Pergamon Psychoneuroendocrinology, Vol. 22, No. 7, pp. 531-538. 1997

© 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain

0306-4530/97 $17.00 + .00

PIh S0306-4530(97)00055-3

D I F F E R E N T C O N T R O L M E C H A N I S M S OF G R O W T H H O R M O N E (GH) S E C R E T I O N B E T W E E N y-AMINO- A N D

y - H Y D R O X Y - B U T Y R I C ACID: N E U R O E N D O C R I N E E V I D E N C E IN P A R K I N S O N ' S D I S E A S E

Riccardo Volpi, t Paolo Chiodera, t Paolo Caffarra, 2 Augusto Scaglioni, 3 Antonel la Saccani t and Vittorio Coiro t

ZDepartment of Internal Medicine, School of Medicine, University of Parma, Via A. Gramsci, 14. 1-43100 Parma, Italy; 2Institute of Neurology, School of Medicine, University of Parma, Via del

Quartiere 4, 1-43100 Parma, Italy; and 3Division of Neurology, Hospital of Fidenza, Via Borghesi I, Fidenza, Italy

(Received 5 December 1996; in final form 17 June 1997)

SUMMARY

The observation that baclofen stimulates growth hormone (GH) secretion in normal men, but not in parkinsonian patients led us to test the GH releasing effect of other y-amino-butyric acid (GABA)ergic agents with different mechanisms of action in Parkinson's disease. For this purpose 10 normal men and 10 de novo parkinsonian patients were tested with sodium valproate (800 mg PO), 7-hydroxybutyric acid (GHB) (25 mg/kg body weight PO) and baclofen (10 mg PO). All drugs induced a significant increment in serum GH levels in the normal controls. On the other hand, GH secretion in parkinsonian patients did not change after baclofen or sodium valproate administration. whereas it showed normal responsiveness to GHB. These data suggest that the mechanism underlying the GH response to GHB is different from that (or those) mediating sodium valproate and/or baclofen action. In addition, the former, but not the latter mechanism appears to be preserved in the parkinsonian brain. © 1997 Elsevier Science Ltd

Keywords--Growth hormone; GABA; y-hydroxy-butyric acid; Parkinson's disease.

INTRODUCTION

Both sodium valproate, which enhances endogenous y-aminobutyric (GABA)ergic tone through inhibition of GABA transaminase (Godin et al., 1969) and by a direct action at pre- and post-synaptic GABA receptor sites (Johnston, 1984), and baclofen (fl-(p-chlorophe- nyl)-y-aminobutyric acid), a GABAB receptor agonist (Fox et al., 1978), are well-known stimulators of growth hormone (GH) secretion in normal men (Racagni et al., 1982). The GH-releasing effect of GABAergic drugs is thought to be exerted at hypothalamic level through mediation of dopaminergic neurotransmission (Koulu et al., 1980; Muller, 1987;

Address correspondence and reprint requests to: Riccardo Volpi, MD, Istituto di Clinica Medica Generale e Terapia Medica, Via A. Gramsci 14, 1-43100 Parma, Italia (Tel/Fax: 0039 521 290786; E-mail: volpi@ipruniv.cce.unipr.it)

531

532 R. Volpi et al.

Racagni et al., 1982). In agreement with this hypothesis, baclofen was unable to stimulate GH secretion in Parkinson's disease (Volpi et al., 1991).

7-hydroxybutyric acid (GHB) is a breakdown product of GABA with neurotransmitter or neuromodulator activity at various levels in the central nervous system (Benavides et al., 1982; Da Prada and Keller, 1976; Snead, 1978). Several neuropharmacological and neurophysiological effects have been described after GHB administration (for review see Snead, 1978), and both similarities and differences have been reported when the effects of the administration of GABAergic drugs and GHB have been compared. Impulse flow in dopaminergic neurones is known to be inhibited by GABA (Keller et al., 1976), baclofen (Da Prada and Keller, 1976) and GHB (Roth et al., 1973). However, the mechanism of action of baclofen and GHB appears to differ from that of GABA. In fact, the administration of either baclofen or GHB in normal rats increased homovanillic acid and dopamine concentrations in the corpus striatum and limbic nucleus (Da Prada and Keller, 1976), suggesting that these substances not only reduce impulse flow in dopaminergic neurones, but also stimulate dopaminergic synthesis (Da Prada and Keller, 1976; Gessa et al., 1968; Keller et al., 1976; Roth et al., 1973).

Like sodium valproate and baclofen, GHB is able to stimulate GH secretion in normal men (Gerra et al., 1994; Racagni et al., 1982; Vescovi and Coiro, 1995).

GABAergic function might play an important role in Parkinson's disease, in fact, GABA concentrations in the substantia nigra have been found to be positively correlated with patients' disability (Rinne et al., 1984). In view of these observations, a better understanding of GABAergic activity in the parkinsonian brain is needed.

In an attempt to gain a better insight into GABA and GABAergic drug mechanisms of action and into the neurotransmitter disturbance affecting Parkinson disease, we tested the GH response to GHB in male patients affected by de novo Parkinson's disease. At different occasions, patients were also tested with sodium valproate and baclofen.

METHODS

Ten men (54-68 years old; mean ± SE: 61.5 ± 1.73) affected by de novo Parkinson's disease were randomly chosen to participate in this study. The Ethical Committee for Clinical Trials of the Institute of Internal Medicine of the University of Parma reviewed the protocol of the study and the subjects gave their informed consent. The diagnosis of Parkinson's disease had been established by the presence of clinical features (resting tremor, bradykinesia and rigidity). The mean (±SE) duration of the disease was 19.3 ± 4.2 months. The severity of the medical stage, assessed with the Hoehn and Yahr (1967) scale, was 1.7 ± 0.10. Patients with secondary parkinsonism or with clinical evidence of other major neuroanatomical system involvements were excluded. All patients were assessed with the Hospital Anxiety and Depression scale (Zigmond and Snaith, 1983), the Research Diagnostic Criteria (Spitzer et al., 1978) and with the Hamilton (1960) depression rating scale. None of the subjects were affected by depression. All of them were within 10% of their ideal body weight. They were fully ambulatory, well nourished and there was no clinical or laboratory evidence of renal, neoplastic or endocrine-metabolic diseases.

Ten normal weight male volunteers aged 56--64 years (mean ± SE: 61.2 ± 0.80) were tested as controls.

All subjects were tested with ~,-hydroxybutyric acid (GHB), sodium valproate, baclofen and placebo. Tests were carried out in random order at weekly intervals.

GABAergic Control of GH Secretion in Parkinson's Disease 533

Experimental Procedure At 0900h on the experimental day, a 19-gauge intravenous indwelling needle was inserted

into an antecubital vein of the subjects who were lying in a recumbent position and who had fasted since the previous evening. The needle was kept patent with a slow infusion of normal saline. A basal blood specimen was taken 15 rain after the insertion of the cannula (time 0).

GHB Test. GHB (Alcover, Laboratorio Farmaceutico CT, Sanremo, Italy) at a dose of 25 mg/kg body weight was administered PO at time 0, just after blood sampling. Further blood samples were taken at 15, 30, 45, 60, 90 and 120 min after GHB administration.

Sodium Valproate Test. At time 0, 800 mg sodium valproate (Depakin, Sigma-Tau, Rome, Italy) was administered PO. Further blood specimens were taken at 30, 60, 90, 120, 150 and 180 min after sodium valproate administration.

Baclofen Test. At time 0, baclofen (Lioresal, Ciba-Geigy, Italy) was administered at a dose of 10 mg just after blood sampling. Further blood samples were taken at time 30, 60, 90, 120 and 150 min after baclofen administration.

Control Test. A placebo was administered at time 0 after blood sampling. Further samples were taken at time 15, 30, 45, 60, 90, 120, 150 and 180 rain after placebo administration.

Sampling time points were established according to previous studies of the effects of GHB (Gerra et al., 1994), sodium valproate (Elias et al., 1982) and baclofen (Koulu et al., 1979) on GH secretion.

Blood pressure and heart rate were monitored at each sampling time.

Assays Blood samples were collected and centrifuged cold; serum was stored at -20 :C until

assayed. Serum GH concentrations were measured with a specific RIA, using materials supplied by Ares Serono (Milan, Italy). All samples were analyzed in the same assay and in duplicate. Sensitivity, intra-assay and inter-assay coefficients of variation were: 0.5 ng/ml, 3.l % and 7.0%, respectively.

Statistical analysis was performed with the Mann-Whitney U-test and analysis of variance (ANOVA) or analysis of covariance (ANCOVA), as appropriate. ANCOVA was performed to exclude the possible interference of variations in severity of the medical stage of Parkinson's disease in the analysis and interpretation of the various tests. Data are reported as the mean ± SE.

RESULTS

In all tests, similar basal serum GH levels were observed in the normal controls and parkinsonian patients (Fig. la, b, c and d). No significant changes (vs basal value) were observed in any group after placebo administration (Fig. ld).

The administration of sodium valproate (Fig. la), baclofen (Fig. lb) or GHB (Fig. lc) induced significant increments in serum GH concentrations in the normal controls (p < .01 vs baseline: sodium valproate test; p < . 0 h baclofen test; p<.01: GHB test). In the parkinsonian patients, the GH response to GHB (p < .01 vs baseline) did not significantly differ from that observed in the normal controls (Fig. lc). In contrast, GH secretion was not stimulated by sodium valproate (Fig. la) or baclofen (Fig. lb) in parkinsonian patients (NS

534 R. Volpi et al.

(a) 6

5

4

3

(b) 5

4

3

2

Sodium Valproate 800 mg p,o.

~ ~ ~ k l S o d i u m V a l p r o a t e Sodium V a l p r o a t e

I 1 I l L I 1 0 30 60 90 120 150 180

_ B a c l o f e n 10 mg p.o.

1 I h I I I 0 30 60 90 120 150

(c)

?_

G H B 25 mg/kg p.o.

-

h h J h 1 1 L I L 0 15 30 45 60 75 90 105 120

(d) 6

"~ 4

3

_ P l a c e b o

_1 • - m - Park P l a c e b o ---43-- N.C. P l a c e b o

I 1 I i 1 I I 0 30 60 90 120 150 180

min.

Fig. 1. Effect o f sodium valproate (a), baclofen (b), 7-hydroxy-butyric acid (GHB) (c) and placebo (d) on serum GH levels in parkinsonian patients and in normal controls. Each point represents the mean -~ SE of 10 observations.

GABAergic Control of GH Secretion in Parkinson's Disease 535

vs baseline and vs control test). During sodium valproate and baclofen tests serum GH levels were significantly lower in parkinsonian patients than in normal controls (F = 21.74, p < .01 ; F = 19.98, p < .01, respectively).

Results of GHB, sodium valproate and baclofen tests did not change when the data were evaluated with ANCOVA to exclude the possible effects of the variation in severity of the medical stage of Parkinson's disease.

No side effects were observed after sodium valproate, baclofen or GHB treatment.

DISCUSSION

In vitro studies did not show any direct effects of GABA or GABAergic drugs on GH secretion from the pituitary gland (Flock et al., 1984; Racagni et al., 1982; Vijayan and McCann, 1978). Therefore, the stimulatory effect of GABA on GH secretion is thought to be mediated at hypothalamic level by stimulation of GH-RH (Growth hormone releasing hormone) or inhibition of somatostatin release into portal circulation (Gamse et al., 1980; Murakami et al., 1985; Stryker et al., 1986; Takahara et al., 1980). This latter hypothesis is more likely and is supported by the observation that GABA agonists induced somatostatin accumulation in the hypothalamus, concomitantly to the increase in plasma GH levels (Racagni et al., 1982). In view of the modulatory role of GABA on monoamine neurotransmissions (Rinne et al., 1989), which in turn exert an important role in the inhibitory control of somatostatin secretion, a monoaminergic (particularly dopaminergic) mediation of the GABAergic stimulatory action on GH secretion has been suggested (Laakmann et al., 1982; Racagni et al., 1982). In fact, L-dopa has been found able to counteract the GABAergic derivative-induced somatostatin accumulation at hypothalamic level, which is consequent to inhibition of somatostatin release (Takahara et al., 1980). In agreement with this hypothesis, dopaminergic alterations in limbic-hypothalamic neuro- transmitter systems are thought to underlie the inability of baclofen to stimulate GH secretion in schizophrenia (Monteleone et al., 1988; Risch and Judd, 1987; Takahara et al., 1980) and Parkinson's disease (Volpi et al., 1991).

The fact that baclofen stimulates GH release and the discovery of [3H]baclofen binding sites in rat hypothalamus (Bowery, 1982) suggested that GABA might stimulate GH secretion through GABAB receptor activation (Fox et al., 1978; Gehlert et al., 1985). However, in contrast with this hypothesis, studies in rats have shown that the GH- stimulating effect of an intraventricular administration of GABA is completely antagonized by the GABAA receptor antagonist bicuculline (Racagni et al., 1982).

The results of the present study show that like baclofen (Volpi et al., 1991), sodium valproate is unable to stimulate GH secretion in parkinsonian patients, suggesting a common mechanism of action of these drugs on GH secretion. Since sodium valproate enhances the endogenous GABAergic tone (Godin et al., 1969), whereas baclofen stimulates GABAB receptors (Fox et al., 1978; Bowery, 1982), the finding that neither sodium valproate nor baclofen administration stimulates GH secretion in parkinsonian patients might represent indirect evidence for the hypothesis that GABA stimulates GH secretion through B receptor activation and that this pathway is defective in Parkinson's disease. However, we cannot exclude that the activation of either GABAA or GABAB receptors independently stimulates GH secretion and that both receptor-mediated neurotransmissions are defective in the parkinsonian brain. Another possibility concerns the inability of dopaminergic neurotrans- mission to mediate GABAergic action in Parkinson's disease.

536 R. Volpi et al.

Surprisingly, in contrast to the other GABAergic drugs, GHB maintained its GH releasing capacity in parkinsonian patients. This discrepancy suggests that the stimulatory effect of GHB on GH secretion is not mediated by a GABAergic pathway, but rather by stimulation of the specific GHB receptors that have been found at various levels in the central nervous system (Benavides et al., 1982). In addition, we may suppose that GHB action on GH secretion is exerted through the mediation of other neurotransmitter(s) than dopamine at hypothalamic level, or through the direct stimulation of the somatotrophs at the pituitary level.

In view of the important stimulatory role played by the serotonergic system in the control of GH secretion, the effect of GHB might be mediated by serotonergic neurotransmission. In fact the involvement of serotonergic neurotransmission has been supposed in mediation of GHB-stimulated GH secretion (Spano and Przengalinski, 1973). However, the possibility that the serotonergic control of GH secretion is preserved in Parkinson's disease is unlikely, because our previous study showed an impaired GH response to serotonergic stimulation in parkinsonian patients (Volpi et al., 1997). In addition, cholinergic neurotransmission plays a role in the control of GH secretion through inhibition of somatostatin release from the hypothalamus (Richardson et al., 1980). It is possible that GHB stimulates GH secretion through a cholinergic pathway, because GHB has profound effects on cholinergic neurotransmission in the brain (Giarman and Schmidt, 1963; Sethy et al., 1976) and high concentrations of GHB receptors have been found in nerve-ending fractions rich in acetylcholine (Maitre et al., 1983). This hypothesis is supported by observations at autopsy that muscarinic receptor density does not decrease in caudate nucleus, putamen, pallidum, substantia nigra and cerebral cortex in the parkinsonian brain (Nishino et al., 1988). In partial agreement with these findings, in autopsied samples other authors showed that muscarinic receptor binding is unaltered in the hippocampus and increased in the frontal and temporal cortex of parkinsonian patients (Rinne et al., 1989).

Whatever the underlying mechanism of the GH response to GHB is, our study shows that it is unaltered in the parkinsonian brain, and indicates the interest and necessity of further studies in this area.

REFERENCES

Benavides, J., Rumigny, J. F., Bourguigon, J. J., Casu, C., Wermuth, C. G., Mandel, P., Vincendon, G. and Maitre, M. (1982) High affinity binding site for ~-hydroxybutyric acid in rat brain. Life Science 30, 953-961.

Bowery, N. G. (1982) Baclofen: 10 years on. Trends in Pharmacological Sciences 3, 400--403. Da Prada, M. and Keller, H. H. (1976) Baclofen and ";-hydroxybutyrate: similar effects on cerebral

dopamine neurons. Life Science 19, 1253-1264. Elias, A. N., Valenta, L. J., Szekeres, A~ V. and Grossman, M. K. (1982) Regulatory role of gamma-

aminobutyric acid in pituitary hormone secretion. Psychoneuroendocrinology 7, 15-30. Flock, J., Acs, Z., Makara, G. B. and Erdo, S. L. (1984) Site of 7-aminobutyric acid (GABA)-mediated

inhibition of growth hormone secretion in the rat. Neuroendocrinology 39, 510-516. Fox, S., Krnjevic, K., Norris, N. E., Puil, E. and Werman, R. (1978) Action of baclofen on mammalian

synaptic transmission. Neuroscience 3, 495--497. Gamse, R., Vaccaro, D. E., Gamse, G., Dipace, M., Fox, T. O. and Leeman, S. E. (1980) Release of

immunoreactive somatostatin from hypothalamic ceils in culture: inhibition by )'-aminobutyric acid. Proceedings of the National Academy of Science 77, 5552-5556.

Gehlert, D. R., Yamamura, H. I. and Wamsley, J. K. (1985) Gamma-aminobutyric acid B receptors in the rat brain. Quantitative autoradiographic localization using [3H]baclofen. Neuroscience Letters $6, 183-187.

GABAergic Control of GH Secretion in Parkinson's Disease 537

Gerra, G., Marcato, A., Fertonani A.ffini, G., Avanzini, P., Maestri, D., Fontanesi, B., Caccavari, R. and Delsignore, R. (1994) Gamma hydroxybutyric acid (GHB) and neuroendocrine function in humans. Neuroendocrinology Letters 7, 55--63.

Gessa, G. L., Crabai, F., Vargiu, L. and Spanu, P. F. (1968) Selective increase of brain dopamine induced by )'-hydroxybutyrate: study of the mechanism of action. Journal of Neurochemistry 15, 377-381.

Giarman, N. J. and Schmidt, K. F. (1963) Some neurochemical aspects of the depressant action of 3,- butyrolactone in the central nervous system. British Journal of Pharmacology 20, 563-568.

Godin, Y., Heiner, L., Mark, J. and Mandez, P. (1969) Effects of di-N-propylacetate, an anticonvulsant compound of GABA metabolism. Journal of Neurochemistry 16, 869-873.

Hamilton, M. (1960) A rating scale for depression. Journal of Neurology and Neurosurgical Psychiatry 23, 56-62.

Hoen, M. M. and Yahr, M. D. (1967) Parkinsonism: onset progression and mortality. Neurology 17, 427-441.

Johnston, D. (1984) Valproic acid: update on its mechanisms of action. Epilepsia 25, 46--52. Keller, H. H., Schaffner, R. and Haefely, W. (1976) Interaction of benzodiazepines with neuroleptics

at central dopamine neurons. Archives of Pharmacology 294, 1-7. Koulu, M., Lammintausta, R. and Dahlstrom, S. (1979) Stimulatory effect of acute baclofen

administration on human growth hormone secretion. Journal of Clinical Endocrinolo~' and Metabolism 48, 1038-1040.

Koulu, M., Lammintausta, R. and Dahlstrom, S. (1980) Effects of some gamma-aminobutyric acid (GABA)-ergic drugs on the dopaminergic control of human growth hormone secretion. Journal of Clinical Endocrinology and Metabolism 51, 124--129.

Laakmann, G., Treusch, J., Eicheier, A., Schmauss, M., Treusch, U. and Wahlster, U. (1982) Inhibitory effect of phentolamine on diazepam-induced growth hormone secretion and lack of effect of diazepam on prolactin secretion in man. Psychoneuroendocrinology 7, 135-139.

Maitre, M., Rumigny, J. F., Cash, C. and Mandel, P. (1983) Subcellular distribution of gammahydroxybutyrate binding sites in the rat. Principal localization in the synaptosomal fraction. Biochemical and Biophysical Research Communications 110, 262-265.

Monteleone, P., Maj, M., lovino, M., Forziati, D., Veltro, F. and Steardo, L. (1988) Baclofen-induced growth hormone secretion is blunted in chronic schizophrenic: neuroendocrine evidence for a GABA disturbance in schizophrenia. Psychiatry Research 26, l-9.

Muller, E. E. (1987) Neural control of somatotropic function. Physiological Review 67, 962-1053. Murakami, Y., Kato, Y., Kabayama, Y., Tojo, K., lnoue, T. and lmura, H. (1985) Involvement of

growth releasing factor in growth hormone secretion induced by gamma-aminobutyric acid in conscious rats. Endocrinology 117, 787-789.

Nishino, N., Fujiwara, H., Noguchi-Kuno, S. A. and Tanaka, C. (1988) GABAA receptor but not muscarinic receptor density was decreased in the brain of patients with Parkinson's disease. Japanese Journal of Pharmacology 48, 331-339.

Racagni, G., Apud, J. A., Cocchi, D., Locatelli, V. and Muller, E. E. (1982) GABAergic control of anterior pituitary hormone secretion. Life Science 31, 823-838.

Richardson, S. B., Hollander, C. S., D'Eletto, R., Greenleaf, P. W. and Thaw, C. (1980) Acetylcholine inhibits the release of somatostatin from the rat hypothalamus in vitro. Endocrinology" 107, 122- 129.

Rinne, J. O., Lonnberg, P., Marjamak, P. and Rinne, U. K. (1989) Brain muscarinic receptor subtypes are differently affected in Alzheimer's disease and Parkinson's disease. Brain Research 483, 402- 406.

Risch, S. C. and Judd, L. L. (1987) Provocative challenges of growth hormone and prolactin secretion in schizophrenic and effective disorders. In Clinical Psychoneuroendocrinology, eds C. Nemerof and P. P. Loosen, pp. 3-37. Guilford Press, New York.

Roth, R. H., Waiters, J. R. and Aghajanian, G. K. (1973) Effect of impulse flow-out on the release and synthesis of dopamine in the rat striatum. In Frontiers in Cathecolamine Research, eds S. H. Snyder and E. Usdin, pp. 567-574. Pergamon Press, New York.

Sethy, V. H., Roth, R. H. and Waiters, J. R. (1976) Effect of anesthetic doses of ~,-hydroxybutyrate on the acetylcholine content of the rat brain. Archives of Pharmacology 295, 9-14.

Snead, O. C. (1978) Gamma hydroxybutyrate in the monkey. Neurology 28, 636--642.

538 R. Volpi et al.

Spano, P. F. and Przengalinski, E. (1973) Stimulation of serotonin synthesis by anesthetic and non- anesthetic doses of gamma-hydroxybutyrate. Pharmacological Research Communications 5, 55-59.

Spitzer, R, L., Endicott, J. and Robins, E. (1978) Research diagnostic criteria: rationale and reliability. Archives of General Psychiatry 35, 773-782.

Stryker, T. D., Conlin, T. and Reichlin, S. (1986) Influence of a benzodiazepine, imidazolam and gamma-aminobutyric acid (GABA) on basal somatostatin secretion from cerebral and diencephalic neurons in dispersed cell culture. Brain Research 362, 339-343.

Takahara, J., Yunoki, S., Hosogi, H., Yakushiji, W., Kageyaha, J. and Ofuji, T. (1980) Concomitant increase in serum growth hormone and hypothalamic somatostatin in rats after injection of gamma- aminobutyric acid, aminooxalacetic and/or gamma-hydroxybutyric acid. Endocrinology 106, 343- 347.

Vescovi, P. P. and Coiro, V. (1995) Gamma-hydroxybutyric acid is unable to stimulate growth hormone secretion in early abstinent alcoholic men. Alcologia 7, 129-132.

Vijayan, E. E. and McCann, S. M. (1978) Effects of intraventricular injection of gamma-aminobutyric acid (GABA) on plasma growth hormone and thyrotropin in conscious ovariectomized rats. Endocrinology 103, 1888-1893.

Volpi, R., Caffarra, P., Scaglioni, A., Boni, S., Saginario, A., Chiodera, P. and Coiro, V. (1997) Defective 5-HTl-receptor-mediated neurotransmission in the control of growth hormone secretion in Parkinson's disease. Neuropsychobiology 35, 79-83.

Voipi, R., Scaglioni, A., Marcato, A., Caffarra, P., Rossi, G., Caffarri, G., Delsignore, R., Chiodera, P. and Coiro, V. (1991) Failure of the gamma-aminobutyric acid (GABA) derivative baciofen, to stimulate growth hormone secretion in Parkinson's disease. Journal of Neural Transmission 4, 259- 264.

Zigmond, A. S. and Snaith, R. P. (1983) The hospital anxiety and depression scale. Acta Psychiatrica Scandinavica 67, 361-370.