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Corticoliberin, somatocrinin and amine contents in normal and parkinsonian human hypothalamus
Neuroscience Letters, 56 (1985) 217-222 Elsevier Scientific Publishers Ireland Ltd.
217
NSL 03296
CORTICOLIBERIN, SOMATOCRININ AND AMINE CONTENTS IN NORMAL AND PARKINSONIAN HUMAN HYPOTHALAMUS
B. C O N T E - D E V O L X 1, M. G R I N O I, A. N I E O U L L O N 2, F. J A V O Y - A G I D 3, E. C A S T A N A S I, V. G U I L L A U M E I, M.C. T O N O N 4, H. V A U D R Y 4 and C. OLIVER 1
lLaboratoire de Neuroendocrinologie Experimentale ( CNRS, UA 560), FacultO de M~deeine Nord, F-13326 Marseille COdex 15; 2CNRS INP 5, Chemin J. Aiguier, F-13277 Marseille COdex 9; 3Laboratoire de MOdecine Exp~rimentale, C.H.U. PitiO-SalpOtriOre, 91 Bd de l'H6pital, F-75634 Paris Ckdex 13; and4Laboratoire d'Endocrinologie, FaeultO de Sciences, CNRS, UA 650, F-76130 Mont-Saint-Aignan (France) (Received December 12th, 1984; Revised version received February 19th, 1985; Accepted February 20th, 1985)
Key words." hypothalamus - Parkinson - monoamines - corticoliberin - somatocrinin - human
We have compared hypothalamic contents of various neurotransmitters (dopamine (DA), norepinephrine and serotonin) and their metabolites (dihydroxyphenyl acetic acid, homovanilic acid, 5-hydroxyindoleacetic acid) in post-mortem h u m a n controls and parkinsonian hypothalami. Neurotransmitters and their metabolites were measured in 0.1 N HC1 hypothalami extracts using electrochemical detection after high performance liquid chromatography. Using specific radioimmunoassays we have also measured corticoliberin and somatocrinin contents in these hypothalami. Despite a 50~o decrease of D A contents in parkinsonian hypothalami, no variations of corticoliberin and somatocrinin contents were found: 16.6-4- 1.78 pg/mg tissue in Parkinson disease vs 16.71 _+ 1.89 in controls for h u m a n corticotropin-releasing factor ( h C R F I 4 0 and 37.38 _+ 11 vs 45.16 for h u m a n growth-hormone-releasing factor (hGRFI44).
Previous investigations [10, 11] have shown that in Parkinson's disease the concentrations of several (but not all) neuropeptides are lowered in discrete brain areas. For example, cholecystokinin-8, substance P and methionine- and leucine-enkephalin levels are decreased in the striatum and substantia nigra, while only somatostatin is lowered in cortical areas. Recently, corticotropin-releasing factor (CRF) [! 7, 19] and growth-hormone-releasing factor (GRF or somatocrinin) [9, 13] which stimulate adrenocorticotropin (ACTH) and growth hormone (GH) secretion respectively, have been identified and synthesized. CRF neurons [4] are mainly localized in the paraventricular nucleus, and their fibers are distributed on the portal capillary plexus, while GRF neurons [3] are concentrated in the mediobasal hypothalamus. The recent development of immunoassays for CRF and GRF has made it possible to quantify these peptides both in control and parkinsonian hypothalamus. Preparation of human tissues. The hypothalami from 16 subjects without known neurological disease (14 women, 2 men) were studied as controls: mean age was 78.7+ 1.4 years, and the mean interval between death and autopsy was 11.5_+ 1.5 h. The group of parkinsonian hypothalami comprised 10 subjects (2 women and 8 men): mean age was 72.5 _+2.2 years, and the mean interval between death and autopsy was 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
218
19.8_+4.5 h; the duration of parkinsonian disease was 11.8 + 3.0 years; all patients had been treated with L-dihydroxyphenylalanine (L-DOPA), but the interval between cessation of treatment and death varied among the patients (1-12 days). Within 2 h after autopsy, the area corresponding to the hypothalamus was dissected and the different samples of hypothalamus from serial sections were pooled, crushed on dry ice and stored at - 7 0 C until peptide and neurotransmitter analysis. Aliquots of each hypothalamus (30-60 rag) were weighed while frozen and homogenized in 1 ml 0.1 N HCI at 4"C. After the homogenate was centrifuged for 10 min at 1500 g, the supernatant fluid was decanted. 200/~1 were saved for amine determinations, and the rest was divided into two aliquots, which were evaporated to dryness under reduced pressure in a Speed Vac (Savant Instrument). Then dry residues were frozen until dissolved in the appropriate buffer and assayed. Assay methods. C R F was measured in acid extracts of the hypothalamus by radioimmunoassay (R1A). The rat CRFI 41 antiserum was obtained in our laboratory after immunization of rabbits which synthetic rat CRFt 41 (Bachem, Marina del Rey, CA, U,S.A.) coupled to bovine serum albumin (BSA) with carbodiimide. Ntyrosyl rat CRFI 41 (Peninsula Labs., U.S.A.) was labeled with 125I using chloramine T and used as a tracer. Synthetic rat CRFI 4L (Peninsula Labs.. U.S.A.) was used
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Fig. 1. Semi-logarithmic plots comparing competitive inhibition of antibody-bound [tZSl]hCRFt 4L with synthetic hCRFI 4~, oCRF~ 4t (ovine) or human hypothalamic extract. 0 , oCRF~ 4~; O, rat hCRFL 41; ml human hypothalamic extract.
219 as a standard. The buffer used for preparing the appropriate dilutions o f tissue extracts, antiserum (final dilution: 1:30,000) and tracer was 0.05 M phosphates - 0.15 M NaCI-0.1% Triton X-100-0.1~o BSA, pH 7.5. The sensitivity of the assay was 5 fmol/tube. This RIA allows the measurement of human CRFI~I ( h C R F l ~ t ) since this peptide appears to be identical with rat CRFI_41 [16, 17]. Indeed, as shown in Fig. 1, immunoreactive C R F in human hypothalamus was similar to synthetic CRF. G R F was quantified in acid extracts o f the hypothalamus by RIA. The hGRFI 40 antiserum was obtained in the Laboratory o f Endocrinology of Rouen by M.C. T o n o n after immunization o f rabbits with synthetic h G R F l ~ 0 (kindly supplied by Dr. J. Rivier, Salk Institute, La Jolla, CA) coupled to BSA with carbodiimide. This antiserum was usable in the assay at 1:10,000 final dilution. It gave a 100~o crossreaction with h G R F I ~ 4 which appears to be the main G R F in the human hypothalamus [13]. h G R F j ~ 4 was labeled with 125I using chloramine T and used as a tracer while synthetic hGRFl_44 was used as a standard (kindly supplied by Sanofi Labs., France). The sensitivity of the assay was 2 fmol/tube. Increasing concentrations of hypothalamic extracts gave a displacement curve of [J25I]hGRF1-44 bound to the antibody that was parallel to that obtained with synthetic hGRFI~4 (Fig. 2).
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Fig. 2. Semi-logarithmic plot comparing competitive antibody-bound [t25I]hGRF~44 with synthetic hGRFI 44, hGRFt 40, rat GRF or human hypothalamic extract. ~, rat GRF; D, hGRFI 44; O,hGRF~ 40; II, human hypothalamic extract.
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Norepinephrine (NE), dopamine (DA), dihydroxyphenyl-acetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5HIAA) were measured by electrochemical detection after high-performance liquid chromatography (HPLC-ECD) according to the method of Lyness [14] after slight modifications. 20 /~1 out of the acid hypothalamic extract were diluted in a final volume of 500 #1 mobile phase (0.1 M sodium acetate (pH 4.5)-2 × 10 -5 M ethylenediaminetetraacetic acid ( E D T A ) - 1 5 0 0 sodium octylsulfate-12'),o methanol) and injected into a Cm HPLC column. Samples were eluted at room temperature at a flow-rate of 1 ml/min. The electrochemical detector was an amperometric glassy-carbon detector (800 mV). The column was calibrated by chromatography of standards individually. The sensitivity of the assay was 20 pg for all amines and metabolites. The inter- and intraassay reproducibility was 5!~i;.Statistical analysis was carried out by Student's t-test. In Parkinson's disease, no changes in CRF and G R F concentrations were observed in the hypothalamus. There was a significant reduction in DA (-45.3~o) and HVA (-42.33~,) in the hypothalamus of patients with Parkinson's disease; in opposition, DOPAC concentrations increased significantly ( + 400/,,) in this group; hypothalamic NE, 5-HT and 5-HIAA were inchanged (Table I). The D O P A C / D A (0.20 vs 0.08), HVA/DA (2.60 vs 1.89) and DOPAC + HVA/ DA (2.80 vs 1.97) were higher in parkinsonian hypothalami than in controls, indicating that the utilization rate of DA was accelerated in this disease. Parkinson's disease is histochemically characterized by a degenerescence of dopaminergic neurons all over the central nervous system (CNS) including the hypothalamus. Our results confirm that the DA concentration in the hypothalamus is significantly reduced [10, 11]. The dopaminergic system is selectively altered at this level
TABLE I C O N C E N T R A T I O N S OF M O N O A M I N E S A N D T H E I R METABOLITES, G R F A N D C R F IN CONTROL AND PARKINSONIAN HYPOTHALAMUS Values are the mean 1 S.E.M. for 16 controls and 10 subjects with Parkinson's disease. *P < 0.05. Control NE DA DOPAC HVA 5-HT 5-HIAA hGRF, 4a hCRFh 4~ "Concentrations ng/mg tissue. "Concentrations pg/mg tissue.
Parkinson's disease
6.04 ± 1.5 (I.64 ± 0.10
4.97 ± 1.7'' 0.35*_+ 0.08 ~
0.05 _+ 0.01 1.21 ± 0.43 0.025± 0.009 0.49 ± 0.08 45.16 +10.7 16.71 _+ 1.89
0.07*± 0.02 ~ 0.91 _+ 0.02 '~ 0.025_+ 0.0D 0.35 ± 0.09 ' 37.38 _+ll" 16.63 + 1.788
221
since NE and 5-HT hypothalamic concentrations do not change. Like other neuropepetides (cholecystokinin octapeptide, substance P, methionine- and leucine-enkephalin, T R H and somatostatin) [10, 11], CRF and G R F are unchanged in parkinsonian hypothalamus. In Parkinson's disease, there are alterations [7], in the secretion of prolactin which is normally under inhibitory control by the hypothalamic DA [21]. No studies are available yet on the secretion of G H and ACTH in untreated Parkinson's disease. It has been established that DA has a stimulating effect on G H secretion in humans [2, 15, 20, 21]. Its mechanism of action is unknown yet: either increased release of G R F or decreased liberation of somatostatin. In the rat, DA alters the release of somatostatin at the level of the hypophysial portal vessels [5]. No significant effect of DOPA and DA on ACTH secretion has been reported in normal subjects [8, 12,
18, 20]. The lack of changes in the hypothalamic content of CRF and G R F may be explained by the following. (1) There is no contact in the hypothalamus between dopaminergic neurons and CRF and/or G R F neurons. Indeed, the paraventricular nucleus of the hypothalamus where CRF cell bodies cell are concentrated is mainly innervated by adrenergic and noradrenergic fibers. No dopaminergic innervation has been demonstrated in this nucleus. G R F and DA-containing neurons are present in the same area of the hypothalamus, i.e. the mediobasal hypothalamus. However, there is no information on the histochemical relationship between both sets of neurons. (2) The dopaminergic system may influence the CRF and/or G R F release. However, it does not affect its synthesis. (3) As in experimental models, the decrease of DA in Parkinson's disease may not be sufficient to induce neuroendocrine changes. Indeed, when a 50~o reduction in hypothalamic DA is induced by the neonatal administration of sodium monoglutamate in rats [6], there is no change in ~melanocyte-stimulating hormone (~-MSH) secretion. However, this peptide is directly under the inhibitory influence by hypothalamic DA and an increased release was expected. (4) The lesions of dopaminergic fibers in Parkinson's disease is followed by an hyperactivity of the remaining dopaminergic neurons. The increased HVA/DA, D O P A C / D A and HVA + D O P A C / D A ratios show an acceleration of the utilization rate of DA [1]. Thus, the decrease in DA levels in the hypothalamus may be masked by the hyperactivity of unaltered dopaminergic neurons, taking into account the lack of changes in C R F and G R F concentrations. This work was supported by INSERM Grants C.R.E. 834007, C.R.E. 846020 and M.R.T. 84-H-1335. 1 Agid, Y., Javoy, F. and Glowinski, J., Hyperactivity of remaining dopaminergic neurones after partial destruction of the nigro-striatal dopaminergic system in the rat, Nature (New Biol.), 245 (1973) 150 151. 2 Bansal, S.A., Lee., L.A. and Woolf, P.D., Dopaminergic stimulation and inhibition of growth hormone secretion in normal man: studies of the pharmacologic specificity, J. Clin. Endocrinol. Metab., 53 (1981) 1273 1277. 3 Bloch, B., Gaillard, R.C., Brazeau, P., Lin, H.D. and Ling, N., Topographical and ontogenetic study
222 of the neurons producing growth-releasing factor in human hypothalamus, Regulat. Pept., 8 (1984) 21-31. 4 Bugnon, C., Fellman, D., Bresson, J.L. and Clavequin, M.C., Etude immunohistochimique du syst6me neuroglandulaire fi CRF chez I'homme, C.R. Acad. Sci. (Paris), 294 (1982) 491-496. 5 Chihara, K., Arimura, A. and Schally, A.V., Effect of intraventricular injection of dopamine, norepinephrine, acetylcholine and 5-hydroxytryptamine on immunoreactive somatostatin release into rat hypophyseal portal blood, Endocrinology, 104 (1979) 1656-1661. 6 Conte-Devolx, B., Giraud, P., Castanas, E., Boudouresque, F., Orlando, M., Gillioz, P. and Oliver, C., Effect of neonatal treatment with monosodium glutamate on the secretion of~-MSH,/~-Endorphin and ACTH in the rat, Neuroendocrinology 33 (1981) 207 21 I. 7 Eisler, T., Thorner, M.O., MacLeod, R.M., Kaiser, D.L. and Calne, D.B., Prolactin secretion in Parkinson disease, Neurology, 31 ( 1981) 1356- 1359. 8 Ganong, W.F., Neurotransmitters and pituitary function: regulation of ACTH secretion, Fed. Proc. 39 (1980) 2923 2930. 9 Guillemim R., Brazeau, P., Bohten, P., Esch, F., Ling, N. and Wehrenberg, W.B., Growth hormonereleasing factor from a human pancreatic tumor that caused acromegly, Science, 218 (1982) 585 587. 10 Javoy-Agid, F., Taquet, M., Cesselin, F., Epelbaum, J., Grouselle, D., Mauborgne, A., Studler, J.M. and Agid, Y.. Neuropeptides in Parkinson's disease, 5th Int. Catecholamine Syrup.. G6teborg, FR.G.. 1983 (abstract). 11 Javoy-Agid, F., Ruberg, M., Pique, L., Bertagna, X., Taquet, M., Stud[er, J.M., Cesselin, F., Epelbaum, J. and Agid, Y., Biochemistry of the hypothalamus in Parkinson disease, Neurology, 2 11984) 672 675. 12 Jones, M.T., Gillham, B., Greenstein, B.D., Abraham, R.R., Dorhnhorst, A., Beckfort, U., Holmes, M.C., Lin, J.H.. Torrellas, A., Bowery, N.G., Direnzo, G., Knowles, F., The role of neurotransmitters and glucocorticoids hormones in the control of adreno-corticotropin hormones and related peptides. In M. Motta, M. Zanisi and F. Piva (Eds.), Pituitary and Related Peptides, Academic Press, New York, 1982, p. 281. 13 I_,ing, N., Esch, F., Bohlen, P., Brazeau, P., Wehrenberg, W.B. and Guillemin, R., Isolation, primary slructure, and synthesis of human hypothalamic somatocrinin: growth-releasing factor. Proc. Natl. Acad. Sci. USA, 81 (1984) 4302-431)6. 14 Lyness, W.H., Simultaneous measurement of dopamine and its metabolites 5-hydroxytryptaminc, 5hydroxyindoleacetic acid and tryptophan in brain tissue using liquid chromatography and electrochemical detection, Life Sci., 31 11982) 1435 1443. 15 Martin, J.B., Functions of central nervous system neurotransmitters in regulation of growth hormone secrction, Fed. Proc., 39 11980) 2902 2906. 16 Rivier, J., Spiess, J. and Vale, W., Characterization of rat hypothalamic corticotropin-releasing factor. Proc. Natl. Acad. Sci. USA, 811(1983)4851 4855. 17 Shibahara, S., Morimoto, Y., Furatani, Y., Notake, M., Takahashi, It., Shimizu, S., ltorikawa, S. and Numa, S., Isolation and sequence analysis of the human corticotropin-releasing factor precursor gcnc, EMBO, J., 2 11983) 775 779. 18 Smythc, G.A., Bradshaw, .I.E. and Vining, R.F., Hypothalamic monoaminc control of stress-induced adrcno-corticotropin rclcasc in the rat, Endocrinology, 113 (1983) 11)62 1071. 19 Vale, W., Spicss, J., Rivicr, C. and Rivier, J., Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and/]-endorphin, Science, 213 ( 1981 ) 1394 1397. 20 Weiner, R.I. and Ganong, W.F., Role of brain monoamines and histamine in regulation of anterior pituitary secretion, Physiol. Rev., 58 (1978) 905 976. 21 Willoughby, J.O. and Day, T.A., Central catecholamine depletion: effects on physiological growth hormone and prolactin sccretion, Neurocndocrinology, 32 11981) 65 69.
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NSL 03296
CORTICOLIBERIN, SOMATOCRININ AND AMINE CONTENTS IN NORMAL AND PARKINSONIAN HUMAN HYPOTHALAMUS
B. C O N T E - D E V O L X 1, M. G R I N O I, A. N I E O U L L O N 2, F. J A V O Y - A G I D 3, E. C A S T A N A S I, V. G U I L L A U M E I, M.C. T O N O N 4, H. V A U D R Y 4 and C. OLIVER 1
lLaboratoire de Neuroendocrinologie Experimentale ( CNRS, UA 560), FacultO de M~deeine Nord, F-13326 Marseille COdex 15; 2CNRS INP 5, Chemin J. Aiguier, F-13277 Marseille COdex 9; 3Laboratoire de MOdecine Exp~rimentale, C.H.U. PitiO-SalpOtriOre, 91 Bd de l'H6pital, F-75634 Paris Ckdex 13; and4Laboratoire d'Endocrinologie, FaeultO de Sciences, CNRS, UA 650, F-76130 Mont-Saint-Aignan (France) (Received December 12th, 1984; Revised version received February 19th, 1985; Accepted February 20th, 1985)
Key words." hypothalamus - Parkinson - monoamines - corticoliberin - somatocrinin - human
We have compared hypothalamic contents of various neurotransmitters (dopamine (DA), norepinephrine and serotonin) and their metabolites (dihydroxyphenyl acetic acid, homovanilic acid, 5-hydroxyindoleacetic acid) in post-mortem h u m a n controls and parkinsonian hypothalami. Neurotransmitters and their metabolites were measured in 0.1 N HC1 hypothalami extracts using electrochemical detection after high performance liquid chromatography. Using specific radioimmunoassays we have also measured corticoliberin and somatocrinin contents in these hypothalami. Despite a 50~o decrease of D A contents in parkinsonian hypothalami, no variations of corticoliberin and somatocrinin contents were found: 16.6-4- 1.78 pg/mg tissue in Parkinson disease vs 16.71 _+ 1.89 in controls for h u m a n corticotropin-releasing factor ( h C R F I 4 0 and 37.38 _+ 11 vs 45.16 for h u m a n growth-hormone-releasing factor (hGRFI44).
Previous investigations [10, 11] have shown that in Parkinson's disease the concentrations of several (but not all) neuropeptides are lowered in discrete brain areas. For example, cholecystokinin-8, substance P and methionine- and leucine-enkephalin levels are decreased in the striatum and substantia nigra, while only somatostatin is lowered in cortical areas. Recently, corticotropin-releasing factor (CRF) [! 7, 19] and growth-hormone-releasing factor (GRF or somatocrinin) [9, 13] which stimulate adrenocorticotropin (ACTH) and growth hormone (GH) secretion respectively, have been identified and synthesized. CRF neurons [4] are mainly localized in the paraventricular nucleus, and their fibers are distributed on the portal capillary plexus, while GRF neurons [3] are concentrated in the mediobasal hypothalamus. The recent development of immunoassays for CRF and GRF has made it possible to quantify these peptides both in control and parkinsonian hypothalamus. Preparation of human tissues. The hypothalami from 16 subjects without known neurological disease (14 women, 2 men) were studied as controls: mean age was 78.7+ 1.4 years, and the mean interval between death and autopsy was 11.5_+ 1.5 h. The group of parkinsonian hypothalami comprised 10 subjects (2 women and 8 men): mean age was 72.5 _+2.2 years, and the mean interval between death and autopsy was 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
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19.8_+4.5 h; the duration of parkinsonian disease was 11.8 + 3.0 years; all patients had been treated with L-dihydroxyphenylalanine (L-DOPA), but the interval between cessation of treatment and death varied among the patients (1-12 days). Within 2 h after autopsy, the area corresponding to the hypothalamus was dissected and the different samples of hypothalamus from serial sections were pooled, crushed on dry ice and stored at - 7 0 C until peptide and neurotransmitter analysis. Aliquots of each hypothalamus (30-60 rag) were weighed while frozen and homogenized in 1 ml 0.1 N HCI at 4"C. After the homogenate was centrifuged for 10 min at 1500 g, the supernatant fluid was decanted. 200/~1 were saved for amine determinations, and the rest was divided into two aliquots, which were evaporated to dryness under reduced pressure in a Speed Vac (Savant Instrument). Then dry residues were frozen until dissolved in the appropriate buffer and assayed. Assay methods. C R F was measured in acid extracts of the hypothalamus by radioimmunoassay (R1A). The rat CRFI 41 antiserum was obtained in our laboratory after immunization of rabbits which synthetic rat CRFt 41 (Bachem, Marina del Rey, CA, U,S.A.) coupled to bovine serum albumin (BSA) with carbodiimide. Ntyrosyl rat CRFI 41 (Peninsula Labs., U.S.A.) was labeled with 125I using chloramine T and used as a tracer. Synthetic rat CRFI 4L (Peninsula Labs.. U.S.A.) was used
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Fig. 1. Semi-logarithmic plots comparing competitive inhibition of antibody-bound [tZSl]hCRFt 4L with synthetic hCRFI 4~, oCRF~ 4t (ovine) or human hypothalamic extract. 0 , oCRF~ 4~; O, rat hCRFL 41; ml human hypothalamic extract.
219 as a standard. The buffer used for preparing the appropriate dilutions o f tissue extracts, antiserum (final dilution: 1:30,000) and tracer was 0.05 M phosphates - 0.15 M NaCI-0.1% Triton X-100-0.1~o BSA, pH 7.5. The sensitivity of the assay was 5 fmol/tube. This RIA allows the measurement of human CRFI~I ( h C R F l ~ t ) since this peptide appears to be identical with rat CRFI_41 [16, 17]. Indeed, as shown in Fig. 1, immunoreactive C R F in human hypothalamus was similar to synthetic CRF. G R F was quantified in acid extracts o f the hypothalamus by RIA. The hGRFI 40 antiserum was obtained in the Laboratory o f Endocrinology of Rouen by M.C. T o n o n after immunization o f rabbits with synthetic h G R F l ~ 0 (kindly supplied by Dr. J. Rivier, Salk Institute, La Jolla, CA) coupled to BSA with carbodiimide. This antiserum was usable in the assay at 1:10,000 final dilution. It gave a 100~o crossreaction with h G R F I ~ 4 which appears to be the main G R F in the human hypothalamus [13]. h G R F j ~ 4 was labeled with 125I using chloramine T and used as a tracer while synthetic hGRFl_44 was used as a standard (kindly supplied by Sanofi Labs., France). The sensitivity of the assay was 2 fmol/tube. Increasing concentrations of hypothalamic extracts gave a displacement curve of [J25I]hGRF1-44 bound to the antibody that was parallel to that obtained with synthetic hGRFI~4 (Fig. 2).
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Fig. 2. Semi-logarithmic plot comparing competitive antibody-bound [t25I]hGRF~44 with synthetic hGRFI 44, hGRFt 40, rat GRF or human hypothalamic extract. ~, rat GRF; D, hGRFI 44; O,hGRF~ 40; II, human hypothalamic extract.
220
Norepinephrine (NE), dopamine (DA), dihydroxyphenyl-acetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5HIAA) were measured by electrochemical detection after high-performance liquid chromatography (HPLC-ECD) according to the method of Lyness [14] after slight modifications. 20 /~1 out of the acid hypothalamic extract were diluted in a final volume of 500 #1 mobile phase (0.1 M sodium acetate (pH 4.5)-2 × 10 -5 M ethylenediaminetetraacetic acid ( E D T A ) - 1 5 0 0 sodium octylsulfate-12'),o methanol) and injected into a Cm HPLC column. Samples were eluted at room temperature at a flow-rate of 1 ml/min. The electrochemical detector was an amperometric glassy-carbon detector (800 mV). The column was calibrated by chromatography of standards individually. The sensitivity of the assay was 20 pg for all amines and metabolites. The inter- and intraassay reproducibility was 5!~i;.Statistical analysis was carried out by Student's t-test. In Parkinson's disease, no changes in CRF and G R F concentrations were observed in the hypothalamus. There was a significant reduction in DA (-45.3~o) and HVA (-42.33~,) in the hypothalamus of patients with Parkinson's disease; in opposition, DOPAC concentrations increased significantly ( + 400/,,) in this group; hypothalamic NE, 5-HT and 5-HIAA were inchanged (Table I). The D O P A C / D A (0.20 vs 0.08), HVA/DA (2.60 vs 1.89) and DOPAC + HVA/ DA (2.80 vs 1.97) were higher in parkinsonian hypothalami than in controls, indicating that the utilization rate of DA was accelerated in this disease. Parkinson's disease is histochemically characterized by a degenerescence of dopaminergic neurons all over the central nervous system (CNS) including the hypothalamus. Our results confirm that the DA concentration in the hypothalamus is significantly reduced [10, 11]. The dopaminergic system is selectively altered at this level
TABLE I C O N C E N T R A T I O N S OF M O N O A M I N E S A N D T H E I R METABOLITES, G R F A N D C R F IN CONTROL AND PARKINSONIAN HYPOTHALAMUS Values are the mean 1 S.E.M. for 16 controls and 10 subjects with Parkinson's disease. *P < 0.05. Control NE DA DOPAC HVA 5-HT 5-HIAA hGRF, 4a hCRFh 4~ "Concentrations ng/mg tissue. "Concentrations pg/mg tissue.
Parkinson's disease
6.04 ± 1.5 (I.64 ± 0.10
4.97 ± 1.7'' 0.35*_+ 0.08 ~
0.05 _+ 0.01 1.21 ± 0.43 0.025± 0.009 0.49 ± 0.08 45.16 +10.7 16.71 _+ 1.89
0.07*± 0.02 ~ 0.91 _+ 0.02 '~ 0.025_+ 0.0D 0.35 ± 0.09 ' 37.38 _+ll" 16.63 + 1.788
221
since NE and 5-HT hypothalamic concentrations do not change. Like other neuropepetides (cholecystokinin octapeptide, substance P, methionine- and leucine-enkephalin, T R H and somatostatin) [10, 11], CRF and G R F are unchanged in parkinsonian hypothalamus. In Parkinson's disease, there are alterations [7], in the secretion of prolactin which is normally under inhibitory control by the hypothalamic DA [21]. No studies are available yet on the secretion of G H and ACTH in untreated Parkinson's disease. It has been established that DA has a stimulating effect on G H secretion in humans [2, 15, 20, 21]. Its mechanism of action is unknown yet: either increased release of G R F or decreased liberation of somatostatin. In the rat, DA alters the release of somatostatin at the level of the hypophysial portal vessels [5]. No significant effect of DOPA and DA on ACTH secretion has been reported in normal subjects [8, 12,
18, 20]. The lack of changes in the hypothalamic content of CRF and G R F may be explained by the following. (1) There is no contact in the hypothalamus between dopaminergic neurons and CRF and/or G R F neurons. Indeed, the paraventricular nucleus of the hypothalamus where CRF cell bodies cell are concentrated is mainly innervated by adrenergic and noradrenergic fibers. No dopaminergic innervation has been demonstrated in this nucleus. G R F and DA-containing neurons are present in the same area of the hypothalamus, i.e. the mediobasal hypothalamus. However, there is no information on the histochemical relationship between both sets of neurons. (2) The dopaminergic system may influence the CRF and/or G R F release. However, it does not affect its synthesis. (3) As in experimental models, the decrease of DA in Parkinson's disease may not be sufficient to induce neuroendocrine changes. Indeed, when a 50~o reduction in hypothalamic DA is induced by the neonatal administration of sodium monoglutamate in rats [6], there is no change in ~melanocyte-stimulating hormone (~-MSH) secretion. However, this peptide is directly under the inhibitory influence by hypothalamic DA and an increased release was expected. (4) The lesions of dopaminergic fibers in Parkinson's disease is followed by an hyperactivity of the remaining dopaminergic neurons. The increased HVA/DA, D O P A C / D A and HVA + D O P A C / D A ratios show an acceleration of the utilization rate of DA [1]. Thus, the decrease in DA levels in the hypothalamus may be masked by the hyperactivity of unaltered dopaminergic neurons, taking into account the lack of changes in C R F and G R F concentrations. This work was supported by INSERM Grants C.R.E. 834007, C.R.E. 846020 and M.R.T. 84-H-1335. 1 Agid, Y., Javoy, F. and Glowinski, J., Hyperactivity of remaining dopaminergic neurones after partial destruction of the nigro-striatal dopaminergic system in the rat, Nature (New Biol.), 245 (1973) 150 151. 2 Bansal, S.A., Lee., L.A. and Woolf, P.D., Dopaminergic stimulation and inhibition of growth hormone secretion in normal man: studies of the pharmacologic specificity, J. Clin. Endocrinol. Metab., 53 (1981) 1273 1277. 3 Bloch, B., Gaillard, R.C., Brazeau, P., Lin, H.D. and Ling, N., Topographical and ontogenetic study
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