© 1990-91 S. Karger AG, Basel 0302-282X/90-91/0243-0129S2.75/0
Neuropsychobiology 1990-91 ;24:1 2 9 -134
Cholinergic Modulation of Growth Hormone-Releasing Hormone Effects on Growth Hormone Secretion in Dementia Giovanni Mnrialdo a , Francesco Zerbib, Ugo Filippi*. Pietro Toscab, Stefano Fonzi*, Enrica Di Paolob, Patrizia Costellia , Savino Porrob, Alessandro PollerP, Faustino Savoldib “Department of Endocrinological and Metabolic Sciences, University o f Genoa: bNeurological Clinic, Center o f Biological Neuropsychiatry. University o f Pavia. Italy
Key Words. Dementia • Growth hormone • Growth hormone-releasing hormone • Cholinergic system
Introduction The more common types of dementia, namely Alz heimer’s disease (AD), senile dementia of the Alzheimer type (SDAT). mixed Alzheimer’s and vascular dementia (MAVD) and multi-infarct dementia (MID) are due to different etiologies [1], The global disturbance of higher mental functions occurring in the various clinical forms of dementia may be considered to depend on largely overlapping central multisystem disorders, which in volve both aminergic and peptidergic neurotransmis sion. The impairment of the cholinergic neurotransmis sion might be considered a prominent neurochemical feature in AD. but also in SDAT and in MID [1.2], The most striking peptidergic changes in AD and SDAT con sist of a cortical decrease in corticotropin-releasing hor
mone and in somatostatin [3, 4], Fine [5] stresses the morphological changes affecting somatostatinergic neu rons. and suggests that this finding might have a possible bearing on the pathogenesis of AD. Both cholinergic and somatostatinergic systems are involved in the regulation of growth hormone (GH) secretion [6]. Cholinergic agents or cholinergic tonus enhancement by cholinesterase inhibitors enhance GH serum levels in humans [7, 8] and GH response to differ ent stimulatory agents, including GH-releasing hormone (GHRH) [8], Since atropine [9. 10] and pirenzepine [11], but not nicotine [10] block these effects, a muscarinic receptor is thought to be involved. The cholinergic effect on GH secretion is mediated by somatostatin: acetylcholine inhibits somatostatin release from rat hypothalami in vitro [ 12] and modulates in vivo
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Abstract. An impairment of cholinergic and somatostatinergic neurotransmission have been reported in demen tia. Both acetylcholine and somatostatin are involved in the regulation of growth hormone (GH) secretion. The effects of GH-releasing hormone (GHRH) 1-44 on GH release have been studied before and after the pretreatment with pyridostigmine or pirenzepine in subjects with senile dementia of the Alzheimer type, multi-infarct dementia and mixed dementia. The data have been compared with those obtained in an age-matched healthy control group. The GH response to GHRH is similar in the patients and in the controls, though the peak occurrence is significantly delayed in dementia. The cholinesterase inhibitor pyridostigmine enhances significantly the GH response to GHRH in both groups. The responses obtained in demented subjects are significantly larger than those found in the controls. Pirenzepine. a muscarinic receptor blocker, inhibits the GHRH effect on GH secretion in both groups. The findings may be interpreted in terms of an underlying impairment of the hypothalamic cholinergic neurotransmission, with an acetylcholine receptor supersensitivity that becomes apparent when the cholinergic tonus is enhanced by the inhibition of cholinesterase by pyridostigmine. No significant differences, due to the type of dementia, have been observed.
Murialdo/Zerbi/Filippi/Tosca/Fonzi/Di Paolo/Costelli/Porro/Polleri/Savoldi
somatostatin effects on GHRH-stimulated GH release in the rat [13, 14]. Changes in GH secretion have been reported in de mentia [15, 16]. The stimulatory effect of GHRH is pre served, though delayed in AD [4] and in SDAT [17]. In sight of these data, the present study aims to inves tigate the role of the cholinergic modulation on GHRH effects on GH secretion in patients with dementia. The group did not include AD subjects.
Materials and Methods Patients and Controls Eleven demented patients (9 females and 2 males) have been studied. Their mean age was 68.6 ± 2.0 (SEM) years. Dementia was diagnosed on the basis of the clinical history, neu rological examination, CT scan and neuropsychological tests. Patients underwent the Hamilton rating scale for depression [18], to exclude pseudodementia, and the Sandoz clinical assess ment geriatric to obtain information on their general behaviour. They exhibited a mini mental state [19] score lower than 24/30. To quantify the deterioration and its severity, the clinical de mentia rating (CDR) [20] was used. In this test, the rating 0 indi cates absence of deterioration, 0.5 a borderline condition, 1 a slight dementia, 2 a moderate dementia and 3 a severe dementia. In addition, all patients were tested by the Wechsler memory scale, the Wechsler adult intelligence scale, Raven’s matrices, the verbal fluence test and the token test for aphasia. The differentiation o f the various forms o f dementia was made by the Hachinski scale [21]. Patients with a score o f 4 or less than 4 were considered to have a primary degenerative dementia; those with a score o f 5 or 6 were considered to have a mixed dementia and those with a score o f 7 or higher were considered to have MID. Accordingly, 5 of the studied patients were found to have SDAT. 4 had MID and 2 MAVD. The essential data of the patients are summarized in table I. Eight healthy subjects (3 men and 5 women), aged between 52 and 81 years (mean age 68.7 ± 3.9 SEM) who had no clinical evi dence o f dementia or of other neuropsychiatrie disorders, were con sidered as the control group. Hormonal Studies All subjects had been fasting since the previous evening and were kept recumbent in the supine position during all tests. Around 8 a.m., a forearm vein was canulaicd 60 min before the test. The canula was maintained patent by a slow-running saline drip. The following tests were performed: Effect o f GURU Administration. GHRH (GRF 1-44, Sanofi Pharma AG, Basle). 1 pg/kg b.w., i.v., was administered in bolus at time O'. Blood samples were drawn at - 3 0 min. just before GHRH injection, and 10, 20. 30. 45. 60. 90 and 120 min thereafter. Effect o f Pyridostigmine Administration on GIIRH Stimulation. Pyridostigmine bromide (Mestinon, Hoffmann-La Roche, Italia), 120 mg, was given orally 30 min before GHRH ( 1 pg/kg b.w.) intra venous infusion. Blood samples were obtained 30 and 0 min before pyridostigmine administration and subsequently according to the above-mentioned GHRH study.
Table 1. Summary o f the studied demented patients Case No.
Sex
Age, years
Diagnosis
CDR
1 2 3 4 5 6 7 8 9 10 11
F F F F M F M F F F F
64 76 74 67 71 68 66 78 52 68 71
MAVD SDAT SDAT MID SDAT MAVD MID SDAT MID SDAT MID
2 i 2 1 1 3 2 2 i 2 1
Table 2. Mean ± SEM values o f GH peaks and areas under the curve (AUC) found in demented patients (DEM) and in healthy controls (Norm) after GHRH, G HR H plus pyridostigmine and GHRH plus pirenzepinc Peak ng/ml
AUC ng/ml X t
GHRH
DEM Norm
7.4± 1.6 8 .0 ± 2.9
4 I5 .7 ± 9 5 .0 412.4 ±139.6
GHRH + Pyridostigmine
DEM Norm
21.7 ± 11.6 13.0± 4.3
1690.8 ±398.6* 729.4 ±208.4
GHRH + Pirenzepine
DEM Norm
2.0 + 0.7 1.7 ± 0 .4
91.6 ± 20.0 126.0±29.8
* p < 0.05; Student’s t test for unpaired data.
Effect o f Pirenzepinc Administration on GURU Stimulation. Pirenzepinc (Gastrozepin, Boehringer Ingelheim. Italia). 0.6 mg/kg b.w. i.v., was administered as a bolus 5 min before GHRH admin istration. Blood samples were obtained as indicated for the GHRH study. Blood samples were rapidly centrifuged and the separated sera stored at - 2 0 °C until assayed. GH was determined by a double antibody RIA method with reagents obtained from Ares Serono, Milan. Italy. The evaluable GH range of concentration was between 0.25 and 30 ng/ml of serum. One nanogram equals 2 pIU WHO 66/217. Intra-assay and interassay coefficients o f variation were between 2.5 and 3.9% and 5.8 and 8.5%, respectively, for values ranging between 1.6 and 20.2 and 0.9 and 18.0 ng/ml, respectively. Analysis o f the Data Statistical analysis o f the data was made using the Student t test and the analysis of variance (ANOVA). The Mann-Whitney U test was used to assess the differences in the GH peak timing in the normal and demented subjects series.
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Cholinergic Modulation of GHRH Effects on GH Secretion in Dementia
Fig. 1. Effect of GHRH 1-44 ( I pg/kg b.w. i.v.) on GH secretion in demented subjects («land in healthy controls (o). Mean values ± SEM.
Results Effects o f GHRH Administration
A GH surge occurred after GHRH administration in demented patients and in normal controls (fig. 1). No significant difference was found between the two series (F = 1.56; p = 0.21; ANOVA). The GH peaks attained after GHRH and the secretory areas under the curve were not significantly different in the two groups (Stu dent’s t test for unpaired data; table 2. However, the GH peaks after GHRH were signifi cantly delayed in demented patients (mean ± SEM: 1,371.4 ± 160.1 s in normal controls and 2,836.3 ± 479.2 s in patients; z = 0.015, Mann-Whitney U test).
Fig. 2. Effect o f GHRH 1-44(1 gg/kg b.w. i.v.) on GH secretion after pretreatment with pyridostigmine bromide (120 mg os) in demented subjects (•) and in healthy controls (o). Mean values ± SEM.
individual variability of responses was found, so that only the areas under the curve, but not the peak values, were significantly larger in demented patients (table 2). Upon pyridostigmine pretreatment, the GH peak timing after GHRH administration was delayed in the two series in respect to what was observed with GHRH alone (mean ± SEM: 3,400.0 ± 570.5 s in the demented group and 2,100.0 ± 441.2 s in the control group), the difference being not significant either between the two series or in respect of the values found after GHRH alone (Mann-Whitney U test). Only in the demented group the GH levels attained after GHRH and pyridostigmine were significantly higher than those obtained after GHRH alone (ta ble 3).
Effects o f GHRH and Pyridostigmine Effects o f GHRH and Pirenzepine
Pirenzepine inhibited the effect of GHRH in both series (fig. 3). No significant changes occurred in the GH levels after pirenzepine and GHRH (F = 2.86; p = 0.0934; ANOVA) in the absolute peak values and in the areas under the curve (p > 0.05, Student’s t test for unpaired data; table 2) found in the demented and in the control group.
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Pyridostigmine was administered 30 min before the GHRH bolus. A small increase in the GH levels (not statistically different versus the basal values or between the two series) occurred in controls during the first part of the experiment when the effect of the cholinesterase inhibitor alone was present (fig. 2). The response to GHRH and pyridostigmine was sig nificantly greater in the demented subjects than in the controls (F = 7.93; p = 0.005; ANOVA) (fig. 2). A wide
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Table 3. ANOVA evaluation (F) o f GHRH effects on GH secre tion in the various experimental conditions in demented patients and in healthy controls Demented patients GHRH vs. GHRH + pyridostigmine 17.7: p < 0.01 GHRH vs. GHRH + pirenzepinc 40.6: p < 0.001 GHRH + pyridostigmine vs. GHRH + pirenzepinc 40.8: p < 0.001
In this instance, GH responses were significantly dif ferent in respect to the effect of GHRH alone or of GHRH and pyridostigmine (table 3). Hormonal Responses and the Clinical Picture
No difference was found between the responses ob served in patients with SDAT or MID and MAVD. The endocrine data were not significantly correlated to the CDR score.
Discussion In this study, concerning demented and age-matched healthy subjects, GH responses to GHRH were investi gated before and after pretreatment with a cholinesterase inhibitor, that enhances cholinergic transmission, or with a blocker of the muscarinic receptor. The demented patients we studied were diagnosed as having SDAT. MID and MAVD. Our study did not include subjects with AD. In our demented group, GH increase after the admin istration of GHRH was similar to the one occurring in the controls. However, the demented subjects exhibited a delay in the time of peak occurrence. A similarly delayed response has been reported by Nemeroff et al. [4] in AD and by Cacabelos et al. [17] in SDAT. GH response to GHRH after pyridostigmine admin istration was significantly larger in our patients, com pared to the changes occurring in controls.
0.08: p = 0.68 12.56;
p < 0.001
10.94: p = 0.001
Parati et al. [22] presented preliminary data indicat ing that the inhibition of acetylcholinesterase by pyrido stigmine induces a slight but significant increment of GH in some patients with SDAT, while in patients with AD. Davidson et al. [23] reported a normal GH response to the anticholinesterase agent edrophonium. Our data induce to believe that an impairment of cho linergic transmission involving also the hypothalamic presynaptic neurons exists in dementia, so that the increase in the available neurotransmitter induced by pyridostigmine reveals a muscarinic receptor supersensi tivity. Studying the effect of atropine on the GHRH-induced GH release, Casanueva et al. [24] have shown that the effect of muscarinic blockade is not exerted at the pitu itary level. A hypothalamic action seems probable. In our study, pirenzepine blunts the GHRH effect both in the normal and in the demented group. An inhi bition of the GH response to GHRH after atropine pre treatment has been reported in SDAT by Parati et al. [ 22].
The selective block of muscarinic receptor blunts the GH release induced by a variety of stimulations [9, 11, 25, 26]. It might be recalled that in another situation, namely anorexia nervosa, pirenzepine fails to antagonize GHRH in the stimulation of GH [27], In the few observed cases, we were not able to find differences in the responses to the various challenges either in connection with the forms of dementia studied, or when the patients were grouped according to CDR. It might be said that the impairment of the cholinergic inhibition of somatostatin modulation, which is believed to influence GH secretion, may be different in the var ious cases and not directly related to the clinical picture on which CDR is based. Kumar et al. [28], studying CSF acetylcholinesterase and choline in AD and SDAT pa
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Fig. 3. Effect of GHRH 1-44 (1 pg/kg b.w. i.v.) on GH secretion after pretreatment with pirenzepinc (0.6 rng/kg b.w. i.v.) in de mented patients (•) and in healthy controls (o). Mean values ± SEM.
Healthy controls
tients, were unable to find a correlation between these neurochemical indices and the clinical picture. In our study, we observed individual differences in the response of GH to GHRH in the various experimen tal conditions stressed by the large SEM values obtained. Similar findings have been described in normal cases [29, 30]. Mazza et al. [31] found that, occasionally, in normal subjects, the response to GHRH after pyridostig mine exhibited individual differences and sometimes was even absent. They explain their findings in terms of differences in the releasable GH pituitary pool, of differ ent responsiveness of GHRH and/or somatostatin recep tors and of variable effects of somatomedins. They point also out that the absorption of oral pyridostigmine may be erratic. Examples of different modulating effects on the GH response to GHRH and pyridostigmine, that may be due to changes in the somatostatinergic or in both the cholin ergic and somatostatinergic tonus, have been reported, respectively, during the night [32] and in obesity [33, 34], Parati et al. [22] found individual differences in GH response to GHRH in their demented subjects. Possibly, the degree of acetylcholine depletion should be considered as a cause of the dishomogeneous GH response in dementia. In conclusion, the stimulatory effect exerted by GHRH on GH secretion, its enhancement after pyrido stigmine and its inhibition by pirenzepine are preserved in dementia. However, in spite of the observed individ ual variations, the enhancement of GH response to GHRH induced by pyridostigmine we have found in demented subjects suggests the existence of a cholinergic supersensitivity related to the acetylcholine depletion.
Acknowledgment The authors thank Dr. Claudio Nava o f Midy S.p.A., Milan, Italy, for the kind supply o f GHRH (GRF 1-44 Sanofi).
References 1 Rossor MN: Dementia. Lancet 1982;ii: 1200—1204. 2 Coyle JT. Singer H, MacKinney M. Price D: Neurotransmitter specific alteration in dementing disorders: Insights from animal models. J Psychiatr Res 1984:18:501-512. 3 Davies P. Katzman R. Terry RD: Reduced somatostatin-like immunoreactivity in cerebral cortex from cases of Alzheimer’s disease and Alzheimer senile dementia. Nature I980;288:279280.
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4 Nemeroff CB. Krishnan KRR. Belkin BM. Ritchie JC. Clark C. Vale WW. Rivier EJ. Thorner MO: Growth hormone response to growth hormone releasing factor in Alzheimer's disease. Neuroendocrinologv 1989:50:663-666. 5 Fine AF: Peptides and Alzheimer’s disease. Nature 1986:319: 537-538. 6 Müller EE: Neural control of somatotrophic function. Physiol Rev 1987:67:962-1053. 7 Soulairac A, Schaub C, Franchimont P. Aymard N, Van Cauwenberge H: Etude de l’activation pharmacologique du pôle cen tral de l’axe hypothalamo-hypophysaire. Ann Endocrinol 1968: 29:45-54. 8 Massara F. Ghigo E. Demislis K, Tangolo D, Mazza E. Locatelli V. Mueller EE, Molinatti GM, Camanni F: Cholinergic involve ment in the growth hormone releasing hormone-induced growth hormone release: Studies in normal and acromegalic subjects. Neuroendocrinology 1986;43:670-675. 9 Bruni JF. Meites J: Effects of cholinergic drugs on growth hor mone release. Life Sci 1978:23:1351-1358. 10 Casanueva FF, Betti R, Ccfa GS, Mueller EE. Mantegazza P: Effect o f agonists and antagonists of cholinergic neurotransmis sion on growth hormone release in the dog. Acta Endocrinol (Copenh) 1983:103:15-20. 11 Massara F. Ghigo E. Goffi S. Molinatti GM. Mueller EE. Camanni F: Blockade o f hp-GRF-40-induced GH release in nor mal men by a cholinergic muscarinic antagonist. J Clin Endocri nol Metab 1984:59:1025-1027. 12 Richardson BS. Hollander SC. D’Eletto R, GreenleafWP. Thaw C: Acetylcholine inhibits the release o f somatostatin from rat hypothalamus in vitro. Endocrinology 1980:107:122-128. 13 Locatelli V, Torsello A. Rcdaclli M. Ghigo E, Massara F. Mueller EE: Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormone releasing hor mone in the rat: Evidence for mediation by somatostatin. J Endocrinol 1986;111:271-278. 14 Torsello A. Panzeri G, Cermenati P. Caroleo MC, Ghigo E, Camanni F. Mueller EE, Locatelli V: Involvement of the soma tostatin and cholinergic systems in the mechanism of growth hormone autofeedback regulation in the rat. J Endocrinol 1988; 117:273-281. 15 Davies P, Levy ML Rosenberg GS, Mathe A, Davis KL: Rela tionship between GH and cortisol and acetylcholine: A Possible neuroendocrine strategy for assessing a cholinergic defect: in Corkin S. Davis KL, Growdon JH. et al. (eds): Alzheimer’s Dis ease: Report on Progress in Research. New York. Raven Press. 1982. pp 204-210. 16 Christie JE, Whally LJ. Dick BH. Blackburn IM, Blackwood DHR, Fink G: Characteristic plasma hormone changes in Alz heimer's disease. Br J Psychiatry 1987;150:674-681. 17 Cacabelos R. Niigawa H. Ikemura Y. Yanagi Y. Tanaka S. Rodriguez-Arnao MD, Gomez Pan A, Nishimura T: GHRHinduced GH response in patients with senile dementia of the Alzheimer type. Acta Endocrinol (Copenh) 1988; 117:295-301. 18 Hamilton M: A rating scale for depression. J Neurol Neurosurg Psychiatry 1960;33:573-581. 19 Folstein MF. Folstein SE. McHugh PR: Mini mental state: A practical method for grading the cognitive state of patients for the clinician. .1 Psychiatr Res 1975;12:189-198. 20 Hughes CP. Berg L. Danziger WC. Coben LA. Martini RL: A new clinical scale for the staging o f dementia. Br J Psychiatry 1982;140:565-572.
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Cholinergic Modulation o f GHRH Effects on GH Secretion in Dementia
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21 Hachinski VC, MIT LD, Zilhka E: Cerebral blood flow in demen tia. Arch Neurol 1975;32:632-637. 22 Parati EA, Cocchi D, Lamperti E, Caraceni T, Mueller EE: Eval uation of hypothalamic somatostatinergic system in patients with senile dementia of the Alzheimer type (abstract). J Endocri nol Invest 1987:10:57. 23 Davidson M, Davis BM. Bastiaens L. Macaluso J, Aryan M. Ryan T, Davis KL: Growth hormone response to edrophonium in patients with Alzheimer’s disease and normal control sub jects. Am J Psychiatry 1988:145:1007-1009. 24 Casanueva FF, Villanueva L. Cabranes JA. Cabezas-Cerrato J. Fcrnandez-Cruz A: Cholinergic mediation o f growth hormone secretion elicited by arginine, clonidine and physical exercise in man. J Clin Endocrinol Metab 1984:59:526-530. 25 Delitala G. Frulio T, Pacifico A, Maiolo M: Participation of muscarinic receptors in glucagone- and arginine-mediated growth hormone secretion. J Clin Endocrinol Metab !982;55: 1231-1233. 26 Casanueva FF, Villanueva L, Dieguez C, Cabranes JA. Diaz Y, Szokc B. Scanlon MF, Schally AV, Fernandez-Cruz A: Atropine blockade of growth hormone (GH)-releasing hormone-induced GH secretion in man is not exerted at pituitary level. J Clin Endocrinol Metab 1986;62:186-191. 27 Tamai H, Komaki G, Matsubayashi S, Kobayashi N, Mori K. Nagakawa T, Truong MPM, Walter RM Jr. Kumagai LF: Effect of cholinergic muscarinic blockade on human growth hormone (GH(-releasing hormone-(I-44)-induccd GH secretion in an orexia nervosa. J Clin Endocrinol Metab 1990;70:738-741. 28 Kumar V. Giacobini E. Markwell S: CSF choline and acetylcho linesterase in early-onset vs. late-onset Alzheimer’s disease pa tients. Acta Neurol Scand 1989;80:461-466. 29 Gelato M. Pcscovitz OH. Cassorla F, Loriaux DL. Mcrriam GR: Dose-response relationships for the effects o f growth hormone releasing factor-(l-44)-NH2 in young adult men and women. J Clin Endocrinol Metab 1984;59:197-201.
30 Penalva A, Muruais C, Casanueva FF. Dieguez C: Effect of enhancement of cholinergic tone with pyridostigmine on the dose-response relationships of growth hormone (GH)-releasing hormone-induced GH secretion in normal subjects. .1 Clin Endo crinol Mctab 1990:70:324-327. 31 Mazza E, Ghigo E, Goffi S, Procopio M. Imperiale E, Arvat E, Bellone J. Boghcn MF. Mueller EE. Camanni F: Effect o f the potentiation o f cholinergic activity on the variability in individ ual GH response to GH-releasing hormone. J Endocrinol Invest 1989:12:795-798. 32 Ghigo E. Arvat E. Mazza E. Mondardini A, Cappa M. Mueller EE, Camanni F: Failure of pyridostigmine to increase both basal and GHRH-induced GH secretion in the night. Acta Endocrinol (Copenh) 1990;122:37-40. 33 Cordido F. Casanueva FF, Dieguez C: Cholinergic receptor acti vation by pyridostigmine restores growth hormone (GH) respon siveness to GH-rclcasing hormone administration in obese sub jects: Evidence for hypothalamic somatostatinergic participa tion in the blunted GH release in obesity. J Clin Endocrinol Mctab 1989;68:290-293. 34 Loche S. Pintor C. Cappa M, Ghigo E, Puggioni R. Locatclli V, Mueller EE: Pyridostigmine counteracts the blunted growth hor mone response to growth hormone-releasing hormone in obese children. Acta Endocrinol (Copenh) 1989:120:624-628.
Giovanni Murialdo, MD Department of Endocrinological and Metabolic Sciences University o f Genoa Vialc Benedetto XV 6 1-16132 Genoa (Italy)
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Neuropsychobiology 1990-91 ;24:1 2 9 -134
Cholinergic Modulation of Growth Hormone-Releasing Hormone Effects on Growth Hormone Secretion in Dementia Giovanni Mnrialdo a , Francesco Zerbib, Ugo Filippi*. Pietro Toscab, Stefano Fonzi*, Enrica Di Paolob, Patrizia Costellia , Savino Porrob, Alessandro PollerP, Faustino Savoldib “Department of Endocrinological and Metabolic Sciences, University o f Genoa: bNeurological Clinic, Center o f Biological Neuropsychiatry. University o f Pavia. Italy
Key Words. Dementia • Growth hormone • Growth hormone-releasing hormone • Cholinergic system
Introduction The more common types of dementia, namely Alz heimer’s disease (AD), senile dementia of the Alzheimer type (SDAT). mixed Alzheimer’s and vascular dementia (MAVD) and multi-infarct dementia (MID) are due to different etiologies [1], The global disturbance of higher mental functions occurring in the various clinical forms of dementia may be considered to depend on largely overlapping central multisystem disorders, which in volve both aminergic and peptidergic neurotransmis sion. The impairment of the cholinergic neurotransmis sion might be considered a prominent neurochemical feature in AD. but also in SDAT and in MID [1.2], The most striking peptidergic changes in AD and SDAT con sist of a cortical decrease in corticotropin-releasing hor
mone and in somatostatin [3, 4], Fine [5] stresses the morphological changes affecting somatostatinergic neu rons. and suggests that this finding might have a possible bearing on the pathogenesis of AD. Both cholinergic and somatostatinergic systems are involved in the regulation of growth hormone (GH) secretion [6]. Cholinergic agents or cholinergic tonus enhancement by cholinesterase inhibitors enhance GH serum levels in humans [7, 8] and GH response to differ ent stimulatory agents, including GH-releasing hormone (GHRH) [8], Since atropine [9. 10] and pirenzepine [11], but not nicotine [10] block these effects, a muscarinic receptor is thought to be involved. The cholinergic effect on GH secretion is mediated by somatostatin: acetylcholine inhibits somatostatin release from rat hypothalami in vitro [ 12] and modulates in vivo
Downloaded by: Temple University 155.247.166.234 - 8/2/2018 3:40:04 PM
Abstract. An impairment of cholinergic and somatostatinergic neurotransmission have been reported in demen tia. Both acetylcholine and somatostatin are involved in the regulation of growth hormone (GH) secretion. The effects of GH-releasing hormone (GHRH) 1-44 on GH release have been studied before and after the pretreatment with pyridostigmine or pirenzepine in subjects with senile dementia of the Alzheimer type, multi-infarct dementia and mixed dementia. The data have been compared with those obtained in an age-matched healthy control group. The GH response to GHRH is similar in the patients and in the controls, though the peak occurrence is significantly delayed in dementia. The cholinesterase inhibitor pyridostigmine enhances significantly the GH response to GHRH in both groups. The responses obtained in demented subjects are significantly larger than those found in the controls. Pirenzepine. a muscarinic receptor blocker, inhibits the GHRH effect on GH secretion in both groups. The findings may be interpreted in terms of an underlying impairment of the hypothalamic cholinergic neurotransmission, with an acetylcholine receptor supersensitivity that becomes apparent when the cholinergic tonus is enhanced by the inhibition of cholinesterase by pyridostigmine. No significant differences, due to the type of dementia, have been observed.
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somatostatin effects on GHRH-stimulated GH release in the rat [13, 14]. Changes in GH secretion have been reported in de mentia [15, 16]. The stimulatory effect of GHRH is pre served, though delayed in AD [4] and in SDAT [17]. In sight of these data, the present study aims to inves tigate the role of the cholinergic modulation on GHRH effects on GH secretion in patients with dementia. The group did not include AD subjects.
Materials and Methods Patients and Controls Eleven demented patients (9 females and 2 males) have been studied. Their mean age was 68.6 ± 2.0 (SEM) years. Dementia was diagnosed on the basis of the clinical history, neu rological examination, CT scan and neuropsychological tests. Patients underwent the Hamilton rating scale for depression [18], to exclude pseudodementia, and the Sandoz clinical assess ment geriatric to obtain information on their general behaviour. They exhibited a mini mental state [19] score lower than 24/30. To quantify the deterioration and its severity, the clinical de mentia rating (CDR) [20] was used. In this test, the rating 0 indi cates absence of deterioration, 0.5 a borderline condition, 1 a slight dementia, 2 a moderate dementia and 3 a severe dementia. In addition, all patients were tested by the Wechsler memory scale, the Wechsler adult intelligence scale, Raven’s matrices, the verbal fluence test and the token test for aphasia. The differentiation o f the various forms o f dementia was made by the Hachinski scale [21]. Patients with a score o f 4 or less than 4 were considered to have a primary degenerative dementia; those with a score o f 5 or 6 were considered to have a mixed dementia and those with a score o f 7 or higher were considered to have MID. Accordingly, 5 of the studied patients were found to have SDAT. 4 had MID and 2 MAVD. The essential data of the patients are summarized in table I. Eight healthy subjects (3 men and 5 women), aged between 52 and 81 years (mean age 68.7 ± 3.9 SEM) who had no clinical evi dence o f dementia or of other neuropsychiatrie disorders, were con sidered as the control group. Hormonal Studies All subjects had been fasting since the previous evening and were kept recumbent in the supine position during all tests. Around 8 a.m., a forearm vein was canulaicd 60 min before the test. The canula was maintained patent by a slow-running saline drip. The following tests were performed: Effect o f GURU Administration. GHRH (GRF 1-44, Sanofi Pharma AG, Basle). 1 pg/kg b.w., i.v., was administered in bolus at time O'. Blood samples were drawn at - 3 0 min. just before GHRH injection, and 10, 20. 30. 45. 60. 90 and 120 min thereafter. Effect o f Pyridostigmine Administration on GIIRH Stimulation. Pyridostigmine bromide (Mestinon, Hoffmann-La Roche, Italia), 120 mg, was given orally 30 min before GHRH ( 1 pg/kg b.w.) intra venous infusion. Blood samples were obtained 30 and 0 min before pyridostigmine administration and subsequently according to the above-mentioned GHRH study.
Table 1. Summary o f the studied demented patients Case No.
Sex
Age, years
Diagnosis
CDR
1 2 3 4 5 6 7 8 9 10 11
F F F F M F M F F F F
64 76 74 67 71 68 66 78 52 68 71
MAVD SDAT SDAT MID SDAT MAVD MID SDAT MID SDAT MID
2 i 2 1 1 3 2 2 i 2 1
Table 2. Mean ± SEM values o f GH peaks and areas under the curve (AUC) found in demented patients (DEM) and in healthy controls (Norm) after GHRH, G HR H plus pyridostigmine and GHRH plus pirenzepinc Peak ng/ml
AUC ng/ml X t
GHRH
DEM Norm
7.4± 1.6 8 .0 ± 2.9
4 I5 .7 ± 9 5 .0 412.4 ±139.6
GHRH + Pyridostigmine
DEM Norm
21.7 ± 11.6 13.0± 4.3
1690.8 ±398.6* 729.4 ±208.4
GHRH + Pirenzepine
DEM Norm
2.0 + 0.7 1.7 ± 0 .4
91.6 ± 20.0 126.0±29.8
* p < 0.05; Student’s t test for unpaired data.
Effect o f Pirenzepinc Administration on GURU Stimulation. Pirenzepinc (Gastrozepin, Boehringer Ingelheim. Italia). 0.6 mg/kg b.w. i.v., was administered as a bolus 5 min before GHRH admin istration. Blood samples were obtained as indicated for the GHRH study. Blood samples were rapidly centrifuged and the separated sera stored at - 2 0 °C until assayed. GH was determined by a double antibody RIA method with reagents obtained from Ares Serono, Milan. Italy. The evaluable GH range of concentration was between 0.25 and 30 ng/ml of serum. One nanogram equals 2 pIU WHO 66/217. Intra-assay and interassay coefficients o f variation were between 2.5 and 3.9% and 5.8 and 8.5%, respectively, for values ranging between 1.6 and 20.2 and 0.9 and 18.0 ng/ml, respectively. Analysis o f the Data Statistical analysis o f the data was made using the Student t test and the analysis of variance (ANOVA). The Mann-Whitney U test was used to assess the differences in the GH peak timing in the normal and demented subjects series.
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Cholinergic Modulation of GHRH Effects on GH Secretion in Dementia
Fig. 1. Effect of GHRH 1-44 ( I pg/kg b.w. i.v.) on GH secretion in demented subjects («land in healthy controls (o). Mean values ± SEM.
Results Effects o f GHRH Administration
A GH surge occurred after GHRH administration in demented patients and in normal controls (fig. 1). No significant difference was found between the two series (F = 1.56; p = 0.21; ANOVA). The GH peaks attained after GHRH and the secretory areas under the curve were not significantly different in the two groups (Stu dent’s t test for unpaired data; table 2. However, the GH peaks after GHRH were signifi cantly delayed in demented patients (mean ± SEM: 1,371.4 ± 160.1 s in normal controls and 2,836.3 ± 479.2 s in patients; z = 0.015, Mann-Whitney U test).
Fig. 2. Effect o f GHRH 1-44(1 gg/kg b.w. i.v.) on GH secretion after pretreatment with pyridostigmine bromide (120 mg os) in demented subjects (•) and in healthy controls (o). Mean values ± SEM.
individual variability of responses was found, so that only the areas under the curve, but not the peak values, were significantly larger in demented patients (table 2). Upon pyridostigmine pretreatment, the GH peak timing after GHRH administration was delayed in the two series in respect to what was observed with GHRH alone (mean ± SEM: 3,400.0 ± 570.5 s in the demented group and 2,100.0 ± 441.2 s in the control group), the difference being not significant either between the two series or in respect of the values found after GHRH alone (Mann-Whitney U test). Only in the demented group the GH levels attained after GHRH and pyridostigmine were significantly higher than those obtained after GHRH alone (ta ble 3).
Effects o f GHRH and Pyridostigmine Effects o f GHRH and Pirenzepine
Pirenzepine inhibited the effect of GHRH in both series (fig. 3). No significant changes occurred in the GH levels after pirenzepine and GHRH (F = 2.86; p = 0.0934; ANOVA) in the absolute peak values and in the areas under the curve (p > 0.05, Student’s t test for unpaired data; table 2) found in the demented and in the control group.
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Pyridostigmine was administered 30 min before the GHRH bolus. A small increase in the GH levels (not statistically different versus the basal values or between the two series) occurred in controls during the first part of the experiment when the effect of the cholinesterase inhibitor alone was present (fig. 2). The response to GHRH and pyridostigmine was sig nificantly greater in the demented subjects than in the controls (F = 7.93; p = 0.005; ANOVA) (fig. 2). A wide
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Table 3. ANOVA evaluation (F) o f GHRH effects on GH secre tion in the various experimental conditions in demented patients and in healthy controls Demented patients GHRH vs. GHRH + pyridostigmine 17.7: p < 0.01 GHRH vs. GHRH + pirenzepinc 40.6: p < 0.001 GHRH + pyridostigmine vs. GHRH + pirenzepinc 40.8: p < 0.001
In this instance, GH responses were significantly dif ferent in respect to the effect of GHRH alone or of GHRH and pyridostigmine (table 3). Hormonal Responses and the Clinical Picture
No difference was found between the responses ob served in patients with SDAT or MID and MAVD. The endocrine data were not significantly correlated to the CDR score.
Discussion In this study, concerning demented and age-matched healthy subjects, GH responses to GHRH were investi gated before and after pretreatment with a cholinesterase inhibitor, that enhances cholinergic transmission, or with a blocker of the muscarinic receptor. The demented patients we studied were diagnosed as having SDAT. MID and MAVD. Our study did not include subjects with AD. In our demented group, GH increase after the admin istration of GHRH was similar to the one occurring in the controls. However, the demented subjects exhibited a delay in the time of peak occurrence. A similarly delayed response has been reported by Nemeroff et al. [4] in AD and by Cacabelos et al. [17] in SDAT. GH response to GHRH after pyridostigmine admin istration was significantly larger in our patients, com pared to the changes occurring in controls.
0.08: p = 0.68 12.56;
p < 0.001
10.94: p = 0.001
Parati et al. [22] presented preliminary data indicat ing that the inhibition of acetylcholinesterase by pyrido stigmine induces a slight but significant increment of GH in some patients with SDAT, while in patients with AD. Davidson et al. [23] reported a normal GH response to the anticholinesterase agent edrophonium. Our data induce to believe that an impairment of cho linergic transmission involving also the hypothalamic presynaptic neurons exists in dementia, so that the increase in the available neurotransmitter induced by pyridostigmine reveals a muscarinic receptor supersensi tivity. Studying the effect of atropine on the GHRH-induced GH release, Casanueva et al. [24] have shown that the effect of muscarinic blockade is not exerted at the pitu itary level. A hypothalamic action seems probable. In our study, pirenzepine blunts the GHRH effect both in the normal and in the demented group. An inhi bition of the GH response to GHRH after atropine pre treatment has been reported in SDAT by Parati et al. [ 22].
The selective block of muscarinic receptor blunts the GH release induced by a variety of stimulations [9, 11, 25, 26]. It might be recalled that in another situation, namely anorexia nervosa, pirenzepine fails to antagonize GHRH in the stimulation of GH [27], In the few observed cases, we were not able to find differences in the responses to the various challenges either in connection with the forms of dementia studied, or when the patients were grouped according to CDR. It might be said that the impairment of the cholinergic inhibition of somatostatin modulation, which is believed to influence GH secretion, may be different in the var ious cases and not directly related to the clinical picture on which CDR is based. Kumar et al. [28], studying CSF acetylcholinesterase and choline in AD and SDAT pa
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Fig. 3. Effect of GHRH 1-44 (1 pg/kg b.w. i.v.) on GH secretion after pretreatment with pirenzepinc (0.6 rng/kg b.w. i.v.) in de mented patients (•) and in healthy controls (o). Mean values ± SEM.
Healthy controls
tients, were unable to find a correlation between these neurochemical indices and the clinical picture. In our study, we observed individual differences in the response of GH to GHRH in the various experimen tal conditions stressed by the large SEM values obtained. Similar findings have been described in normal cases [29, 30]. Mazza et al. [31] found that, occasionally, in normal subjects, the response to GHRH after pyridostig mine exhibited individual differences and sometimes was even absent. They explain their findings in terms of differences in the releasable GH pituitary pool, of differ ent responsiveness of GHRH and/or somatostatin recep tors and of variable effects of somatomedins. They point also out that the absorption of oral pyridostigmine may be erratic. Examples of different modulating effects on the GH response to GHRH and pyridostigmine, that may be due to changes in the somatostatinergic or in both the cholin ergic and somatostatinergic tonus, have been reported, respectively, during the night [32] and in obesity [33, 34], Parati et al. [22] found individual differences in GH response to GHRH in their demented subjects. Possibly, the degree of acetylcholine depletion should be considered as a cause of the dishomogeneous GH response in dementia. In conclusion, the stimulatory effect exerted by GHRH on GH secretion, its enhancement after pyrido stigmine and its inhibition by pirenzepine are preserved in dementia. However, in spite of the observed individ ual variations, the enhancement of GH response to GHRH induced by pyridostigmine we have found in demented subjects suggests the existence of a cholinergic supersensitivity related to the acetylcholine depletion.
Acknowledgment The authors thank Dr. Claudio Nava o f Midy S.p.A., Milan, Italy, for the kind supply o f GHRH (GRF 1-44 Sanofi).
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30 Penalva A, Muruais C, Casanueva FF. Dieguez C: Effect of enhancement of cholinergic tone with pyridostigmine on the dose-response relationships of growth hormone (GH)-releasing hormone-induced GH secretion in normal subjects. .1 Clin Endo crinol Mctab 1990:70:324-327. 31 Mazza E, Ghigo E, Goffi S, Procopio M. Imperiale E, Arvat E, Bellone J. Boghcn MF. Mueller EE. Camanni F: Effect o f the potentiation o f cholinergic activity on the variability in individ ual GH response to GH-releasing hormone. J Endocrinol Invest 1989:12:795-798. 32 Ghigo E. Arvat E. Mazza E. Mondardini A, Cappa M. Mueller EE, Camanni F: Failure of pyridostigmine to increase both basal and GHRH-induced GH secretion in the night. Acta Endocrinol (Copenh) 1990;122:37-40. 33 Cordido F. Casanueva FF, Dieguez C: Cholinergic receptor acti vation by pyridostigmine restores growth hormone (GH) respon siveness to GH-rclcasing hormone administration in obese sub jects: Evidence for hypothalamic somatostatinergic participa tion in the blunted GH release in obesity. J Clin Endocrinol Mctab 1989;68:290-293. 34 Loche S. Pintor C. Cappa M, Ghigo E, Puggioni R. Locatclli V, Mueller EE: Pyridostigmine counteracts the blunted growth hor mone response to growth hormone-releasing hormone in obese children. Acta Endocrinol (Copenh) 1989:120:624-628.
Giovanni Murialdo, MD Department of Endocrinological and Metabolic Sciences University o f Genoa Vialc Benedetto XV 6 1-16132 Genoa (Italy)
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