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Angiotensin converting enzyme in kainic acidЦinjected striata.

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BRIEF COMMUNICATIONS
Angiotensin Converting
Enzyme in Kainic AcidInjected Striata
E. A. Singh, BSc, and E. G. McGeer, P h D
The activity of angiotensin converting enzyme is decreased in kainic acid-injected neostriata of rats, providing another biochemical similarity between this
animal model and Huntington's chorea.
Singh EA, McGeer EG, Angiotensin converting
enzyme in kainic acid-injected striata.
Ann Neurol4:85-86, 1978
Intrastriatal injections of kainic acid are being intensively investigated t o determine whether they may
provide a possible animal model for Huntington's
chorea. The original analogy between Huntington's
chorea and this model was made o n the basis that
both conditions show marked loss of striatal interneurons and are characterized biochemically by loss
of striatal glutamic acid decarboxylase (GAD), striatal
choline acetyltransferase (CAT), and nigral GAD,
with no loss or even an elevation in nigral and striatal
tyrosine hydroxylase [2, 51. Recently, a number of
other biochemical similarities have been demonstrated [ 3 ] involving changes in substance P and in
the receptor binding of various neurotransmitters.
Behavioral similarities have also been reported [ 7 ] .
Marked decreases have been demonstrated in the activity of angiotensin converting enzyme (ACE) in
neostriatal nuclei in patients dying of Huntington's
chorea [ I ] . This paper reports losses in striatal ACE
following irltrastriatal injections of kainic acid.
sected and homogenized at 0°C in 10 vol of 0.32 M
sucrose10.01 M Tris-hydrochloride buffer ( p H 7.4). ACE
activity was measured in samples of homogenate by the
method of Yang and Neff [ 8 ] except that filtration rather
than centrifugation was used to prepare a clear supernatant
fluid for fluorescent measurements. Hip-His-Leu substrate
was obtained from Bachcm Inc, Torrance, CA. Under
these conditions, the reaction was linear with tissue concentrations ranging from 100 to 500 pg of protein per assay
and with time from 10 to 50 minutes. A 30-minute incubation at 37°C was used routinely as suggested by Yang and
Neff. The blanks prepared using heated tissue homogenates were less than 10V of the test assays.
Choline acetyltransferase (CAT) was determined by the
previously reported radioactive method 161 and protein by
the procedure of Lowry e t a1 [4].
Results and Discussion
As indicated in the Figure, there was a significant loss
of ACE in the kainic acid-injected striata in all cases
in which the percentage loss of CAT was 45% o r
greater. Prior work has shown that losses averaging
45% in both CAT and G A D occur when the striatum
is injected with 2.5 to 3 nmoles of kainic acid. In the
present series, the group of animals receiving 2
nmoles of kainic acid showed no significant effect on
ACE despite a loss of CAT averaging over 30%;
Percentage loss of angiotensin converting enzyme (ACE) activity
in kainic acid-injected neostriatum of rats as a function of
the percentage loss oj-chofinearetyltran~
ferase (CAT)activity i n the .fame tiisue. The percentage loss ujaJ. c-afi-ulatedaJ
100 minus the activity in tbe in-jerted neostriatutri per milligram of protein. expressed as a percentaRe of that in the cowtrafateral control neostriatum. Control i'alzles (in nmoles per
mifhgrgm ofprotein per minute) atmeraged2.04 0.1 7JOr
ACEand4.78 2 0.24forCAT(N =21,.Sofidlineiscafculated fine o f linear regression: dashed fine i.c cafcufatednegatii e,
reciprocaf refationship.
*
Method
Male Wistar rats weighing about 300 gm were injected
stereotaxically under Nembutal anesthesia as previously
described [ 5 ] with 2 to 10 nmoles of kainic acid in 1 pl of
physiological saline over a five-minute period. The coordinates used were: AP 9.6, ML 2.8, DV 4.5, as adapted from
Konig and Klippel [3a]. All injections were unilateral, with
contralateral neostriata used as control.
Nine to twelve days after the injection, the rats were
killed by cervical fracture and the neostriatum was dis-
loo.
From the Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia Faculty of
Medicine, Vancouver, BC, Canada.
Accepted for publication Jan 30, 1978.
Address reprint requests to Dr McGeer, Kinsmen Laboratory of
Neurological Research, Department of Psychiatry, University of
British Columbia Faculty of Medicine, Vancouver, BC, Canada
V6T 1W5.
loss in CAl
0364-5134/78/0004-0115.$01.00 @ 1978 by E. A. Singh
85
statistically significant losses of ACE were, however,
found in all rats receiving more than 3 nmoles of
kainic acid. Statistical calculation indicated a significant linear correlation ( Y = 0.78; solid line in the
Figure) between the percentage loss of CAT and that
of ACE in the striatum in this total series of rats
injected with various amounts of kainic acid; the curvilinear line (dashed line in the Figure) given by the
equation 100/CAT = 3.55 - 0.046 (ACE) was
slightly more Significant ( r = 0.80). This and the general distribution of the data seem to indicate that
ACE is not only unaffected by small doses of kainic
acid which are sufficient to cause significant losses of
CAT, but that approximately 40% of the ACE in the
neostriatum is unaffected by injections of kainic acid
in the particular volume and coordinates used. It
must be remembered that these enzyme assays were
done on homogenates of the total neostriatum, so
that the ACE activity left at high doses of kainic acid
may represent preservation of activity at a distance
from the injection site.
The finding that intrastriatal injections of kainic
acid reduce ACE activity in the neostriatum provides
another biochemical similarity between this model
and Huntington’s chorea. Our data are quantitatively
different from those reported for Huntington’s
chorea, however, in that, in human brains studied at
autopsy, a greater loss in ACE than in CAT activity
has been reported in nuclei of the neostriatum, particularly the globus pallidus [l]. It will be of some
interest to determine whether injections of kainic
acid at sites in the neostriatum other than those used
here cause larger losses of ACE relative to those of
CAT. Time sequences may, however, be crucial to
the results since ACE activity was found to be reduced to 35%, as compared to a reduction of CAT to
545+, in 8 rats injected with 2.5 nmoles of kainic acid
two months before sacrifice. The existence of a varie t y of biochemical defects in the neostriatum in
chorea may be one reason why little benefit has generally been attained in clinical trials of cholinergic or
GABAnergic agonists.
disease: correlates with a new animal model. Prog Neuropsychopharmacol 1:13-30, 1977
3a Konig JFR, Klippel RA: The Rat Brain. Huntington, NY,
Krieger, 1967
4. Lowry OH, Rosebrough HF, Farr AL, et al: Protein measurement with the Fohn phenol reagent. J Biol Chem 193:265275, 1951
5 . McGeer EG, McGeer PL: Duplication of biochemical changes
of Himtington’s chorea by intrastriatal injection of glutamic and
kainic acids. Nature 263:517-519, 1976
6. McGeer PL, McGeer EG: Cholinergic enzyme systems in Parkinson’s disease. Arch Neurol 25:265-268, 1971
7 . Sanberg PR, Lehmann J, Fibiger HC: Impaired learning and
memory after kainic acid lesions of the striatum: a behavioral
model of Huntington’s disease. Neurosci Abstr 7:13X, 1977
8. Yang H-YT, Neff NH: Distribution and properties of angiotensin converting enzyme of rat brain. J Neurochem
19~2443-2450, 1972
Identification of Speech
Lateralization by
Intracarotid Injection
of Methohexitd
L. James Willmore, MD, B. Joe Wilder, MD,
Assa Mayersdorf, MD, R. Eugene Ramsay, MD,
and George W. Sypert, MD
Speech lateralization was assessed in 25 patients with
epilepsy by direct carotid injection or carotid
cathqterization from a femoral approach. Hemispheric
anesthesia was induced by intracarotid injection of the
rapidly acting barbiturate methohexital. This technique allowed same-day study of both hemispheres,
permitted repeated injection of either cqotid artery
without accumulation of methohexital sufficient to
cause systemic effects, and added little to the total time
required for three-vessel cerebral angiography. Unilateral seizures were induced by methohexital injection in 4 patients. No other complication secondary t o
the methohexital study was observed.
Willmore LJ, Wilder BJ, Mayersdorf A, et al:
Identification of speech lateralization by
intracarotid injection of methohexital.
Ann Neurol4:86-88, 1978
Supported by grants from the Huntington’s Chorea Foundation,
the Garfield Weston Foundation, and the Medical Research Council of Canada.
References
1. Arregui A, Bennett JP, Bird ED, et al: Huntington’s chorea:
selective depletion of activity of angiotensin converting
enzyme in the corpus striatum. Ann Neurol 2:294-298,
1977
2. Coyle JT,Schwarcz R: Lesion of striatal neurons with kainic
acid provides a model for Huntington’s chorea. Nature
263:244-246, 1976
3. Coyle JT, Schwarcz R, Bennett JP, et al: Clinical
neuropathologic and pharmacologic aspects of Huntington’s
Hemispheric lateralization of speech function can be
identified by intracarotid injection of sodium
amobarbital[6]. Although safe, this procedure, as deFrom the Neurology Service, Veterans Administration Hospital,
and the Departments of Neurology, Neurosurgery, and Neuroscience, University of Florida College of Medicine, Gainesville, FL.
Accepted for publication Feb 10, 1978
Address reprint requests to Dr Wilder, Neurology Service ( 127),
Veterans Administration Hospital, Gainesville, FL 32602.
86 0364-5134/78/0004-0116$01.00 @ 1978 by L. James Willmore
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angiotensins, enzymes, kainic, acidцinjected, striata, converting
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