard in a dose ranging from 1 to 10000 p g / t u b e and I00 #1 of anti-CRF No. 800-3 diluted 1 : 100000 in buffer containing 0.25% normal rabbit serum. The tubes were gently vortexed and incubated at 4°C for a period of 18-24 h when equilibrium was reached. Each tube then received 100/~1 of ice-cold second antibody (diluted in phosphate-buffered saline to assure maximal precipitation of the first antibody) and 500/d of an aqueous solution containing 10% polyethylene glycol and 0.1% Tween 40. Tubes were vortexed and incubation was continued for another hour at 4°C. They were then centrifuged at 3000 × g for 15 rain, decanted and the radioactive content measured.
Peptide extraction Ovine brains were obtained from 5 animals and dissected immediately after killing. The brains were cut into coronal slices approximately 1 cm thick and the areas dissected were weighed and immediately homogenized in a minimum of 20 volumes of 2 M acetic acid. Following centrifugation, the supernatant was lyophilized prior to radioimmunoassay of CRF content whilst the protein content of the pellet was measured according to Lowry et al. [10]
Results
Radioimmunoassay characterization The antibody raised against conjugated CRF we used (No. 800-3) bound approximately 35% of the added radiolabelled CRF at a dilution of 1 : 100000. Under these conditions, a standard curve was generated (Fig. 1) with an EDso of 130 p g / t u b e and a minimum detectable dose of 30 pg/tube. This sensitivity is sufficient to detect small amounts of CRF-like immunoreactivity in various parts of ovine brain. The specificity of the antibody was measured against different peptides. Sauvagine, a peptide isolated from amphibia and sharing some amino acid sequences with C R F showed a cross-reactivity of 0.5%. Other peptides which can be found in various parts of the brain, namely TRH, substance P and neurotensin showed respective cross-reactivities of 0.02, 0.01 and 0.005%. Vasopressin, VIP and oxytocin showed cross-reactivities measureable but smaller than 0.005% whilst other peptides including LHRH, somatostatin, ACTH, a-MSH, fl-endorphin, fl-LPH and y-LPH showed absolutely no cross-reactivity. Extracts from ovine or bovine median eminence and several extrahypothalamic areas of ovine brain exhibited 100% cross-reactivity and complete parallelism with the displacement curve obtained with synthetic CRF. This parallelism, between curves suggests that CRF from ovine and bovine median eminence as well as extrahypothalamic ovine CRF-LI are very similar (Fig. 2). Inter- and intraassay variability were measured as 16 and 11%, respectively on a small number of assays.
192 800-3 1/100,000 ~1 ~ . . . . . . •e...
Bo/T = 35% T C.=20000 S-43%
4~-
N•N
C5
LOGIT Y = 4.132-1.949LOgl0x DE50-132 pg/tube
-
~8-
1
10 1 ~ 0 o
O~j'"
"
~
_
10
100 1000 100004.
3-
O
PG/TUBE Fig. 1. Radioimmunoassay characterization. The anti-CRF (No, 800-3) bound 35% of radiolabelled CRF at a dilution of 1:100000. A standard curve was generated with an EDs0 value of 132 pg/tube and minimum detectable dose of 30 pg/tube.
Too-
100i H
75
MEDIAN EMINENCE TEMPORALCORTEX CEREBELLUM CAUDATENUCLEUS AMYGDALA PREOPTtCAREA
CRF
SAUVAGINE H [D'--C] TRR SUBSTP H NEUROTENSIN VASOPRESSIN VIP OXYTOCIN LHRH SOMATOSTATIN ACTH tr-MSH #-END y-LPH
i
50
25
25
o VOLUME EXTRACT
0 o lo~lo2lo3 lo' ~:~ PEPTIDE (PG/TUBE)
(#L/TUBE)
Fig. 2. Specificity of anti-CRF (No. 800-3). Left: Extracts of ovine median eminence, amygdala, caudate nucleus, preoptic area, temporal cortex or cerebellum exhibited 100% cross-reactivity and complete parallelism with the displacement curve obtained with synthetic CRF. Right: Cross-reactivities of sauvagine, TRH, substance P, neurotensin, vasopressin, VIP, oxytocin, LHRH, somatostatin, ACTH, a-MSH, fl-endorphin and y-LPH with anti-CRF (No. 800-3).
193 TABLE I Distribution of CRF-like immunoreactivity in ovine brain CRF (pg/mg protein) Median eminence Frontal cortex Parietal cortex Temporal cortex Occipital cortex Nucleus accumbens Septum Nucleus caudatus Putamen Globus pallidus Area preoptica Anterior and ventromedian hypothalamus Thalamus dorsalis Thalamus lateralis Thalamus ventralis Corpus amygdaloideum Corpus mamillare Hippocampus Corpus pineale Substantia nigra Colliculus superior Substantia grisea centralis Tegmentum Medulla oblongata Cerebellar vermis Cerebellar hemisphere Ventral spinal cord Dorsal spinal cord
167 500 + 18400 193 + 10 227 + 33 341 + 69 229 + 16 185 + 24 149 + 29 136 + 24 71 + 9 97 + 31 495 + 63 422 + 34 222 + 43 180 + 47 185 + 52 303 + 21 393 + 71 76 + 15 47 + 19 77 + 13 133 + 8 143 + 17 133 + 17 126 + 14 92 + 30 54 + 7 12 ± 1 38 ± 3
Results are the mean ± S.E.M. of values determined individually from 5 animals.
Localization of CRF-like immunoreactivity T h e c o n c e n t r a t i o n s of C R F - l i k e i m m u n o r e a c t i v i t y were d e t e r m i n e d in 28 discrete areas of o v i n e b r a i n as shown in T a b l e I. By far the highest c o n c e n t r a t i o n s of C R F - l i k e i m m u n o r e a c t i v i t y were l o c a t ed in the m e d i a n e m i n e n c e an d c o n c e n t r a t i o n s in o t h er b r a i n areas were at least two orders of m a g n i t u d e lower than in m e d i a n e m i n e n c e a n d did n o t differ so d r a m a t i c a l l y f r o m o n e region to another. Thus, relatively high c o n c e n t r a t i o n s were n o t e d in h y p o t h a l a m i c an d p r e o p t i c areas as well as amygdala, m a m i l l a r y bodies a n d cerebral cortex. S o m e w h a t l o w er c o n c e n t r a t i o n s were seen in t h a l a m u s a n d r h o m b e n c e p h a l i c structures whilst lowest, yet significant, a m o u n t s o c c u r r e d in the basal ganglia, h i p p o c a m p u s , substantia nigra an d cerebellum.
194
Discussion The results document and validate a specific and sensitive radioimmunoassay for CRF. Of numerous peptides tested, only sauvagine, which has a structure similar to that of ovine CRF, showed any cross-reactivity with the antiserum. However, since partial amino acid sequences of CRF were not available, we do not as yet have information on the exact antigenic determinant of our antiserum. Although we cannot be certain of the molecular nature of extracted immunoreactivity, extracts of both ovine and bovine median eminence/hypotha!amus and the CRF-LI of temporal cortex, caudate nucleus, cerebellum and amygdala displaced antibody-bound 125I-labelled CRF parallel to that of authentic CRF standard. Extracts of rat hypothalamus, however, did not cross-react to any appreciable extent, suggesting possible species differences in CRF structure. CRF activity, determined by stimulatory effects on A C T H release in vitro, has been previously localized in hypothalamus and extra-hypothalamic brain [ 11-13], whilst immunohistochemical techniques have shown CRF to be present in rat hypothalamus [14,15]. In agreement with these previous findings, we also noted highest CRF-like immunoreactivity in median eminence, with much lower concentrations elsewhere. Extrahypothalamic concentrations of CRF-like immunoreactivity are comparable to those of other hypothalamic hypophysiotropic hormones [16-19], however, its distribution, with important concentrations found in cerebral cortex, is reminiscent to that of somatostatin but not that of T R H or LHRH. In contrast to other brain peptides, small yet significant concentrations of CRF-like immunoreactivity are found in cerebellum. These results illustrate the wide distribution of CRF-containing neurons in ovine brain and suggest that, in addition to its regulation of corticotropin release, CRF may play a wider role as central nervous system neurotransmitter.
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