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Studies on the functional significance of carbonic anhydrase in central nervous system
BRAIN RESEARCH
185
STUDIES ON T H E F U N C T I O N A L S I G N I F I C A N C E OF CARBONIC A N H Y D R A S E IN C E N T R A L NERVOUS SYSTEM
V. NAIR AND D. BAU Laboratory for Therapeutics Research, Michael Reese Haspital Psychiatric Institute, and Department of Pharmacology, Chicago Medical School, Chicago, Ill. 60616 (U.S.A.)
(Accepted February 24th, 1971)
INTRODUCTION Carbonic anhydrase (CA)* was first demonstrated in the nervous system by Van G o o r in 1940 and since then many investigators have studied the enzyme in detail with respect to its detection, tissue distribution, inhibition, and other properties. However, its physiological role in the nervous system is not completely understood. Our studies with ionizing radiation have provided us with an unique opportunity to pursue this question12,14,15. Earlier, we observed in rats that high doses of cephalic X-irradiation resulted in the production of an apparent anticonvulsant effect as evidenced by the abolition of the maximal electroshock seizure response 14. Acetazolamide (Diamox) is one of the pharmacological agents which inhibits carbonic anhydrase. Millichap et al. 1° have observed an association between the anticonvulsant action of acetazolamide and brain carbonic anhydrase inhibition and postulated that the anticonvulsant action of acetazolamide may be related to the inhibition of this enzyme in brain. Nishimura 17 has also pointed out that the antiepileptic effects of carbonic anhydrase inhibitors might be attributed to checking the propagation of seizure discharges by the inhibition of carbonic anhydrase in brain. Similarly, Gray et al. 5 have reported that the inhibition of brain carbonic anhydrase appeared to be the prime event in the anticonvulsant action of acetazolamide. But, on the basis of more recent studies from their laboratories, Gray and Rauh ~ have concluded that the anticonvulsant action of carbonic anhydrase inhibitors is mediated by disequilibrium of the CO2 buffer system. We thought it worthwhile to determine whether this association between enzyme inhibition and anticonvulsant action holds true in other situations where the seizure suppression resulted from means other than pharmacologic. If such a relationship is found to be a common factor in different situations, no doubt it will strengthen the case for carbonic anhydrase in the regula-
* Carbonate hydro-lyase (EC 4.2.1.1) is the term recommended by the commission on Enzymes of International Union of Biochemistry for the enzyme catalyzing the hydration of CO2, and carbonic anhydrase, the trivial name. Brain Research, 31 (1971) 185-193
186
V. N A I R A N D D. B A U
tion of brain excitability. Accordingly, we have determined the enzyme activity in various brain regions in the following groups of animals: (1) normal, (2) acetazolamide treated, (3) head irradiated, (4) head irradiated and acetazolamide treated. While these studies were in progress, Professor B. Ginsburg*, of the University of Chicago, has made available to us two groups of mice which have been reared in his laboratory of behavioral genetics. One was audiogenic seizure prone (DBA) and the other was audiogenic seizure resistant (C57/6). Brain CA was determined in these mice as well. Finally, it is known that, in a sample of rats selected at random, a small percentage (8-10 ~ ) are seizure resistant when tested by the maximal electroshock seizure method. We have taken this group of naturally seizure resistant rats after prescreening them and examined their brain CA levels. MATERIALS A N D M E T H O D S
Animals
Male Sprague-Dawley rats 70-80 days old were used in these studies. They were housed under controlled environmental conditions (temp. 72°F, light 5 a.m.-7 p.m.). Food and water were given ad libitum. X-irradiation
The head alone was exposed to X-irradiation from a 250 kV, 30 mA GE Maxitron X-ray machine. The filters employed were 0.5 mm Cu and 1 mm A1. The half value layer of the beam was 1.4 mm Cu. During irradiation the animals were unanesthetized and held in lucite tubes provided with a large number of air holes. For shielding, a sheet of lead (3.5 mm thick), shaped to fit the holders, covered the entire body except the region of the head. They were held on a rotating platform (3.5 rev./ min) for uniformity of field. Control animals were sham irradiated. Doses ranging from 50(O10,000 R were employed but this study deals with the animals receiving 10,000 R. A cetazolamide
In the enzyme studies, control rats were given 200 mg/kg acetazolamide intraperitoneally (Diamox, Lederle Labs.). The time of maximal inhibition of enzyme activity was first determined by examining the brain at 1, 2, or 3 h after drug administration. Since there was no difference between the 2 and 3 h period, all animals were sacrificed at 2 h after drug administration. Regional dissection of the brain was performed according to a frozen state dissection method reported earlierll, 1~. The central nervous system (CNS) regions examined were: cerebral cortex, caudate nucleus, hippocampus, cerebellar cortex, medulla and spinal cord. The X-irradiated animals were examined at 3 days postirradiation. They receiv* Presently at the University of Connecticut, Storr, Conn. Brain Research, 31 (1971) 185-193
187
BRAIN CARBONIC ANHYDRASE AND FUNCTION
ed 2.5 mg/kg acetazolamide and were sacrificed 0.5 h later. The time of examination and drug dosage were selected on the basis of our previous findings in the X-irradiated rats 15. In mice, only the caudate, hippocampus and medulla were examined for CA activity. Carbonic anhydrase activity was determined according to the micromethod of Maren 7 as outlined in a previous articlelL M E S resistant rats
Rats were subjected to the MES test according to the method of Toman et al. 19. The apparatus employed to produce the seizures was a constant current stimulator (Wahlquist, Salt Lake City, Utah) of the type described by Woodbury and Davenport 20. Maximal seizure was induced by 150 mA delivered through corneal electrodes for 0.2 sec. Rats were tested every day for 4 days and were then grouped into MES positive and MES resistant ones based on their consistent response for the 4 days. Those responding with tonic extension of the hind limbs were termed MES positives and those failing to show the extensor response as MES resistant ones. Rats were sacrificed for enzyme assay 2 days after the last shock. The procedures for electrical stimulation and EEG recordings were reported in a previous articlelL Mice
Male mice, 35-40 days old, from DBA and C57/6 strains were used. The dissection of the brain regions was performed in a manner similar to that described for rats except that only 3 regions were taken. They were: caudate nucleus, hippocampus and medulla. For each assay, the pooled tissue from 3 brains were used.
TABLE I CARBONIC ANHYDRASEACTIVITYIN RAT C N S (units/g tissue 4- S.E.)
Anatomical region
Frontal cortex Caudate nucleus Hippocampus Cerebellar cortex Medulla Spinal cord
Treatment Control
Control 4acetazolamide* (200 mg/kg)
X-ray
X-ray jr acetazolamide (2.5 mg/kg)
34.4 4- 3.2 30.8 ± 4.4 36.7 4- 2.5 46.5 4- 5.0 97.0 4- 4.3 55.4 4- 3.0
17.4 4- 1.1 4.9 4- 0.9 11.7 4- 1.9 20.4 4- 2.3 61.4 4- 1.6 38.8 4- 1.2
31.5 ± 4.8 7.5 4- 1.6 35.0 4- 0.4 20.3 4- 3.1 50.7 4- 3.8 48.1 4- 1.2
11.4 -b 2.2 4.5 4- 0.4 13.2 4- 0.2 9.9 4- 0.8 33.4 4- 1.6 35.8 4- 1.3
* At the dose of 2.5 mg/kg, acetazolamide produced no significant effects on CA activity in the control rats (see text). Brain Research, 31 (1971) 185-193
188
V. NAIR AND D. BAU ONTROL ~-ETAZOLAMIDE ( 2 . 5 m g / k g ) - R A Y (10,OOO R) + ACETAZOL. (2.5 mg/kg)
100 u) u~
o
I--
! ....
80
S_-_-
60
-I-S_
Z
0
a. 02
"---
i-
a.
20
Fig. 1. Effect of acetazolamide, X-irradiation and the combination on the maximal electroshock seizure response (MES). ~ positive response to MES refers to the percent of animals exhibiting the hind limb tonic extensor response. Acetazolamide (2.5 mg/kg) was given i.p. and the animals examined 0.5 h later. In the irradiated series, the animals were tested at 48-72 h postirradiation (for further details see Nair et a1.15).
IiU.4mAIMIIlII ~maet
1111~. I~1' r
el I 1 ~ I ~ i l ¢ "t"
S1WMATIOII
SllW~mU
Sll/UAIQI
Fig. 2. EEG records. Effect of X-irradiation and irradiation plus acetazolamide before and after electrical stimulation of the sensorimotor cortex of the rat. Stimulation parameters: 30 V, 50 c/sec, 10 sec. The drug (2.5 mg/kg) was given i.p. and stimulus applied 30 rain later (for further details see N air et al.l~).
RESULTS T h e regional d i s t r i b u t i o n of CA in c o n t r o l rat brain is s h o w n in T a b l e I. As reported earlier 11, in the controls, C A activity w a s highest in the m e d u l l a . T w o h o u r s after a c e t a z o l a m i d e a d m i n i s t r a t i o n
(200 mg/kg)
the activity decreased in all the re-
gions. T h e e n z y m e activity w a s l o w e s t in the c a u d a t e n u c l e u s Brain Research, 31 (1971) 185-193
(Table I).
X-irradiation
189
BRAIN CARBONICANHYDRASEAND FUNCTION CARBONIC ANHYDRASE
IN
RAT
CNS
I---:::
b
+
100
100
80
80
60
60
40
40
20
20
F. CORTEX
CAUDATE
HIPPOCAMP.
CEREBELL.
MEDULLA
SP. CORD
Fig. 3. Comparison of the CA levels in the CNS of MES + (exhibiting the hind limb tonic extensor response) and MES- (not showing hind limb tonic extensor response) rats. Data presented are the average of 6 animals. MOUSE
u
_ _
DBA
--80
80 uJ
°
CAUDATE
/ i HIPPOCAMPUS
b
60
40
20
MEDULLA
Fig. 4. Carbonic anhydrase activity in mouse CNS. alone, also lowered the enzyme activity in the different brain regions, with maximal inhibition again being produced in the caudate nucleus. It was observed in our earlier studies that irradiation, by itself, produced a significant degree o f protection against M E S (Fig. 1). Acetazolamide, at a dose level (2.5 mg/kg), which gave little protection (3 9/0) against MES in non-irradiated controls, abolished the tonic extensor response in all the irradiated animals (Fig. 1). Furthermore, this occurred 0.5 h after drug treatment indicating an enhancement o f the speed o f onset as well as intensity o f drug action in the irradiated animals. These effects on Brain Research, 31 (1971) 185-193
190
v. NAIR AND D. BAU
the seizure response have been also supported by EEG records (Fig. 2). it was also found that at this dose level (2.5 mg/kg) of acetazolamide, no significant suppression of CA activity was noted in the non-irradiated controls when examined 0.5 h postdrug treatment. In contrast, in the irradiated animals, acetazolamide enhanced the irradiation induced suppression of enzyme activity in all the brain regions, maximal effect being in the caudate nucleus (Table I). The pattern of CA activity in the CNS of MES resistant rats has also been examined (Fig. 3). The enzyme levels were lower in all the regions, in comparison to the MES positive rats, this decrease being most manifest in the caudate nucleus. Enzyme activity in the caudate nucleus was 59 ~ of that in the MES positive rats. A similar phenomenon is seen also in mice (Fig. 4). The CA activity was markedly lower in the caudate nucleus of the audiogenic seizure resistant mice (C57/6) than in the seizure prone ones (DBA). DISCUSSION Carbonic anhydrase catalyzes the reaction: E
H20 + CO~ ~- H2CO8 ~ H + + HCOsThe second step from H2CO3 to H + and HCOz- is instantaneous so that the enzyme catalyzed reaction is the rate limiting step in the overall process. Single cell determinations by Giacobini 3 in rat brain have demonstrated that the enzyme is concentrated in the glial cells. The nature of the ultimate end products of the decarboxylation reaction in brain is controversial. Natour and Palmer 16 studied mouse brain homogenate and concluded that carbon dioxide was the primary product. Severinghaus et aL as also studied this question using rat brain homogenate, but their results pointed to carbonic acid (HCO3 and H ÷) as the immediate end product. Based on these divergent findings, different interpretations as to the role of carbonic anhydrase in brain have been offered. It would seem that a sound theory regarding the role of carbonic anhydrase in brain should take into account both the clinically well known inhibitory effect of carbon dioxide on seizures and the anticonvulsant action of carbonic anhydrase inhibitors. Davenport z found that in order to reduce the rate of catalyzed uptake of carbon dioxide by blood by 9 0 ~ , carbonic anhydrase in the red cells must be inhibited by more than 99.97~. Similarly, Maren s, from his studies of the enzyme in kidney, stomach, and pancreas, came to the conclusion that CA inhibition is not recognizable physiologically unless about 99 ~ of the tissue enzyme is inactive. It appears that the enzyme concentrations in these tissues, in so far as they have been studied, are in excess of maximum physiological needs. The situation with respect to the brain is the following: Ashley and Schuster I have shown that there is a late appearance of CA in the cerebrum of young animals born in an immature condition (dog, cat, rat and rabbit) while the enzyme is present at birth in the cerebrum of animals more mature when Brain Research,
31 (1971) 185-193
BRAIN CARBONIC ANHYDRASEAND FUNCTION
191
born such as the calf and guinea pig. Their data also suggest a correlation between the appearance of the enzyme in the cerebrum and the beginning of its functioning as indicated by parallel maturation of certain sense organs and EEG data. In humans, according to Gibbs and Gibbs 4, the normal newborn infant shows irregular slow waves and there is nothing to correspond to the 9 or 10/sec waves so common in the adults. By the time the child is 7 years old, the brain waves reach the frequency found in adults. A parallel relationship is revealed in the enzyme development also. Ashley and Schuster found little or no CA in the brain of newborn human, while in the brain of a child, 7 years old, CA content was found to be comparable to the adult level. The work of Millichap 9 has revealed a direct relation between the activity of brain CA and the susceptibility of animals to experimental seizures. Working with newborn animals of different species, he has shown that the development of susceptibility of various experimental seizure patterns is directly related to the level of brain CA. Based on his results, he suggested that brain CA and its specific catalytic activity mat be essential for the propagation of the seizure discharge. Low levels of enzyme activity may be sufficient for focal discharge and the induction of minor seizure patterns, while a relatively high degree o f activity may be required for the propagation of a generalized seizure discharge and the induction of a major tonic seizure. Besides confirming the earlier observations of Millichap, our results go further and suggest that the CA level in the caudate nucleus plays an important role in the regulation of the maximal seizure response. The fact that this association of low enzyme activity in caudate nucleus and anticonvulsant effect is demonstrated in 4 different experimental situations and in two species, makes it, we believe, highly significant. It should be emphasized that the term anticonvulsant action as used here refers to the abolition of the tonic extensor phase of the maximal electroshock seizure response. In all the experimental situations where we found an anticonvulsant effect (as indicated by the suppression of the tonic extensor phase) CA activity was relatively lowest in the caudate nucleus. However, the enzyme level was inhibited to varying degrees in other regions also. The importance of the caudate nucleus is suggested from the fact that it is in this region that CA is relatively lowest in all cases. The maximal electroshock seizure test is commonly employed in the screening of potential anticonvulsant drugs, and the abolition of the tonic hindlimb extension is the frequently used end point in such tests. Clinically recognized antiepileptic agents (anti-grand mal agents in particular) abolish the tonic phase of major seizures even when these drugs fail to raise appreciably the seizure threshold 19. The anatomical pathways and the mechanisms governing the maximal seizure pattern have not been clearly defined. Zablocka and Esplin 21 have studied the mechanisms responsible for the inability of some rats to exhibit tonic hind limb extension. Their results point to inefficiency in the spread of seizure discharge through the brain as the major mechanism responsible for this pattern. This is understandable when one considers that the tonic extensor seizure represents the maximum rate of dissipation of energy of which the brain is capable. The tonic extensor response can be altered by factors which interfere with the intensity or the spread of seizure discharge. The question whether carbonic anhydrase influences both of the above factors Brain Research, 31 (1971) 185-193
192
V. NAIR AND D. BAU
or only one of them specifically can only be answered by further investigations. SUMMARY The role of carbonic anhydrase in the regulation of seizure activity has been studied by comparing the brain enzyme levels of normal rats with those of rats found seizure resistant in the maximal electroshock test. These included rats (1) treated with acetazolamide, an anticonvulsant drug; (2) exposed to head X-irradiation, a procedure known to abolish the tonic extensor response to maximal electroshock; and (3) exposed to a combination of acetazolamide and head X-irradiation. In addition, rats which are congenitally seizure resistant in the maximal electroshock test as well as two strains of mice, one audiogenic seizure prone and the other seizure resistant, were also examined in this study. The enzyme determinations were done in different anatomical regions of the CNS. Our results show a region specific effect. In all the conditions where seizure activity was suppressed, carbonic anhydrase activity was also suppressed with the greatest change occurring in the caudate nucleus. Since it is demonstrated in widely different experimental conditions and in two species, the association of a region specific inhibition of carbonic anhydrase in the caudate nucleus and seizure resistance (anticonvulsant action) is considered highly significant. ACKNOWLEDGEMENTS This work was supported in part by grants from the state of Illinois, Department of Mental Health. The authors wish to thank the University of Chicago Argonne Cancer Hospital and Department of Radiology for use of the radiation facilities and Dr. James Bland for technical assistance with the radiation procedure.
REFERENCES 1 ASHLEY,W., AND SCHUSTER, E. M., Carbonic anhydrase in the brain of the newborn in relation to functional maturity, J. biol. Chem., 184 0950) 109-116. 2 DAVENPORT, H. W., The inhibition of carbonic anhydrase by thiophene-2-sulfonamide and sulfanilamide, J. biol. Chem., 158 0945) 567-571.
3 GIACOBINI,E., A cytochemical study of the localization of carbonic anhydrase in the nervous system, J. Neurochem., 9 (1962) 169-177. 4 GIBBS, F. A., AND GIBBS,E. L., Atlas o f Encephalography, Cummings, Cambridge, Mass., 1941. 5 GRAY, W. O., MAREN, T. H., SISSON, G. M., AND SMITH, F. H., Carbonic anhydrase inhibition. VII. Carbonic anhydrase inhibition and anticonvulsant effect. J. Pharmacol. exp. Ther., 121 (1957) 160-170. 6 GRAY, W. D., AND RAUH, C. E., The anticonvulsant action of carbon dioxide: Interaction with reserpine and inhibitors of carbonic anhydrase, J. Pharmacol. exp. Ther., 163 (1968) 431438. 7 MARES, T. H., A simplified micromethod for the determination of carbonic anhydrase and its inhibitors, J. Pharmacol. exp. Ther., 130 (1960) 26-29. 8 MARES, T. H., The relation between enzyme inhibition and physiologicalresponse in the carbonic anhydrase system, J. Pharmacol. exp. Ther., 139 (1963) 140-153. 9 MILLICHAP, G. J., Seizure patterns in young animals. Significance of brain carbonic anhydrase. II, Proc. Soc. exp. Biol. ( N . Y . ) , 97 (1958) 606-611. Brain Research, 31 (1971) 185-193
BRAIN CARBONIC ANHYDRASE AND FUNCTION
193
10 MILLICHAP,J. G., WOODBURY, D. M., AND GOODMAN, L. S., Mechanism of the anticonvulsant action of acetazolamide, a carbonic anhydrase inhibitor, J. Pharmacol. exp. Ther., 115 (1955) 251-258. 11 NAIR, V., AND BAU, D., Effects of prenatal X-irradiation on the ontogenesis of acetylcholinesterase and carbonic anhydrase in rat central nervous system, Brain Research, 16 (1969) 383-394. 12 NAIR, V., ANO Rorn, L. J., A pharmacological assessment of the changes in the central nervous system following X-irradiation. In T. J. HALEYAND R. S. SNYDER(Eds.), Response of the Nervous System to Ionizing Radiation, Little, Brown, Boston, 1964, pp. 421-447. 13 NAIR, V., AND ROTH, L. J., Penetration of substances into the brain. In L. J. ROTH (Ed.), Isotopes in Experimental Pharmacology, Univ. Chicago Press, 1965, pp. 219-228. 14 NAm, V., SUGANO,H., AND Rowrt, L. J., Recovery of central nervous system functions impaired by lethal head X-irradiation, Proc. Soc. exp. Biol. ( N. Y.), 112 (1963) 273-277. 15 NAm, V., SUGANO,H., AND ROTH, L. J., Enhancement of the anticonvulsant action of acetazolamide after head X-irradiation and its relation to blood-brain changes, Radiat. Res., 23 (1964) 265-281. 16 NATOUR, R. M., AND PALMER, R. F., The immediate product of brain-tissue decarboxylations, Biochem. J., 85 (1962) 110-112. 17 NlSmMURA, T., Carbonic anhydrase inhibitors as antiepileptics, Psychiat. Neurol. yap., 65 (1963) 423-428. 18 SEVERINGHAOS,J. W., HAMmTON,F. N., AND COTEV,S., Carbonic acid production and the role of carbonic anhydrase in decarboxylation in brain, Biochem. J. 114 (1969) 703-705. 19 TOMAN,J. E. P., SWlNYARD,E. A., AND GOODMAN,L. S., Properties of maximal seizures and their alteration by anticonvulsant drugs and other agents, J., Neurophysiol., 9 (1946) 231-239. 20 WOODBURY,L. m., ANDDAVENPORT,V. D., Design and use of a new electroshock seizure apparatus and analysis of factors altering seizure threshold and pattern, Arch. int. Pharmacodyn., 92 (1952) 97-107. 21 ZABLOCKA,B., AND ESPLIN, D. W., Role of seizure spread in determining maximal convulsion pattern in rats, Arch. int. Pharmacodyn., 147 (1964) 525-542.
Brain Research, 31 (1971) 185-193
185
STUDIES ON T H E F U N C T I O N A L S I G N I F I C A N C E OF CARBONIC A N H Y D R A S E IN C E N T R A L NERVOUS SYSTEM
V. NAIR AND D. BAU Laboratory for Therapeutics Research, Michael Reese Haspital Psychiatric Institute, and Department of Pharmacology, Chicago Medical School, Chicago, Ill. 60616 (U.S.A.)
(Accepted February 24th, 1971)
INTRODUCTION Carbonic anhydrase (CA)* was first demonstrated in the nervous system by Van G o o r in 1940 and since then many investigators have studied the enzyme in detail with respect to its detection, tissue distribution, inhibition, and other properties. However, its physiological role in the nervous system is not completely understood. Our studies with ionizing radiation have provided us with an unique opportunity to pursue this question12,14,15. Earlier, we observed in rats that high doses of cephalic X-irradiation resulted in the production of an apparent anticonvulsant effect as evidenced by the abolition of the maximal electroshock seizure response 14. Acetazolamide (Diamox) is one of the pharmacological agents which inhibits carbonic anhydrase. Millichap et al. 1° have observed an association between the anticonvulsant action of acetazolamide and brain carbonic anhydrase inhibition and postulated that the anticonvulsant action of acetazolamide may be related to the inhibition of this enzyme in brain. Nishimura 17 has also pointed out that the antiepileptic effects of carbonic anhydrase inhibitors might be attributed to checking the propagation of seizure discharges by the inhibition of carbonic anhydrase in brain. Similarly, Gray et al. 5 have reported that the inhibition of brain carbonic anhydrase appeared to be the prime event in the anticonvulsant action of acetazolamide. But, on the basis of more recent studies from their laboratories, Gray and Rauh ~ have concluded that the anticonvulsant action of carbonic anhydrase inhibitors is mediated by disequilibrium of the CO2 buffer system. We thought it worthwhile to determine whether this association between enzyme inhibition and anticonvulsant action holds true in other situations where the seizure suppression resulted from means other than pharmacologic. If such a relationship is found to be a common factor in different situations, no doubt it will strengthen the case for carbonic anhydrase in the regula-
* Carbonate hydro-lyase (EC 4.2.1.1) is the term recommended by the commission on Enzymes of International Union of Biochemistry for the enzyme catalyzing the hydration of CO2, and carbonic anhydrase, the trivial name. Brain Research, 31 (1971) 185-193
186
V. N A I R A N D D. B A U
tion of brain excitability. Accordingly, we have determined the enzyme activity in various brain regions in the following groups of animals: (1) normal, (2) acetazolamide treated, (3) head irradiated, (4) head irradiated and acetazolamide treated. While these studies were in progress, Professor B. Ginsburg*, of the University of Chicago, has made available to us two groups of mice which have been reared in his laboratory of behavioral genetics. One was audiogenic seizure prone (DBA) and the other was audiogenic seizure resistant (C57/6). Brain CA was determined in these mice as well. Finally, it is known that, in a sample of rats selected at random, a small percentage (8-10 ~ ) are seizure resistant when tested by the maximal electroshock seizure method. We have taken this group of naturally seizure resistant rats after prescreening them and examined their brain CA levels. MATERIALS A N D M E T H O D S
Animals
Male Sprague-Dawley rats 70-80 days old were used in these studies. They were housed under controlled environmental conditions (temp. 72°F, light 5 a.m.-7 p.m.). Food and water were given ad libitum. X-irradiation
The head alone was exposed to X-irradiation from a 250 kV, 30 mA GE Maxitron X-ray machine. The filters employed were 0.5 mm Cu and 1 mm A1. The half value layer of the beam was 1.4 mm Cu. During irradiation the animals were unanesthetized and held in lucite tubes provided with a large number of air holes. For shielding, a sheet of lead (3.5 mm thick), shaped to fit the holders, covered the entire body except the region of the head. They were held on a rotating platform (3.5 rev./ min) for uniformity of field. Control animals were sham irradiated. Doses ranging from 50(O10,000 R were employed but this study deals with the animals receiving 10,000 R. A cetazolamide
In the enzyme studies, control rats were given 200 mg/kg acetazolamide intraperitoneally (Diamox, Lederle Labs.). The time of maximal inhibition of enzyme activity was first determined by examining the brain at 1, 2, or 3 h after drug administration. Since there was no difference between the 2 and 3 h period, all animals were sacrificed at 2 h after drug administration. Regional dissection of the brain was performed according to a frozen state dissection method reported earlierll, 1~. The central nervous system (CNS) regions examined were: cerebral cortex, caudate nucleus, hippocampus, cerebellar cortex, medulla and spinal cord. The X-irradiated animals were examined at 3 days postirradiation. They receiv* Presently at the University of Connecticut, Storr, Conn. Brain Research, 31 (1971) 185-193
187
BRAIN CARBONIC ANHYDRASE AND FUNCTION
ed 2.5 mg/kg acetazolamide and were sacrificed 0.5 h later. The time of examination and drug dosage were selected on the basis of our previous findings in the X-irradiated rats 15. In mice, only the caudate, hippocampus and medulla were examined for CA activity. Carbonic anhydrase activity was determined according to the micromethod of Maren 7 as outlined in a previous articlelL M E S resistant rats
Rats were subjected to the MES test according to the method of Toman et al. 19. The apparatus employed to produce the seizures was a constant current stimulator (Wahlquist, Salt Lake City, Utah) of the type described by Woodbury and Davenport 20. Maximal seizure was induced by 150 mA delivered through corneal electrodes for 0.2 sec. Rats were tested every day for 4 days and were then grouped into MES positive and MES resistant ones based on their consistent response for the 4 days. Those responding with tonic extension of the hind limbs were termed MES positives and those failing to show the extensor response as MES resistant ones. Rats were sacrificed for enzyme assay 2 days after the last shock. The procedures for electrical stimulation and EEG recordings were reported in a previous articlelL Mice
Male mice, 35-40 days old, from DBA and C57/6 strains were used. The dissection of the brain regions was performed in a manner similar to that described for rats except that only 3 regions were taken. They were: caudate nucleus, hippocampus and medulla. For each assay, the pooled tissue from 3 brains were used.
TABLE I CARBONIC ANHYDRASEACTIVITYIN RAT C N S (units/g tissue 4- S.E.)
Anatomical region
Frontal cortex Caudate nucleus Hippocampus Cerebellar cortex Medulla Spinal cord
Treatment Control
Control 4acetazolamide* (200 mg/kg)
X-ray
X-ray jr acetazolamide (2.5 mg/kg)
34.4 4- 3.2 30.8 ± 4.4 36.7 4- 2.5 46.5 4- 5.0 97.0 4- 4.3 55.4 4- 3.0
17.4 4- 1.1 4.9 4- 0.9 11.7 4- 1.9 20.4 4- 2.3 61.4 4- 1.6 38.8 4- 1.2
31.5 ± 4.8 7.5 4- 1.6 35.0 4- 0.4 20.3 4- 3.1 50.7 4- 3.8 48.1 4- 1.2
11.4 -b 2.2 4.5 4- 0.4 13.2 4- 0.2 9.9 4- 0.8 33.4 4- 1.6 35.8 4- 1.3
* At the dose of 2.5 mg/kg, acetazolamide produced no significant effects on CA activity in the control rats (see text). Brain Research, 31 (1971) 185-193
188
V. NAIR AND D. BAU ONTROL ~-ETAZOLAMIDE ( 2 . 5 m g / k g ) - R A Y (10,OOO R) + ACETAZOL. (2.5 mg/kg)
100 u) u~
o
I--
! ....
80
S_-_-
60
-I-S_
Z
0
a. 02
"---
i-
a.
20
Fig. 1. Effect of acetazolamide, X-irradiation and the combination on the maximal electroshock seizure response (MES). ~ positive response to MES refers to the percent of animals exhibiting the hind limb tonic extensor response. Acetazolamide (2.5 mg/kg) was given i.p. and the animals examined 0.5 h later. In the irradiated series, the animals were tested at 48-72 h postirradiation (for further details see Nair et a1.15).
IiU.4mAIMIIlII ~maet
1111~. I~1' r
el I 1 ~ I ~ i l ¢ "t"
S1WMATIOII
SllW~mU
Sll/UAIQI
Fig. 2. EEG records. Effect of X-irradiation and irradiation plus acetazolamide before and after electrical stimulation of the sensorimotor cortex of the rat. Stimulation parameters: 30 V, 50 c/sec, 10 sec. The drug (2.5 mg/kg) was given i.p. and stimulus applied 30 rain later (for further details see N air et al.l~).
RESULTS T h e regional d i s t r i b u t i o n of CA in c o n t r o l rat brain is s h o w n in T a b l e I. As reported earlier 11, in the controls, C A activity w a s highest in the m e d u l l a . T w o h o u r s after a c e t a z o l a m i d e a d m i n i s t r a t i o n
(200 mg/kg)
the activity decreased in all the re-
gions. T h e e n z y m e activity w a s l o w e s t in the c a u d a t e n u c l e u s Brain Research, 31 (1971) 185-193
(Table I).
X-irradiation
189
BRAIN CARBONICANHYDRASEAND FUNCTION CARBONIC ANHYDRASE
IN
RAT
CNS
I---:::
b
+
100
100
80
80
60
60
40
40
20
20
F. CORTEX
CAUDATE
HIPPOCAMP.
CEREBELL.
MEDULLA
SP. CORD
Fig. 3. Comparison of the CA levels in the CNS of MES + (exhibiting the hind limb tonic extensor response) and MES- (not showing hind limb tonic extensor response) rats. Data presented are the average of 6 animals. MOUSE
u
_ _
DBA
--80
80 uJ
°
CAUDATE
/ i HIPPOCAMPUS
b
60
40
20
MEDULLA
Fig. 4. Carbonic anhydrase activity in mouse CNS. alone, also lowered the enzyme activity in the different brain regions, with maximal inhibition again being produced in the caudate nucleus. It was observed in our earlier studies that irradiation, by itself, produced a significant degree o f protection against M E S (Fig. 1). Acetazolamide, at a dose level (2.5 mg/kg), which gave little protection (3 9/0) against MES in non-irradiated controls, abolished the tonic extensor response in all the irradiated animals (Fig. 1). Furthermore, this occurred 0.5 h after drug treatment indicating an enhancement o f the speed o f onset as well as intensity o f drug action in the irradiated animals. These effects on Brain Research, 31 (1971) 185-193
190
v. NAIR AND D. BAU
the seizure response have been also supported by EEG records (Fig. 2). it was also found that at this dose level (2.5 mg/kg) of acetazolamide, no significant suppression of CA activity was noted in the non-irradiated controls when examined 0.5 h postdrug treatment. In contrast, in the irradiated animals, acetazolamide enhanced the irradiation induced suppression of enzyme activity in all the brain regions, maximal effect being in the caudate nucleus (Table I). The pattern of CA activity in the CNS of MES resistant rats has also been examined (Fig. 3). The enzyme levels were lower in all the regions, in comparison to the MES positive rats, this decrease being most manifest in the caudate nucleus. Enzyme activity in the caudate nucleus was 59 ~ of that in the MES positive rats. A similar phenomenon is seen also in mice (Fig. 4). The CA activity was markedly lower in the caudate nucleus of the audiogenic seizure resistant mice (C57/6) than in the seizure prone ones (DBA). DISCUSSION Carbonic anhydrase catalyzes the reaction: E
H20 + CO~ ~- H2CO8 ~ H + + HCOsThe second step from H2CO3 to H + and HCOz- is instantaneous so that the enzyme catalyzed reaction is the rate limiting step in the overall process. Single cell determinations by Giacobini 3 in rat brain have demonstrated that the enzyme is concentrated in the glial cells. The nature of the ultimate end products of the decarboxylation reaction in brain is controversial. Natour and Palmer 16 studied mouse brain homogenate and concluded that carbon dioxide was the primary product. Severinghaus et aL as also studied this question using rat brain homogenate, but their results pointed to carbonic acid (HCO3 and H ÷) as the immediate end product. Based on these divergent findings, different interpretations as to the role of carbonic anhydrase in brain have been offered. It would seem that a sound theory regarding the role of carbonic anhydrase in brain should take into account both the clinically well known inhibitory effect of carbon dioxide on seizures and the anticonvulsant action of carbonic anhydrase inhibitors. Davenport z found that in order to reduce the rate of catalyzed uptake of carbon dioxide by blood by 9 0 ~ , carbonic anhydrase in the red cells must be inhibited by more than 99.97~. Similarly, Maren s, from his studies of the enzyme in kidney, stomach, and pancreas, came to the conclusion that CA inhibition is not recognizable physiologically unless about 99 ~ of the tissue enzyme is inactive. It appears that the enzyme concentrations in these tissues, in so far as they have been studied, are in excess of maximum physiological needs. The situation with respect to the brain is the following: Ashley and Schuster I have shown that there is a late appearance of CA in the cerebrum of young animals born in an immature condition (dog, cat, rat and rabbit) while the enzyme is present at birth in the cerebrum of animals more mature when Brain Research,
31 (1971) 185-193
BRAIN CARBONIC ANHYDRASEAND FUNCTION
191
born such as the calf and guinea pig. Their data also suggest a correlation between the appearance of the enzyme in the cerebrum and the beginning of its functioning as indicated by parallel maturation of certain sense organs and EEG data. In humans, according to Gibbs and Gibbs 4, the normal newborn infant shows irregular slow waves and there is nothing to correspond to the 9 or 10/sec waves so common in the adults. By the time the child is 7 years old, the brain waves reach the frequency found in adults. A parallel relationship is revealed in the enzyme development also. Ashley and Schuster found little or no CA in the brain of newborn human, while in the brain of a child, 7 years old, CA content was found to be comparable to the adult level. The work of Millichap 9 has revealed a direct relation between the activity of brain CA and the susceptibility of animals to experimental seizures. Working with newborn animals of different species, he has shown that the development of susceptibility of various experimental seizure patterns is directly related to the level of brain CA. Based on his results, he suggested that brain CA and its specific catalytic activity mat be essential for the propagation of the seizure discharge. Low levels of enzyme activity may be sufficient for focal discharge and the induction of minor seizure patterns, while a relatively high degree o f activity may be required for the propagation of a generalized seizure discharge and the induction of a major tonic seizure. Besides confirming the earlier observations of Millichap, our results go further and suggest that the CA level in the caudate nucleus plays an important role in the regulation of the maximal seizure response. The fact that this association of low enzyme activity in caudate nucleus and anticonvulsant effect is demonstrated in 4 different experimental situations and in two species, makes it, we believe, highly significant. It should be emphasized that the term anticonvulsant action as used here refers to the abolition of the tonic extensor phase of the maximal electroshock seizure response. In all the experimental situations where we found an anticonvulsant effect (as indicated by the suppression of the tonic extensor phase) CA activity was relatively lowest in the caudate nucleus. However, the enzyme level was inhibited to varying degrees in other regions also. The importance of the caudate nucleus is suggested from the fact that it is in this region that CA is relatively lowest in all cases. The maximal electroshock seizure test is commonly employed in the screening of potential anticonvulsant drugs, and the abolition of the tonic hindlimb extension is the frequently used end point in such tests. Clinically recognized antiepileptic agents (anti-grand mal agents in particular) abolish the tonic phase of major seizures even when these drugs fail to raise appreciably the seizure threshold 19. The anatomical pathways and the mechanisms governing the maximal seizure pattern have not been clearly defined. Zablocka and Esplin 21 have studied the mechanisms responsible for the inability of some rats to exhibit tonic hind limb extension. Their results point to inefficiency in the spread of seizure discharge through the brain as the major mechanism responsible for this pattern. This is understandable when one considers that the tonic extensor seizure represents the maximum rate of dissipation of energy of which the brain is capable. The tonic extensor response can be altered by factors which interfere with the intensity or the spread of seizure discharge. The question whether carbonic anhydrase influences both of the above factors Brain Research, 31 (1971) 185-193
192
V. NAIR AND D. BAU
or only one of them specifically can only be answered by further investigations. SUMMARY The role of carbonic anhydrase in the regulation of seizure activity has been studied by comparing the brain enzyme levels of normal rats with those of rats found seizure resistant in the maximal electroshock test. These included rats (1) treated with acetazolamide, an anticonvulsant drug; (2) exposed to head X-irradiation, a procedure known to abolish the tonic extensor response to maximal electroshock; and (3) exposed to a combination of acetazolamide and head X-irradiation. In addition, rats which are congenitally seizure resistant in the maximal electroshock test as well as two strains of mice, one audiogenic seizure prone and the other seizure resistant, were also examined in this study. The enzyme determinations were done in different anatomical regions of the CNS. Our results show a region specific effect. In all the conditions where seizure activity was suppressed, carbonic anhydrase activity was also suppressed with the greatest change occurring in the caudate nucleus. Since it is demonstrated in widely different experimental conditions and in two species, the association of a region specific inhibition of carbonic anhydrase in the caudate nucleus and seizure resistance (anticonvulsant action) is considered highly significant. ACKNOWLEDGEMENTS This work was supported in part by grants from the state of Illinois, Department of Mental Health. The authors wish to thank the University of Chicago Argonne Cancer Hospital and Department of Radiology for use of the radiation facilities and Dr. James Bland for technical assistance with the radiation procedure.
REFERENCES 1 ASHLEY,W., AND SCHUSTER, E. M., Carbonic anhydrase in the brain of the newborn in relation to functional maturity, J. biol. Chem., 184 0950) 109-116. 2 DAVENPORT, H. W., The inhibition of carbonic anhydrase by thiophene-2-sulfonamide and sulfanilamide, J. biol. Chem., 158 0945) 567-571.
3 GIACOBINI,E., A cytochemical study of the localization of carbonic anhydrase in the nervous system, J. Neurochem., 9 (1962) 169-177. 4 GIBBS, F. A., AND GIBBS,E. L., Atlas o f Encephalography, Cummings, Cambridge, Mass., 1941. 5 GRAY, W. O., MAREN, T. H., SISSON, G. M., AND SMITH, F. H., Carbonic anhydrase inhibition. VII. Carbonic anhydrase inhibition and anticonvulsant effect. J. Pharmacol. exp. Ther., 121 (1957) 160-170. 6 GRAY, W. D., AND RAUH, C. E., The anticonvulsant action of carbon dioxide: Interaction with reserpine and inhibitors of carbonic anhydrase, J. Pharmacol. exp. Ther., 163 (1968) 431438. 7 MARES, T. H., A simplified micromethod for the determination of carbonic anhydrase and its inhibitors, J. Pharmacol. exp. Ther., 130 (1960) 26-29. 8 MARES, T. H., The relation between enzyme inhibition and physiologicalresponse in the carbonic anhydrase system, J. Pharmacol. exp. Ther., 139 (1963) 140-153. 9 MILLICHAP, G. J., Seizure patterns in young animals. Significance of brain carbonic anhydrase. II, Proc. Soc. exp. Biol. ( N . Y . ) , 97 (1958) 606-611. Brain Research, 31 (1971) 185-193
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10 MILLICHAP,J. G., WOODBURY, D. M., AND GOODMAN, L. S., Mechanism of the anticonvulsant action of acetazolamide, a carbonic anhydrase inhibitor, J. Pharmacol. exp. Ther., 115 (1955) 251-258. 11 NAIR, V., AND BAU, D., Effects of prenatal X-irradiation on the ontogenesis of acetylcholinesterase and carbonic anhydrase in rat central nervous system, Brain Research, 16 (1969) 383-394. 12 NAIR, V., ANO Rorn, L. J., A pharmacological assessment of the changes in the central nervous system following X-irradiation. In T. J. HALEYAND R. S. SNYDER(Eds.), Response of the Nervous System to Ionizing Radiation, Little, Brown, Boston, 1964, pp. 421-447. 13 NAIR, V., AND ROTH, L. J., Penetration of substances into the brain. In L. J. ROTH (Ed.), Isotopes in Experimental Pharmacology, Univ. Chicago Press, 1965, pp. 219-228. 14 NAm, V., SUGANO,H., AND Rowrt, L. J., Recovery of central nervous system functions impaired by lethal head X-irradiation, Proc. Soc. exp. Biol. ( N. Y.), 112 (1963) 273-277. 15 NAm, V., SUGANO,H., AND ROTH, L. J., Enhancement of the anticonvulsant action of acetazolamide after head X-irradiation and its relation to blood-brain changes, Radiat. Res., 23 (1964) 265-281. 16 NATOUR, R. M., AND PALMER, R. F., The immediate product of brain-tissue decarboxylations, Biochem. J., 85 (1962) 110-112. 17 NlSmMURA, T., Carbonic anhydrase inhibitors as antiepileptics, Psychiat. Neurol. yap., 65 (1963) 423-428. 18 SEVERINGHAOS,J. W., HAMmTON,F. N., AND COTEV,S., Carbonic acid production and the role of carbonic anhydrase in decarboxylation in brain, Biochem. J. 114 (1969) 703-705. 19 TOMAN,J. E. P., SWlNYARD,E. A., AND GOODMAN,L. S., Properties of maximal seizures and their alteration by anticonvulsant drugs and other agents, J., Neurophysiol., 9 (1946) 231-239. 20 WOODBURY,L. m., ANDDAVENPORT,V. D., Design and use of a new electroshock seizure apparatus and analysis of factors altering seizure threshold and pattern, Arch. int. Pharmacodyn., 92 (1952) 97-107. 21 ZABLOCKA,B., AND ESPLIN, D. W., Role of seizure spread in determining maximal convulsion pattern in rats, Arch. int. Pharmacodyn., 147 (1964) 525-542.
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