00?2-3042~79/0701-002910?00 0
Journal of Ncurochumicrry Vol. 33. pp. 29 to 33 Pergamon Press Ltd 1979. Printed In Great Britain
0 International Society lor Neurocliemistry Ltd
EFFECT OF DRUGS ON RAT BRAIN, CEREBROSPINAL FLUID AND BLOOD GABA CONTENT JOHN W. FERKANY, IAN J. BUTLERand S. J. ENNA' Departments of Pharmacology, Neurobiology and Anatomy, Neurology and Pediatrics, University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77025, U.S.A.
(Received 24 November 1978. Accepted 9 January 1979)
Abstract-Acute administration of GABA transaminase inhibitors to rats results in a dose-dependent increase in both brain and blood GABA content and administration of isonicotinic acid hydrazide (INH), at a dose which decreases the amount of brain GABA, also lowers blood levels of this amino acid. Chronic treatment (10 days) with INH (20 mg/kg), y-acetylenic-GABA (10 mg/kg) or aminooxyacetic acid (AOAA)(10 mg/kg) results in a significant elevation in both rat brain and blood GABA concentrations. A t the doses studied, only AOAA caused a significant elevation in CSF GABA content. Co-administration of pyridoxal phosphate (2 mg/kg) blocks the chronic INH-induced rise in blood GABA but does not affect the increase in brain content of this amino acid. Chronic administration of di-n-propylacetate (20mg/kg) did not significantly alter brain, blood or CSF GABA levels. The results suggest that, under the proper conditions, changes in blood GABA levels after administration of inhibitors of GABA synthesis or degradation may be an indirect indicator of changes in the brain content of this amino acid. Blood GABA determinations may be useful for studying the biochemical effectiveness of GABA transaminase inhibitors in man.
ALTERATIONS in the y-aminobutyric acid (GABA) neurotransmitter system have been implicated in a variety of neurological and psychiatric disorders (WOOD& PEESKER, 1972a; PERRY et al., 1973; BIRD & IVERSEN, 1974; ANLEZARKet al., 1976; ENNA et al., 1977a; VAN KAMMEN,1977). In particular, studies have suggested that increasing brain GABA content may be beneficial in epilepsy and Huntington's disease (PERRYet al., 1977; SCHWARCZet al., 1977; SCHECHTER ef a/., 1977; WOOD et al., 1977). In these studies, brain GABA levels are increased by the administration of agents known to inhibit 4-aminobutyrate : 2-oxoglutarate aminotransferase (EC 2.6.1.19; GABA-T), the enzyme which catalyzes the degradation of GABA. However, while in laboratory studies the effectiveness of GABA-T inhibitors can be readily determined by directly measuring the brain content of GABA, there is no clinical procedure capable of monitoring the biochemical effectiveness of these agents. Such a clinical test may be useful in determining the mechanism of action of these agents in man and in characterizing the biochemical abnormalities underlying these disorders. Recently, methods have been developed which are capable of measuring GABA in human cerebrospinal
fluid (CSF) (GLAESER& HARE, 1975; ENNA rt al., 1977c; HUIZINGA et a!., 1977; BOHLENet a!., 1978). In additon, a procedure has been described which is capable of detecting the trace quantities of GABA in blood (FERKANY et al., 1978). While CSF GABA appears to originate from brain (ENNAet al., 19776, c), the primary source of blood GABA is unknown. However, preliminary studies indicated that administration of a GABA-T inhibitor raises blood GABA levels as well as brain GABA content, suggesting that blood GABA determinations may be a useful clinical tool to indirectly monitor the biochemical effectiveness of drugs which are known to inhibit this enzyme (FERKANY et al., 1978). In the present investigation this possibility is further explored by comparing changes in CSF and blood GABA content with brain GABA levels after either acute or chronic administration of drugs which alter GABA metabolism. The results indicate that, under the proper conditions, blood GABA determinations may be used as an indirect monitor of brain GABA variations in response to these drugs.
MATERIALS AND METHODS
' To whom reprint requests should be addressed, at the
Drug administration and sample collection. Male Sprague-
Departments of Pharmacology and of Neurobiology and Anatomy. Abbreuiations used: GABA, y-aminobutyric acid; AOAA, aminooxyacetic acid; GAD, L-glutamic acid decarboxylase (EC 4.1.1.15); GABA-T, Caminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19); INH, isonicotinic acid hydrazide; DPA, di-n-propylacetic acid
Dawley rats (200-25Og) were used and drugs, dissolved in lactated sterile Ringers solution (Travenol Labs, Deerfield, IL) just prior to treatment, were injected intraperitoneally. Control animals received an equivalent volume of vehicle. Prior to sample collection, animals were tightly anesthetized with chloral hydrate (Sigma Chemical Co, St. Louis.
29
30
IANJ. BUTLER and S. J. ENNA JOHNW. FERKANY,
MO; 400 mg/kg, i.p.). For CSF collection, a 25 gauge Butterfly intravenous needle with a blunted bevel was carefully inserted into the cisterna magna and the CSF drained spontaneously into an ice-chilled 1 ml glass microfuge tube by way of a cannula attached to the needle. Over a 3 W 5 min period. 100-300 pl of CSF were collected and then stored at -20°C until assayed. After withdrawal by cardiac puncture, blood was transferred into I5 ml Sorvall centrifuge tubes which contained an equal volume of 7% perchloric acid. Following a brief vortex mixing, the samples were centrifuged at 48,000g for 20 min at 4°C. After centrifugation, the clear supernatant was decanted into another Sorvall centrifuge tube and was carefully added to bring the pH sufficient ~ N - K O H to 7.0. Neutralized supernatant was centrifuged at 48,000 g for 20 min at 4°C to separate the insoluble KCIO,. Following centrifugation, the supernatant was decanted into 5 ml glass culture tubes and stored at 2°C until assayed. Immediately after blood collection the whole brain was rapidly removed and frozen in a dry iceeacetone bath. These frozen samples were stored at -20°C until assayed. In experiments where CSF was not taken animals were lightly anesthetized with ether prior to removing blood and brain samples. Sample analysis. Blood, CSF and brain GABA content were measured by radioreceptor assay using C3H]GABA 1976; ENNA et a/., 1 9 77 ~ ; as a ligand (ENNA& SNYDER. et al., 1978). The principle of this assay is related FERKANY to the fact that the quantity of C3H]GABA bound to rat brain membranes is a function of the amount of unlabeled GABA present in the incubation medium (ENNA, 1978). For assay, portions of the neutralized blood extract were analyzed directly. Frozen brain samples were homogenized in 20 vol 5% trichloroacetic acid with a Brinkman Polytron PT-10. The homogenate was centrifuged at 48,OOOg for 10min and a measured portion of the supernatant was diluted 2500-fold with water. A 10-5Opl portion of this diluent was used for analysis. The GABA content of CSF was determined by analyzing 75-100 pl portions of the untreated fluid. In all cases, dilutions were made such that between 30 and 70% of the bound radioactivity was displaced by the sample. Standard curves for the displacement of ['HIGABA by known quantities of unlabeled GABA were generated daily. Each sample was analyzed on two separate occasions, and each analysis was performed in duplicate. The significance of differences between means was determined using a two-tailed Student's t-test. Chemicals. Di-n-propylacetate (DPA) was obtained from Dr. JUDYWALT~RS, y-acetylenic-GABA from Merrell International (Strasbourg, France), isonicotinic acid hydrazide from Eastman Kodak (Rochester, NY), aminooxyacetic acid from Sigma Chemical Co. (St. Louis, MO), and pyridoxal-5-phosphate from Calbiochem (La Jolla, CA). ['H]yAminobutyric acid (54.1 Ci/mmol) was purchased from Amersham Corporation (Arlington Heights, IL). RESULTS
Dose-response studies The concentration of GABA in brain and blood rose in a dose-dependent manner after administration of either y-acetylenic-GABA or AOAA (Tables 1 and 2). However, for both drugs, a t a given dose, the increase in brain GABA was always greater than
T A B L1.~DOSE-RESPONSE OF BRAIN A N D BLOOD GABA CONTENT TO Y-ACETYLENIC-GABA Dose (mg/kg) Control (13) 10 (4) 50 (4) 100 (4)
GABA concentration* Brain Blood (vnolig) (nmol/ml)
*
2.2 0. t 4.1 i 0.7$ 6.4 k 0.63 8.6 k 0.21
0.7 k 0.04 0.9 2 0.021 1.1 k 0.07t 1.2 f 0.14t
Values are the mean ~ s . E . Mof. the number of experiments shown in parentheses. * Analyzed 5 h after i.p. injection. 1P < 0.05. $ P < 0.001. TABLE2. DOSE-RESPONSE OF BRAIN
AND BLOOD TENT TO AMINOOXYACETIC ACID
Dose (mg/kg) Control (6) 5 (4) 15 (4) 40 (4)
GABA
CON-
GABA concentration* Brain Blood (pmoI/g) (nmol/ml) 1.7 k 0.2 2.8 0.37 4.3 0.31 6.8 k 0.2$
+
0.7 k 0.05 1.0 k 0.08t 1.6 2 0.043 1.7 f 0.053
Values are the mean ~ s . E . M . of the number of experiments shown in parentheses. * Analyzed 4 h after i.p. injection. t P < 0.01. 3 P < 0.001. the increase in the blood content, with y-acetylenic-
GABA increasing the brain concentration 24-fold over a dose range of 1G-100mg/kg, whereas the blood GABA concentration in these same animals did not quite double (Table 1). Similarly, AOAA induced a 4-fold increase in brain GABA over a dose range of 5 4 0 m g / k g , but only a 2-fold increase in blood GABA was observed (Table 2). A significant decrease in both brain and blood
GABA was observed a t 9 0 m i n after the administration of 300 mg/kg INH (Table 3) with a similar magnitude of change in brain and blood GABA content, being reduced approx 30% in brain and approx 40% in blood at this dose. Also, with INH. blood but not
3. DOSE-RESPONSE OF BRAIN TABLL
A N D BLOOD GABA CONTENT TO ISONICOTINIC ACID HYDKAZIDC
Dose (mg/kg) Control (6) 50 (4) 100 (4) 300 (4)
GABA concentration* Brain Blood (mol/g) (nmol/ml) 1.7 f 0.2 1.7 0.2 1.6 0.1 1.2 O.1t
*
0.8 f 0.07 0.8 & 0.07
0.6 $- 0.031 0.5 f 0.031
Values are the mean ~ s . E . Mof. the number of experiments shown in parentheses. * Analyzed 90 min after i.p. injection. t P < 0.05. $ P < 0.001.
31
Brain. cerebrospinal fluid and blood GABA
were significantly elevated, being 3-fold and 4-fold higher than control levels, respectively. E j k t of chronic drug administration on brain, CSF und blood G A B A content
m a
0
a
a m
1.0
30
150
90
300
TIME (min.)
FIG. 1. Time course of the effect of a single i.p. dose of AOAA (40mg/kg) on rat brain (A) and blood (B) GABA content. Each point is the mean k S.E.M. of 3 experiments.
brain GABA was significantly reduced at a dose of 100 mg/kg (Table 3). Timi. course of response to A O A A
Analysis of blood and brain GABA content at various times after administration of a single dose (40 mg/kg) of AOAA revealed that while the brain GABA concentration steadily increased over a 5 h period, there was no significant change in blood GABA content up to 90min after injection (Fig. 1A and B). Between 90 and 150 min after injection, blood GABA levels increase at a rate similar to that observed for brain GABA. By 5 h after treatment with AOAA, both blood and brain GABA concentrations TABLE 4. BLOOD,CSF
Drug Control (25) DPA (4) INH (6) y- Acetylenic-
Chronic administration of INH, y-acetylenicGABA and AOAA significantly elevated brain and blood GABA levels (Table 4). Concentrations of GABA in CSF, although increased in all cases, were significantly elevated only after chronic administration of AOAA. No increase in brain, CSF or blood GABA was detected in animals treated chronically with 20 mg/kg DPA (Table 4). As opposed to the results observed after acute administration, chronic administration of the effective drugs resulted in an elevation of blood GABA similar to or, in the case of AOAA, greater than the elevation in brain GABA content. Thus, after 10 days of chronic administration of INH (20 mg/kg, b.i.d.), brain GABA increased 26% relative to saline treated controls whereas blood GABA increased 45%. With y-acetylenic-GABA (10 mg/kg, bid.), a 10 day treatment resulted in brain GABA levels 40% higher than controls with blood GABA levels being elevated 77%. A similar 10 day treatment with AOAA (IOmg/kg once daily) induced increases in brain and blood GABA content of 67% and 164%, respectively (Table 4). It is noteworthy that even though CSF GABA was significantly increased only after AOAA, the percentage increase in CSF GABA observed after the various drug treatments was similar to the percentage increase in brain content. Thus, after INH, CSF GABA increased 26%, after y-acetylenic-GABA, 50% and after AOAA, SS%, values which compare favorably with the 26, 40 and 67% increases in brain GABA found after administration of these three drugs.
A N D BRAIN GABA CONTENT FOLLOWING CHRONIC TION OF VARIOUS DRUGS
Dose (mg/kg) ~
Brain (Pmolig) 1.9
+ 0.1
ADMINISTRA-
GABA concentration Blood CSF (nmoliml) (pmoliml) 0.8 -t 0.09*
* 22 93 * 20
100
20 b.i.d.
2.3 k 0.2
0.8
k 0.10
20 b.i.d.
2.4 k 0.2*
1.1
*
0.1*
130 k 47
10 b.i.d.
2.7 & 0.2t
1.4 k 0.3*
160 & 35
3.2
2.0 k 0.4t
200
GABA (5)
AOAA
10 once daily
0.23
k 16*
(6)
Values are the mean ~ s . E . M . of the number of experiments shown in parentheses. All drugs were administered for 10 days and the animals were killed 24 h following the final dose. * P < 0.05. 'r P < 0.01. 1P < 0.001.
32
JOHN
W. FERKANY, IAN J. B U T L ~ and R S. J. ENNA
TABLE 5. EFFECTOF PYRIDOXINE ON
BLOOD, CSF A N D BRAIN GABA CONTENT FOLLOWING CHRONIC ADMINISTRATION OF INH
Drug treatment
Dose (mg/kg)
1.5
Control
+ 0.2
GABA concentration Blood CSF (nmol/ml) (pmol/ml) 1.2 k 0.07
88 k 14
2
1.5 k 0.2
1.2
* 0.1
80 2 7
2 20
2.3 f 0.2'
1.3
k 0.1
130 k 28
(5)
Pyridoxine
Brain (Pmolig)
(61
Pyridoxine
+ INH
(5)
Values are the mean ~ s . E . Mof. the number of experiments shown in parentheses. Drugs were administered twice daily for 8 days and the animals killed 16 h following the final dose. * P i0.025.
e f f e c t of pyridoxine on INH-induced elevations of GABA
When used clinically, INH is administered with pyridoxine to counter INH-induced pyridoxine defiet a/., 1977) and it ciency (WEINSTEIN, 1975; PERRY has been reported that co-administration of pyridoxine alters the biochemical response to INH (WOOD & PEESKER, 1972b). To test the interaction of these two compounds on brain, CSF and blood GABA content, animals were treated chronically with either pyridoxine alone or pyridoxine in combination with INH (Table 5). The results indicate that while pyridoxine itself has no effect on GABA content, this compound does inhibit the increase in blood, but not brain, GABA levels observed after chronic INH treatment (Tables 4 and 5).
DISCUSSION Rccent clinical studies with GABA-T inhibitors have revealed a need for a simple method to determine the biochemical effectiveness of these drugs in 1978; PERRY & HANSEN,1978). The man (PERRY, results of the present investigation indicate that blood GABA analysis may be such a procedure. Thus, after acute administration of either y-acetylenic-GABA or AOAA to rats, both brain and blood GABA content increased in a dose-dependent manner. Furthermore, a decrease in brain GABA induced by acute INH treatment was also mirrored by a decrease in blood GABA content. This lowering of brain and blood GABA levels by INH is presumably due to the fact that, in addition to inhibiting GABA-T, this drug inhibits glutamic acid decarboxylase (GAD) (WOOD& PEESKER, 1973), the enzyme which catalyzes the synthesis of GABA, and under the conditions of this experiment the inhibition of GAD predominated. It is important to note, however, that at the l00mg/kg dose of INH, blood GABA was significantly reduced at a time when brain GABA 'was unchanged. This finding suggests that peripheral GAD is more sensi-
tive to inhibition by this drug than brain GAD. Other evidence for this interpretation is provided by the results obtained with the time course study of AOAA. Like INH, both y-acetylenic-GABA and AOAA inhibit GAD as well as GABA-T, with the effect on the latter enzyme being more prominent at lower doses (WOOD& F'EESKER, 1973; WOOD et al., 1977; ENNA & MAGGI, 1979). Accordingly, the delayed increase in blood GABA after AOAA treatment may reflect the action of this drug on peripheral GAD at a time when systemic levels of this drug are highest. As the content of drug decreases in the periphery. the inhibition of GABA-T predominates and blood content increases. O n the other hand, the immediate increase in brain GABA levels after this dose of AOAA suggests that either AOAA does not attain a high enough concentration in brain to significantly inhibit GAD or that brain GAD is less sensitive to the action of this drug than peripheral GAD. Along these lines it is noteworthy that a recent report indicated that GABA-T and GAD activities in various brain regions are differentially affected following the systemic administration of AOAA to rats, with the differences apparently being due to the brain distribution of the drug (WALTERS et a/., 1978). Thus it is likely that the present results are a reflection of the higher concentration of AOAA in the periphery vis-a-vis the brain rather than to kinetic differences in the sensitivity of the enzymes in the different tissues. That peripheral enzymes are more readily affected than those in the brain is also indicated by the finding that co-administration of pyridoxine abolishes the INH-induced increase in blood GABA without affecting the increase in brain content of this amino acid. The ineffectiveness of pyridoxine to modify brain GABA after INH treatment confirms an earlier report (PERRYet a/., 1974). This finding also suggests that blood GABA determinations are of no value as a monitor for brain GABA increases in subjects treated with carbonyl-trapping agents, such as INH or AOAA, together with pyridoxine.
Brain, cerebrospinal fluid and blood GABA With regard t o CSF GABA analysis, the results of the present study suggest that it may be a more direct indicator of increases in brain GABA levels than blood analysis since there was a n excellent correlation between the percentage increase in CSF GABA
33
ENNAS. J., WOODJ. H. & SNYDER S. H. (1977~)y-Aminobutyric acid (GABA) in human cerebrospinal fluid: radioreceptor assay. J . Neurochem. 28, 1121-1 124. FERKANY J. W., SMITHL. A,, SEIFERT W. E.. CAPRIOLI R. M. & ENNAS. J. (1978) Measurement of gamma-aminobutyric acid in blood. L f e Sci. 22, 2121-2128. relative t o the percentage increase in brain GABA. GLAESER B. S. & HAM T. A. (1975) Measurement of GABA However, CSF GABA levels tend t o be more variable in human cerebrospinal fluid. Biochrm. M r d . 12. than blood GABA levels, making significant changes 274-281. in CSF GABA content difficult to determine. HLIIZINGA J. C., TEtLKtN A. w., MUSKETF. A., VAN D I N It is noteworthy that brain, blood and CSF GABA MEULEN J. & WOLTHEKS B. G. (1977) Identification of levels are not significantly increased after the chronic GABA in human cerebrospinal fluid by gas liquid chromatography and mass spectrometry. N e w Enyl. J . administration of D P A to rats in a dose which is Med. 296. 692. comparable to that used clinically. This substantiates P ~ R RT. Y L. (1978) Isoniazid and Huntington’s chorea. the suggestion that the primary mechanism of action N e w Enyl. J . Med. 298, 1092-1093. of this antiepileptic drug is probably unrelated to its PERRYT. L. & HANSENS. (1978) Biochemical effects i n ability t o inhibit GABA-T (PERRY & HANSEN,1978). man and rat of three drugs which can increase brain This may also explain why this drug was ineffective GABA content. J . Neurochem. 30, 679-684. in the treatment of Huntington’s disease (SHOULSON PERRY T. L.. HANSEN S. & KLOSTER M. (1973) Huntingct ( J / . , 1976). ton’s chorea: deficiency of y-aminobutyric acid in brain. In conclusion, the present study provides evidence N e w Enyl. J . M e d . 288, 337-342. that, under appropriate conditions, blood and CSF PERRYT. L., URQUHART N.. HANSENs. & Kk:NNt,l)Y J. (1974) y-Aminobutyric acid: drug induced elevation in GABA analysis may be useful tools for both experimonkey brain. J . Nrurochrrii. 23, 443445. mental and clinical studies with GABA-T inhibitors. PERRY T. L., MACLtol) P. M. & HANSEN S. (1977) Treatment of Huntington’s chorea with isoniazid. N r w Engl. AckjiowlPdyejnrnts-This study was supported in part by J . Med. 297, 840. grants from the Pharmaceutical Manufacturers AssociSCHECHTER P. J., TRANIIX Y.. J U N G M. H. & SJOIXIXMA ation, the Huntington’s Chorea Foundation, USPHS A. (1977) Antiseizure activity of p-acetylenic y-aminobuMH-29739, NS-13803. RCDA NS-00335 (S.J.E.),and a pretyric acid: a catalytic, irreversible inhibitor of y-aminodoctoral fellowship MH-07688 (J.W.F.). butyric acid transaminase. J . Phurmuc. rxp. Ther. 201, 606-612. SCHWARCZ R.,BENNETT J. P. & COYLE J. T. (1977) Inhibitors of GABA metabolism: implications for HuntingREFERENCES ton’s disease. Ann. Neurol. 2, 299-303. ANLIZARK G., HORTON R. W.. Mk.LI)Kl:M B. S. & SAWAYASHOULSON I., KARTZINEL R. & CHASET. N. (1976) HuntingM. C. B. (1976) Anticonvulsant action of ethanolamineton’s disease: treatment with dipropylacetic acid and o-sulphate and di-n-propylacetate and the metabolism gamma-aminobutyric acid. Neurology 26, 61-63. of y-aminobutyric acid (GABA) in mice with audiogenic VAN KAMMEN D. P. (1977) y-Aminobutyric acid (GABA) seizures. Biochem. Phurmuc. 25, 413-417. and the dopamine hypothesis of schizophrenia. Am. J. BIRDE. D. & IVERSEN L. L. (1974) Huntington’s chorea: Psychiut. 134, 138- 143. postmortem measurement of glutamic acid decarboxy- WALTERS J. R., ENC N., PERICIC D. & MILLER L. P. (1978) lase. choline acetyltransferase and dopamine in basal Effects of aminooxyacetic acid and L-glutamic acid-yganglia. Bruin 97, 4577472. hydrazide on GABA metabolism in specific brain BOHLFNP., SCHECHTER P. J., VAN DAMMt w., COQUILLAT regions. J . Nrurochem. 30, 759-766. G., DOSCHJ. C. & KOCH-WASER J. (1978) Automated WEINSTEIN L. (1975) Antimicrobial agents: drugs used in assay of gamma-aminobutyric acid in human cerebrothe chemotherapy of tuberculosis and leprosy, in Thc spinal fluid. Clin. Chmi. 24, 256-260. L. S. Phurmucoloyicul Busis of Therapeutics (GOODMAN ENNAS. J. (1978) Radioreceptor assay techniques for & GILMAN A., eds.) 5th Ed., pp. 1201-1223. Macmillan, neurotransmitters and drugs, in Neurotransmitter RecepNew York. tor Binding (YAMAMURAH., ENNAS. J. & KUHARM., WOODJ. D. & PEESKER S. J. (1972~)A correlation between eds.) pp. 127-139. Raven Press, New York. changes in GABA metabolism and isonicotinic acid hyS. H. (1976) A simple. sensitive and ENNAS. J. & SNYDER drazide-induced seizures. Brain Res. 45, 489498. specific radioreceptor assay for endogenous GABA in WOOD J. D. & PEESKER S. J. (19726) The effect on GABA brain tissue. J . Nrurocheni. 26. 221 -224. metabolism in brain of isonicotinic acid hydrazide and ENNAS . J. & MAGGIA. (1979) Biochemical pharmacology pyridoxine as a function of time after administration. of GABAergic agonists. Llfe Sci. 24, 1727-1738. J . Neurochem. 19, 1527-1537. ENNAS. J., STLRN L. 2.. W A S T ~GK. J. & YAMAMURAH. I. WOOD J. D. & PEESKER S. J. (1973) The role of GABA (1977~)Neurobiology and pharmacology of Huntingmetabolism in the convulsant and anticonvulsant actions ton’s disease. L f e Sci. 20, 205-212. of aminooxyacetic acid. J . Neurochem. 20. 379-387. ENNAS. I., STERNL. Z., WASTEK G. J. & YAMAMURAH. I. WOODJ. D., DURHAM J . S. & PEESKER S. J. (1977) Effect (1977h) Cerebrospinal fluid y-aminobutyric acid variof di-n-propylacetate and y-acetylenic GABA on hyperations in neurological disorders. Archs Neurol. 34, baric oxygen-induced seizures and GABA metabolism. 683-685. Neurochem. Res. 2, 707-715.
Journal of Ncurochumicrry Vol. 33. pp. 29 to 33 Pergamon Press Ltd 1979. Printed In Great Britain
0 International Society lor Neurocliemistry Ltd
EFFECT OF DRUGS ON RAT BRAIN, CEREBROSPINAL FLUID AND BLOOD GABA CONTENT JOHN W. FERKANY, IAN J. BUTLERand S. J. ENNA' Departments of Pharmacology, Neurobiology and Anatomy, Neurology and Pediatrics, University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77025, U.S.A.
(Received 24 November 1978. Accepted 9 January 1979)
Abstract-Acute administration of GABA transaminase inhibitors to rats results in a dose-dependent increase in both brain and blood GABA content and administration of isonicotinic acid hydrazide (INH), at a dose which decreases the amount of brain GABA, also lowers blood levels of this amino acid. Chronic treatment (10 days) with INH (20 mg/kg), y-acetylenic-GABA (10 mg/kg) or aminooxyacetic acid (AOAA)(10 mg/kg) results in a significant elevation in both rat brain and blood GABA concentrations. A t the doses studied, only AOAA caused a significant elevation in CSF GABA content. Co-administration of pyridoxal phosphate (2 mg/kg) blocks the chronic INH-induced rise in blood GABA but does not affect the increase in brain content of this amino acid. Chronic administration of di-n-propylacetate (20mg/kg) did not significantly alter brain, blood or CSF GABA levels. The results suggest that, under the proper conditions, changes in blood GABA levels after administration of inhibitors of GABA synthesis or degradation may be an indirect indicator of changes in the brain content of this amino acid. Blood GABA determinations may be useful for studying the biochemical effectiveness of GABA transaminase inhibitors in man.
ALTERATIONS in the y-aminobutyric acid (GABA) neurotransmitter system have been implicated in a variety of neurological and psychiatric disorders (WOOD& PEESKER, 1972a; PERRY et al., 1973; BIRD & IVERSEN, 1974; ANLEZARKet al., 1976; ENNA et al., 1977a; VAN KAMMEN,1977). In particular, studies have suggested that increasing brain GABA content may be beneficial in epilepsy and Huntington's disease (PERRYet al., 1977; SCHWARCZet al., 1977; SCHECHTER ef a/., 1977; WOOD et al., 1977). In these studies, brain GABA levels are increased by the administration of agents known to inhibit 4-aminobutyrate : 2-oxoglutarate aminotransferase (EC 2.6.1.19; GABA-T), the enzyme which catalyzes the degradation of GABA. However, while in laboratory studies the effectiveness of GABA-T inhibitors can be readily determined by directly measuring the brain content of GABA, there is no clinical procedure capable of monitoring the biochemical effectiveness of these agents. Such a clinical test may be useful in determining the mechanism of action of these agents in man and in characterizing the biochemical abnormalities underlying these disorders. Recently, methods have been developed which are capable of measuring GABA in human cerebrospinal
fluid (CSF) (GLAESER& HARE, 1975; ENNA rt al., 1977c; HUIZINGA et a!., 1977; BOHLENet a!., 1978). In additon, a procedure has been described which is capable of detecting the trace quantities of GABA in blood (FERKANY et al., 1978). While CSF GABA appears to originate from brain (ENNAet al., 19776, c), the primary source of blood GABA is unknown. However, preliminary studies indicated that administration of a GABA-T inhibitor raises blood GABA levels as well as brain GABA content, suggesting that blood GABA determinations may be a useful clinical tool to indirectly monitor the biochemical effectiveness of drugs which are known to inhibit this enzyme (FERKANY et al., 1978). In the present investigation this possibility is further explored by comparing changes in CSF and blood GABA content with brain GABA levels after either acute or chronic administration of drugs which alter GABA metabolism. The results indicate that, under the proper conditions, blood GABA determinations may be used as an indirect monitor of brain GABA variations in response to these drugs.
MATERIALS AND METHODS
' To whom reprint requests should be addressed, at the
Drug administration and sample collection. Male Sprague-
Departments of Pharmacology and of Neurobiology and Anatomy. Abbreuiations used: GABA, y-aminobutyric acid; AOAA, aminooxyacetic acid; GAD, L-glutamic acid decarboxylase (EC 4.1.1.15); GABA-T, Caminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19); INH, isonicotinic acid hydrazide; DPA, di-n-propylacetic acid
Dawley rats (200-25Og) were used and drugs, dissolved in lactated sterile Ringers solution (Travenol Labs, Deerfield, IL) just prior to treatment, were injected intraperitoneally. Control animals received an equivalent volume of vehicle. Prior to sample collection, animals were tightly anesthetized with chloral hydrate (Sigma Chemical Co, St. Louis.
29
30
IANJ. BUTLER and S. J. ENNA JOHNW. FERKANY,
MO; 400 mg/kg, i.p.). For CSF collection, a 25 gauge Butterfly intravenous needle with a blunted bevel was carefully inserted into the cisterna magna and the CSF drained spontaneously into an ice-chilled 1 ml glass microfuge tube by way of a cannula attached to the needle. Over a 3 W 5 min period. 100-300 pl of CSF were collected and then stored at -20°C until assayed. After withdrawal by cardiac puncture, blood was transferred into I5 ml Sorvall centrifuge tubes which contained an equal volume of 7% perchloric acid. Following a brief vortex mixing, the samples were centrifuged at 48,000g for 20 min at 4°C. After centrifugation, the clear supernatant was decanted into another Sorvall centrifuge tube and was carefully added to bring the pH sufficient ~ N - K O H to 7.0. Neutralized supernatant was centrifuged at 48,000 g for 20 min at 4°C to separate the insoluble KCIO,. Following centrifugation, the supernatant was decanted into 5 ml glass culture tubes and stored at 2°C until assayed. Immediately after blood collection the whole brain was rapidly removed and frozen in a dry iceeacetone bath. These frozen samples were stored at -20°C until assayed. In experiments where CSF was not taken animals were lightly anesthetized with ether prior to removing blood and brain samples. Sample analysis. Blood, CSF and brain GABA content were measured by radioreceptor assay using C3H]GABA 1976; ENNA et a/., 1 9 77 ~ ; as a ligand (ENNA& SNYDER. et al., 1978). The principle of this assay is related FERKANY to the fact that the quantity of C3H]GABA bound to rat brain membranes is a function of the amount of unlabeled GABA present in the incubation medium (ENNA, 1978). For assay, portions of the neutralized blood extract were analyzed directly. Frozen brain samples were homogenized in 20 vol 5% trichloroacetic acid with a Brinkman Polytron PT-10. The homogenate was centrifuged at 48,OOOg for 10min and a measured portion of the supernatant was diluted 2500-fold with water. A 10-5Opl portion of this diluent was used for analysis. The GABA content of CSF was determined by analyzing 75-100 pl portions of the untreated fluid. In all cases, dilutions were made such that between 30 and 70% of the bound radioactivity was displaced by the sample. Standard curves for the displacement of ['HIGABA by known quantities of unlabeled GABA were generated daily. Each sample was analyzed on two separate occasions, and each analysis was performed in duplicate. The significance of differences between means was determined using a two-tailed Student's t-test. Chemicals. Di-n-propylacetate (DPA) was obtained from Dr. JUDYWALT~RS, y-acetylenic-GABA from Merrell International (Strasbourg, France), isonicotinic acid hydrazide from Eastman Kodak (Rochester, NY), aminooxyacetic acid from Sigma Chemical Co. (St. Louis, MO), and pyridoxal-5-phosphate from Calbiochem (La Jolla, CA). ['H]yAminobutyric acid (54.1 Ci/mmol) was purchased from Amersham Corporation (Arlington Heights, IL). RESULTS
Dose-response studies The concentration of GABA in brain and blood rose in a dose-dependent manner after administration of either y-acetylenic-GABA or AOAA (Tables 1 and 2). However, for both drugs, a t a given dose, the increase in brain GABA was always greater than
T A B L1.~DOSE-RESPONSE OF BRAIN A N D BLOOD GABA CONTENT TO Y-ACETYLENIC-GABA Dose (mg/kg) Control (13) 10 (4) 50 (4) 100 (4)
GABA concentration* Brain Blood (vnolig) (nmol/ml)
*
2.2 0. t 4.1 i 0.7$ 6.4 k 0.63 8.6 k 0.21
0.7 k 0.04 0.9 2 0.021 1.1 k 0.07t 1.2 f 0.14t
Values are the mean ~ s . E . Mof. the number of experiments shown in parentheses. * Analyzed 5 h after i.p. injection. 1P < 0.05. $ P < 0.001. TABLE2. DOSE-RESPONSE OF BRAIN
AND BLOOD TENT TO AMINOOXYACETIC ACID
Dose (mg/kg) Control (6) 5 (4) 15 (4) 40 (4)
GABA
CON-
GABA concentration* Brain Blood (pmoI/g) (nmol/ml) 1.7 k 0.2 2.8 0.37 4.3 0.31 6.8 k 0.2$
+
0.7 k 0.05 1.0 k 0.08t 1.6 2 0.043 1.7 f 0.053
Values are the mean ~ s . E . M . of the number of experiments shown in parentheses. * Analyzed 4 h after i.p. injection. t P < 0.01. 3 P < 0.001. the increase in the blood content, with y-acetylenic-
GABA increasing the brain concentration 24-fold over a dose range of 1G-100mg/kg, whereas the blood GABA concentration in these same animals did not quite double (Table 1). Similarly, AOAA induced a 4-fold increase in brain GABA over a dose range of 5 4 0 m g / k g , but only a 2-fold increase in blood GABA was observed (Table 2). A significant decrease in both brain and blood
GABA was observed a t 9 0 m i n after the administration of 300 mg/kg INH (Table 3) with a similar magnitude of change in brain and blood GABA content, being reduced approx 30% in brain and approx 40% in blood at this dose. Also, with INH. blood but not
3. DOSE-RESPONSE OF BRAIN TABLL
A N D BLOOD GABA CONTENT TO ISONICOTINIC ACID HYDKAZIDC
Dose (mg/kg) Control (6) 50 (4) 100 (4) 300 (4)
GABA concentration* Brain Blood (mol/g) (nmol/ml) 1.7 f 0.2 1.7 0.2 1.6 0.1 1.2 O.1t
*
0.8 f 0.07 0.8 & 0.07
0.6 $- 0.031 0.5 f 0.031
Values are the mean ~ s . E . Mof. the number of experiments shown in parentheses. * Analyzed 90 min after i.p. injection. t P < 0.05. $ P < 0.001.
31
Brain. cerebrospinal fluid and blood GABA
were significantly elevated, being 3-fold and 4-fold higher than control levels, respectively. E j k t of chronic drug administration on brain, CSF und blood G A B A content
m a
0
a
a m
1.0
30
150
90
300
TIME (min.)
FIG. 1. Time course of the effect of a single i.p. dose of AOAA (40mg/kg) on rat brain (A) and blood (B) GABA content. Each point is the mean k S.E.M. of 3 experiments.
brain GABA was significantly reduced at a dose of 100 mg/kg (Table 3). Timi. course of response to A O A A
Analysis of blood and brain GABA content at various times after administration of a single dose (40 mg/kg) of AOAA revealed that while the brain GABA concentration steadily increased over a 5 h period, there was no significant change in blood GABA content up to 90min after injection (Fig. 1A and B). Between 90 and 150 min after injection, blood GABA levels increase at a rate similar to that observed for brain GABA. By 5 h after treatment with AOAA, both blood and brain GABA concentrations TABLE 4. BLOOD,CSF
Drug Control (25) DPA (4) INH (6) y- Acetylenic-
Chronic administration of INH, y-acetylenicGABA and AOAA significantly elevated brain and blood GABA levels (Table 4). Concentrations of GABA in CSF, although increased in all cases, were significantly elevated only after chronic administration of AOAA. No increase in brain, CSF or blood GABA was detected in animals treated chronically with 20 mg/kg DPA (Table 4). As opposed to the results observed after acute administration, chronic administration of the effective drugs resulted in an elevation of blood GABA similar to or, in the case of AOAA, greater than the elevation in brain GABA content. Thus, after 10 days of chronic administration of INH (20 mg/kg, b.i.d.), brain GABA increased 26% relative to saline treated controls whereas blood GABA increased 45%. With y-acetylenic-GABA (10 mg/kg, bid.), a 10 day treatment resulted in brain GABA levels 40% higher than controls with blood GABA levels being elevated 77%. A similar 10 day treatment with AOAA (IOmg/kg once daily) induced increases in brain and blood GABA content of 67% and 164%, respectively (Table 4). It is noteworthy that even though CSF GABA was significantly increased only after AOAA, the percentage increase in CSF GABA observed after the various drug treatments was similar to the percentage increase in brain content. Thus, after INH, CSF GABA increased 26%, after y-acetylenic-GABA, 50% and after AOAA, SS%, values which compare favorably with the 26, 40 and 67% increases in brain GABA found after administration of these three drugs.
A N D BRAIN GABA CONTENT FOLLOWING CHRONIC TION OF VARIOUS DRUGS
Dose (mg/kg) ~
Brain (Pmolig) 1.9
+ 0.1
ADMINISTRA-
GABA concentration Blood CSF (nmoliml) (pmoliml) 0.8 -t 0.09*
* 22 93 * 20
100
20 b.i.d.
2.3 k 0.2
0.8
k 0.10
20 b.i.d.
2.4 k 0.2*
1.1
*
0.1*
130 k 47
10 b.i.d.
2.7 & 0.2t
1.4 k 0.3*
160 & 35
3.2
2.0 k 0.4t
200
GABA (5)
AOAA
10 once daily
0.23
k 16*
(6)
Values are the mean ~ s . E . M . of the number of experiments shown in parentheses. All drugs were administered for 10 days and the animals were killed 24 h following the final dose. * P < 0.05. 'r P < 0.01. 1P < 0.001.
32
JOHN
W. FERKANY, IAN J. B U T L ~ and R S. J. ENNA
TABLE 5. EFFECTOF PYRIDOXINE ON
BLOOD, CSF A N D BRAIN GABA CONTENT FOLLOWING CHRONIC ADMINISTRATION OF INH
Drug treatment
Dose (mg/kg)
1.5
Control
+ 0.2
GABA concentration Blood CSF (nmol/ml) (pmol/ml) 1.2 k 0.07
88 k 14
2
1.5 k 0.2
1.2
* 0.1
80 2 7
2 20
2.3 f 0.2'
1.3
k 0.1
130 k 28
(5)
Pyridoxine
Brain (Pmolig)
(61
Pyridoxine
+ INH
(5)
Values are the mean ~ s . E . Mof. the number of experiments shown in parentheses. Drugs were administered twice daily for 8 days and the animals killed 16 h following the final dose. * P i0.025.
e f f e c t of pyridoxine on INH-induced elevations of GABA
When used clinically, INH is administered with pyridoxine to counter INH-induced pyridoxine defiet a/., 1977) and it ciency (WEINSTEIN, 1975; PERRY has been reported that co-administration of pyridoxine alters the biochemical response to INH (WOOD & PEESKER, 1972b). To test the interaction of these two compounds on brain, CSF and blood GABA content, animals were treated chronically with either pyridoxine alone or pyridoxine in combination with INH (Table 5). The results indicate that while pyridoxine itself has no effect on GABA content, this compound does inhibit the increase in blood, but not brain, GABA levels observed after chronic INH treatment (Tables 4 and 5).
DISCUSSION Rccent clinical studies with GABA-T inhibitors have revealed a need for a simple method to determine the biochemical effectiveness of these drugs in 1978; PERRY & HANSEN,1978). The man (PERRY, results of the present investigation indicate that blood GABA analysis may be such a procedure. Thus, after acute administration of either y-acetylenic-GABA or AOAA to rats, both brain and blood GABA content increased in a dose-dependent manner. Furthermore, a decrease in brain GABA induced by acute INH treatment was also mirrored by a decrease in blood GABA content. This lowering of brain and blood GABA levels by INH is presumably due to the fact that, in addition to inhibiting GABA-T, this drug inhibits glutamic acid decarboxylase (GAD) (WOOD& PEESKER, 1973), the enzyme which catalyzes the synthesis of GABA, and under the conditions of this experiment the inhibition of GAD predominated. It is important to note, however, that at the l00mg/kg dose of INH, blood GABA was significantly reduced at a time when brain GABA 'was unchanged. This finding suggests that peripheral GAD is more sensi-
tive to inhibition by this drug than brain GAD. Other evidence for this interpretation is provided by the results obtained with the time course study of AOAA. Like INH, both y-acetylenic-GABA and AOAA inhibit GAD as well as GABA-T, with the effect on the latter enzyme being more prominent at lower doses (WOOD& F'EESKER, 1973; WOOD et al., 1977; ENNA & MAGGI, 1979). Accordingly, the delayed increase in blood GABA after AOAA treatment may reflect the action of this drug on peripheral GAD at a time when systemic levels of this drug are highest. As the content of drug decreases in the periphery. the inhibition of GABA-T predominates and blood content increases. O n the other hand, the immediate increase in brain GABA levels after this dose of AOAA suggests that either AOAA does not attain a high enough concentration in brain to significantly inhibit GAD or that brain GAD is less sensitive to the action of this drug than peripheral GAD. Along these lines it is noteworthy that a recent report indicated that GABA-T and GAD activities in various brain regions are differentially affected following the systemic administration of AOAA to rats, with the differences apparently being due to the brain distribution of the drug (WALTERS et a/., 1978). Thus it is likely that the present results are a reflection of the higher concentration of AOAA in the periphery vis-a-vis the brain rather than to kinetic differences in the sensitivity of the enzymes in the different tissues. That peripheral enzymes are more readily affected than those in the brain is also indicated by the finding that co-administration of pyridoxine abolishes the INH-induced increase in blood GABA without affecting the increase in brain content of this amino acid. The ineffectiveness of pyridoxine to modify brain GABA after INH treatment confirms an earlier report (PERRYet a/., 1974). This finding also suggests that blood GABA determinations are of no value as a monitor for brain GABA increases in subjects treated with carbonyl-trapping agents, such as INH or AOAA, together with pyridoxine.
Brain, cerebrospinal fluid and blood GABA With regard t o CSF GABA analysis, the results of the present study suggest that it may be a more direct indicator of increases in brain GABA levels than blood analysis since there was a n excellent correlation between the percentage increase in CSF GABA
33
ENNAS. J., WOODJ. H. & SNYDER S. H. (1977~)y-Aminobutyric acid (GABA) in human cerebrospinal fluid: radioreceptor assay. J . Neurochem. 28, 1121-1 124. FERKANY J. W., SMITHL. A,, SEIFERT W. E.. CAPRIOLI R. M. & ENNAS. J. (1978) Measurement of gamma-aminobutyric acid in blood. L f e Sci. 22, 2121-2128. relative t o the percentage increase in brain GABA. GLAESER B. S. & HAM T. A. (1975) Measurement of GABA However, CSF GABA levels tend t o be more variable in human cerebrospinal fluid. Biochrm. M r d . 12. than blood GABA levels, making significant changes 274-281. in CSF GABA content difficult to determine. HLIIZINGA J. C., TEtLKtN A. w., MUSKETF. A., VAN D I N It is noteworthy that brain, blood and CSF GABA MEULEN J. & WOLTHEKS B. G. (1977) Identification of levels are not significantly increased after the chronic GABA in human cerebrospinal fluid by gas liquid chromatography and mass spectrometry. N e w Enyl. J . administration of D P A to rats in a dose which is Med. 296. 692. comparable to that used clinically. This substantiates P ~ R RT. Y L. (1978) Isoniazid and Huntington’s chorea. the suggestion that the primary mechanism of action N e w Enyl. J . Med. 298, 1092-1093. of this antiepileptic drug is probably unrelated to its PERRYT. L. & HANSENS. (1978) Biochemical effects i n ability t o inhibit GABA-T (PERRY & HANSEN,1978). man and rat of three drugs which can increase brain This may also explain why this drug was ineffective GABA content. J . Neurochem. 30, 679-684. in the treatment of Huntington’s disease (SHOULSON PERRY T. L.. HANSEN S. & KLOSTER M. (1973) Huntingct ( J / . , 1976). ton’s chorea: deficiency of y-aminobutyric acid in brain. In conclusion, the present study provides evidence N e w Enyl. J . M e d . 288, 337-342. that, under appropriate conditions, blood and CSF PERRYT. L., URQUHART N.. HANSENs. & Kk:NNt,l)Y J. (1974) y-Aminobutyric acid: drug induced elevation in GABA analysis may be useful tools for both experimonkey brain. J . Nrurochrrii. 23, 443445. mental and clinical studies with GABA-T inhibitors. PERRY T. L., MACLtol) P. M. & HANSEN S. (1977) Treatment of Huntington’s chorea with isoniazid. N r w Engl. AckjiowlPdyejnrnts-This study was supported in part by J . Med. 297, 840. grants from the Pharmaceutical Manufacturers AssociSCHECHTER P. J., TRANIIX Y.. J U N G M. H. & SJOIXIXMA ation, the Huntington’s Chorea Foundation, USPHS A. (1977) Antiseizure activity of p-acetylenic y-aminobuMH-29739, NS-13803. RCDA NS-00335 (S.J.E.),and a pretyric acid: a catalytic, irreversible inhibitor of y-aminodoctoral fellowship MH-07688 (J.W.F.). butyric acid transaminase. J . Phurmuc. rxp. Ther. 201, 606-612. SCHWARCZ R.,BENNETT J. P. & COYLE J. T. (1977) Inhibitors of GABA metabolism: implications for HuntingREFERENCES ton’s disease. Ann. Neurol. 2, 299-303. ANLIZARK G., HORTON R. W.. Mk.LI)Kl:M B. 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