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GABA receptor binding and hemodynamic responses to ICV GABA in adult spontaneously hypertensive rats.
Life Sciences, Vol. 33, pp. 701-709 Printed in the U.S.A.
Pergamon Press
GABA RECEPTOR BINDING AND HEMODYNAMIC RESPONSES TO ICY GABA IN A D U L T SPONTANEOUSLY HYPERTENSIVE RATS. T.J.
Brennan, J.R.
Haywood, and M . K . T i c k u
Department of Pharmacology, U n i v e r s i t y of Texas Health Science Center San A n t o n i o , T X 78284 (Received in final form June 6, 1983) Summary
Blood p r e s s u r e and heart rate responses a f t e r central admini s t r a t i o n of GABA (100-1000 tJg, ICV) and a f t e r g a n g l i o n i c blockade w i t h hexamethonium (25 m g / k g , i . a . ) were compared in conscious 12 week-old spontaneously h y p e r t e n s i v e (SH) and age-matched WistarKyoto (WKY) rats. Ganglionic blockade produced an e q u i v a l e n t change in mean a r t e r i a l p r e s s u r e between SH and WKY rats. Thus1 the total f u n c t i o n a l sympathetic n e r v o u s system c o n t r i b u t i o n to blood p r e s s u r e is e q u i v a l e n t in these two s t r a i n s . Intraventricularlyadministered GABA produced a g r e a t e r absolute decrease in mean a r t e r i a l p r e s s u r e in the SHR compared to the WKY. However, the p e r c e n t changes in a r t e r i a l p r e s s u r e produced by GABA were not d i f f e r e n t between these s t r a i n s . T h i s g r e a t e r absolute depressor e f f e c t of GABA does not appear to be due to d i f f e r e n c e s in GABA receptor b i n d i n g constants since GABA receptor a f f i n i t y and number of b i n d i n g sites were not s i g n i f i c a n t l y d i f f e r e n t in the f o r e b r a i n , cerebellum or pons-medulla between SH and WKY rats. A l t h o u g h several factors have been implicated in the development and maintenance of h i g h blood p r e s s u r e in the spontaneously h y p e r t e n s i v e rat ( S H R ) , a considerable amount of a t t e n t i o n has been devoted to the c o n t r i b u t i o n of the sympathetic n e r v o u s system. J u d y et al. (1) have reported an elevated g r e a t e r s p l a n c h n i c n e r v e a c t i v i t y in conscious SH compared to normotensive WKY rats. However, r e d u c t i o n s in p e r i p h e r a l sympathetic n e r v o u s system f u n c t i o n by immunosympathectomy (2) or chemical sympathectomy ( 3 , 4 ) decreased a r t e r i a l p r e s s u r e b u t did not p r e v e n t the development of h y p e r t e n sion in the SHR. Blockade of the autonomic n e r v o u s system w i t h pharmacologic agents like hexamethonium or p h e n o x y b e n z a m i n e normalized blood p r e s s u r e between a d u l t SH and WKY rats while conscious (5) and anesthetized ( 1 ) . These r e s u l t s are in disagreement w i t h those of Touw et al. (6) w h i c h demonstrated t h a t hexamethonium produces an e q u i v a l e n t r e d u c t i o n in mean a r t e r i a l p r e s s u r e (MAP) in conscious a d u l t normotensive and h y p e r t e n s i v e rats. The i n v o l v e m e n t of the sympathetic n e r v o u s system in spontaneous h y p e r tension is also suggested b y studies of central n e u r o g e n i c f a c t o r s . For example, c e n t r a l 6 - h y d r o x y d o p a m i n e treatment reduces blood p r e s s u r e to a g r e a t e r e x t e n t in h y p e r t e n s i v e rats compared to age-matched normotensive controis (7,8,9). In a d d i t i o n , c l o n i d i n e , a c e n t r a l l y acting a n t i h y p e r t e n s i v e agent (see r e v i e w b y K o r n e r et a l . ) (10), produces a g r e a t e r fall in blood p r e s s u r e in the SH compared to the WKY rat (11). C h r o n i c a d m i n i s t r a t i o n of diazepam, has also been shown to attenuate the development of h y p e r t e n s i o n in the SH rat ( 1 2 ) . Diazepam, a benzodiazepine, appears to act in the central n e r v o u s 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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Cardiovascular
Effects of GABA in SHR
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system to f a c i l i t a t e i n h i b i t o r y transmission mediated by gamma-aminobutyric acid ( G A B A ) (13). It has also been shown t h a t i n t r a - c e r e b r o v e n t r i c u l a r ( I C V ) a d m i n i s t r a t i o n of GABA decreases blood p r e s s u r e by r e d u c i n g sympathetic vasomotor tone t h r o u g h a central mechanism in cats (14,15,16) and rats ( t 7 ) . The following studies were u n d e r t a k e n to compare the h y p o t e n s i v e action of the p e r i p h e r a l s y m p a t h o l y t i c compound, hexamethonium, and c e n t r a l l y administered GABA in conscious a d u l t (12 week) SH and WKY rats to determine if augmented sympathetic n e r v o u s system f u n c t i o n or d i f f e r e n c e s in ICV GABAinduced h y p o t e n s i o n are observed in this model of genetic h y p e r t e n s i o n . F i n a l l y , in separate g r o u p s of a d u l t SH and WKY rats, GABA receptor b i n d i n g was measured in the f o r e b r a i n , cerebellum, and pons-medulla to determine if d i f f e r e n c e s e x i s t in GABA receptor b i n d i n g constants between SH and WKY rats. Methods Twelve week old SH and WKY rats ( w e i g h i n g 220-280g) were purchased from Taconic Farms of New Y o r k . Rats were housed in i n d i v i d u a l l y hanging wire mesh cages in a t e m p e r a t u r e c o n t r o l l e d room with a twelve hour l i g h t / d a r k cycle. Standard lab chow and water were administered ad libitum. Hemodynamic Studies. T w e n t y - t h r e e gauge stainless steel guide cannulae were implanted into the lateral v e n t r i c l e of chloral h y d r a t e anesthetized rats using the c o - o r d i n a t e s 0.8mm. p o s t e r i o r , 1.3mm. lateral to bregma and 6.0 mm. below the surface of the skull. T h r e e days a f t e r cannulae implantation, rats were again anesthetized with chloral h y d r a t e and t y g o n catheters with teflon tips were inserted into the femoral a r t e r y . The h e p a r i n - f i t l e d catheters were t u n n e l l e d s u b c u t a n e o u s l y to e x i t at the back of the neck where t h e y were s u t u r e d in place. The n e x t day rats were placed in experimental cages w i t h o u t food and water. Mean a r t e r i a l pressure (MAP) was recorded with an Ailtech Pressure T r a n s d u c e r (Model MS20) connected to the a r t e r i a l catheter by p o l y e t h y l e n e (PE SO) t u b i n g . Heart rate (HR) was recorded from the pulse pressure using a Beckman 9857B cardiotachometer. GABA was dissolved in a r t i f i c i a l CSF as described by Merlis (18) and administered randomly in doses of 0,100,300,600 and 1000 IJg in a volume of 2-5 ~1 in conscious, f r e e l y moving SH and WKY rats. Two hours were allowed between each dose of GABA. ICV injections of GABA produced a biphasic response, i n c l u d i n g a s h o r t latency small d epr es s or effect and a long latency, pronounced d e p r e s s o r response lasting a p p r o x i m a t e l y t h i r t y minutes. From the long latency response, the peak h y p o t e n s i v e effect and the hear t rate at t h a t peak h y p o t e n s i v e period were r e c o r d e d . Two hours a f t e r the last injection of GABA, hexamethonium (25 m g / k g , i n t r a - a r t e r i a l ) was slowly infused into normotensive and h y p e r t e n s i v e rats to assess the autonomic component of blood p r e s s u r e and h e a r t rate. Ten minutes a f t e r autonomic blockade, blood pressure and h e a r t rate were recorded. V e n t r i c u l a r cannuta placement was v e r i f i e d one day before GABA administ r a t i o n by an a n g i o te n s i n d r i n k i n g test. D e l i v e r y of small doses of angiotensin II to the v e n t r i c l e s produces rapid d r i n k i n g responses (19). H y d r a t e d SH and WKY rats r a p i d l y d r i n k i n g at least 2.5 ml of water a f t e r ICV injection of 100 ng of angiotensin II were used the fo l l o w i n g day f o r central GABA injections. At the t e r m i n a t i o n of the e x p e r i m e n t Evan's blue dye was injected into the v e n t r i c u l a r cannulae. Brains were removed and o n l y data from rats in which staining of the v e n t r i c l e s a f t e r dye injection are included in this s t u d y . B i n d i n g Studies. T w e l v e week-old male SH and WKY rats were also purchased from Taconic Farms of New Y o r k f o r b i n d i n g studies. Polyethylene
Vol. 33, No. 8, 1983
Cardiovascular
Effects of GABA in SHR
703
(PE 50) t u b i n g was inserted into the femoral a r t e r y u n d e r ether anesthesia as described f o r hemodynamic s t u d i e s . T w o h o u r s later MAP and HR were recorded f o r 15 minutes. The a r t e r i a l p r e s s u r e of 12-week-old SH and WKY rats used f o r b i n d i n g studies tended to be s i g n i f i c a n t l y less than those used for hemodynamic e x p e r i m e n t s . T h i s d i f f e r e n c e is most l i k e l y due to incomplete r e c o v e r y from e t h e r anesthesia. On the n e x t d a y , rats were decapitated and t h e i r b r a i n s were r a p i d l y removed and kept in iced 0.32M sucrose. Brains were dissected into ponsmedulla, cerebellum, and f o r e b r a i n and pooled (6 b r a i n s regions per g r o u p ) for binding studies. As described p r e v i o u s l y (20), tissue from pooled brain regions was prepared to obtain the c r u d e mitochondrial plus microsomal (P2 + P3) f r a c t i o n s . The tissue was washed, frozen o v e r n i g h t , in 0.05M T r i s - c i t r a t e (pH 7 . 1 ) , and rewashed in b u f f e r the f o l l o w i n g day. On the day of the e x p e r i m e n t , tissue was washed t h r e e times in b u f f e r , and resuspended in the b u f f e r f o r assays. The b i n d i n g of [ 3 H ] G A B A and [3H]muscimol (New England Nuclear) to its receptors was assayed b y a modification of the methods of Z u k i n et al. (21) and Enna and S n y d e r (22) as described b y T i c k u (20). B i n d i n g assays were performed by i n c u b a t i n g aliquots of P2 + P3 homogenate (0.5 - 1.0 mg/ml p r o t e i n ) w i t h v a r i o u s c o n c e n t r a t i o n s of [ 3 H ] G A B A (58 Ci/mmol) or [3H]muscimol (7.13 Ci/mmol) w i t h or w i t h o u t excess n o n - r a d i o a c t i v e GABA (1 retool). The samples were c e n t r i f u g e d and the pellets were r a p i d l y rinsed w i t h b u f f e r , solubilized o v e r n i g h t with 0.3 ml Soluene-350, and r a d i o a c t i v i t y was counted in 3 ml of toluene c o n t a i n i n g d i p h e n y l o x a z o l e . Counting efficiency measured by the i n t e r n a l standard ( [ 3 H ] t o l u e n e ) method was a p p r o x i m a t e l y
40-~. Specific b i n d i n g was obtained b y s u b t r a c t i n g from the total r a d i o a c t i v i t y t h a t w h i c h was not displaced by excess n o n - r a d i o a c t i v e G A B A . B i n d i n g assays were performed in t r i p l i c a t e . Protein was estimated b y the method of L o w r y et al. ( 2 3 ) . T h i r t e e n p o i n t [ 3 H ] G A B A and [3H]muscimol scatchard plots were determined. The b i n d i n g a f f i n i t i e s ( K D ) and number of b i n d i n g sites ( B m a x ) were calculated f o r two sites b y linear regression w i t h Feldman (24) analysis. The effects of GABA compared to a r t i f i c i a l CSF were analyzed b y analysis of v a r i a n c e . Differences among repeated measures were determined w i t h a S t u d e n t Newman-Keuls range test. Differences between SHR an WKY were analyzed by an u n p a i r e d S t u d e n t s T - t e s t . S i g n i f i c a n c e was taken at the P<.05 level. Results Hemodynamic Studies Before central injections of G A B A , mean a r t e r i a l p r e s s u r e and h e a r t rate were 178-+8 mmHg and 365-+14 beats per minute (BPM) in the SHR (n=8) and 129.+3 mmHg and 339+19 BPM in the WKY (n=8) as shown in Table 1. Figure 1 r e p r e s e n t s a t y p i c a l p o l y g r a p h r e c o r d i n g of the mean a r t e r i a l p r e s s u r e and h e a r t rate a f t e r the a d m i n i s t r a t i o n of 600 pg of GABA ICV in conscious SH and WKY rats. An a p p a r e n t biphasic response o c c u r r e d f o l l o w i n g central a d m i n i s t r a t i o n of G A B A . T h e r e was an i n i t i a l small depressor effect of a p p r o x i m a t e l y 10 mmHg in both g r o u p s of rats t h a t was accompanied b y a r e f l e x increase in h e a r t rate. In a d d i t i o n t h e r e was a long latency, more p r o n o u n c e d d e p r e s s o r e f f e c t w i t h a noticeable absence of a r e f l e x t a c h y c a r d i a . The responses are a r e s u l t of the i n f l u e n c e of GABA on the central n e r v o u s
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Cardiovascular
Effects of GABA in SHR
Vol.
33, No. 8, 1983
system since i n t r a v e n o u s a d m i n i s t r a t i o n of 1000 IJg of GABA did not a l t e r MAP or HR. Before and a f t e r the a d m i n i s t r a t i o n of all doses of G A B A , the a r t e r i a l p r e s s u r e of the SH rat was always s i g n i f i c a n t l y elevated above t h a t of the WKY ( T a b l e 1). ICY GABA was not able to normalize p r e s s u r e between the two g r o u p s of r a t s . The depressor response of 100 IJg of GABA ICY was not d i f f e r e n t between the two g r o u p s . Injection of 300, 600, and 1000 lag ICV produced a g r e a t e r absolute decrease in a r t e r i a l p r e s s u r e in the h y p e r t e n s i v e rats as shown in Table 1. However, t h e r e were no s i g n i f i c a n t d i f f e r e n c e s in the p e r c e n t decrease in a r t e r i a l p r e s s u r e produced by all doses of GABA between SH and WKY rats. Heart rate was not s i g n i f i c a n t l y d i f f e r e n t between normotensive and h y p e r t e n s i v e rats before GABA a d m i n i s t r a t i o n . Central injection of 100 and 300 Iag GABA did not a l t e r h e a r t rate s i g n i f i c a n t l y in e i t h e r g r o u p . However, injection of 600 and 1000 IJg GABA produced a s i g n i f i c a n t l y g r e a t e r absolute and p e r c e n t decrease in h e a r t rate in the SHR while not s i g n i f i c a n t l y c h a n g i n g h e a r t rate in the WKY ( T a b l e I ) .
240] WKY MAP (mmHg)
HR
(bpm)
1801
4801
3601 240J
MAP (mm Hq)
HR (bpm)
21801 4011201SHR
6001~ GABA ICV
~
~
..............
60] 3601 240 J
., 0
I0
.$ : -
--.--_t
20
30
- "~,~--'~'. ~ : ~ ' ~ 40
50
Time ( rain )
F i g u r e 1. Effect of 600 IJg of GABA ( I C Y ) on mean a r t e r i a l p r e s s u r e (MAP) and h e a r t rate ( H R ) of conscious SH ( l o w e r panel) and WKY ( u p p e r panel) rats.
Vol. 33, No. 8, 1983
Cardiovascular Effects of GABA in SHR
705
In t h e same a n i m a l s , b e f o r e a u t o n o m i c b l o c k a d e , mean a r t e r i a l p r e s s u r e was 185 -+ 4 mmHg in t h e SHR a n d 133 -+ 1 in t h e WKY r a t s . After hexamet h o n i u m a d m i n i s t r a t i o n (25 m g / k g ) t h e a r t e r i a l p r e s s u r e o f t h e SHR was s t i l l s i g n i f i c a n t l y g r e a t e r t h a n t h e WKY ( T a b l e 1 ) . As s h o w n in T a b l e 1, a u t o n o m i c b l o c k a d e p r o d u c e d an e q u i v a l e n t a b s o l u t e a n d p e r c e n t c h a n g e in mean a r t e r i a l p r e s s u r e in n o r m o t e n s i v e a n d h y p e r t e n s i v e r a t s . B e f o r e h e x a m e t h o n i u m a d m i n i s t r a t i o n h e a r t r a t e was 382 + 14 b e a t s p e r m i n u t e ( B P M ) in t h e SHR a n d 321 -+ 18 BPM in t h e W K Y . At the time these data were recorded, h e a r t r a t e was s i g n i f i c a n t l y e l e v a t e d in t h e SH r a t . H e x a m e t h o n i u m d e c r e a s e d h e a r t r a t e (42 -+ 17 B P M ) in t h e SHR b u t i n c r e a s e d h e a r t r a t e (43 -+ 22 B P M ) in t h e WKY ( P < . 0 5 ) as s h o w n in T a b l e I . Ganglionic b l o c k a d e a l s o p r o d u c e d a g r e a t e r a b s o l u t e a n d p e r c e n t d e c r e a s e in h e a r t r a t e in t h e S H R . H e a r t r a t e b e t w e e n t h e t w o g r o u p s was n o t s i g n i f i c a n t l y d i f f e r e n t after ganglionic blockade. Binding Studies• Mean a r t e r i a l p r e s s u r e a n d h e a r t r a t e o f t h e 12 w e e k o l d SH r a t s u s e d f o r b i n d i n g s t u d i e s was 157-+12 mmHg a n d 407_+29 b e a t s p e r minute (BPM) respectively (n=18). A r t e r i a l p r e s s u r e o f WKY r a t s was 119-+11 m m H g , h e a r t r a t e was 365_+30 BPM. B l o o d p r e s s u r e was g r e a t e r in t h e SH r a t (P<.05); heart rates were not different. GABA receptor sensitivity in t h e f o r e b r a i n c e r e b e l l u m , a n d p o n s - m e d u l l a was m e a s u r e d b y u s i n g [ 3 H ] m u s c i m o l as t h e r a d i o l i g a n d . Scatchard analysis of the binding data indicate the existence of two receptor sites. The affinities of t h e t w o r e c e p t o r s i t e s u s i n g [ 3 H ] m u s c i m o l a r e in a g r e e m e n t w i t h r e p o r t e d v a l u e s in o t h e r s p e c i e s ( 2 5 ) . The K_ and B values of the two GABA receptor sites were not ax slgntflcantly d l f f e r e n r ~ in f o r e b r a i n , c e r e b e l l u m a n d p o n s - m e d u l l a o f SH a n d WKY r a t s ( T a b l e 2 ) . Due t o t h e l a r g e v a r i a t i o n w i t h i n r e p l i c a t e s a n d h i g h n o n s p e c i f i c b i n d i n g in t h e p o n s - m e d u l l a , t h e G A B A r e c e p t o r s e n s i t i v i t y was m e a s u r e d in t h i s r e g i o n u s i n g b o t h [ 3 H ] G A B A a n d [ 3 H ] m u s c i m o l . [3H] GABA was u s e d b e c a u s e t h e h i g h e r s p e c i f i c a c t i v i t y o f t h i s l i g a n d i m p r o v e d t h e accuracy of the Scatchard plot. Table 2 shows that the binding constants w e r e n o t s i g n i f i c a n t l y d i f f e r e n t in t h e b r a i n s t e m o f t h e s e t w o s t r a i n s . •
,
.
U
.
Discussion If t h e h i g h b l o o d p r e s s u r e seen in t h e SHR is m a i n t a i n e d b y a g r e a t e r a c t i v i t y o f t h e s y m p a t h e t i c n e r v o u s s y s t e m , t h e n h e x a m e t h o n i u m w o u l d be e x p e c t e d t o l o w e r b l o o d p r e s s u r e m o r e in t h e SH r a t t h a n in t h e W K Y . In o u r s t u d i e s , h e x a m e t h o n i u m l o w e r e d MAP 50-+7 a n d 45-+6 mmHg in SH a n d WKY r a t s , respectively• T h e s i m i l a r c h a n g e in a r t e r i a l p r e s s u r e a f t e r h e x a m e t h o n i u m in c o n s c i o u s n o r m o t e n s i v e a n d h y p e r t e n s i v e r a t s is in a g r e e m e n t w i t h t h e r e s u l t s o f T o u w e t a l . ( 6 ) b u t in d i s a g r e e m e n t w i t h t h e f i n d i n g s o f Numao e t a l . ( 5 ) and Judy et al. (I). Our studies support the contention that the sympathetic n e r v o u s s y s t e m is n o t m a i n t a i n i n g b l o o d p r e s s u r e t o a g r e a t e r e x t e n t in t h e a d u l t SHR r e l a t i v e t o n o r m o t e n s i v e r a t s . G a n g l i o n i c b l o c k a d e w i t h h e x a m e t h o n i u m d e c r e a s e d h e a r t r a t e in t h e SHR f r o m 382_+14 t o 340_+7 BPM ( P < . 0 S ) . In t h e WKY h e x a m e t h o n i u m i n c r e a s e d h e a r t r a t e f r o m 321_+18 t o 364_+20 BPM ( P < . 0 5 ) . These results suggest that the s y m p a t h e t i c c o n t r i b u t i o n t o h e a r t r a t e is g r e a t e r in t h e SH r a t c o m p a r e d t o t h e WKY a n d v a g a l i n p u t t o t h e h e a r t is g r e a t e r in t h e WKY r a t s r e l a t i v e t o t h e SHR. F o r t h e s e r e a s o n s , h e x a m e t h o n i u m i n c r e a s e s h e a r t r a t e in t h e WKY a n d d e c r e a s e s h e a r t r a t e in t h e SHR ( T a b l e I ) . However intrinsic heart rate ( a f t e r a u t o n o m i c b l o c k a d e ) is e q u i v a l e n t in b o t h g r o u p s o f r a t s ( T a b l e I ) .
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Cardiovascular Effects of GABA in SHR
Vol. 33, No. 8, 1983
TABLE i
EFFECTS OF GABA AND HEXAMETHONIUM ON MEAN ARTERIAL PRESSURE AND HEART RATE IN SH AND WKY RATS MAP SHR
HR WKY
SHR
WKY
CSF
Pre Post A %A
180 180 0 0
t ± ± ±
8* 8* 0 0
133 132 -i -i
t ± ± ±
3 2 0 0
340 340 -I 0
I00 pg GABA, ICV
Pre Post A
178 ± 172 ± -6 ±
9* 9* 2
129 ± 123 ± -6 ±
3 4 2
370 ± 14 361 ± 14 -9 ± 5
%Z~
-3 ±
1
-5 ±
2
Post A %Z~
154 + -24 ± -14 ±
2?
117 ± -13 ± -11 ±
55 ~ ~
346 ± 10 - I i ± I0 -3 ± 3
353 ± 15 +20 ± 16 +7 ± 6
Pre Post A
180 ± 8 ~ 144 ± 11.? -35 ± ~
128 ± 107 ± -21+
3? 4? ~
369 ± 14 337 ± 18 -30 ± 13".~
337 ± 19 350 ± 19 +12 + 12
%A
-20 ±
-16 ±
?
Pre Post
177 ± 9*? 137 ± 10"~
128 ± 100 ±
2? 5~
363 ± 17 316 ± 147~
334 ± 22 347 ± 14
-40 ±
+13 ± I0
GABA, ICV
600 pg GABA, ICV
1000 pg GABA, ICV
Hexamethonium (25mg/kg, I.A.)
A= * = =
Pre Post A ~L~
t
~$
± 9 + I0 ± 1 ± 0
-2 ±
-9 ±
1
3*T
-29 ±
3
-47 ±
8 *T
-23 ±
-22 ±
3?
-13 ±
2*~
185 133 -50 -27
133 87 -46 -34
± 4" ± 12" ± 7 ± 4
± 10 ± 6 ± 6 ± 4
382 340 -42 -11
± 14" ± 7 ± 17" ± 3*
342 343 -I 0
± 19 ± 19 ± 0 ± 1
348 ± 16 348 ± 15 0 ± 5
0 ±
+4 ±
+5 ± 321 364 +43 +14
1
3
3
± 10 ± 20 ± 22 ± 6
absolute change in mean a r t e r i a l pressure (MAP, mmHg) or heart rate (HR, beats per minute). ~w~= percent change in MAP or HR. P<.05 vs. WKY rats (unpaired t - t e s t ) denotes a s i g n i f i c a n t difference within each group vs. a r t i f i c i a l CSF injection (ANOVA) of P<.05.
Central administration of GABA reduces the sympathetic component of blood pressure and heart rate in both cats (14,15,16) and rats (17). In our studies with conscious SH and WKY rats, ICV GABA produces a biphasic response (Fig. I ) . The immediate depressor effect of all doses of ICV GABA ( a p p r o x i m a t e l y 10 mmHg in both groups of r a t s ) is buffered by an increase in heart rate. This response presumably occurs when GABA acts on s t r u c t u r e s r a p i d l y accessible to lateral v e n t r i c l e administration. The longer latency, profound depressor effect is not accompanied by a r e f l e x tachycardia. In both strains heart rate o r d i n a r i l y decreases or does not change d u r i n g the prolonged depressor response. This longer latency response is similar to t h a t observed in the cat (115) when the GABA agonist muscimol is perfused in the
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TABLE 2 GABA RECEPTOR SENSITIVITY IN SH AND WKY RATS
Bmax
KD(nM)
(pmol/mg protein)
KD1
SHR Forebrain (n=3) Cerebellum (n=3) Pons-Medulla
WKY Forebrain (n=3) Cerebellum (n=3) Pons-Medulla
KD2
BmaxI
Bmax2
3.4 -+ 0.4
90
+ 25
1.1 +- 0.1
8.1 _+ 1.8
2.4 + 0.9
44
-+ 9
1.6 + 0.8
6.2 -+ 1.0
1.6 5.6*
85 141"
0.2 O. I *
2.3 1.8*
5.1 -+ 0.5
66
+
7
1.4 _+ 0.2
6.6 + 1.5
2.7 + 1.6
55
-+ 18
1.6 -+ 0.7
6.5 -+ 0.9
1.1 5.4*
100 203*
1.2 O. 1"
2.3 1.8*
[3H] GABA binding is denoted by * Binding of [3H] muscimol and [3H] GABA* was measured as described in METHODS, K. and B values were determined from i n d i v i d u a l s a t u r a t i o n isotherms using ~u., max~ . relaman analysls (23). The values represent the mean ± S.D. of the number of experiments indicated in the parentheses. Each value represents analysis of i n d i v i d u a l isotherm containing brain regions pooled from 6 rats.
fourth ventricle. in t h e h i n d b r a i n
Therefore, the prolonged depressor effect presumably occurs as s u g g e s t e d ( 1 5 ) .
I C Y G A B A d e c r e a s e s h e a r t r a t e in t h e S H r a t , b u t h e a r t r a t e does n o t c h a n g e s i g n i f i c a n t l y a f t e r G A B A a d m i n i s t r a t i o n in t h e WKY ( T a b l e I ) . Based on o u r p o s t u l a t i o n t h a t s y m p a t h e t i c o u t f l o w to t h e h e a r t in t h e SHR is g r e a t e r t h a n in t h e WKY r a t , ICV G A B A w o u l d be e x p e c t e d to l o w e r h e a r t r a t e t o a g r e a t e r e x t e n t in t h e h y p e r t e n s i v e r a t . In c o n t r a s t , i f v a g a l t o n e to t h e h e a r t is g r e a t e r in t h e WKY r a t r e l a t i v e to t h e SHR, as we h a v e s u g g e s t e d , a r e d u c t i o n in r e s t i n g v a g a l t o n e f o l l o w i n g t h e ~ d e p r e s s o r s t i m u l u s ( i . e . ICY G A B A ) w o u l d t e n d to r a i s e h e a r t r a t e m o r e in t h e WKY t h a n t h e SHR. Theref o r e , c e n t r a l l y a d m i n i s t e r e d G A B A causes h e a r t r a t e to d e c r e a s e m o r e in t h e SHR b y r e d u c i n g s y m p a t h e t i c o u t f l o w to a h e a r t w h o s e r a t e is m a i n t a i n e d b y a g r e a t e r s y m p a t h e t i c a n d r e d u c e d v a g a l t o n e r e l a t i v e t o t h e WKY. C e n t r a l G A B A a d m i n i s t r a t i o n p r o d u c e s a g r e a t e r a b s o l u t e d e c r e a s e in b l o o d p r e s s u r e in t h e h y p e r t e n s i v e r a t . T h i s is in a g r e e m e n t w i t h a n o t h e r report that intra-arterial m u s c i m o l , a G A B A a g o n i s t , also r e d u c e s b l o o d p r e s s u r e m o r e in SH r a t s ( 2 6 ) ; h o w e v e r , a c e n t r a l s i t e o f a c t i o n was n o t demonstrated. In a d d i t i o n , Baum e t al. ( 2 7 ) h a v e d e m o n s t r a t e d a p r o n o u n c e d
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d e p r e s s o r e f f e c t of I V T muscimol in SH r a t s . When t h e p e a k d e p r e s s o r e f f e c t of G A B A is a n a l y z e d b y t h e p e r c e n t c h a n g e in a r t e r i a l p r e s s u r e , s i g n i f i c a n t d i f f e r e n c e s b e t w e e n SH and WKY r a t s w e r e n o t o b s e r v e d . Therefore, the g r e a t e r a b s o l u t e c h a n g e in b l o o d p r e s s u r e p r o d u c e d b y G A B A a p p e a r s t o be r e l a t e d to t h e h i g h b a s e l i n e a r t e r i a l p r e s s u r e of SH r a t s . T h e b i n d i n g s t u d i e s d e m o n s t r a t e no m a j o r d i f f e r e n c e s in r e g i o n a l G A B A a g o n i s t r e c e p t o r b i n d i n g b e t w e e n a d u l t SH and WKY r a t s . T h e s e r e s u l t s also d e m o n s t r a t e t h a t e n h a n c e d a b s o l u t e d e p r e s s o r r e s p o n s e to G A B A in SH r a t s r e l a t i v e to WKY r a t s can n o t be a t t r i b u t e d t o d i f f e r e n c e s in t h e G A B A r e c e p t o r binding constants between these two strains. We h a v e also f o u n d no d i f f e r ences in G A B A a g o n i s t b i n d i n g b e t w e e n 4 and 8 w e e k - o l d SH and WKY r a t s (27). Since we a r e m e a s u r i n g r e c e p t o r b i n d i n g in g r o s s b r a i n r e g i o n s , p e r h a p s o n l y a small n u m b e r of t h e t o t a l r e c e p t o r p o p u l a t i o n p a r t i c i p a t e in cardiovascular function. T h e r e f o r e , i t is p o s s i b l e t h a t r e c e p t o r c h a n g e s o c c u r t h a t a r e n o t d e t e c t a b l e in t i s s u e h o m o g e n a t e p r e p a r a t i o n s . In c o n c l u s i o n , o u r s t u d i e s i n d i c a t e t h a t t h e t o t a l f u n c t i o n a l c o n t r i b u t i o n of t h e s y m p a t h e t i c n e r v o u s s y s t e m in b l o o d p r e s s u r e m a i n t e n a n c e in t h e a d u l t SH r a t is e q u i v a l e n t to t h a t o f t h e W K Y . D e s p i t e t h e g r e a t e r a b s o l u t e c h a n g e in a r t e r i a l p r e s s u r e p r o d u c e d b y G A B A in t h e SH r a t s , i t a p p e a r s t h a t t h e f u n c t i o n a l c o n t r i b u t i o n of t h e s y m p a t h e t i c n e r v o u s s y s t e m s e n s i t i v e to G A B A is e q u i v a l e n t b e t w e e n t h e h y p e r t e n s i v e and n o r m o t e n s i v e s t r a i n s . Acknowledgements T h i s s t u d y was s u p p o r t e d b y f u n d s f r o m t h e A m e r i c a n H e a r t A s s o c i a t i o n ( T e x a s A f f i l i a t e ) g r a n t s to J . R . H . and M . K . T . T h e a u t h o r s also w i s h to t h a n k Ms. M a r y J o y n e r and Ms. J a n e t Wall f o r t h e i r e x c e l l e n t s e c r e t a r i a l assistance. References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15.
W.V. JUDY, A.M. WATANABE, D . P . H E N R Y , H . R . BESCH, W . R . BESCH and G.M. HOCKEL, C i r c . Res. ( S u p p l I I ) II 21 ( 1 9 7 6 ) . B. FOLKOW, M. H A L L B E C K , Y . JUNDGREN and L. WEISS, Acta P h y s i o l . S c a n d 84 512 ( 1 9 7 2 ) . Y . Y A M O N , H. Y A M A B E , W. DEJONG, W. L O V E N B E R G and A . SJOERDSMA, E u r J. P h a r m a c o l 17 135 ( 1 9 7 2 ) . A . P . P R O V O S T a n d W. DEJONG, C l i n E x p H y p e r t e n s i o n 1 177 ( 1 9 7 8 ) . Y . NUMAO, S. H I R O Y U K I and J. I R I U C H I J I M A , Jap H e a r t J 16 719 (1975). K . B . TOUW, J . R . H A Y W O O D , R . A . SHAFFER and M . J . B R O D Y , H y p e r t e n s i o n 2 408 ( 1 9 8 0 ) . G. H A E U S L E R , In Recjulation o f B l o o d P r e s s u r e b y t h e C e n t r a l N e r v o u s S y s t e m , e d i t e d b y O n e s t i G . , F e r n a n d e z M . , and Kim E . K . New Y o r k , G r u n e & S t r a t t o n 53, ( 1 9 7 6 ) . T . K U B O , a n d M. H A S H I M O T O , A c t a P h y s i o l Scand 8 512 ( 1 9 7 2 ) . L. E R I N O F F , A . HELLER and S. O P A R I L , Proc Soc E x p Biol Med 150 748 (1975). P . I . KORNER a n d J . A . ANGUS P h a r m a c T h e r 13 321 ( 1 9 8 1 ) . Y . O H K U S U , J p n . C i r c . J. 35 1175 ( 1 9 7 1 ) . R.M. SCHICKEN, P e d i a t . Res. 13 992 ( 1 9 7 9 ) . J . F . T A L L M A N , S. P A U L , P. SK---OLNICK and D. G A L L A G H E R , Science 207 274 ( 1 9 8 0 ) . C.~. SWEET, H . C . WENGER and D . M . GROSS, Can J. P h y s i o l . P h a r m a c o l 57 600 ( 1 9 7 8 ) . M.J. ANTO-NACClO, L. KERWlN and D . G . T A Y L O R , N e u r o p h a r m a c o l o g y 17 783 ( 1 9 7 8 ) .
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16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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D . G . WILLIFORD, B . L . H A M I L T O N , J. DIAS SOUZA, T . P . WILLIAMS J . A . DIMICCO and R . A . G I L L I S , C i r c . Res. 47 80 (1980). B. PERSSON, A r c h . Pharmacl 313 255 (1980). J . K . MERLIS, Am. J. P h y s i o l . 131 67 (1940). A . K . JOHNSON and A . N . EPSTEIN, B r a i n Research 86 399-418 (1975). M . K . T I C K U , B r . J. Pharmac 70 403 (1980). S . R . Z U K I N , A . B . YOUNG and S . H . SNYDER, Proc. Natl. Acad. Sci ( U S A ) 71 4802 (1974). S . J . ENNA and S . H . SNYDER, B r a i n Res. 100 81 (1975). O . H . LOWRY, N . J . ROSEBROUGH, A . L . FARR and R . I . R A N D A L L , J. Biol. Chem 193 265 (1951). H . A . FELDMAN, A n a l y t i c a l Biochem 48 317 (1972). K. BEAUMONT, W.S. C H I L T O N , H. YAMAMURA and S . J . ENNA, Brain Res. 148 153 (1978). G . , FEUERSTEIN, I. YAMAGUCHI and I . J . KOPIN, Clin. Exp. H y p e r t e n s i o n 3 207 (1978). T. BAU~I AND F . T . BECKER, J. C a r d i o v a s c u l a r Pharmacol 4 165 (1982). T. BRENNAN, J . R . HAYWOOD and M . K . T I C K U , Fed. Proc. 532 (1981).
Pergamon Press
GABA RECEPTOR BINDING AND HEMODYNAMIC RESPONSES TO ICY GABA IN A D U L T SPONTANEOUSLY HYPERTENSIVE RATS. T.J.
Brennan, J.R.
Haywood, and M . K . T i c k u
Department of Pharmacology, U n i v e r s i t y of Texas Health Science Center San A n t o n i o , T X 78284 (Received in final form June 6, 1983) Summary
Blood p r e s s u r e and heart rate responses a f t e r central admini s t r a t i o n of GABA (100-1000 tJg, ICV) and a f t e r g a n g l i o n i c blockade w i t h hexamethonium (25 m g / k g , i . a . ) were compared in conscious 12 week-old spontaneously h y p e r t e n s i v e (SH) and age-matched WistarKyoto (WKY) rats. Ganglionic blockade produced an e q u i v a l e n t change in mean a r t e r i a l p r e s s u r e between SH and WKY rats. Thus1 the total f u n c t i o n a l sympathetic n e r v o u s system c o n t r i b u t i o n to blood p r e s s u r e is e q u i v a l e n t in these two s t r a i n s . Intraventricularlyadministered GABA produced a g r e a t e r absolute decrease in mean a r t e r i a l p r e s s u r e in the SHR compared to the WKY. However, the p e r c e n t changes in a r t e r i a l p r e s s u r e produced by GABA were not d i f f e r e n t between these s t r a i n s . T h i s g r e a t e r absolute depressor e f f e c t of GABA does not appear to be due to d i f f e r e n c e s in GABA receptor b i n d i n g constants since GABA receptor a f f i n i t y and number of b i n d i n g sites were not s i g n i f i c a n t l y d i f f e r e n t in the f o r e b r a i n , cerebellum or pons-medulla between SH and WKY rats. A l t h o u g h several factors have been implicated in the development and maintenance of h i g h blood p r e s s u r e in the spontaneously h y p e r t e n s i v e rat ( S H R ) , a considerable amount of a t t e n t i o n has been devoted to the c o n t r i b u t i o n of the sympathetic n e r v o u s system. J u d y et al. (1) have reported an elevated g r e a t e r s p l a n c h n i c n e r v e a c t i v i t y in conscious SH compared to normotensive WKY rats. However, r e d u c t i o n s in p e r i p h e r a l sympathetic n e r v o u s system f u n c t i o n by immunosympathectomy (2) or chemical sympathectomy ( 3 , 4 ) decreased a r t e r i a l p r e s s u r e b u t did not p r e v e n t the development of h y p e r t e n sion in the SHR. Blockade of the autonomic n e r v o u s system w i t h pharmacologic agents like hexamethonium or p h e n o x y b e n z a m i n e normalized blood p r e s s u r e between a d u l t SH and WKY rats while conscious (5) and anesthetized ( 1 ) . These r e s u l t s are in disagreement w i t h those of Touw et al. (6) w h i c h demonstrated t h a t hexamethonium produces an e q u i v a l e n t r e d u c t i o n in mean a r t e r i a l p r e s s u r e (MAP) in conscious a d u l t normotensive and h y p e r t e n s i v e rats. The i n v o l v e m e n t of the sympathetic n e r v o u s system in spontaneous h y p e r tension is also suggested b y studies of central n e u r o g e n i c f a c t o r s . For example, c e n t r a l 6 - h y d r o x y d o p a m i n e treatment reduces blood p r e s s u r e to a g r e a t e r e x t e n t in h y p e r t e n s i v e rats compared to age-matched normotensive controis (7,8,9). In a d d i t i o n , c l o n i d i n e , a c e n t r a l l y acting a n t i h y p e r t e n s i v e agent (see r e v i e w b y K o r n e r et a l . ) (10), produces a g r e a t e r fall in blood p r e s s u r e in the SH compared to the WKY rat (11). C h r o n i c a d m i n i s t r a t i o n of diazepam, has also been shown to attenuate the development of h y p e r t e n s i o n in the SH rat ( 1 2 ) . Diazepam, a benzodiazepine, appears to act in the central n e r v o u s 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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system to f a c i l i t a t e i n h i b i t o r y transmission mediated by gamma-aminobutyric acid ( G A B A ) (13). It has also been shown t h a t i n t r a - c e r e b r o v e n t r i c u l a r ( I C V ) a d m i n i s t r a t i o n of GABA decreases blood p r e s s u r e by r e d u c i n g sympathetic vasomotor tone t h r o u g h a central mechanism in cats (14,15,16) and rats ( t 7 ) . The following studies were u n d e r t a k e n to compare the h y p o t e n s i v e action of the p e r i p h e r a l s y m p a t h o l y t i c compound, hexamethonium, and c e n t r a l l y administered GABA in conscious a d u l t (12 week) SH and WKY rats to determine if augmented sympathetic n e r v o u s system f u n c t i o n or d i f f e r e n c e s in ICV GABAinduced h y p o t e n s i o n are observed in this model of genetic h y p e r t e n s i o n . F i n a l l y , in separate g r o u p s of a d u l t SH and WKY rats, GABA receptor b i n d i n g was measured in the f o r e b r a i n , cerebellum, and pons-medulla to determine if d i f f e r e n c e s e x i s t in GABA receptor b i n d i n g constants between SH and WKY rats. Methods Twelve week old SH and WKY rats ( w e i g h i n g 220-280g) were purchased from Taconic Farms of New Y o r k . Rats were housed in i n d i v i d u a l l y hanging wire mesh cages in a t e m p e r a t u r e c o n t r o l l e d room with a twelve hour l i g h t / d a r k cycle. Standard lab chow and water were administered ad libitum. Hemodynamic Studies. T w e n t y - t h r e e gauge stainless steel guide cannulae were implanted into the lateral v e n t r i c l e of chloral h y d r a t e anesthetized rats using the c o - o r d i n a t e s 0.8mm. p o s t e r i o r , 1.3mm. lateral to bregma and 6.0 mm. below the surface of the skull. T h r e e days a f t e r cannulae implantation, rats were again anesthetized with chloral h y d r a t e and t y g o n catheters with teflon tips were inserted into the femoral a r t e r y . The h e p a r i n - f i t l e d catheters were t u n n e l l e d s u b c u t a n e o u s l y to e x i t at the back of the neck where t h e y were s u t u r e d in place. The n e x t day rats were placed in experimental cages w i t h o u t food and water. Mean a r t e r i a l pressure (MAP) was recorded with an Ailtech Pressure T r a n s d u c e r (Model MS20) connected to the a r t e r i a l catheter by p o l y e t h y l e n e (PE SO) t u b i n g . Heart rate (HR) was recorded from the pulse pressure using a Beckman 9857B cardiotachometer. GABA was dissolved in a r t i f i c i a l CSF as described by Merlis (18) and administered randomly in doses of 0,100,300,600 and 1000 IJg in a volume of 2-5 ~1 in conscious, f r e e l y moving SH and WKY rats. Two hours were allowed between each dose of GABA. ICV injections of GABA produced a biphasic response, i n c l u d i n g a s h o r t latency small d epr es s or effect and a long latency, pronounced d e p r e s s o r response lasting a p p r o x i m a t e l y t h i r t y minutes. From the long latency response, the peak h y p o t e n s i v e effect and the hear t rate at t h a t peak h y p o t e n s i v e period were r e c o r d e d . Two hours a f t e r the last injection of GABA, hexamethonium (25 m g / k g , i n t r a - a r t e r i a l ) was slowly infused into normotensive and h y p e r t e n s i v e rats to assess the autonomic component of blood p r e s s u r e and h e a r t rate. Ten minutes a f t e r autonomic blockade, blood pressure and h e a r t rate were recorded. V e n t r i c u l a r cannuta placement was v e r i f i e d one day before GABA administ r a t i o n by an a n g i o te n s i n d r i n k i n g test. D e l i v e r y of small doses of angiotensin II to the v e n t r i c l e s produces rapid d r i n k i n g responses (19). H y d r a t e d SH and WKY rats r a p i d l y d r i n k i n g at least 2.5 ml of water a f t e r ICV injection of 100 ng of angiotensin II were used the fo l l o w i n g day f o r central GABA injections. At the t e r m i n a t i o n of the e x p e r i m e n t Evan's blue dye was injected into the v e n t r i c u l a r cannulae. Brains were removed and o n l y data from rats in which staining of the v e n t r i c l e s a f t e r dye injection are included in this s t u d y . B i n d i n g Studies. T w e l v e week-old male SH and WKY rats were also purchased from Taconic Farms of New Y o r k f o r b i n d i n g studies. Polyethylene
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(PE 50) t u b i n g was inserted into the femoral a r t e r y u n d e r ether anesthesia as described f o r hemodynamic s t u d i e s . T w o h o u r s later MAP and HR were recorded f o r 15 minutes. The a r t e r i a l p r e s s u r e of 12-week-old SH and WKY rats used f o r b i n d i n g studies tended to be s i g n i f i c a n t l y less than those used for hemodynamic e x p e r i m e n t s . T h i s d i f f e r e n c e is most l i k e l y due to incomplete r e c o v e r y from e t h e r anesthesia. On the n e x t d a y , rats were decapitated and t h e i r b r a i n s were r a p i d l y removed and kept in iced 0.32M sucrose. Brains were dissected into ponsmedulla, cerebellum, and f o r e b r a i n and pooled (6 b r a i n s regions per g r o u p ) for binding studies. As described p r e v i o u s l y (20), tissue from pooled brain regions was prepared to obtain the c r u d e mitochondrial plus microsomal (P2 + P3) f r a c t i o n s . The tissue was washed, frozen o v e r n i g h t , in 0.05M T r i s - c i t r a t e (pH 7 . 1 ) , and rewashed in b u f f e r the f o l l o w i n g day. On the day of the e x p e r i m e n t , tissue was washed t h r e e times in b u f f e r , and resuspended in the b u f f e r f o r assays. The b i n d i n g of [ 3 H ] G A B A and [3H]muscimol (New England Nuclear) to its receptors was assayed b y a modification of the methods of Z u k i n et al. (21) and Enna and S n y d e r (22) as described b y T i c k u (20). B i n d i n g assays were performed by i n c u b a t i n g aliquots of P2 + P3 homogenate (0.5 - 1.0 mg/ml p r o t e i n ) w i t h v a r i o u s c o n c e n t r a t i o n s of [ 3 H ] G A B A (58 Ci/mmol) or [3H]muscimol (7.13 Ci/mmol) w i t h or w i t h o u t excess n o n - r a d i o a c t i v e GABA (1 retool). The samples were c e n t r i f u g e d and the pellets were r a p i d l y rinsed w i t h b u f f e r , solubilized o v e r n i g h t with 0.3 ml Soluene-350, and r a d i o a c t i v i t y was counted in 3 ml of toluene c o n t a i n i n g d i p h e n y l o x a z o l e . Counting efficiency measured by the i n t e r n a l standard ( [ 3 H ] t o l u e n e ) method was a p p r o x i m a t e l y
40-~. Specific b i n d i n g was obtained b y s u b t r a c t i n g from the total r a d i o a c t i v i t y t h a t w h i c h was not displaced by excess n o n - r a d i o a c t i v e G A B A . B i n d i n g assays were performed in t r i p l i c a t e . Protein was estimated b y the method of L o w r y et al. ( 2 3 ) . T h i r t e e n p o i n t [ 3 H ] G A B A and [3H]muscimol scatchard plots were determined. The b i n d i n g a f f i n i t i e s ( K D ) and number of b i n d i n g sites ( B m a x ) were calculated f o r two sites b y linear regression w i t h Feldman (24) analysis. The effects of GABA compared to a r t i f i c i a l CSF were analyzed b y analysis of v a r i a n c e . Differences among repeated measures were determined w i t h a S t u d e n t Newman-Keuls range test. Differences between SHR an WKY were analyzed by an u n p a i r e d S t u d e n t s T - t e s t . S i g n i f i c a n c e was taken at the P<.05 level. Results Hemodynamic Studies Before central injections of G A B A , mean a r t e r i a l p r e s s u r e and h e a r t rate were 178-+8 mmHg and 365-+14 beats per minute (BPM) in the SHR (n=8) and 129.+3 mmHg and 339+19 BPM in the WKY (n=8) as shown in Table 1. Figure 1 r e p r e s e n t s a t y p i c a l p o l y g r a p h r e c o r d i n g of the mean a r t e r i a l p r e s s u r e and h e a r t rate a f t e r the a d m i n i s t r a t i o n of 600 pg of GABA ICV in conscious SH and WKY rats. An a p p a r e n t biphasic response o c c u r r e d f o l l o w i n g central a d m i n i s t r a t i o n of G A B A . T h e r e was an i n i t i a l small depressor effect of a p p r o x i m a t e l y 10 mmHg in both g r o u p s of rats t h a t was accompanied b y a r e f l e x increase in h e a r t rate. In a d d i t i o n t h e r e was a long latency, more p r o n o u n c e d d e p r e s s o r e f f e c t w i t h a noticeable absence of a r e f l e x t a c h y c a r d i a . The responses are a r e s u l t of the i n f l u e n c e of GABA on the central n e r v o u s
704
Cardiovascular
Effects of GABA in SHR
Vol.
33, No. 8, 1983
system since i n t r a v e n o u s a d m i n i s t r a t i o n of 1000 IJg of GABA did not a l t e r MAP or HR. Before and a f t e r the a d m i n i s t r a t i o n of all doses of G A B A , the a r t e r i a l p r e s s u r e of the SH rat was always s i g n i f i c a n t l y elevated above t h a t of the WKY ( T a b l e 1). ICY GABA was not able to normalize p r e s s u r e between the two g r o u p s of r a t s . The depressor response of 100 IJg of GABA ICY was not d i f f e r e n t between the two g r o u p s . Injection of 300, 600, and 1000 lag ICV produced a g r e a t e r absolute decrease in a r t e r i a l p r e s s u r e in the h y p e r t e n s i v e rats as shown in Table 1. However, t h e r e were no s i g n i f i c a n t d i f f e r e n c e s in the p e r c e n t decrease in a r t e r i a l p r e s s u r e produced by all doses of GABA between SH and WKY rats. Heart rate was not s i g n i f i c a n t l y d i f f e r e n t between normotensive and h y p e r t e n s i v e rats before GABA a d m i n i s t r a t i o n . Central injection of 100 and 300 Iag GABA did not a l t e r h e a r t rate s i g n i f i c a n t l y in e i t h e r g r o u p . However, injection of 600 and 1000 IJg GABA produced a s i g n i f i c a n t l y g r e a t e r absolute and p e r c e n t decrease in h e a r t rate in the SHR while not s i g n i f i c a n t l y c h a n g i n g h e a r t rate in the WKY ( T a b l e I ) .
240] WKY MAP (mmHg)
HR
(bpm)
1801
4801
3601 240J
MAP (mm Hq)
HR (bpm)
21801 4011201SHR
6001~ GABA ICV
~
~
..............
60] 3601 240 J
., 0
I0
.$ : -
--.--_t
20
30
- "~,~--'~'. ~ : ~ ' ~ 40
50
Time ( rain )
F i g u r e 1. Effect of 600 IJg of GABA ( I C Y ) on mean a r t e r i a l p r e s s u r e (MAP) and h e a r t rate ( H R ) of conscious SH ( l o w e r panel) and WKY ( u p p e r panel) rats.
Vol. 33, No. 8, 1983
Cardiovascular Effects of GABA in SHR
705
In t h e same a n i m a l s , b e f o r e a u t o n o m i c b l o c k a d e , mean a r t e r i a l p r e s s u r e was 185 -+ 4 mmHg in t h e SHR a n d 133 -+ 1 in t h e WKY r a t s . After hexamet h o n i u m a d m i n i s t r a t i o n (25 m g / k g ) t h e a r t e r i a l p r e s s u r e o f t h e SHR was s t i l l s i g n i f i c a n t l y g r e a t e r t h a n t h e WKY ( T a b l e 1 ) . As s h o w n in T a b l e 1, a u t o n o m i c b l o c k a d e p r o d u c e d an e q u i v a l e n t a b s o l u t e a n d p e r c e n t c h a n g e in mean a r t e r i a l p r e s s u r e in n o r m o t e n s i v e a n d h y p e r t e n s i v e r a t s . B e f o r e h e x a m e t h o n i u m a d m i n i s t r a t i o n h e a r t r a t e was 382 + 14 b e a t s p e r m i n u t e ( B P M ) in t h e SHR a n d 321 -+ 18 BPM in t h e W K Y . At the time these data were recorded, h e a r t r a t e was s i g n i f i c a n t l y e l e v a t e d in t h e SH r a t . H e x a m e t h o n i u m d e c r e a s e d h e a r t r a t e (42 -+ 17 B P M ) in t h e SHR b u t i n c r e a s e d h e a r t r a t e (43 -+ 22 B P M ) in t h e WKY ( P < . 0 5 ) as s h o w n in T a b l e I . Ganglionic b l o c k a d e a l s o p r o d u c e d a g r e a t e r a b s o l u t e a n d p e r c e n t d e c r e a s e in h e a r t r a t e in t h e S H R . H e a r t r a t e b e t w e e n t h e t w o g r o u p s was n o t s i g n i f i c a n t l y d i f f e r e n t after ganglionic blockade. Binding Studies• Mean a r t e r i a l p r e s s u r e a n d h e a r t r a t e o f t h e 12 w e e k o l d SH r a t s u s e d f o r b i n d i n g s t u d i e s was 157-+12 mmHg a n d 407_+29 b e a t s p e r minute (BPM) respectively (n=18). A r t e r i a l p r e s s u r e o f WKY r a t s was 119-+11 m m H g , h e a r t r a t e was 365_+30 BPM. B l o o d p r e s s u r e was g r e a t e r in t h e SH r a t (P<.05); heart rates were not different. GABA receptor sensitivity in t h e f o r e b r a i n c e r e b e l l u m , a n d p o n s - m e d u l l a was m e a s u r e d b y u s i n g [ 3 H ] m u s c i m o l as t h e r a d i o l i g a n d . Scatchard analysis of the binding data indicate the existence of two receptor sites. The affinities of t h e t w o r e c e p t o r s i t e s u s i n g [ 3 H ] m u s c i m o l a r e in a g r e e m e n t w i t h r e p o r t e d v a l u e s in o t h e r s p e c i e s ( 2 5 ) . The K_ and B values of the two GABA receptor sites were not ax slgntflcantly d l f f e r e n r ~ in f o r e b r a i n , c e r e b e l l u m a n d p o n s - m e d u l l a o f SH a n d WKY r a t s ( T a b l e 2 ) . Due t o t h e l a r g e v a r i a t i o n w i t h i n r e p l i c a t e s a n d h i g h n o n s p e c i f i c b i n d i n g in t h e p o n s - m e d u l l a , t h e G A B A r e c e p t o r s e n s i t i v i t y was m e a s u r e d in t h i s r e g i o n u s i n g b o t h [ 3 H ] G A B A a n d [ 3 H ] m u s c i m o l . [3H] GABA was u s e d b e c a u s e t h e h i g h e r s p e c i f i c a c t i v i t y o f t h i s l i g a n d i m p r o v e d t h e accuracy of the Scatchard plot. Table 2 shows that the binding constants w e r e n o t s i g n i f i c a n t l y d i f f e r e n t in t h e b r a i n s t e m o f t h e s e t w o s t r a i n s . •
,
.
U
.
Discussion If t h e h i g h b l o o d p r e s s u r e seen in t h e SHR is m a i n t a i n e d b y a g r e a t e r a c t i v i t y o f t h e s y m p a t h e t i c n e r v o u s s y s t e m , t h e n h e x a m e t h o n i u m w o u l d be e x p e c t e d t o l o w e r b l o o d p r e s s u r e m o r e in t h e SH r a t t h a n in t h e W K Y . In o u r s t u d i e s , h e x a m e t h o n i u m l o w e r e d MAP 50-+7 a n d 45-+6 mmHg in SH a n d WKY r a t s , respectively• T h e s i m i l a r c h a n g e in a r t e r i a l p r e s s u r e a f t e r h e x a m e t h o n i u m in c o n s c i o u s n o r m o t e n s i v e a n d h y p e r t e n s i v e r a t s is in a g r e e m e n t w i t h t h e r e s u l t s o f T o u w e t a l . ( 6 ) b u t in d i s a g r e e m e n t w i t h t h e f i n d i n g s o f Numao e t a l . ( 5 ) and Judy et al. (I). Our studies support the contention that the sympathetic n e r v o u s s y s t e m is n o t m a i n t a i n i n g b l o o d p r e s s u r e t o a g r e a t e r e x t e n t in t h e a d u l t SHR r e l a t i v e t o n o r m o t e n s i v e r a t s . G a n g l i o n i c b l o c k a d e w i t h h e x a m e t h o n i u m d e c r e a s e d h e a r t r a t e in t h e SHR f r o m 382_+14 t o 340_+7 BPM ( P < . 0 S ) . In t h e WKY h e x a m e t h o n i u m i n c r e a s e d h e a r t r a t e f r o m 321_+18 t o 364_+20 BPM ( P < . 0 5 ) . These results suggest that the s y m p a t h e t i c c o n t r i b u t i o n t o h e a r t r a t e is g r e a t e r in t h e SH r a t c o m p a r e d t o t h e WKY a n d v a g a l i n p u t t o t h e h e a r t is g r e a t e r in t h e WKY r a t s r e l a t i v e t o t h e SHR. F o r t h e s e r e a s o n s , h e x a m e t h o n i u m i n c r e a s e s h e a r t r a t e in t h e WKY a n d d e c r e a s e s h e a r t r a t e in t h e SHR ( T a b l e I ) . However intrinsic heart rate ( a f t e r a u t o n o m i c b l o c k a d e ) is e q u i v a l e n t in b o t h g r o u p s o f r a t s ( T a b l e I ) .
706
Cardiovascular Effects of GABA in SHR
Vol. 33, No. 8, 1983
TABLE i
EFFECTS OF GABA AND HEXAMETHONIUM ON MEAN ARTERIAL PRESSURE AND HEART RATE IN SH AND WKY RATS MAP SHR
HR WKY
SHR
WKY
CSF
Pre Post A %A
180 180 0 0
t ± ± ±
8* 8* 0 0
133 132 -i -i
t ± ± ±
3 2 0 0
340 340 -I 0
I00 pg GABA, ICV
Pre Post A
178 ± 172 ± -6 ±
9* 9* 2
129 ± 123 ± -6 ±
3 4 2
370 ± 14 361 ± 14 -9 ± 5
%Z~
-3 ±
1
-5 ±
2
Post A %Z~
154 + -24 ± -14 ±
2?
117 ± -13 ± -11 ±
55 ~ ~
346 ± 10 - I i ± I0 -3 ± 3
353 ± 15 +20 ± 16 +7 ± 6
Pre Post A
180 ± 8 ~ 144 ± 11.? -35 ± ~
128 ± 107 ± -21+
3? 4? ~
369 ± 14 337 ± 18 -30 ± 13".~
337 ± 19 350 ± 19 +12 + 12
%A
-20 ±
-16 ±
?
Pre Post
177 ± 9*? 137 ± 10"~
128 ± 100 ±
2? 5~
363 ± 17 316 ± 147~
334 ± 22 347 ± 14
-40 ±
+13 ± I0
GABA, ICV
600 pg GABA, ICV
1000 pg GABA, ICV
Hexamethonium (25mg/kg, I.A.)
A= * = =
Pre Post A ~L~
t
~$
± 9 + I0 ± 1 ± 0
-2 ±
-9 ±
1
3*T
-29 ±
3
-47 ±
8 *T
-23 ±
-22 ±
3?
-13 ±
2*~
185 133 -50 -27
133 87 -46 -34
± 4" ± 12" ± 7 ± 4
± 10 ± 6 ± 6 ± 4
382 340 -42 -11
± 14" ± 7 ± 17" ± 3*
342 343 -I 0
± 19 ± 19 ± 0 ± 1
348 ± 16 348 ± 15 0 ± 5
0 ±
+4 ±
+5 ± 321 364 +43 +14
1
3
3
± 10 ± 20 ± 22 ± 6
absolute change in mean a r t e r i a l pressure (MAP, mmHg) or heart rate (HR, beats per minute). ~w~= percent change in MAP or HR. P<.05 vs. WKY rats (unpaired t - t e s t ) denotes a s i g n i f i c a n t difference within each group vs. a r t i f i c i a l CSF injection (ANOVA) of P<.05.
Central administration of GABA reduces the sympathetic component of blood pressure and heart rate in both cats (14,15,16) and rats (17). In our studies with conscious SH and WKY rats, ICV GABA produces a biphasic response (Fig. I ) . The immediate depressor effect of all doses of ICV GABA ( a p p r o x i m a t e l y 10 mmHg in both groups of r a t s ) is buffered by an increase in heart rate. This response presumably occurs when GABA acts on s t r u c t u r e s r a p i d l y accessible to lateral v e n t r i c l e administration. The longer latency, profound depressor effect is not accompanied by a r e f l e x tachycardia. In both strains heart rate o r d i n a r i l y decreases or does not change d u r i n g the prolonged depressor response. This longer latency response is similar to t h a t observed in the cat (115) when the GABA agonist muscimol is perfused in the
Vol. 33, No. 8, 1983
Cardiovascular Effects of GABA in SHR
707
TABLE 2 GABA RECEPTOR SENSITIVITY IN SH AND WKY RATS
Bmax
KD(nM)
(pmol/mg protein)
KD1
SHR Forebrain (n=3) Cerebellum (n=3) Pons-Medulla
WKY Forebrain (n=3) Cerebellum (n=3) Pons-Medulla
KD2
BmaxI
Bmax2
3.4 -+ 0.4
90
+ 25
1.1 +- 0.1
8.1 _+ 1.8
2.4 + 0.9
44
-+ 9
1.6 + 0.8
6.2 -+ 1.0
1.6 5.6*
85 141"
0.2 O. I *
2.3 1.8*
5.1 -+ 0.5
66
+
7
1.4 _+ 0.2
6.6 + 1.5
2.7 + 1.6
55
-+ 18
1.6 -+ 0.7
6.5 -+ 0.9
1.1 5.4*
100 203*
1.2 O. 1"
2.3 1.8*
[3H] GABA binding is denoted by * Binding of [3H] muscimol and [3H] GABA* was measured as described in METHODS, K. and B values were determined from i n d i v i d u a l s a t u r a t i o n isotherms using ~u., max~ . relaman analysls (23). The values represent the mean ± S.D. of the number of experiments indicated in the parentheses. Each value represents analysis of i n d i v i d u a l isotherm containing brain regions pooled from 6 rats.
fourth ventricle. in t h e h i n d b r a i n
Therefore, the prolonged depressor effect presumably occurs as s u g g e s t e d ( 1 5 ) .
I C Y G A B A d e c r e a s e s h e a r t r a t e in t h e S H r a t , b u t h e a r t r a t e does n o t c h a n g e s i g n i f i c a n t l y a f t e r G A B A a d m i n i s t r a t i o n in t h e WKY ( T a b l e I ) . Based on o u r p o s t u l a t i o n t h a t s y m p a t h e t i c o u t f l o w to t h e h e a r t in t h e SHR is g r e a t e r t h a n in t h e WKY r a t , ICV G A B A w o u l d be e x p e c t e d to l o w e r h e a r t r a t e t o a g r e a t e r e x t e n t in t h e h y p e r t e n s i v e r a t . In c o n t r a s t , i f v a g a l t o n e to t h e h e a r t is g r e a t e r in t h e WKY r a t r e l a t i v e to t h e SHR, as we h a v e s u g g e s t e d , a r e d u c t i o n in r e s t i n g v a g a l t o n e f o l l o w i n g t h e ~ d e p r e s s o r s t i m u l u s ( i . e . ICY G A B A ) w o u l d t e n d to r a i s e h e a r t r a t e m o r e in t h e WKY t h a n t h e SHR. Theref o r e , c e n t r a l l y a d m i n i s t e r e d G A B A causes h e a r t r a t e to d e c r e a s e m o r e in t h e SHR b y r e d u c i n g s y m p a t h e t i c o u t f l o w to a h e a r t w h o s e r a t e is m a i n t a i n e d b y a g r e a t e r s y m p a t h e t i c a n d r e d u c e d v a g a l t o n e r e l a t i v e t o t h e WKY. C e n t r a l G A B A a d m i n i s t r a t i o n p r o d u c e s a g r e a t e r a b s o l u t e d e c r e a s e in b l o o d p r e s s u r e in t h e h y p e r t e n s i v e r a t . T h i s is in a g r e e m e n t w i t h a n o t h e r report that intra-arterial m u s c i m o l , a G A B A a g o n i s t , also r e d u c e s b l o o d p r e s s u r e m o r e in SH r a t s ( 2 6 ) ; h o w e v e r , a c e n t r a l s i t e o f a c t i o n was n o t demonstrated. In a d d i t i o n , Baum e t al. ( 2 7 ) h a v e d e m o n s t r a t e d a p r o n o u n c e d
708
Cardiovascular
Effects of GABA in SHR
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d e p r e s s o r e f f e c t of I V T muscimol in SH r a t s . When t h e p e a k d e p r e s s o r e f f e c t of G A B A is a n a l y z e d b y t h e p e r c e n t c h a n g e in a r t e r i a l p r e s s u r e , s i g n i f i c a n t d i f f e r e n c e s b e t w e e n SH and WKY r a t s w e r e n o t o b s e r v e d . Therefore, the g r e a t e r a b s o l u t e c h a n g e in b l o o d p r e s s u r e p r o d u c e d b y G A B A a p p e a r s t o be r e l a t e d to t h e h i g h b a s e l i n e a r t e r i a l p r e s s u r e of SH r a t s . T h e b i n d i n g s t u d i e s d e m o n s t r a t e no m a j o r d i f f e r e n c e s in r e g i o n a l G A B A a g o n i s t r e c e p t o r b i n d i n g b e t w e e n a d u l t SH and WKY r a t s . T h e s e r e s u l t s also d e m o n s t r a t e t h a t e n h a n c e d a b s o l u t e d e p r e s s o r r e s p o n s e to G A B A in SH r a t s r e l a t i v e to WKY r a t s can n o t be a t t r i b u t e d t o d i f f e r e n c e s in t h e G A B A r e c e p t o r binding constants between these two strains. We h a v e also f o u n d no d i f f e r ences in G A B A a g o n i s t b i n d i n g b e t w e e n 4 and 8 w e e k - o l d SH and WKY r a t s (27). Since we a r e m e a s u r i n g r e c e p t o r b i n d i n g in g r o s s b r a i n r e g i o n s , p e r h a p s o n l y a small n u m b e r of t h e t o t a l r e c e p t o r p o p u l a t i o n p a r t i c i p a t e in cardiovascular function. T h e r e f o r e , i t is p o s s i b l e t h a t r e c e p t o r c h a n g e s o c c u r t h a t a r e n o t d e t e c t a b l e in t i s s u e h o m o g e n a t e p r e p a r a t i o n s . In c o n c l u s i o n , o u r s t u d i e s i n d i c a t e t h a t t h e t o t a l f u n c t i o n a l c o n t r i b u t i o n of t h e s y m p a t h e t i c n e r v o u s s y s t e m in b l o o d p r e s s u r e m a i n t e n a n c e in t h e a d u l t SH r a t is e q u i v a l e n t to t h a t o f t h e W K Y . D e s p i t e t h e g r e a t e r a b s o l u t e c h a n g e in a r t e r i a l p r e s s u r e p r o d u c e d b y G A B A in t h e SH r a t s , i t a p p e a r s t h a t t h e f u n c t i o n a l c o n t r i b u t i o n of t h e s y m p a t h e t i c n e r v o u s s y s t e m s e n s i t i v e to G A B A is e q u i v a l e n t b e t w e e n t h e h y p e r t e n s i v e and n o r m o t e n s i v e s t r a i n s . Acknowledgements T h i s s t u d y was s u p p o r t e d b y f u n d s f r o m t h e A m e r i c a n H e a r t A s s o c i a t i o n ( T e x a s A f f i l i a t e ) g r a n t s to J . R . H . and M . K . T . T h e a u t h o r s also w i s h to t h a n k Ms. M a r y J o y n e r and Ms. J a n e t Wall f o r t h e i r e x c e l l e n t s e c r e t a r i a l assistance. References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15.
W.V. JUDY, A.M. WATANABE, D . P . H E N R Y , H . R . BESCH, W . R . BESCH and G.M. HOCKEL, C i r c . Res. ( S u p p l I I ) II 21 ( 1 9 7 6 ) . B. FOLKOW, M. H A L L B E C K , Y . JUNDGREN and L. WEISS, Acta P h y s i o l . S c a n d 84 512 ( 1 9 7 2 ) . Y . Y A M O N , H. Y A M A B E , W. DEJONG, W. L O V E N B E R G and A . SJOERDSMA, E u r J. P h a r m a c o l 17 135 ( 1 9 7 2 ) . A . P . P R O V O S T a n d W. DEJONG, C l i n E x p H y p e r t e n s i o n 1 177 ( 1 9 7 8 ) . Y . NUMAO, S. H I R O Y U K I and J. I R I U C H I J I M A , Jap H e a r t J 16 719 (1975). K . B . TOUW, J . R . H A Y W O O D , R . A . SHAFFER and M . J . B R O D Y , H y p e r t e n s i o n 2 408 ( 1 9 8 0 ) . G. H A E U S L E R , In Recjulation o f B l o o d P r e s s u r e b y t h e C e n t r a l N e r v o u s S y s t e m , e d i t e d b y O n e s t i G . , F e r n a n d e z M . , and Kim E . K . New Y o r k , G r u n e & S t r a t t o n 53, ( 1 9 7 6 ) . T . K U B O , a n d M. H A S H I M O T O , A c t a P h y s i o l Scand 8 512 ( 1 9 7 2 ) . L. E R I N O F F , A . HELLER and S. O P A R I L , Proc Soc E x p Biol Med 150 748 (1975). P . I . KORNER a n d J . A . ANGUS P h a r m a c T h e r 13 321 ( 1 9 8 1 ) . Y . O H K U S U , J p n . C i r c . J. 35 1175 ( 1 9 7 1 ) . R.M. SCHICKEN, P e d i a t . Res. 13 992 ( 1 9 7 9 ) . J . F . T A L L M A N , S. P A U L , P. SK---OLNICK and D. G A L L A G H E R , Science 207 274 ( 1 9 8 0 ) . C.~. SWEET, H . C . WENGER and D . M . GROSS, Can J. P h y s i o l . P h a r m a c o l 57 600 ( 1 9 7 8 ) . M.J. ANTO-NACClO, L. KERWlN and D . G . T A Y L O R , N e u r o p h a r m a c o l o g y 17 783 ( 1 9 7 8 ) .
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