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Baclofen-induced growth hormone secretion is blunted in chronic schizophrenics: Neuroendocrine evidence for a GABA disturbance in schizophrenia
Psychiatry Research, 26, l-9 Elsevier
Baclofen-Induced Growth Hormone Secretion Is Blunted in Chronic Schizophrenics: Neuroendocrine Evidence for a GABA Disturbance in Schizophrenia. Palmiero Monteleone, Mario Maj, Michele Franc0 Veltro, and Luca Steardo
lovino,
Domenico
Forziati,
Received March 20. 1987; revised version received July 13, 1987; accepted September
4, 1987.
Abstract. To substantiate a previously reported disturbance of y-aminobutyric acid (GABA) in chronic schizophrenia, plasma growth hormone (GH) response to a direct GABA agonist (baclofen, IO mg) was assessed in 12 unmedicated chronic schizophrenic males and IO sex- and age-matched healthy controls. Baclofen and placebo were administered orally, in a double-blind design, and blood samples were collected before and 30, 60,90, 120, 180, and 240 min after drug or placebo administration. Baclofen induced a clear-cut rise in plasma GH levels over baseline values, but the GH increase observed in the patients was significantly smaller than that in controls. These results support the idea that GABA mechanisms may be impaired in chronic schizophrenia.
Key Words. Baclofen,
y-aminobutyric
acid, growth
hormone,
schizophrenia.
The “dopamine hypothesis” of schizophrenia, which postulates that “positive” or “productive” symptoms of this condition are linked to an overactivity of dopaminergic mesolimbic and mesocortical systems, has largely influenced psychobiological research for more than 20 years and has received substantial, although mainly indirect, support from experimental studies (Rodnight, 1983; Seeman, 1986). In recent years, however, the validity of this hypothesis has been repeatedly questioned. Several authors have emphasized the clinical and, most probably, biological heterogeneity of schizophrenic disorders, as well as the possible involvement of other neuromediators in their pathogenesis (Rodnight, 1983; Crow, 1985). As part of this reexamination, the inhibitory neurotransmitter y-aminobutyric acid (GABA) has drawn some attention. To date, however, conflicting results have emerged from studies measuring GABAergic activity in post-mortem brain or in cerebrospinal fluid (CSF) from schizophrenic patients (McGeer and McGeer,
Palmiero Monteleone, M.D., is Clinical Assistant, Mental Health Service U.S.L. 41, Regione Campania, Naples, Italy. Mario Maj, M.D., is Professor of Mental Hygiene, Institute of Medical Psychology and Psychiatry, First Medical School, University of Naples, Italy. Michele lovino, M.D., is Staff Neurologist, Department of Neurology, Second Medical School, University of Naples, Italy. Domenico Forziati, M.D., is Clinical Assistant, Psychiatric Hospital “Frullone,” Naples, Italy. Franc0 Veltro, M.D., is Staff Psychiatrist, Institute of Medical Psychology and Psychiatry, First Medical School, University of Naples, Italy. Luca Steardo, M.D., is Visiting Scientist, Experimental Therapeutics Branch, NINCDS, NIH, Bethesda, MD, USA, and Professor of Neurochemistry, Department of Neurology, Second Medical School, University of Naples, Italy. (Reprint requests to Dr. P. Monteleone, Servizio di Salute Mentale, U.S.L. 41 della Regione Campania, c/o Fondazione Ruggiero, via Fondo Cancello, 80145 Napoli, Italy.) 01651781/88/$03.50
@ 1988 Elsevier Scientific
Publishers Ireland
Ltd.
2 1979; Perry et al., 1979; Spokes et al., 1980; Gerner and Hare, 1981; Van Kammen et al., 1982). Findings of clinical trials with drugs affecting GABA transmission (Fredericksen, 1975; Linnoila et al., 1976; Lautin et al., 1980; Morselli et al., 1980) have also been inconsistent. Thus, at present, evidence supporting a GABA disturbance in the pathophysiology of schizophrenia is far from conclusive. A neuroendocrine approach to the issue was adopted by our group in two previous reports (Monteleone et al., 1985, 1986) showing that in chronic schizophrenics, unlike healthy controls, the indirect GABAergic drug sodium valproate fails to affect basal plasma prolactin (PRL) and growth hormone (GH) secretion. Although we were not able to clarify whether the abnormal responsiveness to GABAergic stimulation represented a primary defect or a secondary alteration of the endogenous GABA system in schizophrenia, our results strongly supported the idea that GABA mechanisms are impaired in this illness. Thus, to substantiate these findings, we decided to assess the GH response to a direct GABA agonist in a sample of untreated chronic schizophrenics compared to sex- and age-matched healthy controls. Thep-chlorophenyl derivative of GABA, baclofen, which freely crosses the bloodbrain barrier (Naik et al., 1976) was selected because it has been reported to increase basal GH levels in healthy humans (Koulu et al., 1979; Passariello et al., 1982; Chiodera et al., 1983).
Methods Subjects participating in this study were I2 male patients in a large psychiatric hospital who met Research Diagnostic Criteria (RDC) (Spitzer et al., 1978) for chronic schizophrenia and IO healthy male volunteers. The patient group consisted of seven paranoid, three disorganized, and two undifferentiated schizophrenics, aged 45-69 years (mean f SD = 59.1 f 7.8) who had been drug free for at least 6 weeks. Four of them had been treated with electroconvulsive therapy (ECT) several years before. The mean duration of their illness was 32.4 + 18.8 years (range 15-50). Patients’ psychopathological state was assessed with the Brief Psychiatric Rating Scale (BPRS) (Overall and Gorham, 1962) and the Scale for the Assessment of Negative Symptoms (SANS) (Andreasen, 1981) by two psychiatrists who were blind to the patients’ GH response. The control group comprised IO healthy men, aged 40-68 years (mean f SD f 60.4 f 7.0) who were carefully screened to rule out significant psychiatric symptoms and family history of mental illness. They had been drug free for at least 4 weeks at the time of the experiments. Electrocardiograms, electroencephalograms, and clinical screening were carried out to exclude medical diseases. Both schizophrenics and healthy volunteers were nonobese and had no family history of diabetes. The GH stimulation test was carried out at 9 a.m. after an overnight fast under basal metabolic conditions. A butterfly cannula was inserted into an antecubital vein and was kept open with dilute heparinized saline, which was discarded before sampling. Thirty (T = -15) and 45 (T = 0) min later, baseline blood samples were drawn. Then, in a double-blind design, each subject received orally IO mg of baclofen or a placebo tablet on 2 different days, I week apart. Further blood samples were collected 30,60,90, 120, 180, and 240 min after the drug or placebo administration. Plasma was separated by centrifugation at 4,000 rpm and stored at -20°C until assayed for GH. Plasma GH levels were determined by radioimmunoassay using commercial kits supplied by Biodata (Milan, Italy). Interassay and intra-assay coefficients of variation were 7.2% and 49?,, respectively, at GH concentration of 3 ng/ ml. The results were statistically analyzed by two-way analysis of variance (ANOVA) with repeated measures for each test condition. One-way ANOVA, Dunnett’s test, and Student’s
3 test were also performed where appropriate. In schizophrenics, correlations between peak GH responses and GH areas under the curve (AUC) following baclofen administration, on the one hand, and clinical and demographic variables, on the other, were done by Pearson productmoment coefficient.
Results Demographic
and
clinical
characteristics
of the patients
are shown
in Table
I.
Table 1. Demographic and clinical characteristics of schizophrenic patients
Patient No.
Age (years)
Duration of illness (years)
History of ECT
Schizophrenic subtype (RDC)
40
63
+
Disorganized
36
65
Undifferentiated
110
31
43
+ -
160
41
34
+
Paranoid
132
55
79
Paranoid
67
47
88
+ -
15
60
38
51
_
Paranoid
50
215
36
53
_
Paranoid
25
130
53
87
-
Undifferentiated
30
120
55
84
_
Paranoid
36
145
47
59
_
Disorganized
41
70
40
51
-
Disorganized
133.9
43.2
64.7
54.8
8.3
15.8
12.
66 64 60 63 58 45 69 64 62 59 66
Mean
59.1
32.4
7.8
18.8
SD
SANS total score
168
49
11.
BPRS total score
230
25 40 39 25 25 38
1.
2. 3. 4. 5. 6. 7. 8. 9. 10.
Duration of treatment (months)
Note. BPRS = Brief Psychiatric Rating Scale. SANS = Scale for the Assessment ECT = electroconvulsive therapy. RDC = Research Diagnostic Criteria.
Paranoid
Paranoid
of Negative
Symptoms.
In healthy volunteers, baclofen induced a clear-cut rise in plasma GH levels, whereas placebo did not (Fig. 1). Two-way ANOVA with repeated measures revealed a significant difference between baclofen and placebo treatment (F= 23.30; df= 1, 18;p
4
Fig. 1. Plasma growth hormone (GH) levels in normal and chronic schizophrenic subjects following baclofen (10 mgp.0.) or placebo administration 4
2 .
2 io c Y
I
Chronic
Eaclofen
rchizophrenice o---o
Placebo
I 6_ (I : 4_ a i 2_
0,
Healthy
-i5 i
subjects
il
s’o 9’0 lb TIME
Cminutael
Data represent mean f SEM averaged from 10 healthy male volunteers and 12 schizophrenic * ** t 0
p p p p
< < < <
0.01 (Dunnett’s 0.05 (Dunnett’s 0.005 (Dunnett’s 0.001 (Dunnett’s
2;o
1itJ
patients.
test). test). test). test).
levels was observed, achieving a statistical significance of p < 0.0 I (Dunnett’s test) at T = 30,60,90, and 120 min and p < 0.05 at T = 180 min. In the schizophrenic subjects, however, the rise of plasma GH levels after baclofen administration was not so sharp as in healthy controls. In fact, from a mean I!I SD baselinevalueof2.1 +0.5ng/ml,plasmaGH roseto3.3+0.7,3.7+0.6,3.7+0.8, 3.6 * 0.9, and 3. I AI 0.8 ng/ ml at T = 30,60,90, 120, and I80 min, respectively. The increases in GH levels (values observed at each time point from T = 30 to T = 240 min minus baseline value) were significantly lower in schizophrenics than in controls (p < 0.02 at T = 60 and p < 0.0000 at T = 90 and 120 min, Student’s t test). Fig. 2 shows the % increase of plasma GH over baseline values, which was significantly lower in the schizophrenic patients than in controls @ < 0.01 at T = 60 and p < 0.005 at T = 90 and 120 min, Student’s t test). No significant difference was observed in and controls. Following baclofen basal GH levels between schizophrenics administration, GH AUCs were significantly different (p < 0.01, Student’s t test) in the two groups: 785.5 f 176.2 ng/ml/ min (mean + SD) in schizophrenic patients versus 1036.5 + 261.6 ng/ml/min in healthy subjects. Finally, in the schizophrenic group, no significant correlation was found between peak GH responses and BPRS scores (r = -0.37) or SANS total scores (r = -0.08) or between AUC and the same
5 scores (BPRS: r = 0.13; SANS: r = 0.22). No correlation was found between hormonal parameters and age, duration of illness, or duration of treatment.
these
Fig. 2. Increase (%) of plasma growth hormone (GH) over baseline value in healthy controls and schizophrenic patients following acute oral administration of baclofen (10 mg) *
0
Cant
*
EJ
Schizophrenics
rols
minutes * **
p < 0.01, Student’s t test. p < 0.005, Student’s t test.
Discussion In line with previous observations (Koulu et al., 1979; Passariello et al., 1982; Chiodera et al., 1983), our results show that acute baclofen administration increases plasma GH concentrations in man, under basal conditions. It is commonly believed that baclofen, which easily crosses the blood-brain barrier (Naik et al., 1976) exerts pharmacological effects in the central nervous system similar to GABA, acting as a specific GABAa receptor agonist (Kelly and Moore, 1978; Bowery et al., 1980). Thus, the influence of baclofen on GH secretion may reflect the physiological action of GABA. Data reported by our group (Monteleone et al., 1986, 1987; Steardo et al., 1986) and by others (Takahara et al., 1977; Cavagnini et al., 1980a, 1980h) showed that either pharmacological activation of endogenous GABA system or direct administration of GABA itself enhanced basal GH secretion in humans, suggesting that GABA may exert a modulatory role on GH release. There is evidence to suggest that GABA may predominantly act at the hypothalamic level by inhibiting somatostatin release into portal circulation, which consequently raises plasma GH levels. In fact, administration of muscimol in the preoptic/anterior hypothalamic area, where somatostatin neurons are located, stimulates GH release, whereas
6 bicuculline inhibits spontaneously occurring GH secretion in the rat, suggesting a tonic modulation of somatostatin neurons by GABA (Willoughby et al., 1986). In addition, Takahara et al. (1980) reported that intracerebroventricular injection of GABA in the rat increased both serum GH and hypothalamic somatostatin content. More recently, 3H-baclofen binding sites have been described in rat hypothalamus (Gehlert et al., 1985), and Stryker et al. (1986) have postulated that GABA may influence somatostatin secretion through the calcium-dependent GABAs receptors. This suggestion is consistent with the stimulatory action of the putative GABAs receptor agonist baclofen on basal GH release observed in this study. On the other hand, a peripheral action of baclofen on pituitary somatotrophs seems to be excluded by the demonstration that this drug does not affect GH release from the rat pituitary gland in vitro (Anderson and Mitchell, 1986). Recently, some investigators have questioned the specificity of baclofen as a pure GABAs agonist. For instance, Fung et al. (1985) reported that this compound may act as an agonist at the a,-adrenoceptors. An activation of these receptors might be an alternative explanation of the stimulatory effect of baclofen on human GH release since an increase of basal GH after a-adrenergic stimulation has been shown in various species, including man (Tuomisto and Mannisto, 1985). In the present investigation, the baclofen-induced GH increase was blunted in unmedicated chronic schizophrenic patients. These data, together with our previous observations (Monteleone et al., 1985, 1986), may suggest an alteration of the hypothalamic GABAergic system in chronic schizophrenia. Alternatively, in line with the report of Fung et al. (1985), a subsensitivity of hypothalamic a2adrenoceptors may be hypothesized. This last interpretation would be in agreement with the report by Rosen et al. (1985) of a subsensitivity of platelet a,-adrenergic receptors in a sample of schizophrenic patients with prominent negative symptoms. Nevertheless, it must be emphasized that the GH response to clonidine, a specific a,-agonist, has been reported to be normal (Asnis and Ryan, 1983) or even exaggerated (Matussek et al., 1980) in chronic schizophrenia, so that it seems unlikely that a subsensitivity of cx,-adrenoceptors could be involved in the blunted baclofen-induced increase of GH in our patients. On the other hand, since GABA has been shown to interact with other hypothalamic neurotransmitter systems modulating GH secretion (Cavagnini et al., 1980~; Laakman et al., 1982), it cannot be definitively concluded that the present findings do not reflect primary or secondary changes in these neuronal pathways. Finally, we cannot rule out previous history of chronic neuroleptic treatment as an explanation for our results, since antipsychotic drugs profoundly alter many neurotransmitter systems, including GABA mechanisms. In this regard, Cleghorn et al. (1983) reported that the GH response to apomorphine hydrocloride in schizophrenic patients varied according to the evolution of the disease and to the previous therapy. Moreover, reductions of glutamic acid decarboxylase (GAD) activity in certain brain areas (Gunne and Haggstrom, 1983; Itoh, 1983; Gunne et al., 1984), as well as anomalous behavioral responses to GABA agonists (Freed et al., 1980), have been reported in animals chronically treated with haloperidol. To the best of our knowledge, however, no subsensitivity of GABAs binding sites following chronic neuroleptic exposure has been demonstrated either in experimental animals nr irl man.
7 In conclusion, if one accepts the working hypothesis of psychoneuroendocrinology that alterations in the levels of pituitary hormones, either at baseline or in response to various challenges, may reflect a dysfunction of one or more neurotransmitter systems in central brain areas, our finding of hyporesponsiveness of GH to baclofen, together with previously reported neuroendocrine abnormalities following sodium valproate administration in schizophrenic subjects (Monteleone et al., 1985, 1986) suggests a GABA disturbance in chronic schizophrenia. Acknowledgments. gift of baclofen
The authors thank Dr. Vierti Giovanni (Ciba-Geigy) for the generous and Mr. Antonio Daria and Michele Scarca for technical assistance.
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8 Gunne, L.M., Haggstrom, J.E., and Sjoqvist, B. Association with persistent neurolepticinduced dyskinesia of regional changes in brain GABA synthesis. Nature, 309, 347 (1984). Itoh, M. Effect of haloperidol on glutamate decarboxylase activity in discrete brain areas of rat. Psychopharmacology, 79, I69 ( 1983). Kelly, P.H., and Moore, K.E. Dopamine concentrations in the rat brain following injections into the substantia nigra of baclofen, gamma-aminobutyric acid, apomorphine and amphetamine. Neuropharmacology, 17, 169 (1978). Koulu, M., Lammintausta, R., and Dahlstrom, S. Stimulatory effect of acute baclofen administration on human growth hormone secretion. Journal qf’clinical Endocrinology and Metabolism, 48, IO38 ( 1979). Laakman, G., Treusch, J., Eichmeier, A., Schmauss, M., Treusch, U., and Wahlster, U. Inhibitory effect of phentolamine on diazepam-induced growth hormone secretion and lack of effect of diazepam on prolactin secretion in man. fsychoneuroendocrinolo~y, 7, I35 (1982). Lautin, A., Angrist, B., Stanley, M., Gershon, S., Heckl, K., and Karo Bath, M. Sodium valproate in schizophrenia: Some biochemical correlations. British Journalof’f’sychiutry, 137, 240 ( 1980). Linnoila, M., Viukari, M., and Hietala, 0. Effect of sodium valproate on tardive dyskinesia. British Journal of’ Psvchiutry. 129, I I4 ( 1976). Matussek, N., Ackenheil, M., Hippius, H., Miiller, F., Schroder, H.-Th., Schultes, H., and Wasilewski, B. Effect of clonidine on growth hormone release in psychiatric patients and controls. fs_vchiatry Research, 2, 25 (1980). McGeer, E., and McGeer, P. GABA-containing neurons in schizophrenia, Huntington’s chorea and normal aging. In: Krogsgaard-Larsen, P., Scheel-Kruger, J., and Koford, H., eds. GA BA Neurotransmitters: Pharmacochemical, Biochemical and Pharmacological Aspects. Munksgaard, Copenhagen (1979). Monteleone, P., lovino, M., Orio, F., and Steardo, 1,. Impaired growth hormone response to sodium valproate in normal aging. Psychopharmacology, 91, IO (1987). Monteleone, P., Maj, M., lovino, M., and Steardo, L. Growth hormone response to sodium valproate in chronic schizophrenia. Biological Psychiatry, 21, 588 (1986). Monteleone, P., Zontini, G., and Steardo, L. Failure of the GABAergic drug, sodium valproate, to reduce basal plasma prolactin secretion in chronic schizophrenia. ~~ychoneuroendocrinology, 10, 475 (1985). Morselli, P., Bossi, L., Henry, J., Zarifian, E., and Bartholini, G. On the therapeutic action of SL-76-002, a new GABA-mimetic agent: Preliminary observation in neuropsychiatric disorders. Bruin Research Bulletin, 5 (Suppl. 2) 95 (1980). Naik, S.R., Guidotti, A., and Costa, E. Central GABA receptor agonists: Comparison of muscimol and baclofen. Neuropharmacology, 15,479 ( 1976). Overall, J.E., and Gorham, D.R. The Brief Psychiatric Rating Scale. Psychological Reports, 10, 799 ( 1962).
9 Passariello, N., Giugliano, D., Torella, R., Sgambato, S., Coppola, L., and Frascolla, N. A possible role for gamma-aminobutyric acid in the control of endocrine pancreas. Journal of Clinical Endocrinology and Metabolism, 54, I I45 ( 1982). Perry, T., Buchanan, J., Kish, S., and Hausen, S. Gamma-aminobutyric acid deficiency in brain of schizophrenic patients. Lancet, 1, 237 (1979). Rodnight, R. Schizophrenia: Some current neurochemical approaches. Journal of Neurochemistry, 41, I2 ( 1983). Rosen, J., Silk, K.R., Rice, H.E., and Smith, C.B. Platelet alpha,-adrenergic dysfunction in negative symptom schizophrenia: A preliminary study. Biological Psychiatry, 20, 539 (1985). Seeman, P. Dopamine/neuroleptic receptors in schizophrenia. In: Burrows, G.D., Norman, T.R., and Rubinstein, G., eds. Handbook of Studies in Schizophrenia, Part 2. Elsevier Science Publishers, Amsterdam (1986). Spitzer, R.L., Endicott, J., and Robins, E. Research Diagnostic Criteria: Rationale and reliability. Archives of General Psychiatry, 35, 773 (1978). Spokes, E.G.S., Garrett, N.J., Rossor, M.N., and Iversen, L.L. Distribution of GABA in post-mortem brain tissue from control, psychotic and Huntington’s chorea subjects. Journal qf’ Neurological Sciences, 48, 303 ( 1980). Steardo, L., lovino, M., Monteleone, P., Agrusta, M., and Orio, F. Pharmacological evidence for a dual GABAergic regulation of growth hormone release in humans. Life Sciences, 39,979 ( 1986). Stryker, T.D., Conlin, T., and Reichlin, S. Influence of a benzodiazepine, midazolam, and gamma-aminobutyric acid (GABA) on basal somatostatin secretion from cerebral and diencephalic neurons in dispersed cell culture. Bruin Research. 362,339 (1986). Takahara, J., Yunoki, S., Yakushiji, W., Yamaguchi, J., Yamane, Y., and Ofuji, T. Stimulatory effects of gamma-hydroxybutyric acid on growth hormone and prolactin release in humans. Journal of Clinical Endocrinology and Metabolism, 44, 1014 (1977). Takahara, J., Yunoki, S., Hosogi, H., Yakushiji, W., Kageyama, J., and Ofuji, T. Concomitant increases in serum growth hormone and hypothalamic somatostatin in rats after injection of gamma-aminobutyric acid, aminooxyacetic acid or gamma-hydroxybutyric acid. Endocrinology. 106, 343 ( 1980). Tuomisto, J., and Mannisto, P. Neurotransmitter regulation of anterior pituitary hormones. Pharmacological Reviews, 37,249 (1985). Van Kammen, D.P., Sternberg, D.S., Hare, T.A., Waters, R.N., and Bunney, W.E., Jr. CSF levels of gamma-aminobutyric acid in schizophrenia: Low values in recently ill patients. Archives of General Psychiutry, 39, 9 I (I 982). Willoughby, J.O., Jervois, P.M., Menadue, M.F., and Blessing, W.N. Activation of GABA receptors in the hypothalamus stimulates secretion of growth hormone and prolactin. Bruin Research, 374, I I9 ( 1986).
Baclofen-Induced Growth Hormone Secretion Is Blunted in Chronic Schizophrenics: Neuroendocrine Evidence for a GABA Disturbance in Schizophrenia. Palmiero Monteleone, Mario Maj, Michele Franc0 Veltro, and Luca Steardo
lovino,
Domenico
Forziati,
Received March 20. 1987; revised version received July 13, 1987; accepted September
4, 1987.
Abstract. To substantiate a previously reported disturbance of y-aminobutyric acid (GABA) in chronic schizophrenia, plasma growth hormone (GH) response to a direct GABA agonist (baclofen, IO mg) was assessed in 12 unmedicated chronic schizophrenic males and IO sex- and age-matched healthy controls. Baclofen and placebo were administered orally, in a double-blind design, and blood samples were collected before and 30, 60,90, 120, 180, and 240 min after drug or placebo administration. Baclofen induced a clear-cut rise in plasma GH levels over baseline values, but the GH increase observed in the patients was significantly smaller than that in controls. These results support the idea that GABA mechanisms may be impaired in chronic schizophrenia.
Key Words. Baclofen,
y-aminobutyric
acid, growth
hormone,
schizophrenia.
The “dopamine hypothesis” of schizophrenia, which postulates that “positive” or “productive” symptoms of this condition are linked to an overactivity of dopaminergic mesolimbic and mesocortical systems, has largely influenced psychobiological research for more than 20 years and has received substantial, although mainly indirect, support from experimental studies (Rodnight, 1983; Seeman, 1986). In recent years, however, the validity of this hypothesis has been repeatedly questioned. Several authors have emphasized the clinical and, most probably, biological heterogeneity of schizophrenic disorders, as well as the possible involvement of other neuromediators in their pathogenesis (Rodnight, 1983; Crow, 1985). As part of this reexamination, the inhibitory neurotransmitter y-aminobutyric acid (GABA) has drawn some attention. To date, however, conflicting results have emerged from studies measuring GABAergic activity in post-mortem brain or in cerebrospinal fluid (CSF) from schizophrenic patients (McGeer and McGeer,
Palmiero Monteleone, M.D., is Clinical Assistant, Mental Health Service U.S.L. 41, Regione Campania, Naples, Italy. Mario Maj, M.D., is Professor of Mental Hygiene, Institute of Medical Psychology and Psychiatry, First Medical School, University of Naples, Italy. Michele lovino, M.D., is Staff Neurologist, Department of Neurology, Second Medical School, University of Naples, Italy. Domenico Forziati, M.D., is Clinical Assistant, Psychiatric Hospital “Frullone,” Naples, Italy. Franc0 Veltro, M.D., is Staff Psychiatrist, Institute of Medical Psychology and Psychiatry, First Medical School, University of Naples, Italy. Luca Steardo, M.D., is Visiting Scientist, Experimental Therapeutics Branch, NINCDS, NIH, Bethesda, MD, USA, and Professor of Neurochemistry, Department of Neurology, Second Medical School, University of Naples, Italy. (Reprint requests to Dr. P. Monteleone, Servizio di Salute Mentale, U.S.L. 41 della Regione Campania, c/o Fondazione Ruggiero, via Fondo Cancello, 80145 Napoli, Italy.) 01651781/88/$03.50
@ 1988 Elsevier Scientific
Publishers Ireland
Ltd.
2 1979; Perry et al., 1979; Spokes et al., 1980; Gerner and Hare, 1981; Van Kammen et al., 1982). Findings of clinical trials with drugs affecting GABA transmission (Fredericksen, 1975; Linnoila et al., 1976; Lautin et al., 1980; Morselli et al., 1980) have also been inconsistent. Thus, at present, evidence supporting a GABA disturbance in the pathophysiology of schizophrenia is far from conclusive. A neuroendocrine approach to the issue was adopted by our group in two previous reports (Monteleone et al., 1985, 1986) showing that in chronic schizophrenics, unlike healthy controls, the indirect GABAergic drug sodium valproate fails to affect basal plasma prolactin (PRL) and growth hormone (GH) secretion. Although we were not able to clarify whether the abnormal responsiveness to GABAergic stimulation represented a primary defect or a secondary alteration of the endogenous GABA system in schizophrenia, our results strongly supported the idea that GABA mechanisms are impaired in this illness. Thus, to substantiate these findings, we decided to assess the GH response to a direct GABA agonist in a sample of untreated chronic schizophrenics compared to sex- and age-matched healthy controls. Thep-chlorophenyl derivative of GABA, baclofen, which freely crosses the bloodbrain barrier (Naik et al., 1976) was selected because it has been reported to increase basal GH levels in healthy humans (Koulu et al., 1979; Passariello et al., 1982; Chiodera et al., 1983).
Methods Subjects participating in this study were I2 male patients in a large psychiatric hospital who met Research Diagnostic Criteria (RDC) (Spitzer et al., 1978) for chronic schizophrenia and IO healthy male volunteers. The patient group consisted of seven paranoid, three disorganized, and two undifferentiated schizophrenics, aged 45-69 years (mean f SD = 59.1 f 7.8) who had been drug free for at least 6 weeks. Four of them had been treated with electroconvulsive therapy (ECT) several years before. The mean duration of their illness was 32.4 + 18.8 years (range 15-50). Patients’ psychopathological state was assessed with the Brief Psychiatric Rating Scale (BPRS) (Overall and Gorham, 1962) and the Scale for the Assessment of Negative Symptoms (SANS) (Andreasen, 1981) by two psychiatrists who were blind to the patients’ GH response. The control group comprised IO healthy men, aged 40-68 years (mean f SD f 60.4 f 7.0) who were carefully screened to rule out significant psychiatric symptoms and family history of mental illness. They had been drug free for at least 4 weeks at the time of the experiments. Electrocardiograms, electroencephalograms, and clinical screening were carried out to exclude medical diseases. Both schizophrenics and healthy volunteers were nonobese and had no family history of diabetes. The GH stimulation test was carried out at 9 a.m. after an overnight fast under basal metabolic conditions. A butterfly cannula was inserted into an antecubital vein and was kept open with dilute heparinized saline, which was discarded before sampling. Thirty (T = -15) and 45 (T = 0) min later, baseline blood samples were drawn. Then, in a double-blind design, each subject received orally IO mg of baclofen or a placebo tablet on 2 different days, I week apart. Further blood samples were collected 30,60,90, 120, 180, and 240 min after the drug or placebo administration. Plasma was separated by centrifugation at 4,000 rpm and stored at -20°C until assayed for GH. Plasma GH levels were determined by radioimmunoassay using commercial kits supplied by Biodata (Milan, Italy). Interassay and intra-assay coefficients of variation were 7.2% and 49?,, respectively, at GH concentration of 3 ng/ ml. The results were statistically analyzed by two-way analysis of variance (ANOVA) with repeated measures for each test condition. One-way ANOVA, Dunnett’s test, and Student’s
3 test were also performed where appropriate. In schizophrenics, correlations between peak GH responses and GH areas under the curve (AUC) following baclofen administration, on the one hand, and clinical and demographic variables, on the other, were done by Pearson productmoment coefficient.
Results Demographic
and
clinical
characteristics
of the patients
are shown
in Table
I.
Table 1. Demographic and clinical characteristics of schizophrenic patients
Patient No.
Age (years)
Duration of illness (years)
History of ECT
Schizophrenic subtype (RDC)
40
63
+
Disorganized
36
65
Undifferentiated
110
31
43
+ -
160
41
34
+
Paranoid
132
55
79
Paranoid
67
47
88
+ -
15
60
38
51
_
Paranoid
50
215
36
53
_
Paranoid
25
130
53
87
-
Undifferentiated
30
120
55
84
_
Paranoid
36
145
47
59
_
Disorganized
41
70
40
51
-
Disorganized
133.9
43.2
64.7
54.8
8.3
15.8
12.
66 64 60 63 58 45 69 64 62 59 66
Mean
59.1
32.4
7.8
18.8
SD
SANS total score
168
49
11.
BPRS total score
230
25 40 39 25 25 38
1.
2. 3. 4. 5. 6. 7. 8. 9. 10.
Duration of treatment (months)
Note. BPRS = Brief Psychiatric Rating Scale. SANS = Scale for the Assessment ECT = electroconvulsive therapy. RDC = Research Diagnostic Criteria.
Paranoid
Paranoid
of Negative
Symptoms.
In healthy volunteers, baclofen induced a clear-cut rise in plasma GH levels, whereas placebo did not (Fig. 1). Two-way ANOVA with repeated measures revealed a significant difference between baclofen and placebo treatment (F= 23.30; df= 1, 18;p
4
Fig. 1. Plasma growth hormone (GH) levels in normal and chronic schizophrenic subjects following baclofen (10 mgp.0.) or placebo administration 4
2 .
2 io c Y
I
Chronic
Eaclofen
rchizophrenice o---o
Placebo
I 6_ (I : 4_ a i 2_
0,
Healthy
-i5 i
subjects
il
s’o 9’0 lb TIME
Cminutael
Data represent mean f SEM averaged from 10 healthy male volunteers and 12 schizophrenic * ** t 0
p p p p
< < < <
0.01 (Dunnett’s 0.05 (Dunnett’s 0.005 (Dunnett’s 0.001 (Dunnett’s
2;o
1itJ
patients.
test). test). test). test).
levels was observed, achieving a statistical significance of p < 0.0 I (Dunnett’s test) at T = 30,60,90, and 120 min and p < 0.05 at T = 180 min. In the schizophrenic subjects, however, the rise of plasma GH levels after baclofen administration was not so sharp as in healthy controls. In fact, from a mean I!I SD baselinevalueof2.1 +0.5ng/ml,plasmaGH roseto3.3+0.7,3.7+0.6,3.7+0.8, 3.6 * 0.9, and 3. I AI 0.8 ng/ ml at T = 30,60,90, 120, and I80 min, respectively. The increases in GH levels (values observed at each time point from T = 30 to T = 240 min minus baseline value) were significantly lower in schizophrenics than in controls (p < 0.02 at T = 60 and p < 0.0000 at T = 90 and 120 min, Student’s t test). Fig. 2 shows the % increase of plasma GH over baseline values, which was significantly lower in the schizophrenic patients than in controls @ < 0.01 at T = 60 and p < 0.005 at T = 90 and 120 min, Student’s t test). No significant difference was observed in and controls. Following baclofen basal GH levels between schizophrenics administration, GH AUCs were significantly different (p < 0.01, Student’s t test) in the two groups: 785.5 f 176.2 ng/ml/ min (mean + SD) in schizophrenic patients versus 1036.5 + 261.6 ng/ml/min in healthy subjects. Finally, in the schizophrenic group, no significant correlation was found between peak GH responses and BPRS scores (r = -0.37) or SANS total scores (r = -0.08) or between AUC and the same
5 scores (BPRS: r = 0.13; SANS: r = 0.22). No correlation was found between hormonal parameters and age, duration of illness, or duration of treatment.
these
Fig. 2. Increase (%) of plasma growth hormone (GH) over baseline value in healthy controls and schizophrenic patients following acute oral administration of baclofen (10 mg) *
0
Cant
*
EJ
Schizophrenics
rols
minutes * **
p < 0.01, Student’s t test. p < 0.005, Student’s t test.
Discussion In line with previous observations (Koulu et al., 1979; Passariello et al., 1982; Chiodera et al., 1983), our results show that acute baclofen administration increases plasma GH concentrations in man, under basal conditions. It is commonly believed that baclofen, which easily crosses the blood-brain barrier (Naik et al., 1976) exerts pharmacological effects in the central nervous system similar to GABA, acting as a specific GABAa receptor agonist (Kelly and Moore, 1978; Bowery et al., 1980). Thus, the influence of baclofen on GH secretion may reflect the physiological action of GABA. Data reported by our group (Monteleone et al., 1986, 1987; Steardo et al., 1986) and by others (Takahara et al., 1977; Cavagnini et al., 1980a, 1980h) showed that either pharmacological activation of endogenous GABA system or direct administration of GABA itself enhanced basal GH secretion in humans, suggesting that GABA may exert a modulatory role on GH release. There is evidence to suggest that GABA may predominantly act at the hypothalamic level by inhibiting somatostatin release into portal circulation, which consequently raises plasma GH levels. In fact, administration of muscimol in the preoptic/anterior hypothalamic area, where somatostatin neurons are located, stimulates GH release, whereas
6 bicuculline inhibits spontaneously occurring GH secretion in the rat, suggesting a tonic modulation of somatostatin neurons by GABA (Willoughby et al., 1986). In addition, Takahara et al. (1980) reported that intracerebroventricular injection of GABA in the rat increased both serum GH and hypothalamic somatostatin content. More recently, 3H-baclofen binding sites have been described in rat hypothalamus (Gehlert et al., 1985), and Stryker et al. (1986) have postulated that GABA may influence somatostatin secretion through the calcium-dependent GABAs receptors. This suggestion is consistent with the stimulatory action of the putative GABAs receptor agonist baclofen on basal GH release observed in this study. On the other hand, a peripheral action of baclofen on pituitary somatotrophs seems to be excluded by the demonstration that this drug does not affect GH release from the rat pituitary gland in vitro (Anderson and Mitchell, 1986). Recently, some investigators have questioned the specificity of baclofen as a pure GABAs agonist. For instance, Fung et al. (1985) reported that this compound may act as an agonist at the a,-adrenoceptors. An activation of these receptors might be an alternative explanation of the stimulatory effect of baclofen on human GH release since an increase of basal GH after a-adrenergic stimulation has been shown in various species, including man (Tuomisto and Mannisto, 1985). In the present investigation, the baclofen-induced GH increase was blunted in unmedicated chronic schizophrenic patients. These data, together with our previous observations (Monteleone et al., 1985, 1986), may suggest an alteration of the hypothalamic GABAergic system in chronic schizophrenia. Alternatively, in line with the report of Fung et al. (1985), a subsensitivity of hypothalamic a2adrenoceptors may be hypothesized. This last interpretation would be in agreement with the report by Rosen et al. (1985) of a subsensitivity of platelet a,-adrenergic receptors in a sample of schizophrenic patients with prominent negative symptoms. Nevertheless, it must be emphasized that the GH response to clonidine, a specific a,-agonist, has been reported to be normal (Asnis and Ryan, 1983) or even exaggerated (Matussek et al., 1980) in chronic schizophrenia, so that it seems unlikely that a subsensitivity of cx,-adrenoceptors could be involved in the blunted baclofen-induced increase of GH in our patients. On the other hand, since GABA has been shown to interact with other hypothalamic neurotransmitter systems modulating GH secretion (Cavagnini et al., 1980~; Laakman et al., 1982), it cannot be definitively concluded that the present findings do not reflect primary or secondary changes in these neuronal pathways. Finally, we cannot rule out previous history of chronic neuroleptic treatment as an explanation for our results, since antipsychotic drugs profoundly alter many neurotransmitter systems, including GABA mechanisms. In this regard, Cleghorn et al. (1983) reported that the GH response to apomorphine hydrocloride in schizophrenic patients varied according to the evolution of the disease and to the previous therapy. Moreover, reductions of glutamic acid decarboxylase (GAD) activity in certain brain areas (Gunne and Haggstrom, 1983; Itoh, 1983; Gunne et al., 1984), as well as anomalous behavioral responses to GABA agonists (Freed et al., 1980), have been reported in animals chronically treated with haloperidol. To the best of our knowledge, however, no subsensitivity of GABAs binding sites following chronic neuroleptic exposure has been demonstrated either in experimental animals nr irl man.
7 In conclusion, if one accepts the working hypothesis of psychoneuroendocrinology that alterations in the levels of pituitary hormones, either at baseline or in response to various challenges, may reflect a dysfunction of one or more neurotransmitter systems in central brain areas, our finding of hyporesponsiveness of GH to baclofen, together with previously reported neuroendocrine abnormalities following sodium valproate administration in schizophrenic subjects (Monteleone et al., 1985, 1986) suggests a GABA disturbance in chronic schizophrenia. Acknowledgments. gift of baclofen
The authors thank Dr. Vierti Giovanni (Ciba-Geigy) for the generous and Mr. Antonio Daria and Michele Scarca for technical assistance.
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