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Evidence for involvement of amino acid neurotransmitters in anesthesia and naloxone induced reversal of respiratory paralysis
Life Sciences, Vol. 54, No. 25, pp. 2021-2033, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All fights reserved 0024-3205/94 $6.00 + .00
Pergamon 0024-3205-(94) E0056-W
EVIDENCE FOR INVOLVEMENT OF A M I N O ACID N E U R O T R A N S M I T T E R S IN A N E S T H E S I A AND N A L O X O N E INDUCED REVERSAL OF R E S P I R A T O R Y PARALYSIS
Amin Suria and Fauzia Rasheed
Department of Pharmacology Faculty of Health Sciences Aga Khan U n i v e r s i t y Medical College Karachi-74800, Pakistan (Received in final form April
11, 1994)
Summary General anesthetics render a person unconscious and may p r o d u c e r e s p i r a t o r y paralysis at therapeutic doses. No p h a r m a c o l o g i c a l agent is available to restore r e s p i r a t i o n and the m e c h a n i s m / s of anesthesia or apnea is not clearly understood. In this report, we present evidence to show that naloxone reversed respiratory failure induced by thiopental, ketamine, halothane but not that induced by p h e n o b a r b i t a l . F u r t h e r m o r e , 25 mg/kg, i.v. thiopental, 140 mg/kg, i.v. ketamine, and 3% halothane produced a n e s t h e s i a without significantly altering r e s p i r a t o r y rate, increased GABA and decreased glutamate (except k e t a m i n e and phenobarbital) levels in rat b r a i n stem and cortex, but not in caudate and cerebellum. Aspartate, g l y c i n e and alanine levels were not affected in four b r a i n regions studied. Pretreatment with TSC for 30 m i n u t e s did not change GABA or glutamate contents, but a b o l i s h e d the anesthetic as well as the r e s p i r a t o r y d e p r e s s a n t actions of the anesthetics. Increasing the doses of anesthetics produced respiratory failure with further rise in GABA and fall in glutamate in brain stem and cortex. Naloxone reversed respiratory paralysis and r e s t o r e d G A B A close to control values in rat brain stem and cortex with no changes in caudate or cerebellum. Data p r e s e n t e d here suggest that GABA may be n e c e s s a r y to p r o d u c e loss of consciousness and naloxone reverses a n e s t h e t i c induced respiratory failure.
Key Words:
anesthetics, G A B A , glutamate, naloxone, respiration
General a n e s t h e t i c s given by inhalation or intravenous route render a p e r s o n u n c o n s c i o u s and may produce respiratory d e p r e s s i o n at t h e r a p e u t i c doses. At higher doses, these agents may p r o d u c e r e s p i r a t o r y arrest and death. The mechanism/s by w h i c h a n e s t h e s i a
Corresponding author: Professor Amin Suria, Department of Pharmacology, The Aga Khan U n i v e r s i t y Hospital, S t a d i u m Road, P.O.Box 3500, Karachi, Pakistan
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or r e s p i r a t o r y a r r e s t is p r o d u c e d is not u n d e r s t o o d . In r e l a t i o n to anesthesia, anesthetics have been proposed to increase the t h i c k n e s s of l i p i d b i l a y e r s (I), a l t e r b r a i n m e t a b o l i c a c t i v i t y (2,3,4), p e r t u r b the f l u i d i t y of cell m e m b r a n e s (5) a n d p r o d u c e functional loss of m e m b r a n e p r o t e i n s (6,7). Recently, however, investigators have focused their attention on the e f f e c t s of anesthetics on neuronal or synaptic function, thinking that i n h i b i t i o n of n e u r o n a l a c t i v i t y w o u l d i n h i b i t m a n y b r a i n f u n c t i o n s (8,9). In p a r t i c u l a r , n e u r o t r a n s m i t t e r a m i n o acids, g l u t a m a t e a n d GABA (Gamma aminobutyric acid) involvement in the a c t i o n of a n e s t h e t i c s has b e e n e x p l o r e d (8). A n e s t h e t i c s , as a group, h a v e been demonstrated to p r o f o u n d l y affect the function of G A B A activated chloride conductances (9). W i t h r e s p e c t to r e s p i r a t i o n , a n u m b e r of n e u r o t r a n s m i t t e r s including epinephrine, norepinephrine and s e r o t o n i n have been implicated (I0). Neurotransmitter amino acids have also been p r o p o s e d to p l a y an a c t i v e role in the c o n t r o l of v e n t i l a t i o n (II) . It has b e e n h y p o t h e s i z e d that the final n e u r a l o u t p u t d e t e r m i n i n g the c e n t r a l d r i v e w i l l d e p e n d on the b a l a n c e b e t w e e n e x c i t a t i o n b y g l u t a m a t e a n d i n h i b i t i o n b y G A B A (11,12). Thus, these t r a n s m i t t e r s m a y be m o d i f i e d in r e s p i r a t o r y d e p r e s s i o n or p a r a l y s i s p r o d u c e d b y general anesthetics. Although, naloxone has b e e n shown to b l o c k the g e n e r a l a n e s t h e t i c a c t i o n of h a l o t h a n e a n d e n f l u r a n e (13). It is n o t e w o r t h y that no agent is a v a i l a b l e that can m o d i f y the respiratory d e p r e s s a n t e f f e c t s of a n e s t h e t i c s . D i n g l e d i n e et al. (14) w o r k i n g w i t h m i c e s h o w e d that h i g h d o s e n a l o x o n e i n d u c e d c o n v u l s i o n s c o u l d be a n t a g o n i z e d b y d i a z e p a m and p r o p o s e d that n a l o x o n e a c t s as a GABAantagonist. We h a v e s h o w n that t h i o p e n t a l i n d u c e d r e s p i r a t o r y arrest, as w e l l as i n c r e a s e in G A B A a n d d e c r e a s e in g l u t a m a t e c o n t e n t s in rat b r a i n s t e m a n d c o r t e x can be r e v e r s e d b y n a l o x o n e . In this report, we p r e s e n t f u r t h e r e v i d e n c e to d e m o n s t r a t e that i.v., as w e l l as i n h a l a t i o n a n e s t h e t i c s at t h e r a p e u t i c d o s e s p r o d u c e a fall in g l u t a m a t e w i t h c o n c o m i t a n t rise in G A B A c o n t e n t s in rat b r a i n s t e m a n d cortex. We a l s o show that h i g h e r d o s e s of a n e s t h e t i c s p r o d u c e r e s p i r a t o r y paralysis, and a f u r t h e r d e c r e a s e in g l u t a m a t e a n d an i n c r e a s e in G A B A levels. N a l o x o n e r e v e r s e s r e s p i r a t o r y a r r e s t a n d r e s t o r e s the G A B A l e v e l s b a c k to its c o n t r o l v a l u e s e x c e p t in c a s e of k e t a m i n e and r e s t a b l i s h e s g l u t a m a t e l e v e l s o n l y in t h i o p e n t a l t r e a t e d group.
Materials
and Methods
Adult Sprague-Dawley rats (150-200 g) of e i t h e r sex w e r e e m p l o y e d for all e x p e r i m e n t s . To m o n i t o r the e f f e c t s of a n e s t h e t i c s a n d o t h e r d r u g s on r e s p i r a t i o n , a n i m a l s w e r e first o b s e r v e d for c o n t r o l r e s p i r a t o r y rate, t h e n i n j e c t e d i n t r a v e n o u s l y , w i t h e i t h e r thiopental (25 mg/kg), or k e t a m i n e (140 mg/kg), or p h e n o b a r b i t a l (250 mg/kg) or 3 % h a l o t h a n e . T h e s e doses p r o d u c e d anesthesia w i t h o u t a p p r e c i a b l y a l t e r i n g the r e s p i r a t o r y rate. At this point, the t r a c h e a was exposed, i n c i s e d and a glass T - s h a p e d c a n n u l a was inserted. O n e side of the c a n n u l a was o p e n to r o o m a i r a n d the other was connected to a differential transducer (PT-120, B i o s c i e n c e ) that was c o n n e c t e d to a B i o s c i e n c e two
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c h a n n e l r e c o r d e r v i a A C c o u p l e r (FC 137, B i o s c i e n c e ) for r e c o r d i n g r e s p i r a t o r y rate. A n a d d i t i o n a l d o s e of 25 m g / k g t h i o p e n t a l or 40 m g / k g k e t a m i n e or 150 m g / k g p h e n o b a r b i t a l or h a l o t h a n e was t h e n s l o w l y i n j e c t e d or g i v e n b y i n h a l a t i o n till the a n i m a l s t o p p e d b r e a t h i n g . I m m e d i a t e l y , n a l o x o n e (2.5 mg/kg, i.v.) was i n j e c t e d to the s a m e a n i m a l as r e s p i r a t i o n was c o n t i n u o u s l y m o n i t o r e d . A total of I0 a n i m a l s w e r e u s e d for m o n i t o r i n g the e f f e c t s of e a c h drug. E f f e c t s of n a l o x o n e a l o n e w e r e a l s o m o n i t o r e d in a n o t h e r g r o u p of I0 rats. To s t u d y the e f f e c t s of g e n e r a l a n e s t h e t i c s on a m i n o a c i d neurotransmitters l e v e l s in the b r a i n a n i m a l s w e r e k e p t in a 12hour light-dark cycle a n d w e r e fed c h o w ad libitum. Rats were d i v i d e d i n t o 5 g r o u p s w i t h e a c h g r o u p c o n t a i n i n g five animals. F i r s t g r o u p s e r v e d as a c o n t r o l a n d r e c e i v e d i.v. s a l i n e or just r o o m air. S e c o n d g r o u p was s u b j e c t e d to e i t h e r i.v. t h i o p e n t a l (25 mg/kg) or k e t a m i n e (140 mg/kg) or p h e n o b a r b i t a l (250 mg/kg) or was g i v e n i n h a l a t i o n anesthetic, halothane (3 %). H a l o t h a n e w a s g i v e n t h r o u g h an a n e s t h e s i a m a c h i n e (Boyles, U. K.), the h o s e of w h i c h was f i t t e d to the w h o l e m o u t h of the animal. T h e s e d o s e s of various anesthetics produced complete surgical anesthesia. Third group received either thiopental (50 mg/kg) or k e t a m i n e (180 mg/kg) or p h e n o b a r b i t a l (400 m g / kg) or 5 % h a l o t h a n e . At t h e s e d o s e s a n i m a l s s t o p p e d b r e a t h i n g . G r o u p f o u r was first g i v e n h i g h d o s e s of a n e s t h e t i c s , as in g r o u p 3. As s o o n as the a n i m a l s stopped breathing i.v. n a l o x o n e (2.5 mg/kg) was a d m i n i s t e r e d . G r o u p five r e c e i v e d n a l o x o n e alone. Rats were then sacrificed by a focused beam microwave (General M e d i c a l E n g i n e e r i n g Corp., Peabody, M.A.) irradiation (output 2.5 KW) w i t h an e x p o s u r e time of 4 s e c o n d s to e n s u r e the i n a c t i v a t i o n of all enzymes. T h e y w e r e t h e n d e c a p i t a t e d , c r a n i u m s w e r e r e m o v e d a n d b r a i n s w e r e isolated. C e r e b e l l u m , caudate, c o r t e x a n d b r a i n s t e m r e g i o n s w e r e d i s s e c t e d out and all s a m p l e s w e r e stored at -70°C u n t i l ready for a n a l y s i s . At the time of analysis, s a m p l e s w e r e thawed, homogenized in 1 ml h i g h g r a d e purity water in g l a s s homogenizing tubes w i t h g l a s s pestles (Kontes, Vineland, N.J.). Homogenates were centrifuged for 5 m i n u t e s at I000 x g. S u p e r n a t a n t s w e r e d r a w n off a n d i0 ul of e a c h s a m p l e w a s i n j e c t e d in HPLC. P e l l e t s w e r e d i s s o l v e d in 1 ml of 0.i N N a O H a n d s u b j e c t e d to p r o t e i n e s t i m a t i o n b y the m e t h o d of L o w r y et al. (15). T h e H P L C s y s t e m (Waters A s s o c i a t e s , U.S.A.) c o n s i s t e d of 2 pumps, M - 5 i 0 a n d M - 6 0 0 0 A; F l u o r e s c e n c e d e t e c t o r 420 AC; W I S P 710 B i n j e c t o r ; D a t a M o d u l e 730 and a S y s t e m C o n t r o l l e r 720. A m i c r o bondapak C-18 column (Novapak, 25 cm x 2.1 m m i.d., Waters A s s o c i a t e s , USA) at a m b i e n t t e m p e r a t u r e was u s e d for s e p a r a t i o n of v a r i o u s a m i n o acids. A m i n o acids a n a l y s i s w a s c a r r i e d out w i t h "AUTO TAG' m e t h o d d e s c r i b e d in W a t e r s A s s o c i a t e s Manual, First Edition, (16). T w o b u f f e r s y s t e m s w e r e u t i l i z e d . B u f f e r A was 2:2:96 methanol:Tetrahydrofuran:Water w i t h 0.05 M N a O A C (Sodium acetate) a n d 0.05 M Na2HPO 4 ( D i s o d i u m h y d r o g e n p h o s p h a t e ) ; pH adjusted to 7.5 with acetic acid. Buffer B was 65:35 Methanol:Water. G r a d i e n t s y s t e m w i t h a s t a r t i n g f l o w r a t e of 0.I m l / m i n a n d g o i n g to 1.3 ml / m i n was u s e d w i t h total e l u t i o n t i m e of 30 mins. for e a c h sample. All
solvents
used
were
of u l t r a
high purity
HPLC
grade.
All
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solvents, N a O A C a n d Na2HPO 4 w e r e a l s o o b t a i n e d f r o m E. M e r c k (FRG). All a m i n o a c i d s standards, TSC, naloxone, a n d o - p h t h a l d e h y d e w e r e purchased from Sigma C h e m i c a l s ( U . S . A ). T h i o p e n t a l s o d i u m was o b t a i n e d f r o m A b b o t t ( P a k i s t a n ) Ltd. K e t a m i n e ( G e d e o n R i c h t e r Ltd., Hungary ) and Halothane ( ICI P a k i s t a n Ltd.) w e r e used. S t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d u s i n g S t u d e n t ' s t-test.
Results
In the first set of e x p e r i m e n t s , e f f e c t s of v a r i o u s g e n e r a l anesthetics on r e s p i r a t i o n w e r e m o n i t o r e d as d e s c r i b e d in the Methods section. A Group of rats received 25 mg/kg., i.v. t h i o p e n t a l . A n e s t h e s i a was p r o d u c e d but r e s p i r a t o r y rate was not a l t e r e d . At this p o i n t a n i m a l was dissected, t r a c h e a was i s o l a t e d a n d i n c i s e d for i n s e r t i n g T - c a n n u l a and c o n n e c t e d to p r e s s u r e transducer. Control respiratory rate was recorded. Then an a d d i t i o n a l d o s e of 25 m g / k g t h i o p e n t a l was injected. R e s p i r a t i o n b e c a m e d e e p e r a n d slower. W i t h i n 2-3 m i n u t e s the a n i m a l s s t o p p e d b r e a t h i n g . At this p o i n t i.v. n a l o x o n e was i m m e d i a t e l y g i v e n to the s a m e animal. Respiratory paralysis reversed (Fig.l) like p r e v i o u s l y r e p o r t e d (17). The f r e q u e n c y a n d d e p t h of r e s p i r a t i o n
25 mg/Kg (i.v)
Thiotpental 25 mg/Kg 3 mins later after 5 mins of first in ection
T Naloxone2.5mg/Kgi.v
J I I I I I J I I I
5 mins later T
li
®2 O:
10 mins after Naloxone
Fig.1 N a l o x o n e i n d u c e d r e v e r s a l of r e s p i r a t o r y a r r e s t p r o d u c e d b y 50 mg/Kg, i.v. thiopental. At the c e s s a t i o n of respiration after thiopental,naloxone was i n j e c t e d in the same a n i m a l . ( n=10 ) was r e s t o r e d to n o r m a l a f t e r about 60 m i n u t e s . W i t h o u t n a l o x o n e adminstration, the a n i m a l died within 5 minutes. Similarly, n a l o x o n e p r o d u c e d r e v e r s a l of a p n e a i n d u c e d by h i g h d o s e k e t a m i n e (Fig.2), halothane (Fig.3). Each figure is r e p r e s e n t a t i v e of r e c o r d s o b t a i n e d in i0 animals. A g r o u p of rats was a l s o i n j e c t e d w i t h 250 m g / k g p h e n o b a r b i t a l . It p r o d u c e d a n e s t h e s i a and with increasing dose to 400 mg/kg respiratory arrest ensued. Administration of n a l o x o n e to this g r o u p of animals did not reverse respiratory paralysis (Fig.4B) . Similarly, i.v. s a l i n e d i d not r e v e r s e a p n e a (Fig.4A) . I n c r e a s i n g n a l o x o n e c o n c e n t r a t i o n to 8 m g / k g a l s o h a d no e f f e c t on r e s p i r a t o r y arrest.
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GABA, Anesthesia and Respiratory Arrest
2025
,, ~o,,~o7o~,, i
IIIIlIIlli!!ii!III , il ~"~:, I~ilit I Ketamine
140 mg/Kg (i.v.)
}
i Ketamlne 40 mglKg after 5 mins of first injection
Naloxone 2.5 mg/kg
3 rains later , 5 r a i n s l a t e r
110 mins after Naloxone
Fig.2 N a l o x o n e i n d u c e d r e v e r s a l of r e s p i r a t o r y a r r e s t p r o d u c e d by 180 mg/kg, i.v. ketamine. At the cessation of r e s p i r a t i o n a f t e r ketamine, n a l o x o n e was i n j e c t e d in the same animal. ( n=10
Hslothane
3%
l Halothane
3 mins.
5 mins.
5%
Nsloxone 2.5 mglkg(l.,.)
II]l
lip
=o sEco,os
Naloxone, lO rains, after
Fig.3 N a l o x o n e i n d u c e d r e v e r s a l of r e s p i r a t o r y a r r e s t p r o d u c e d b y 5% H a l o t h a n e . At the c e s s a t i o n of r e s p i r a t i o n a f t e r halothane, n a l o x o n e was i n j e c t e d in the same animal. ( n=10 ) T h e s e d a t a d e m o n s t r a t e that i n h a l a t i o n or i.vo a n e s t h e t i c s , are n o n - b a r b i t u r a t e type, i n d u c e d a p n e a can be r e v e r s e d b y naloxone.
that i.v.
A n o t h e r set of e x p e r i m e n t s w e r e c o n d u c t e d in e x a c t l y t h e same m a n n e r as d e s c r i b e d above, e x c e p t that a n i m a l s w e r e p r e t r e a t e d with Thiosemicarbazide (TSC), ( 40 m g / k g i.p., or 19 m g / k g i.v.) for 30 m i n u t e s . In the p r e s e n c e of TSC, a n e s t h e t i c s t e s t e d w e r e
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A l Thiopental 25 mg/Kg (i.v)
I Thiopental, 25 mg/Kg (i.v)
I 3 mins.later
I Saline 0.3 ml (i,v)
after 5 mlns of first injection
B
10 mins after
,, ,'°;'7"°',,,
IPhenobarbftctIPhenob,rbUal
Naloxone 2.5 mglKg (i.v)
t 3 rniml, lllter
250 mg/Kg (i.v,) 150 mg/kg (l.v.) after 5 mln= of
~I0 mins iller Naloxone
first injection
Fig.4 Effects of s a l i n e on t h i o p e n t a l induced respiratory arrest (A) a n d e f f e c t s of n a l o x o n e on p h e n o b a r b i t a l i n d u c e d a p n e a (B).
IBRAIN
STEM
[]
CORTEX
THIOPENTAL . p < 0.001 v s Control o~Q. 2 5 0
-- p < 0.001 vs Control *,', p < 0.001 vs Control
o~ 2 0 0
E Q) 1 5 0 c
W
100
,< p-
50
3
(.9
0 x %
,v
Fig.5
E f f e c t s of T h i o p e n t a l a n d n a l o x o n e on g l u t a m a t e l e v e l s in rat b r a i n s t e m a n d cortex. E a c h b a r r e p r e s e n t s a m e a n of 5 e x p e r i m e n t s . u n a b l e to p r o d u c e a n e s t h e s i a or r e s p i r a t o r y p a r a l y s i s , at the d o s e s m e n t i o n e d above. T h e s e d a t a d e m o n s t r a t e that the p r e s e n c e of G A B A m a y b e a p r e r e q u i s i t e for a n e s t h e t i c s to e x e r t t h e i r a c t i v i t y a n d t h a t G A B A m a y a l s o b e i n v o l v e d in the c o n t r o l of v e n t i l a t i o n . In an a t t e m p t to c o r r e l a t e the c h a n g e s in r e s p i r a t i o n w i t h neurotransmitter a m i n o a c i d s l e v e l s in rat b r a i n a f t e r v a r i o u s d r u g m a n i p u l a t i o n s , n e x t set of e x p e r i m e n t s w e r e c o n d u c t e d . R a t s were divided into 5 groups. Group 1 received normal saline and
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GABA, Anesthesia and Respiratory Arrest
A
2027
KETAM~'qE p ~ 0 co i~s control
,
200
n=5
},
Ioo
B
~OT~NE
~_p
. p < 0 0 0 1 v s Control , . p < 0 001 vs Control • ** p < 0 0 0 1 vs C ~ I r o l
........... ~00
."
Z: : • :.
,
s Control
3%
5%
5% Na~xone Nal~one~,. 2 5 m g / K g 2 5 mg/Kg
Fig.
6
E f f e c t s of K e t a m i n e (A), H a l o t h a n e (B) a n d n a l o x o n e o n g l u t a m a t e l e v e l s in rat b r a i n s t e m a n d cortex. E a c h b a r r e p r e s e n t s a m e a n of 5 e x p e r i m e n t s .
B CORTEX []BRAIN
STEM
THIOPENTAL ,, p < 0 001 v s C o n t r o l
351
:
~ p < 0.001 v s C o n t r o t
~o ~ 30 ~. 25
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E f f e c t s of T h i o p e n t a l a n d n a l o x o n e on G A B A l e v e l s in rat b r a i n s t e m a n d cortex. E a c h b a r r e p r e s e n t s a m e a n of 5 experiments. s e r v e d as c o n t r o l . G r o u p 2 r e c e i v e d 25 m g / k g t h i o p e n t a l o r 140 m g / k g k e t a m i n e or 250 m g / k g p h e n o b a r b i t a l or 3 % h a l o t h a n e . A f t e r treatment, r a t s w e r e s a c r i f i c e d as s o o n as s u r g i c a l a n e s t h e s i a e n s u e d . B r a i n s w e r e i s o l a t e d a n d d i s s e c t e d to s e p a r a t e cortex, brain stem, cerebellum and caudate. Brain parts were then h o m o g e n i z e d in 0 . 4 N p e r c h l o r a t e a n d s u b j e c t e d to H P L C a n a l y s i s f o r neurotransmitter a m i n o a c i d s levels. In this group, we f o u n d a s i g n i f i c a n t r e d u c t i o n in g l u t a m a t e (Fig.5 a n d 6) a n d an e l e v a t i o n in G A B A l e v e l s (Fig.7 a n d 8) in b r a i n s t e m a n d cortex, e x c e p t k e t a m i n e t h a t d i d n o t h a v e a n y a f f e c t on g l u t a m a t e levels. T h e
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o t h e r t w o a r e a s d i d not s h o w a n y c h a n g e in a m i n o a c i d s levels. Also, a s p a r t a t e a n d g l y c i n e w e r e not a l t e r e d in a n y b r a i n r e g i o n studied. In a d d i t i o n , p h e n o b a r b i t a l w a s f o u n d to h a v e no e f f e c t on glutamate o r GABA l e v e l s in any brain region. Next, group 3 was treated with 50 m g / K g t h i o p e n t a l o r 180 m g / K g k e t a m i n e o r 5% halothane. This dose produced complete anesthesia and respiratory paralysis. At this point of respiratory arrest, animals were sacrificed and amino acids levels were measured. We f o u n d t h a t thiopental, nitrous oxide and halothane further abated glutamate levels and raised GABA l e v e l s . A
KETAMINE
4O
:
. p
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Fig. 8 E f f e c t s of K e t a m i n e (A), H a l o t h a n e (B) a n d n a l o x o n e on G A B A l e v e l s in rat b r a i n s t e m a n d cortex. Each bar r e p r e s e n t s a m e a n of 5 e x p e r i m e n t s .
H o w e v e r , k e t a m i n e d i d not a l t e r g l u t a m a t e b u t i n c r e a s e d G A B A c o n t e n t in b r a i n s t e m a n d cortex. P h e n o b a r b i t a l h a d no e f f e c t on any neurotransmitter a m i n o a c i d s (data not shown). F u r t h e r m o r e , g r o u p 4 w a s t r e a t e d in e x a c t l y the same f a s h i o n as g r o u p 3, e x c e p t t h a t n a l o x o n e w a s i n j e c t e d i•v. as s o o n as the a n i m a l s s t o p p e d breathing. B r a i n s w e r e t h e n t a k e n out a n d v a r i o u s p a r t s w e r e a s s a y e d . N a l o x o n e w a s f o u n d to r e v e r s e the i n c r e a s e in G A B A l e v e l s (Fig.7 a n d 8 ) , e x c e p t in p h e n o b a r b i t a l t r e a t e d a n i m a l s . H o w e v e r , g l u t a m a t e d i d n o t s h o w r e v e r s a l w i t h the e x c e p t i o n of t h i o p e n t a l t r e a t e d a n i m a l s (Fig.5 a n d 6). G r o u p 5 w a s t r e a t e d w i t h n a l o x o n e alone. In this case, n a l o x o n e d i d not i n d u c e a n y s i g n i f i c a n t c h a n g e in GABA, h o w e v e r d e c r e a s e d g l u t a m a t e c o n t e n t s in b r a i n s t e m a n d cortex.
Discussion G A B A is a m a j o r a n d m o s t a b u n d a n t n e u r o t r a n s m i t t e r f o u n d in the m a m m a l i a n c e n t r a l n e r v o u s system• Its f u n c t i o n a r e d e f i n e d to b e i n h i b i t o r y a n d f a c i l i t a t i o n of G A B A - e r g i c t r a n s m i s s i o n p r o d u c e s a n i n h i b i t i o n of n e u r o n a l output. G e n e r a l a n e s t h e t i c s h a v e b e e n
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GABA, Anesthesia and Respiratory Arrest
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p o s t u l a t e d to exert an affect/s that results in the f a c i l i t a t i o n of this i n h i b i t o r y p a t h w a y (for review, see Keane and Biziere, 1987), that leads to anesthetic state (8). Previously, we have shown that thiopental decreases the excitatory amino acid g l u t a m a t e and, increases G A B A contents in rat b r a i n stem and cortex (17). T h i o p e n t a l has also been reported to inhibit G A B A transaminase (GABA-T), an enzyme that breakdown GABA, thus e l e v a t i n g G A B A levels (18). We p r o p o s e d that i n h i b i t i o n of G A B A - T m a y not b e the sole m e c h a n i s m by w h i c h thiopental acts and that p e r t u r b a t i o n of G A B A receptors may be involved. The G A B A r e c e p t o r m e c h a n i s m s in the CNS have recently been reviewed (19). I n c r e a s e d synaptic effects of G A B A have been r e p o r t e d w i t h CNS d e p r e s s a n t s like b e n z o d i a z e p i n e s (20), b a r b i t u r a t e s (21) and general anesthetics (22). We have extended our studies to i n v e s t i g a t e the effects of i.v. and inhalation a n e s t h e t i c s on G A B A and g l u t a m a t e levels in different regions of rat brain. In this study we have found that thiopental, ketamine and halothane, but not phenobarbital, augment GABA levels in rat b r a i n stem and cortex (Fig.7 and 8). It is n o t e w o r t h y that G A B A has b e e n i m p l i c a t e d in the action of phenobarbital (21). These a n e s t h e t i c s have r e c e n t l y been reported to have no effect on the release or u p t a k e of G A B A (23). Furthermore, with the e x c e p t i o n of k e t a m i n e and phenobarbital, these agents also produce a reduction in g l u t a m a t e levels. However these agents have b e e n p o s t u l a t e d to have some p o s s i b l e selectivity of action for e x c i t a t o r y amino acids release m e c h a n i s m (23). The failure of these agents to exert an a n e s t h e t i c and r e s p i r a t o r y paralysis effects in the p r e s e n c e of TSC p r o v i d e s an evidence that GABA indeed plays an important role in a n e s t h e s i a and v e n t i l a t o r y responses. This role of G A B A in the action of a n e s t h e t i c s is further strengthened by the findings that GABA-mimetics loose their efficacy in the p r e s e n c e of general anesthetics (24). This would m e a n that anesthetics, in e x e r t i n g their action, activate G A B A receptors to their m a x i m u m and they cannot be further a c t i v a t e d by G A B A agonists. This a c t i v a t i o n of G A B A s y s t e m seems specific for inhalation and i.v. a n e s t h e t i c s but not for phenobarbital, since it fails to induce any change in G A B A or glutamate. However, b a r b i t u r a t e s have been p o s t u l a t e d to affect G A B A synaptic m e c h a n i s m s (25). In addition, anesthetics have b e e n shown to p r o d u c e hypoxia and G A B A levels have been r e p o r t e d to increase d u r i n g hypoxic r e s p i r a t o r y d e p r e s s i o n in both m a m m a l s (26) and lower v e r t e b r a t e s (27). Therefore, it is c o n c e i v a b l e that a n e s t h e t i c s first produce hypoxia that results in a l t e r a t i o n in the levels of GABA. With the e x c e p t i o n of ketamine, all a n e s t h e t i c s tested d e c r e a s e d g l u t a m a t e levels as well in the b r a i n stem and cortex. K e t a m i n e has b e e n p o s t u l a t e d to antagonize one of the g l u t a m a t e receptors, N M D A (28.29). Our findings in no way challenges those data but p r o v i d e additional evidence that ketamine exerts a G A B A f a c i l i t a t o r y effect, that will result in the g e n e r a l i z e d i n h i b i t i o n of CNS. Our o b s e r v a t i o n s that the anesthetic efficacy of these agents is lost in the absence of G A B A (rats p r e t r e a t e d with TSC) show that G A B A is n e c e s s a r y for anesthetics to exert their activity. The m o d i f i c a t i o n of the amino acid n e u r o t r a n s m i t t e r s by a n e s t h e t i c s does not n e c e s s a r i l y m e a n that the site of synthesis of these agents m a y be in the same nerve ending. It is p o s s i b l e that the
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site may not even be in the same neuron. W h a t e v e r the site of release, it is clear that the released transmitters would interact with their specific receptors. Glutamate has been p o s t u l a t e d to interact with m u l t i p l e receptors, while GABA has been p r o p o s e d to interact with GABA A and GABA B sites. GABA A sites are thought to be coupled to chloride ionophores (30,31) and GABA B sites are r e c o g n i z e d to be a s s o c i a t e d with K+ or Ca++ and / or adenylate cyclase (32,33). Our data do not show the types of G A B A or g l u t a m a t e receptors that anesthetics interact with. However, p r e l i m i n a r y experiments in our lab have shown that i.v. THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-01), a specific GABA A receptor stimulant ( 3 4 ) produces anesthesia without a f f e c t i n g respiration. These data are in agreement with those r e p o r t e d by Cheng and Brunner (35). The anesthetic effect of THIP is obtunded in rats pretreated with TSC or bicuculline (Unpublished observations). Moreover, i.v. baclofen, a GABA B receptor a c t i v a t o r (36,37) induces sleep and r e s p i r a t o r y failure, but does not produce anesthesia as tail pinch and corneal reflexes are present. The sleep inducing effects of b a c l o f e n are a b o l i s h e d by p h a c l o f e n or 2 - h y d r o x y s a c l o f e n (Our u n p u b l i s h e d observations). Furthermore, studies of Waldrop et ai.(38) have d e m o n s t r a t e d that m i c r o i n j e c t i o n of GABA antagonists into the p o s t e r i o r h y p o t h a l a m u s stimulates v e n t i l a t i o n and in their studies a GABA agonist d e p r e s s e d breathing. It is tempting to speculate that G A B A receptors involved in anesthesia are different from those m e d i a t i n g the d e p r e s s i o n of respiration. Similar conclusions have been drawn by Yeadon and Kitchen ( 3 9 ) for analgesia and r e s p i r a t i o n from the data of M c G i l l a r d and Takemore, (40), who r e p o r t e d significant differences in pA2 values with agonists and antagonists, in their experiments on mice for analgesia and respiration. Our data provides evidence for the requirement of amino acid n e u r o t r a n s m i t t e r s in p r o d u c i n g anesthesia and/or r e s p i r a t o r y paralysis. The present study also provides evidence that naloxone is able to reverse the respiratory failure p r o d u c e d by general anesthetics. It is interesting that naloxone does not reverse p h e n o b a r b i t a l induced apnea. Naloxone has been reported to reverse r e s p i r a t o r y d e p r e s s i o n p r o d u c e d by b u p r e n o r p h i n e (41), morphine (42) and antagonize morphine receptors ( 4 3 ) and inhibit GABA m e d i a t e d nerve transmission (14). However, the antagonistic actions of naloxone on apnea induced by general anesthetics have not been demonstrated. No data are available to correlate the effect of m o r p h i n e induced respiratory d e p r e s s i o n and GABA levels in the brain. It can be speculated that naloxone may activate some p e p t i d e that indirectly involve GABA in its p r o t e c t i v e action. The role p l a y e d by G A B A is not clear. It may also be possible that a n e s t h e t i c s first m o b i l i z e GABA, that in turn affects other n e u r o t r a n s m i t t e r s involved in the control of ventilation. Besides G A B A (44), N o r e p i n e p h r i n e (45), Dopamine (46), Substance P (47) and opioid peptides (48) have been p o s t u l a t e d to be involved in the control of ventilation. Thus p o s s i b i l i t y cannot be entirely ruled out that the resultant changes in GABA are the consequences of v a r i a t i o n s in other putative neurotransmitters. Although g l u t a m a t e has been shown to stimulate v e n t i l a t i o n (49), the decrease in glutamate levels by naloxone in our study suggests that n a l o x o n e may not involve gluamate in its p r o t e c t i v e action. N a l o x o n e treatment did not produce adverse effects on respiration. K e e p i n g this in view, the significant decrease in glutamate
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GABA, Anesthesia and RespiratoryArrest
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content observed in n a l o x o n e treated group might reflect some s e c o n d a r y or i n d i r e c t action. However, this o b s e r v a t i o n in t u r n r e i n f o r c e s the G A B A e r g i c p o t e n t i a t i o n as a f a c t o r in r e s p i r a t o r y d e p r e s s i o n p r o d u c e d b y a n a e s t h e t i c s . T h i s is a l s o e v i d e n t b y the fact that G A B A l e v e l s i n c r e a s e d d u r i n g r e s p i r a t o r y d e p r e s s i o n a n d d e c r e a s e d in rats in w h i c h r e s p i r a t o r y d e p r e s s i o n was r e v e r s e d b y n a l o x o n e . Thus, the d a t a p r e s e n t e d h e r e c l e a r l y d e m o n s t r a t e that G A B A m a y be i n v o l v e d in p r o d u c i n g anesthesia and r e s p i r a t o r y paralysis by general anesthetics. Anesthetics loose their efficacy in the a b s e n c e of GABA. F u r t h e r m o r e , that n a l o x o n e is a b l e to r e v e r s e the r e s p i r a t o r y a r r e s t p r o d u c e d b y i.v. or i n h a l a t i o n anesthetic, but not p r o d u c e d b y p h e n o b a r b i t a l . Future studies could be directed to r e c o g n i z i n g the n a t u r e of a m i n o acid neurotransmitter r e c e p t o r s in the a c t i o n of general anesthetics a n d the m e c h a n i s m of n a l o x o n e in r e v e r s i n g r e s p i r a t o r y failure.
Acknowledgements T h i s w o r k was s u p p o r t e d b y funds H e a l t h Sciences, A g a K h a n U n i v e r s i t y ,
provided Karachi,
b y the F a c u l t y Pakistan.
of
T h e a u t h o r s w i s h to e x p r e s s t h e i r s i n c e r e t h a n k s to Prof. Rehana Kamal, Dr. F a u z i a K h a n a n d Dr. T. Bhatti, consultant a n e s t h e s i o l o g i s t s , A g a K h a n U n i v e r s i t y Hospital, for t h e i r h e l p f u l suggestions and expert assistance.
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GABA, Anesthesia and Respiratory Arrest
4 9. J. MITRA, N.R. P R A B H A R K A R , J.L. O V E R H O L T a n d N. S. C H E R N I A C K , Res. Bull.l_88 6 8 1 - 6 8 4 (1987).
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Brain
Pergamon 0024-3205-(94) E0056-W
EVIDENCE FOR INVOLVEMENT OF A M I N O ACID N E U R O T R A N S M I T T E R S IN A N E S T H E S I A AND N A L O X O N E INDUCED REVERSAL OF R E S P I R A T O R Y PARALYSIS
Amin Suria and Fauzia Rasheed
Department of Pharmacology Faculty of Health Sciences Aga Khan U n i v e r s i t y Medical College Karachi-74800, Pakistan (Received in final form April
11, 1994)
Summary General anesthetics render a person unconscious and may p r o d u c e r e s p i r a t o r y paralysis at therapeutic doses. No p h a r m a c o l o g i c a l agent is available to restore r e s p i r a t i o n and the m e c h a n i s m / s of anesthesia or apnea is not clearly understood. In this report, we present evidence to show that naloxone reversed respiratory failure induced by thiopental, ketamine, halothane but not that induced by p h e n o b a r b i t a l . F u r t h e r m o r e , 25 mg/kg, i.v. thiopental, 140 mg/kg, i.v. ketamine, and 3% halothane produced a n e s t h e s i a without significantly altering r e s p i r a t o r y rate, increased GABA and decreased glutamate (except k e t a m i n e and phenobarbital) levels in rat b r a i n stem and cortex, but not in caudate and cerebellum. Aspartate, g l y c i n e and alanine levels were not affected in four b r a i n regions studied. Pretreatment with TSC for 30 m i n u t e s did not change GABA or glutamate contents, but a b o l i s h e d the anesthetic as well as the r e s p i r a t o r y d e p r e s s a n t actions of the anesthetics. Increasing the doses of anesthetics produced respiratory failure with further rise in GABA and fall in glutamate in brain stem and cortex. Naloxone reversed respiratory paralysis and r e s t o r e d G A B A close to control values in rat brain stem and cortex with no changes in caudate or cerebellum. Data p r e s e n t e d here suggest that GABA may be n e c e s s a r y to p r o d u c e loss of consciousness and naloxone reverses a n e s t h e t i c induced respiratory failure.
Key Words:
anesthetics, G A B A , glutamate, naloxone, respiration
General a n e s t h e t i c s given by inhalation or intravenous route render a p e r s o n u n c o n s c i o u s and may produce respiratory d e p r e s s i o n at t h e r a p e u t i c doses. At higher doses, these agents may p r o d u c e r e s p i r a t o r y arrest and death. The mechanism/s by w h i c h a n e s t h e s i a
Corresponding author: Professor Amin Suria, Department of Pharmacology, The Aga Khan U n i v e r s i t y Hospital, S t a d i u m Road, P.O.Box 3500, Karachi, Pakistan
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or r e s p i r a t o r y a r r e s t is p r o d u c e d is not u n d e r s t o o d . In r e l a t i o n to anesthesia, anesthetics have been proposed to increase the t h i c k n e s s of l i p i d b i l a y e r s (I), a l t e r b r a i n m e t a b o l i c a c t i v i t y (2,3,4), p e r t u r b the f l u i d i t y of cell m e m b r a n e s (5) a n d p r o d u c e functional loss of m e m b r a n e p r o t e i n s (6,7). Recently, however, investigators have focused their attention on the e f f e c t s of anesthetics on neuronal or synaptic function, thinking that i n h i b i t i o n of n e u r o n a l a c t i v i t y w o u l d i n h i b i t m a n y b r a i n f u n c t i o n s (8,9). In p a r t i c u l a r , n e u r o t r a n s m i t t e r a m i n o acids, g l u t a m a t e a n d GABA (Gamma aminobutyric acid) involvement in the a c t i o n of a n e s t h e t i c s has b e e n e x p l o r e d (8). A n e s t h e t i c s , as a group, h a v e been demonstrated to p r o f o u n d l y affect the function of G A B A activated chloride conductances (9). W i t h r e s p e c t to r e s p i r a t i o n , a n u m b e r of n e u r o t r a n s m i t t e r s including epinephrine, norepinephrine and s e r o t o n i n have been implicated (I0). Neurotransmitter amino acids have also been p r o p o s e d to p l a y an a c t i v e role in the c o n t r o l of v e n t i l a t i o n (II) . It has b e e n h y p o t h e s i z e d that the final n e u r a l o u t p u t d e t e r m i n i n g the c e n t r a l d r i v e w i l l d e p e n d on the b a l a n c e b e t w e e n e x c i t a t i o n b y g l u t a m a t e a n d i n h i b i t i o n b y G A B A (11,12). Thus, these t r a n s m i t t e r s m a y be m o d i f i e d in r e s p i r a t o r y d e p r e s s i o n or p a r a l y s i s p r o d u c e d b y general anesthetics. Although, naloxone has b e e n shown to b l o c k the g e n e r a l a n e s t h e t i c a c t i o n of h a l o t h a n e a n d e n f l u r a n e (13). It is n o t e w o r t h y that no agent is a v a i l a b l e that can m o d i f y the respiratory d e p r e s s a n t e f f e c t s of a n e s t h e t i c s . D i n g l e d i n e et al. (14) w o r k i n g w i t h m i c e s h o w e d that h i g h d o s e n a l o x o n e i n d u c e d c o n v u l s i o n s c o u l d be a n t a g o n i z e d b y d i a z e p a m and p r o p o s e d that n a l o x o n e a c t s as a GABAantagonist. We h a v e s h o w n that t h i o p e n t a l i n d u c e d r e s p i r a t o r y arrest, as w e l l as i n c r e a s e in G A B A a n d d e c r e a s e in g l u t a m a t e c o n t e n t s in rat b r a i n s t e m a n d c o r t e x can be r e v e r s e d b y n a l o x o n e . In this report, we p r e s e n t f u r t h e r e v i d e n c e to d e m o n s t r a t e that i.v., as w e l l as i n h a l a t i o n a n e s t h e t i c s at t h e r a p e u t i c d o s e s p r o d u c e a fall in g l u t a m a t e w i t h c o n c o m i t a n t rise in G A B A c o n t e n t s in rat b r a i n s t e m a n d cortex. We a l s o show that h i g h e r d o s e s of a n e s t h e t i c s p r o d u c e r e s p i r a t o r y paralysis, and a f u r t h e r d e c r e a s e in g l u t a m a t e a n d an i n c r e a s e in G A B A levels. N a l o x o n e r e v e r s e s r e s p i r a t o r y a r r e s t a n d r e s t o r e s the G A B A l e v e l s b a c k to its c o n t r o l v a l u e s e x c e p t in c a s e of k e t a m i n e and r e s t a b l i s h e s g l u t a m a t e l e v e l s o n l y in t h i o p e n t a l t r e a t e d group.
Materials
and Methods
Adult Sprague-Dawley rats (150-200 g) of e i t h e r sex w e r e e m p l o y e d for all e x p e r i m e n t s . To m o n i t o r the e f f e c t s of a n e s t h e t i c s a n d o t h e r d r u g s on r e s p i r a t i o n , a n i m a l s w e r e first o b s e r v e d for c o n t r o l r e s p i r a t o r y rate, t h e n i n j e c t e d i n t r a v e n o u s l y , w i t h e i t h e r thiopental (25 mg/kg), or k e t a m i n e (140 mg/kg), or p h e n o b a r b i t a l (250 mg/kg) or 3 % h a l o t h a n e . T h e s e doses p r o d u c e d anesthesia w i t h o u t a p p r e c i a b l y a l t e r i n g the r e s p i r a t o r y rate. At this point, the t r a c h e a was exposed, i n c i s e d and a glass T - s h a p e d c a n n u l a was inserted. O n e side of the c a n n u l a was o p e n to r o o m a i r a n d the other was connected to a differential transducer (PT-120, B i o s c i e n c e ) that was c o n n e c t e d to a B i o s c i e n c e two
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GABA, Anesthesia and Respiratory Arrest
2023
c h a n n e l r e c o r d e r v i a A C c o u p l e r (FC 137, B i o s c i e n c e ) for r e c o r d i n g r e s p i r a t o r y rate. A n a d d i t i o n a l d o s e of 25 m g / k g t h i o p e n t a l or 40 m g / k g k e t a m i n e or 150 m g / k g p h e n o b a r b i t a l or h a l o t h a n e was t h e n s l o w l y i n j e c t e d or g i v e n b y i n h a l a t i o n till the a n i m a l s t o p p e d b r e a t h i n g . I m m e d i a t e l y , n a l o x o n e (2.5 mg/kg, i.v.) was i n j e c t e d to the s a m e a n i m a l as r e s p i r a t i o n was c o n t i n u o u s l y m o n i t o r e d . A total of I0 a n i m a l s w e r e u s e d for m o n i t o r i n g the e f f e c t s of e a c h drug. E f f e c t s of n a l o x o n e a l o n e w e r e a l s o m o n i t o r e d in a n o t h e r g r o u p of I0 rats. To s t u d y the e f f e c t s of g e n e r a l a n e s t h e t i c s on a m i n o a c i d neurotransmitters l e v e l s in the b r a i n a n i m a l s w e r e k e p t in a 12hour light-dark cycle a n d w e r e fed c h o w ad libitum. Rats were d i v i d e d i n t o 5 g r o u p s w i t h e a c h g r o u p c o n t a i n i n g five animals. F i r s t g r o u p s e r v e d as a c o n t r o l a n d r e c e i v e d i.v. s a l i n e or just r o o m air. S e c o n d g r o u p was s u b j e c t e d to e i t h e r i.v. t h i o p e n t a l (25 mg/kg) or k e t a m i n e (140 mg/kg) or p h e n o b a r b i t a l (250 mg/kg) or was g i v e n i n h a l a t i o n anesthetic, halothane (3 %). H a l o t h a n e w a s g i v e n t h r o u g h an a n e s t h e s i a m a c h i n e (Boyles, U. K.), the h o s e of w h i c h was f i t t e d to the w h o l e m o u t h of the animal. T h e s e d o s e s of various anesthetics produced complete surgical anesthesia. Third group received either thiopental (50 mg/kg) or k e t a m i n e (180 mg/kg) or p h e n o b a r b i t a l (400 m g / kg) or 5 % h a l o t h a n e . At t h e s e d o s e s a n i m a l s s t o p p e d b r e a t h i n g . G r o u p f o u r was first g i v e n h i g h d o s e s of a n e s t h e t i c s , as in g r o u p 3. As s o o n as the a n i m a l s stopped breathing i.v. n a l o x o n e (2.5 mg/kg) was a d m i n i s t e r e d . G r o u p five r e c e i v e d n a l o x o n e alone. Rats were then sacrificed by a focused beam microwave (General M e d i c a l E n g i n e e r i n g Corp., Peabody, M.A.) irradiation (output 2.5 KW) w i t h an e x p o s u r e time of 4 s e c o n d s to e n s u r e the i n a c t i v a t i o n of all enzymes. T h e y w e r e t h e n d e c a p i t a t e d , c r a n i u m s w e r e r e m o v e d a n d b r a i n s w e r e isolated. C e r e b e l l u m , caudate, c o r t e x a n d b r a i n s t e m r e g i o n s w e r e d i s s e c t e d out and all s a m p l e s w e r e stored at -70°C u n t i l ready for a n a l y s i s . At the time of analysis, s a m p l e s w e r e thawed, homogenized in 1 ml h i g h g r a d e purity water in g l a s s homogenizing tubes w i t h g l a s s pestles (Kontes, Vineland, N.J.). Homogenates were centrifuged for 5 m i n u t e s at I000 x g. S u p e r n a t a n t s w e r e d r a w n off a n d i0 ul of e a c h s a m p l e w a s i n j e c t e d in HPLC. P e l l e t s w e r e d i s s o l v e d in 1 ml of 0.i N N a O H a n d s u b j e c t e d to p r o t e i n e s t i m a t i o n b y the m e t h o d of L o w r y et al. (15). T h e H P L C s y s t e m (Waters A s s o c i a t e s , U.S.A.) c o n s i s t e d of 2 pumps, M - 5 i 0 a n d M - 6 0 0 0 A; F l u o r e s c e n c e d e t e c t o r 420 AC; W I S P 710 B i n j e c t o r ; D a t a M o d u l e 730 and a S y s t e m C o n t r o l l e r 720. A m i c r o bondapak C-18 column (Novapak, 25 cm x 2.1 m m i.d., Waters A s s o c i a t e s , USA) at a m b i e n t t e m p e r a t u r e was u s e d for s e p a r a t i o n of v a r i o u s a m i n o acids. A m i n o acids a n a l y s i s w a s c a r r i e d out w i t h "AUTO TAG' m e t h o d d e s c r i b e d in W a t e r s A s s o c i a t e s Manual, First Edition, (16). T w o b u f f e r s y s t e m s w e r e u t i l i z e d . B u f f e r A was 2:2:96 methanol:Tetrahydrofuran:Water w i t h 0.05 M N a O A C (Sodium acetate) a n d 0.05 M Na2HPO 4 ( D i s o d i u m h y d r o g e n p h o s p h a t e ) ; pH adjusted to 7.5 with acetic acid. Buffer B was 65:35 Methanol:Water. G r a d i e n t s y s t e m w i t h a s t a r t i n g f l o w r a t e of 0.I m l / m i n a n d g o i n g to 1.3 ml / m i n was u s e d w i t h total e l u t i o n t i m e of 30 mins. for e a c h sample. All
solvents
used
were
of u l t r a
high purity
HPLC
grade.
All
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solvents, N a O A C a n d Na2HPO 4 w e r e a l s o o b t a i n e d f r o m E. M e r c k (FRG). All a m i n o a c i d s standards, TSC, naloxone, a n d o - p h t h a l d e h y d e w e r e purchased from Sigma C h e m i c a l s ( U . S . A ). T h i o p e n t a l s o d i u m was o b t a i n e d f r o m A b b o t t ( P a k i s t a n ) Ltd. K e t a m i n e ( G e d e o n R i c h t e r Ltd., Hungary ) and Halothane ( ICI P a k i s t a n Ltd.) w e r e used. S t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d u s i n g S t u d e n t ' s t-test.
Results
In the first set of e x p e r i m e n t s , e f f e c t s of v a r i o u s g e n e r a l anesthetics on r e s p i r a t i o n w e r e m o n i t o r e d as d e s c r i b e d in the Methods section. A Group of rats received 25 mg/kg., i.v. t h i o p e n t a l . A n e s t h e s i a was p r o d u c e d but r e s p i r a t o r y rate was not a l t e r e d . At this p o i n t a n i m a l was dissected, t r a c h e a was i s o l a t e d a n d i n c i s e d for i n s e r t i n g T - c a n n u l a and c o n n e c t e d to p r e s s u r e transducer. Control respiratory rate was recorded. Then an a d d i t i o n a l d o s e of 25 m g / k g t h i o p e n t a l was injected. R e s p i r a t i o n b e c a m e d e e p e r a n d slower. W i t h i n 2-3 m i n u t e s the a n i m a l s s t o p p e d b r e a t h i n g . At this p o i n t i.v. n a l o x o n e was i m m e d i a t e l y g i v e n to the s a m e animal. Respiratory paralysis reversed (Fig.l) like p r e v i o u s l y r e p o r t e d (17). The f r e q u e n c y a n d d e p t h of r e s p i r a t i o n
25 mg/Kg (i.v)
Thiotpental 25 mg/Kg 3 mins later after 5 mins of first in ection
T Naloxone2.5mg/Kgi.v
J I I I I I J I I I
5 mins later T
li
®2 O:
10 mins after Naloxone
Fig.1 N a l o x o n e i n d u c e d r e v e r s a l of r e s p i r a t o r y a r r e s t p r o d u c e d b y 50 mg/Kg, i.v. thiopental. At the c e s s a t i o n of respiration after thiopental,naloxone was i n j e c t e d in the same a n i m a l . ( n=10 ) was r e s t o r e d to n o r m a l a f t e r about 60 m i n u t e s . W i t h o u t n a l o x o n e adminstration, the a n i m a l died within 5 minutes. Similarly, n a l o x o n e p r o d u c e d r e v e r s a l of a p n e a i n d u c e d by h i g h d o s e k e t a m i n e (Fig.2), halothane (Fig.3). Each figure is r e p r e s e n t a t i v e of r e c o r d s o b t a i n e d in i0 animals. A g r o u p of rats was a l s o i n j e c t e d w i t h 250 m g / k g p h e n o b a r b i t a l . It p r o d u c e d a n e s t h e s i a and with increasing dose to 400 mg/kg respiratory arrest ensued. Administration of n a l o x o n e to this g r o u p of animals did not reverse respiratory paralysis (Fig.4B) . Similarly, i.v. s a l i n e d i d not r e v e r s e a p n e a (Fig.4A) . I n c r e a s i n g n a l o x o n e c o n c e n t r a t i o n to 8 m g / k g a l s o h a d no e f f e c t on r e s p i r a t o r y arrest.
Vol. 54, No. 25, 1994
GABA, Anesthesia and Respiratory Arrest
2025
,, ~o,,~o7o~,, i
IIIIlIIlli!!ii!III , il ~"~:, I~ilit I Ketamine
140 mg/Kg (i.v.)
}
i Ketamlne 40 mglKg after 5 mins of first injection
Naloxone 2.5 mg/kg
3 rains later , 5 r a i n s l a t e r
110 mins after Naloxone
Fig.2 N a l o x o n e i n d u c e d r e v e r s a l of r e s p i r a t o r y a r r e s t p r o d u c e d by 180 mg/kg, i.v. ketamine. At the cessation of r e s p i r a t i o n a f t e r ketamine, n a l o x o n e was i n j e c t e d in the same animal. ( n=10
Hslothane
3%
l Halothane
3 mins.
5 mins.
5%
Nsloxone 2.5 mglkg(l.,.)
II]l
lip
=o sEco,os
Naloxone, lO rains, after
Fig.3 N a l o x o n e i n d u c e d r e v e r s a l of r e s p i r a t o r y a r r e s t p r o d u c e d b y 5% H a l o t h a n e . At the c e s s a t i o n of r e s p i r a t i o n a f t e r halothane, n a l o x o n e was i n j e c t e d in the same animal. ( n=10 ) T h e s e d a t a d e m o n s t r a t e that i n h a l a t i o n or i.vo a n e s t h e t i c s , are n o n - b a r b i t u r a t e type, i n d u c e d a p n e a can be r e v e r s e d b y naloxone.
that i.v.
A n o t h e r set of e x p e r i m e n t s w e r e c o n d u c t e d in e x a c t l y t h e same m a n n e r as d e s c r i b e d above, e x c e p t that a n i m a l s w e r e p r e t r e a t e d with Thiosemicarbazide (TSC), ( 40 m g / k g i.p., or 19 m g / k g i.v.) for 30 m i n u t e s . In the p r e s e n c e of TSC, a n e s t h e t i c s t e s t e d w e r e
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GABA, Anesthesia and Respiratory Arrest
Vol. 54, No. 25, 1994
A l Thiopental 25 mg/Kg (i.v)
I Thiopental, 25 mg/Kg (i.v)
I 3 mins.later
I Saline 0.3 ml (i,v)
after 5 mlns of first injection
B
10 mins after
,, ,'°;'7"°',,,
IPhenobarbftctIPhenob,rbUal
Naloxone 2.5 mglKg (i.v)
t 3 rniml, lllter
250 mg/Kg (i.v,) 150 mg/kg (l.v.) after 5 mln= of
~I0 mins iller Naloxone
first injection
Fig.4 Effects of s a l i n e on t h i o p e n t a l induced respiratory arrest (A) a n d e f f e c t s of n a l o x o n e on p h e n o b a r b i t a l i n d u c e d a p n e a (B).
IBRAIN
STEM
[]
CORTEX
THIOPENTAL . p < 0.001 v s Control o~Q. 2 5 0
-- p < 0.001 vs Control *,', p < 0.001 vs Control
o~ 2 0 0
E Q) 1 5 0 c
W
100
,< p-
50
3
(.9
0 x %
,v
Fig.5
E f f e c t s of T h i o p e n t a l a n d n a l o x o n e on g l u t a m a t e l e v e l s in rat b r a i n s t e m a n d cortex. E a c h b a r r e p r e s e n t s a m e a n of 5 e x p e r i m e n t s . u n a b l e to p r o d u c e a n e s t h e s i a or r e s p i r a t o r y p a r a l y s i s , at the d o s e s m e n t i o n e d above. T h e s e d a t a d e m o n s t r a t e that the p r e s e n c e of G A B A m a y b e a p r e r e q u i s i t e for a n e s t h e t i c s to e x e r t t h e i r a c t i v i t y a n d t h a t G A B A m a y a l s o b e i n v o l v e d in the c o n t r o l of v e n t i l a t i o n . In an a t t e m p t to c o r r e l a t e the c h a n g e s in r e s p i r a t i o n w i t h neurotransmitter a m i n o a c i d s l e v e l s in rat b r a i n a f t e r v a r i o u s d r u g m a n i p u l a t i o n s , n e x t set of e x p e r i m e n t s w e r e c o n d u c t e d . R a t s were divided into 5 groups. Group 1 received normal saline and
Vol. 54, No. 25, 1994
GABA, Anesthesia and Respiratory Arrest
A
2027
KETAM~'qE p ~ 0 co i~s control
,
200
n=5
},
Ioo
B
~OT~NE
~_p
. p < 0 0 0 1 v s Control , . p < 0 001 vs Control • ** p < 0 0 0 1 vs C ~ I r o l
........... ~00
."
Z: : • :.
,
s Control
3%
5%
5% Na~xone Nal~one~,. 2 5 m g / K g 2 5 mg/Kg
Fig.
6
E f f e c t s of K e t a m i n e (A), H a l o t h a n e (B) a n d n a l o x o n e o n g l u t a m a t e l e v e l s in rat b r a i n s t e m a n d cortex. E a c h b a r r e p r e s e n t s a m e a n of 5 e x p e r i m e n t s .
B CORTEX []BRAIN
STEM
THIOPENTAL ,, p < 0 001 v s C o n t r o l
351
:
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E f f e c t s of T h i o p e n t a l a n d n a l o x o n e on G A B A l e v e l s in rat b r a i n s t e m a n d cortex. E a c h b a r r e p r e s e n t s a m e a n of 5 experiments. s e r v e d as c o n t r o l . G r o u p 2 r e c e i v e d 25 m g / k g t h i o p e n t a l o r 140 m g / k g k e t a m i n e or 250 m g / k g p h e n o b a r b i t a l or 3 % h a l o t h a n e . A f t e r treatment, r a t s w e r e s a c r i f i c e d as s o o n as s u r g i c a l a n e s t h e s i a e n s u e d . B r a i n s w e r e i s o l a t e d a n d d i s s e c t e d to s e p a r a t e cortex, brain stem, cerebellum and caudate. Brain parts were then h o m o g e n i z e d in 0 . 4 N p e r c h l o r a t e a n d s u b j e c t e d to H P L C a n a l y s i s f o r neurotransmitter a m i n o a c i d s levels. In this group, we f o u n d a s i g n i f i c a n t r e d u c t i o n in g l u t a m a t e (Fig.5 a n d 6) a n d an e l e v a t i o n in G A B A l e v e l s (Fig.7 a n d 8) in b r a i n s t e m a n d cortex, e x c e p t k e t a m i n e t h a t d i d n o t h a v e a n y a f f e c t on g l u t a m a t e levels. T h e
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o t h e r t w o a r e a s d i d not s h o w a n y c h a n g e in a m i n o a c i d s levels. Also, a s p a r t a t e a n d g l y c i n e w e r e not a l t e r e d in a n y b r a i n r e g i o n studied. In a d d i t i o n , p h e n o b a r b i t a l w a s f o u n d to h a v e no e f f e c t on glutamate o r GABA l e v e l s in any brain region. Next, group 3 was treated with 50 m g / K g t h i o p e n t a l o r 180 m g / K g k e t a m i n e o r 5% halothane. This dose produced complete anesthesia and respiratory paralysis. At this point of respiratory arrest, animals were sacrificed and amino acids levels were measured. We f o u n d t h a t thiopental, nitrous oxide and halothane further abated glutamate levels and raised GABA l e v e l s . A
KETAMINE
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:
. p
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Fig. 8 E f f e c t s of K e t a m i n e (A), H a l o t h a n e (B) a n d n a l o x o n e on G A B A l e v e l s in rat b r a i n s t e m a n d cortex. Each bar r e p r e s e n t s a m e a n of 5 e x p e r i m e n t s .
H o w e v e r , k e t a m i n e d i d not a l t e r g l u t a m a t e b u t i n c r e a s e d G A B A c o n t e n t in b r a i n s t e m a n d cortex. P h e n o b a r b i t a l h a d no e f f e c t on any neurotransmitter a m i n o a c i d s (data not shown). F u r t h e r m o r e , g r o u p 4 w a s t r e a t e d in e x a c t l y the same f a s h i o n as g r o u p 3, e x c e p t t h a t n a l o x o n e w a s i n j e c t e d i•v. as s o o n as the a n i m a l s s t o p p e d breathing. B r a i n s w e r e t h e n t a k e n out a n d v a r i o u s p a r t s w e r e a s s a y e d . N a l o x o n e w a s f o u n d to r e v e r s e the i n c r e a s e in G A B A l e v e l s (Fig.7 a n d 8 ) , e x c e p t in p h e n o b a r b i t a l t r e a t e d a n i m a l s . H o w e v e r , g l u t a m a t e d i d n o t s h o w r e v e r s a l w i t h the e x c e p t i o n of t h i o p e n t a l t r e a t e d a n i m a l s (Fig.5 a n d 6). G r o u p 5 w a s t r e a t e d w i t h n a l o x o n e alone. In this case, n a l o x o n e d i d not i n d u c e a n y s i g n i f i c a n t c h a n g e in GABA, h o w e v e r d e c r e a s e d g l u t a m a t e c o n t e n t s in b r a i n s t e m a n d cortex.
Discussion G A B A is a m a j o r a n d m o s t a b u n d a n t n e u r o t r a n s m i t t e r f o u n d in the m a m m a l i a n c e n t r a l n e r v o u s system• Its f u n c t i o n a r e d e f i n e d to b e i n h i b i t o r y a n d f a c i l i t a t i o n of G A B A - e r g i c t r a n s m i s s i o n p r o d u c e s a n i n h i b i t i o n of n e u r o n a l output. G e n e r a l a n e s t h e t i c s h a v e b e e n
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GABA, Anesthesia and Respiratory Arrest
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p o s t u l a t e d to exert an affect/s that results in the f a c i l i t a t i o n of this i n h i b i t o r y p a t h w a y (for review, see Keane and Biziere, 1987), that leads to anesthetic state (8). Previously, we have shown that thiopental decreases the excitatory amino acid g l u t a m a t e and, increases G A B A contents in rat b r a i n stem and cortex (17). T h i o p e n t a l has also been reported to inhibit G A B A transaminase (GABA-T), an enzyme that breakdown GABA, thus e l e v a t i n g G A B A levels (18). We p r o p o s e d that i n h i b i t i o n of G A B A - T m a y not b e the sole m e c h a n i s m by w h i c h thiopental acts and that p e r t u r b a t i o n of G A B A receptors may be involved. The G A B A r e c e p t o r m e c h a n i s m s in the CNS have recently been reviewed (19). I n c r e a s e d synaptic effects of G A B A have been r e p o r t e d w i t h CNS d e p r e s s a n t s like b e n z o d i a z e p i n e s (20), b a r b i t u r a t e s (21) and general anesthetics (22). We have extended our studies to i n v e s t i g a t e the effects of i.v. and inhalation a n e s t h e t i c s on G A B A and g l u t a m a t e levels in different regions of rat brain. In this study we have found that thiopental, ketamine and halothane, but not phenobarbital, augment GABA levels in rat b r a i n stem and cortex (Fig.7 and 8). It is n o t e w o r t h y that G A B A has b e e n i m p l i c a t e d in the action of phenobarbital (21). These a n e s t h e t i c s have r e c e n t l y been reported to have no effect on the release or u p t a k e of G A B A (23). Furthermore, with the e x c e p t i o n of k e t a m i n e and phenobarbital, these agents also produce a reduction in g l u t a m a t e levels. However these agents have b e e n p o s t u l a t e d to have some p o s s i b l e selectivity of action for e x c i t a t o r y amino acids release m e c h a n i s m (23). The failure of these agents to exert an a n e s t h e t i c and r e s p i r a t o r y paralysis effects in the p r e s e n c e of TSC p r o v i d e s an evidence that GABA indeed plays an important role in a n e s t h e s i a and v e n t i l a t o r y responses. This role of G A B A in the action of a n e s t h e t i c s is further strengthened by the findings that GABA-mimetics loose their efficacy in the p r e s e n c e of general anesthetics (24). This would m e a n that anesthetics, in e x e r t i n g their action, activate G A B A receptors to their m a x i m u m and they cannot be further a c t i v a t e d by G A B A agonists. This a c t i v a t i o n of G A B A s y s t e m seems specific for inhalation and i.v. a n e s t h e t i c s but not for phenobarbital, since it fails to induce any change in G A B A or glutamate. However, b a r b i t u r a t e s have been p o s t u l a t e d to affect G A B A synaptic m e c h a n i s m s (25). In addition, anesthetics have b e e n shown to p r o d u c e hypoxia and G A B A levels have been r e p o r t e d to increase d u r i n g hypoxic r e s p i r a t o r y d e p r e s s i o n in both m a m m a l s (26) and lower v e r t e b r a t e s (27). Therefore, it is c o n c e i v a b l e that a n e s t h e t i c s first produce hypoxia that results in a l t e r a t i o n in the levels of GABA. With the e x c e p t i o n of ketamine, all a n e s t h e t i c s tested d e c r e a s e d g l u t a m a t e levels as well in the b r a i n stem and cortex. K e t a m i n e has b e e n p o s t u l a t e d to antagonize one of the g l u t a m a t e receptors, N M D A (28.29). Our findings in no way challenges those data but p r o v i d e additional evidence that ketamine exerts a G A B A f a c i l i t a t o r y effect, that will result in the g e n e r a l i z e d i n h i b i t i o n of CNS. Our o b s e r v a t i o n s that the anesthetic efficacy of these agents is lost in the absence of G A B A (rats p r e t r e a t e d with TSC) show that G A B A is n e c e s s a r y for anesthetics to exert their activity. The m o d i f i c a t i o n of the amino acid n e u r o t r a n s m i t t e r s by a n e s t h e t i c s does not n e c e s s a r i l y m e a n that the site of synthesis of these agents m a y be in the same nerve ending. It is p o s s i b l e that the
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site may not even be in the same neuron. W h a t e v e r the site of release, it is clear that the released transmitters would interact with their specific receptors. Glutamate has been p o s t u l a t e d to interact with m u l t i p l e receptors, while GABA has been p r o p o s e d to interact with GABA A and GABA B sites. GABA A sites are thought to be coupled to chloride ionophores (30,31) and GABA B sites are r e c o g n i z e d to be a s s o c i a t e d with K+ or Ca++ and / or adenylate cyclase (32,33). Our data do not show the types of G A B A or g l u t a m a t e receptors that anesthetics interact with. However, p r e l i m i n a r y experiments in our lab have shown that i.v. THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-01), a specific GABA A receptor stimulant ( 3 4 ) produces anesthesia without a f f e c t i n g respiration. These data are in agreement with those r e p o r t e d by Cheng and Brunner (35). The anesthetic effect of THIP is obtunded in rats pretreated with TSC or bicuculline (Unpublished observations). Moreover, i.v. baclofen, a GABA B receptor a c t i v a t o r (36,37) induces sleep and r e s p i r a t o r y failure, but does not produce anesthesia as tail pinch and corneal reflexes are present. The sleep inducing effects of b a c l o f e n are a b o l i s h e d by p h a c l o f e n or 2 - h y d r o x y s a c l o f e n (Our u n p u b l i s h e d observations). Furthermore, studies of Waldrop et ai.(38) have d e m o n s t r a t e d that m i c r o i n j e c t i o n of GABA antagonists into the p o s t e r i o r h y p o t h a l a m u s stimulates v e n t i l a t i o n and in their studies a GABA agonist d e p r e s s e d breathing. It is tempting to speculate that G A B A receptors involved in anesthesia are different from those m e d i a t i n g the d e p r e s s i o n of respiration. Similar conclusions have been drawn by Yeadon and Kitchen ( 3 9 ) for analgesia and r e s p i r a t i o n from the data of M c G i l l a r d and Takemore, (40), who r e p o r t e d significant differences in pA2 values with agonists and antagonists, in their experiments on mice for analgesia and respiration. Our data provides evidence for the requirement of amino acid n e u r o t r a n s m i t t e r s in p r o d u c i n g anesthesia and/or r e s p i r a t o r y paralysis. The present study also provides evidence that naloxone is able to reverse the respiratory failure p r o d u c e d by general anesthetics. It is interesting that naloxone does not reverse p h e n o b a r b i t a l induced apnea. Naloxone has been reported to reverse r e s p i r a t o r y d e p r e s s i o n p r o d u c e d by b u p r e n o r p h i n e (41), morphine (42) and antagonize morphine receptors ( 4 3 ) and inhibit GABA m e d i a t e d nerve transmission (14). However, the antagonistic actions of naloxone on apnea induced by general anesthetics have not been demonstrated. No data are available to correlate the effect of m o r p h i n e induced respiratory d e p r e s s i o n and GABA levels in the brain. It can be speculated that naloxone may activate some p e p t i d e that indirectly involve GABA in its p r o t e c t i v e action. The role p l a y e d by G A B A is not clear. It may also be possible that a n e s t h e t i c s first m o b i l i z e GABA, that in turn affects other n e u r o t r a n s m i t t e r s involved in the control of ventilation. Besides G A B A (44), N o r e p i n e p h r i n e (45), Dopamine (46), Substance P (47) and opioid peptides (48) have been p o s t u l a t e d to be involved in the control of ventilation. Thus p o s s i b i l i t y cannot be entirely ruled out that the resultant changes in GABA are the consequences of v a r i a t i o n s in other putative neurotransmitters. Although g l u t a m a t e has been shown to stimulate v e n t i l a t i o n (49), the decrease in glutamate levels by naloxone in our study suggests that n a l o x o n e may not involve gluamate in its p r o t e c t i v e action. N a l o x o n e treatment did not produce adverse effects on respiration. K e e p i n g this in view, the significant decrease in glutamate
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content observed in n a l o x o n e treated group might reflect some s e c o n d a r y or i n d i r e c t action. However, this o b s e r v a t i o n in t u r n r e i n f o r c e s the G A B A e r g i c p o t e n t i a t i o n as a f a c t o r in r e s p i r a t o r y d e p r e s s i o n p r o d u c e d b y a n a e s t h e t i c s . T h i s is a l s o e v i d e n t b y the fact that G A B A l e v e l s i n c r e a s e d d u r i n g r e s p i r a t o r y d e p r e s s i o n a n d d e c r e a s e d in rats in w h i c h r e s p i r a t o r y d e p r e s s i o n was r e v e r s e d b y n a l o x o n e . Thus, the d a t a p r e s e n t e d h e r e c l e a r l y d e m o n s t r a t e that G A B A m a y be i n v o l v e d in p r o d u c i n g anesthesia and r e s p i r a t o r y paralysis by general anesthetics. Anesthetics loose their efficacy in the a b s e n c e of GABA. F u r t h e r m o r e , that n a l o x o n e is a b l e to r e v e r s e the r e s p i r a t o r y a r r e s t p r o d u c e d b y i.v. or i n h a l a t i o n anesthetic, but not p r o d u c e d b y p h e n o b a r b i t a l . Future studies could be directed to r e c o g n i z i n g the n a t u r e of a m i n o acid neurotransmitter r e c e p t o r s in the a c t i o n of general anesthetics a n d the m e c h a n i s m of n a l o x o n e in r e v e r s i n g r e s p i r a t o r y failure.
Acknowledgements T h i s w o r k was s u p p o r t e d b y funds H e a l t h Sciences, A g a K h a n U n i v e r s i t y ,
provided Karachi,
b y the F a c u l t y Pakistan.
of
T h e a u t h o r s w i s h to e x p r e s s t h e i r s i n c e r e t h a n k s to Prof. Rehana Kamal, Dr. F a u z i a K h a n a n d Dr. T. Bhatti, consultant a n e s t h e s i o l o g i s t s , A g a K h a n U n i v e r s i t y Hospital, for t h e i r h e l p f u l suggestions and expert assistance.
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GABA, Anesthesia and Respiratory Arrest
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