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Central noradrenergic-cholinergic interaction in regulation of gastric acid secretion in rats
Life Sciences, Vol. 32, pp. 1363-1370 Printed in the U.S.A.
Pergamon Press
CENTRAL NORADRENERGIC-CHOLINERGIC I N T E R A C T I O N IN R E G U L A T I O N OF G A S T R I C A C I D S E C R E T I O N IN RATS Yasunobu
Okuma, Y o s h i t s u g u Osumi, Toshio and M i t s u h i r o Nagata
Department
Ishikawa
of Pharmacology, Kochi M e d i c a l Nankoku, Kochi 781-51, Japan
School,
(Received in final form December 13, 1982) Summary P o s s i b l e roles of n o r a d r e n a l i n e (NA) and a c e t y l c h o l i n e (ACh) w i t h i n the lateral h y p o t h a l a m i c area (LHA) in r e g u l a t i o n of gastric acid s e c r e t i o n were e x a m i n e d in urethane a n e s t h e t i z e d rats. When NA 30 nmoles was given into the LHA, the gastric acid o u t p u t d e c r e a s e d and this i n h i b i t o r y effect of NA was p o t e n t i a t e d in rats p r e t r e a t e d w i t h r e s e r p i n e (2 mg/kg, i.p., 20 hr). E v e n in a dose of 3 nmoles w h i c h was w i t h o u t e f f e c t in n o n - t r e a t e d control animals, there was a r e m a r k a b l e d e c r e a s e in acid output. In these r e s e r p i n i z e d animals, A C h in a dose of 30 nmoles induced a r e m a r k a b l e increase in acid output, while in the controls this A C h - i n d u c e d increase was o b s e r v e d only w i t h a i0 times h i g h e r dose. In the rats not given reserpine, the c h o l i n e r g i c m u s c a r i n i c a g o n i s t b e t h a n e c h o l (i0 nmoles) i n c r e a s e d the gastric acid output while n i c o t i n e (30 nmoles) was w i t h o u t effect. Therefore, in rats, the c e n t r a l n o r a d r e n e r g i c inhibitory m e c h a n i s m s r e l a t e d to r e g u l a t i o n of gastric function m a y be p r e s e n t at the level of LHA as well as at the ala c i n e r e a (area of the dorsal m o t o r nucleus of vagi and the nucleus tractus solitarius). In addition, in the LHA, a c h o l i n e r gic m u s c a r i n i c m e c h a n i s m w h i c h elevates gastric acid s e c r e t i o n m a y be a n t a g o n i z e d by a n o r a d r e n e r g i c inhibitory mechanism. The role of the central nervous s y s t e m in r e g u l a t i o n of gastric f u n c t i o n has been e x t e n s i v e l y e x a m i n e d at the level of the hypothalamus (1-4) and other regions of the brain (5,6). Nevertheless, there remains a p a u c i t y in i n f o r m a t i o n c o n c e r n i n g tentative roles of central n e u r o t r a n s m i t t e r c a n d i d a t e s such as m o n o amines, a c e t y l c h o l i n e (ACh) and GABA, in the r e g u l a t i o n of gastric functions. Osumi et al. r e p o r t e d the e x i s t e n c e of a central nora d r e n e r g i c i n h i b i t o r y m e c h a n i s m in r e g u l a t i o n of gastric functions and s u g g e s t e d that the sites of action were p r o b a b l y the lateral h y p o t h a l a m i c area (LHA) and/or the ala c i n e r e a (7,8). Recently, we noted the e x i s t e n c e of this n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m w i t h i n the ala cinerea, of w h i c h n o r a d r e n e r g i c nerve t e r m i n a l s o r i g i n a t e from the locus c o e r u l e u s (9). Thus, we a t t e m p t e d to d e t e r m i n e w h e t h e r the i n h i b i t o r y role of n o r a d r e n a l i n e (NA) in r e g u l a t i o n of gastric acid s e c r e t i o n m a y 0024-3205/83/121363-08503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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be p r e s e n t at the level of the LHA. The n o r a d r e n e r g i c - c h o l i n e r g i c i n t e r a c t i o n w i t h i n the LHA and the p o s s i b l e role of o t h e r m o n o amines and G A B A in r e g u l a t i o n of g a s t r i c acid s e c r e t i o n w e r e also examined. Materials
and M e t h o d s
M a l e W i s t a r rats w e i g h i n g 220-250 g w e r e m a i n t a i n e d at 22-24 °C u n d e r a c o n s t a n t d a y - n i g h t r h y t h m for 7 to i0 days and g i v e n food ( l a b o l a t o r y chow, CA-l, J a p a n C L E A Co.) and tap w a t e r ad libitum. P r i o r to e a c h e x p e r i m e n t , all food but n o t w a t e r w-as w i t h h e l d for 16 hr. U n d e r u r e t h a n e a n e s t h e s i a (I g/kg, i.p.), the a b d o m e n was o p e n e d by a t r a n s v e r s e i n c i s i o n and a r o u n d - t i p cannula (5 cm long, 0.5 cm o u t e r diameter) c o n n e c t e d to a p o l y e t h y lene tube was i n s e r t e d into the s t o m a c h via an i n c i s i o n into the d u o d e n u m (i cm d i s t a l from the p y l o r i c s p h i n c t e r ) . The tip of the c a n n u l a lay just above the p y l o r i c s p h i n c t e r and was h e l d in p l a c e by two l i g a t u r e s a r o u n d the duodenum, one at the p o i n t of i n c i s i o n and the o t h e r c l o s e to the pylorus, as d e s c r i b e d by M a i n and W h i t t l e (i0). To r e m o v e the solid contents, the s t o m a c h was f l u s h e d w i t h saline, t a k i n g care to a v o i d d i s t e n t i o n . A f t e r rep e a t e d w a s h i n g s , two ml of s o l u t i o n p r e w a r m e d to 38°C was p l a c e d in the s t o m a c h at the b e g i n n i n g of e a c h 15 m i n c o l l e c t i o n period. The c o m p o s i t i o n of this s o l u t i o n was a 1:5 (v/v) m i x t u r e of g l y c ine and m a n n i t o l a d j u s t e d to 300 m o s M and pH 3.5 by a d d i t i o n of 0.i N HCI, a c c o r d i n g to the m e t h o d of B l a i r et al. (ii). Determ i n a t i o n s in 2 c o n s e c u t i v e 1 5 - m i n - c o l l e c t i o n s of g a s t r i c juice w e r e then m a d e to e s t a b l i s h the basal value. In the o t h e r series of e x p e r i m e n t s , the rats had b e e n g i v e n an i n t r a p e r i t o n e a l (i.p.) a d m i n i s t r a t i o n of r e s e r p i n e 2 mg/kg, 20 hr b e f o r e the e x p e r i m e n t . The p u r p o s e of this p r o c e d u r e was to i n v e s t i g a t e the e f f e c t of A C h on g a s t r i c functions, in the a b s e n c e of a n o r a d r e n e r g i c inhibitory m e c h a n i s m . The test s u b s t a n c e to be m i c r o i n j e c t e d w a s a d j u s t e d to 300 m o s M w i t h N a C I solution. A s t a i n l e s s steel m i c r o p i p e t t e (outer d i a m e t e r 0.35 mm) was then i n s e r t e d into the L H A (AP: 5.0, L: 1.5, H: 2.0) f o l l o w i n g the B r a i n A t r a s by K o n l g and K l i p p e l (12), and 1 ~i of test s u b s t a n c e was a d m i n i s t e r e d d u r i n g 60 sec. This m i c r o p i p e t t e also s e r v e d as the anode side of the b i p o l a r electrode. The d i a m e t e r of the o t h e r c a t h o d e side of the b i p o l a r e l e c t r o d e was 0.i mm. For e l e c t r i c a l s t i m u l a t i o n of the r e s t r i c t ed region, into w h i c h the test s u b s t a n c e had b e e n m i c r o i n j e c t e d , b i p h a g i c s q u a r e - w a v e p u l s e s of 0.5 mA, 2 msec, i0 c y c l e s / s e c w e r e a p p l i e d for i0 m i n by m e a n s of a e l e c t r o n i c s t i m u l a t o r (Model 3 2 0 ~ N i h o n K o d e n LTD., Japan). rd
,
A c i d o u t p u t was d e t e r m i n e d by t i t r a t i o n of g a s t r i c samples to pH 7.0 w i t h 0.01 N NaOH, u s i n g a pH meter. At t e r m i n a t i o n of the e x p e r i m e n t , the b r a i n was r e m o v e d and f r o z e n s e c t i o n s cut at 30 ~un w e r e s t a i n e d w i t h c r e s y l v i o l e t for m i c r o s c o p i c study of the sites of m i c r o i n j e c t i o n . The e x p e r i m e n t a l p r o c e d u r e for m i c r o i n j e c t i o n of test s u b s t a n c e into the L H A is r e p r e s e n t e d in Fig. i. N i n e t y to 120 m i n after the test s u b s t a n c e (in case of this rat, N A 30 nmoles) was g i v e n into the LHA, u n i l a t e r a l e l e c t r i c a l stimulation w a s p e r f o r m e d . W h e n i n c r e a s e in the a c i d o u t p u t w i t h electrical s t i m u l a t i o n occurred, and w h e n the tip of the p i p e t t e p l a c e d w i t h i n the L H A was h i s t o l o g i c a l l y confirmed, the r e s u l t s of m i c r o i n j e c t i o n of the test s u b s t a n c e s w e r e s u m m a r i z e d for s t a t i s -
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NA and ACh in Gastric Acid Secretion
tistical calculation. S t a t i s t i c a l s i g n i f i c a n c e of the data was Student's t-test for paired comparison.
calculated
1365
using
1
"~15 E uD
\ O" U.I
=.10
Fig.
O.
The e x p e r i m e n t a l p r o c e d u r e for microinjection; effect on gastric acid output. NA 30 nmoles was m i c r o i n j e c t e d into the LHA of rats in a total volume of 1 91. ES: e l e c t r i c a l stimulation given at i0 cycles/sec, 2 msec, 0.5 mA for i0 min.
=1 0
t 0
t i
i
60
i
1
!
i
120 rain Results
M e a n basal gastric acid output o b t a i n e d from rats in the control series was 6.4 ± 0.5 ~ E q / 1 5 min (n=78). In the r e s e r p i n e p r e t r e a t e d animals, this basal level was 17.2 ± 2.5 ; E q / 1 5 min (n=31). Effects of NA m i c r o i n j e c t e d into the LHA on gastric acid output. The a d m i n i s t r a t i o n of saline alone into the LHA did not slgn--iTicantly alter the basal acid output, as shown in Fig. 2. NA 30 nmoles but not 3 nmoles a p p l i e d into the LHA d e c r e a s e d gastric acid output. A c i d output in this NA 30 n m o l e s - t r e a t e d group d e c r e a s e d to the lowest level at the 45-min c o l l e c t i o n (56.5 ± 9.4 % of the basal value, n=6) and then r e t u r n e d toward the basal level. This d e c r e a s e d level was s i g n i f i c a n t l y different, as compared to the r e s p e c t i v e basal values, just before the a p p l i c a t i o n of N A (p < 0.01). In the r e s e r p i n e p r e t r e a t e d animals, NA 3 nmoles a dose w i t h o u t effect in n o n - r e s e r p i n i z e d animals, s i g n i f i c a n t l y d e c r e a s e d gastric acid output. Effect of A C h m i c r o i n j e c t e d into the LHA on gastric acid output. M i c r o i n j e c t i o n of A C h 300 nmoles into the LHA significantly i n c r e a s e d the gastric acid o u t p u t (Fig. 3). Acid output i n c r e a s e d to the h i g h e s t level at the 45-min c o l l e c t i o n p e r i o d (186.8 ± 24.3 % of the basal level, n=10), and r e t u r n e d toward the basal level. In the r e s e r p i n e p r e t r e a t e d animals, the A C h - i n d u c e d increase in acid o u t p u t was potentiated, A C h 30 nmoles a dose w i t h o u t effect in n o n - r e s e r p i n i z e d animals, s i g n i f i c a n t l y increased the gastric acid output.
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A
C:
v
'10
0
'
30 Fig.
60
min
90
2
E f f e c t of N A g i v e n into the LHA on g a s t r i c acid output. In n o n - r e s e r p i n i z e d rats: O O : s a l i n e (n=6), D D : NA 3 n m o l e s (n=4), ~ : N A 30 nmoles (n=6). In r e s e r p i n i z e d rats: O----O: NA 3 n m o l e s (n=8). ,: p < 0.05, **: p < 0.01 ( s t a t i s t i c a l l y signif i c a n t as c o m p a r e d to the r e s p e c t i v e b a s a l v a l u e s just b e f o r e the a p p l i c a t i o n of NA.
E f f e c t s of c h o l i n e r g i c agonists, d o p a m i n e , s e r o t o n i n and G A B A m i c r o i n j e c t e d into the L H A on g a s t r i c acid output. A c h o l i n e r g i c m u s c a r i n i c agonist, b e t h a n e c h o l i0 nmoles b u t not 3 n m o l e s g i v e n i n t o the LHA i n d u c e d a r e m a r k a b l e i n c r e a s e in gastric a c i d o u t p u t (Fig. 4). However, the c h o l i n e r g i c n i c o t i n i c agonist, nicotine, did not s i g n i f i c a n t l y a f f e c t g a s t r i c a c i d output, e v e n in a d o s e of 30 nmoles. D o p a m i n e 30 nmoles, s e r o t o n i n 30 n m o l e s and G A B A 300 n m o l e s g i v e n into the L H A h a d no s i g n i f i c a n t e f f e c t on g a s t r i c acid o u t p u t (Data not shown). Discussion The a d m i n i s t r a t i o n of N A into the L H A d e c r e a s e d g a s t r i c acid output, w h i l e A C h i n c r e a s e d it, a n d the r e s p o n s e s i n d u c e d by N A and A C h w e r e p o t e n t i a t e d in the r e s e r p i n e - p r e t r e a t e d animals. We h a v e a l r e a d y r e p o r t e d that a n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m seems to be p r e s e n t w i t h i n the ala cinerea, of w h i c h
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NA and ACh in Gastric Acid Secretion
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20
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or iii
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60
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rain 90
3
E f f e c t of A C h g i v e n into the LHA on g a s t r i c acid output. In n o n - r e s e r p i n i z e d rats: O O : A C h 30 n m o l e s (n=7), H : ACh 300 n m o l e s (n=10). In r e s e r p i n i z e d rats: O----O : A C h 30 nmoles (n=6). ,: p < 0.05, **: p < 0.01 ( s t a t i s t i c a l l y s i g n i f i c a n t as c o m p a r e d to the r e s p e c t i v e basal v a l u e s just b e f o r e the a p p l i c a tion of ACh).
n o r a d r e n e r g i c nerve t e r m i n a l s o r i g i n a t e d f r o m the locus c o e r u l e u s (9). A c c o r d i n g to d a t a o b t a i n e d in h i s t o c h e m i c a l and n e u r o c h e m ical studies, the L H A has a large n u m b e r of NA n e r v e t e r m i n a l s (~, 14) and these n o r a d r e n e r g i c t e r m i n a l s w i t h i n the L H A p r o b a b l y o r i g i n a t e from AI, A 2 cell groups t h r o u g h n o r a d r e n e r g i c v e n t r a l bundles, but not the locus c o e r u l e u s (15-17). In l i g h t of all these evidences, there m a y be a c e n t r a l n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m w h i c h r e g u l a t e s g a s t r i c functions, at the level of the L H A as w e l l as the ala cinerea, as we s u g g e s t e d e l s e w h e r e (7).
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NA and ACh in Gastric Acid Secretion
20
Vol. 32, No. 12, 1983
////~ .°
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A
E
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-,.. = 0"
I III I I 0 0
0
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L
30
60 Fig.
I
90
1~0 min
4
E f f e c t s of b e t h a n e c h o l and n i c o t i n e on g a s t r i c acid o u t p u t following a d m i n i s t r a t i o n into the LHA. B----g : b e t h a n e c h o l 3 n m o l e s (n=6), D----O : b e t h a n e c h o l i0 n m o l e s (n=5), O O : n i c o t i n e 30 nmoles (n=7). ,: p < 0.05, **: p < 0.01 ( s t a t i s t i c a l l y s i g n i f i c a n t as c o m p a r e d to the r e s p e c t i v e b a s a l v a l u e s just b e f o r e the a p p l i c a t i o n of b e t h a n e c h o l ) .
As for the c e n t r a l c h o l i n e r g i c r e g u l a t i o n of g a s t r i c functions, the e f f e c t s of A C h and o t h e r c h o l i n e r g i c a g o n i s t s on gastric a c i d s e c r e t i o n have b e e n r e p o r t e d (18-21). Intracerebrov e n t r i c u l a r or i n t r a c a r o t i d i n f u s i o n of A C h i n c r e a s e d g a s t r i c sec r e t i o n (18,19) and p i l o c a r p i n e and m e t h a c h o l i n e e n h a n c e d g a s t r i c s e c r e t i o n w h e n i n j e c t e d into the i n f e r i o r c e r e b e l l a r p e d u n c l e or s u p r a o p t i c n u c l e u s (20,21). However, the e f f e c t on g a s t r i c functions of c h o l i n e r g i c a g o n i s t s a p p l i e d into the L H A w a s not d e t e r mined. The L H A is d e f i n e d as the s o - c a l l e d "feeding center", and e l e c t r i c a l s t i m u l a t i o n of this area in rats e x h i b i t s a s i g n i f i c a n t i n c r e a s e in the v o l u m e of g a s t r i c s e c r e t i o n and acid o u t p u t (3). In the p r e s e n t study, it was c l e a r l y d e m o n s t r a t e d that A C h applied into the LHA r e s u l t e d in an i n c r e a s e in g a s t r i c acid secretion,
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therefore, it is p r o b a b l y the m u s c a r i n i c r e c e p t o r s w h i c h are activated by ACh, since the same r e s p o n s e was o b t a i n e d w i t h a small dose of the pure m u s c a r i n i c r e c e p t o r agonist, bethanechol. Nicotine was w i t h o u t effect, even in a larger dose than that used in our p r e v i o u s study (22). In this previous study, we r e p o r t e d that n i c o t i n e given into the caudal p o r t i o n of the v e n t r o m e d i a l hypothalamus increases gastric acid s e c r e t i o n (22). Therefore, both c h o l i n e r g i c m u s c a r i n i c receptors w i t h i n the LHA and n i c o t i n i c receptors w i t h i n the caudal p o r t i o n of v e n t r o m e d i a l h y p o t h a l a m u s may be related to the central e x c i t a t o r y mechanism, in r e g u l a t i o n of gastric functions. In r e s e r p i n e p r e t r e a t e d animals, the N A - i n d u c e d d e c r e a s e in acid output was p o t e n t i a t e d p r o b a b l y b e c a u s e of the p o s t s y n a p t i c NA receptors s u p e r s e n s i t i v i t y w i t h i n the LHA. The levels of N A and d o p a m i n e in various brain regions of the r e s e r p i n e - t r e a t e d rats were found to be r e m a r k a b l y lower than in the r e s p e c t i v e controls (9) and the basal gastric acid o u t p u t in these rats was m u c h higher than in the n o n - t r e a t e d controls (9,23). Thus, the inhibitory effect on gastric acid s e c r e t i o n of NA given into the ala cinerea was r e a d i l y m a n i f e s t e d in these N A - s u p e r s e n s i t i v e animals (9). It is i n t e r e s t i n g that the e x c i t a t o r y e f f e c t of A C h was also p o t e n t i a t e d in these r e s e r p i n i z e d animals. A C h in a dose of 30 nmoles (which was w i t h o u t effect in the n o n - r e s e r p i n i z e d rats), induced a r e m a r k a b l e increase in acid output. This p o t e n t i a t i o n cannot be a t t r i b u t e d to a d e c r e a s e d s y m p a t h e t i c o u t f l o w at the level of gastric A u e r b a c h ' s plexus, since the increase in acid output induced by i n t r a h y p o t h a l a m i c a l l y a p p l i e d n i c o t i n e was not p o t e n t i a t e d in the r e s e r p i n i z e d animals (22). Therefore, one has to consider w h e t h e r the effect of ACh was perhaps p o t e n t i a t e d by d y s f u n c t i o n s of the central n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m in the r e s e r p i n i z e d animals. Such a functional i n t e r a c t i o n b e t w e e n n o r a d r e n e r g i c and c h o l i n e r g i c neuron systems w i t h i n the brain is well known. P r e t r e a t m e n t w i t h 6 - h y d r o x y d o p a m i n e b l o c k e d the cataleptic effects of pilocarpine, a m u s c a r i n i c c h o l i n e r g i c a g o n i s t and p o t e n t i a t e d the l o c o m o t o r s t i m u l a n t effects induced by scopolamine, a m u s c a r i n i c a n t a g o n i s t (24,25). Methacholine increased h y p o t h a l a m i c blood flow, and this increase was b l o c k e d by chemical s y m p a t h e c t o m y of the hypothalamus, using 6 - h y d r o x y d o p a m i n e (26). S t i m u l a t i o n of the i n t r a c e r e b r a l n o r a d r e n e r g i c p a t h w a y increases h y p o t h a l a m i c blood flow, and this increase was a b o l i s h e d by an i n t r a h y p o t h a l a m i c injection of the m u s c a r i n i c antagonist, atropine and by a n i c o t i n i c antagonist, m e c a m y l a m i n e (27). We t e n t a t i v e l y conclude that the central n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m in r e g u l a t i o n of gastric functions is present at the level of the LHA as well as the ala cinerea. Furthermore, the c h o l i n e r g i c m u s c a r i n i c system in the LHA has an e x i t a t o r y action on gastric acid s e c r e t i o n w h i c h is a n t a g o n i z e d by n o r a d r e n ergic i n h i b i t o r y mechanisms. Acknowledgements Thanks are due to M. Ohara for p r e p a r i n g this m a n u s c r i p t . This w o r k was s u p p o r t e d by a G r a n t - i n A i d for S c i e n t i f i c Research, No. 457060 from the M i n i s t r y of Education, Science and Culture, Japan, and by a Grant from the Japan T o b a c c o and Salt public Cor-
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poration. References 1. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
R.W. P O R T E R , H.J. M O V I U S a n d J.D. F R E N C H , S u r g e r y . 1 8 8 7 5 880 (1953). P.T. R I D L E Y a n d F.P. B R O O K S , Am. J. P h y s i o l . 209 3 1 9 - 3 2 3 (1965) . A. M I S H E R a n d F.P. B R O O K S , Am. J. P h y s i o l . 211 4 0 3 - 4 0 6 (1966). G.R. M A S O N a n d T.S. N E L S E N , Am. J. P h y s i o l . 217 1 7 7 1 - 1 7 7 5 (1969) . F . W . L . K E R R a n d R.M. P R E S H A W , J. P h y s i o l . 205 4 0 5 - 4 1 5 (1969). M. K A D E K A R O , C. T I M O - I A R I A a n d M . L . M . V I C E N T I N I , J. P h y s i o l . 299 3 9 7 - 4 0 7 (1980). Y. O S U M I , S. A I B A R A , K. S A K A E and M. F U J I W A R A , L i f e Sci. 20 1 4 0 7 - 1 4 1 6 (1977). ~ . O S U M I , Y. N A G A S A K A , W A N G L.H. FU a n d M. F U J I W A R A , L i f e Sci. 23 2 2 7 5 - 2 2 8 0 (1978). Y. O S U M I , T. I S H I K A W A , Y. O K U M A , Y. N A G A S A K A and M. F U J I W A R A , Eur. J. P h a r m a c o l . 75 2 7 - 3 5 (1981). I.H.M. M A I N a n d B . J . R . W H I T T L E , B--{. J. P h a r m a c o l . 49 5 3 4 542 (1973). E.L. B L A I R , E.R. G R U N D , J.D. REED, D.J. S A N D E R S , G. S A N G E R a n d B. SHAW, J. P h y s i o l . 253 4 9 3 - 5 0 4 (1975). J . F . R . K O N I G a n d R.A. K L I P P E L , T h e R a t B r a i n , W i l l i a m s a n d Wilkins, Baltimore. (1963). K. F U X E , A c t a P h y s i o l . S c a n d . 64 s u p p l . 247 3 6 - 8 5 (1965). D.M. J A C O B O W I T S a n d M. P A L C O V I T S , J. C o m p . N e u r o l . 157 1328 (1974). U. U N G E R S T E D T , A c t a P h y s i o l . S c a n d . 82 s u p p l . 367 1-48 (1971). R.M. K O B A Y A S H I , M. P A L C O V I T S , I.J. K O P I N a n d D.M. J A C O B O W I T S . B r a i n Res. 77 2 6 9 - 2 7 9 (1974). M. P A L C O V I T S , L. Z~BORSZ-KY, A. F E M I N G E R , ~. M E Z E Y , M . I . K . F E K E T E , J.P. H E R M A N , B. K A N Y I C S K A a n d D. S Z A B 6 , B r a i n Res. 191 1 6 1 - 1 7 1 (1980). S.V. A N I C H K O V a n d L.L. G R E C H I S H K I N , Arch. Int. P h a r m a c o d y n . 166 4 1 7 - 4 2 3 (1967). N. E M M E L I N and G.S. K A H L S O N , A c t a P h y s i o l . S c a n d . 8 2 8 9 - 3 0 4 (1944). E.J. Z A W O I S K I , Exp. N e u r o l . 34 3 5 5 - 3 7 1 (1972). E.J. Z A W O I S K I and I. K O P L O V I T S , Exp. N e u r o l . 55 1 2 2 - 1 3 2 (1977). T. I S H I K A W A , Y. O S U M I , M. F U J I W A R A a n d M. N A G A T A , Eur. J. P h a r m a c o l . 80 3 3 1 - 3 3 6 (1982). I. Y A M A G U C H I , J. H I R O I , H. F U K E a n d S. K U M A D A , J. P h a r m a c o l . Exp. Ther. 205 7 1 0 - 7 1 7 (1978). S.T. M A S O N a n d H.C. F I B I G E R , N a t u r e 277 3 9 6 - 3 9 7 (1979). S.T. M A S O N , Eur. J. P h a r m a c o l . 56 1 3 1 - 1 3 7 (1979). K.P. K L U G M A N , G. M I T C H E L L a n d C. R O S E N D O R F F , Br. J. P h a r m a col. 66 2 1 7 - 2 2 1 (1979). K.P. K L U G M A N , G. M I T C H E L L a n d C. R O S E N D O R F F , S t r o k e ii 5 2 2 527 (1980).
Pergamon Press
CENTRAL NORADRENERGIC-CHOLINERGIC I N T E R A C T I O N IN R E G U L A T I O N OF G A S T R I C A C I D S E C R E T I O N IN RATS Yasunobu
Okuma, Y o s h i t s u g u Osumi, Toshio and M i t s u h i r o Nagata
Department
Ishikawa
of Pharmacology, Kochi M e d i c a l Nankoku, Kochi 781-51, Japan
School,
(Received in final form December 13, 1982) Summary P o s s i b l e roles of n o r a d r e n a l i n e (NA) and a c e t y l c h o l i n e (ACh) w i t h i n the lateral h y p o t h a l a m i c area (LHA) in r e g u l a t i o n of gastric acid s e c r e t i o n were e x a m i n e d in urethane a n e s t h e t i z e d rats. When NA 30 nmoles was given into the LHA, the gastric acid o u t p u t d e c r e a s e d and this i n h i b i t o r y effect of NA was p o t e n t i a t e d in rats p r e t r e a t e d w i t h r e s e r p i n e (2 mg/kg, i.p., 20 hr). E v e n in a dose of 3 nmoles w h i c h was w i t h o u t e f f e c t in n o n - t r e a t e d control animals, there was a r e m a r k a b l e d e c r e a s e in acid output. In these r e s e r p i n i z e d animals, A C h in a dose of 30 nmoles induced a r e m a r k a b l e increase in acid output, while in the controls this A C h - i n d u c e d increase was o b s e r v e d only w i t h a i0 times h i g h e r dose. In the rats not given reserpine, the c h o l i n e r g i c m u s c a r i n i c a g o n i s t b e t h a n e c h o l (i0 nmoles) i n c r e a s e d the gastric acid output while n i c o t i n e (30 nmoles) was w i t h o u t effect. Therefore, in rats, the c e n t r a l n o r a d r e n e r g i c inhibitory m e c h a n i s m s r e l a t e d to r e g u l a t i o n of gastric function m a y be p r e s e n t at the level of LHA as well as at the ala c i n e r e a (area of the dorsal m o t o r nucleus of vagi and the nucleus tractus solitarius). In addition, in the LHA, a c h o l i n e r gic m u s c a r i n i c m e c h a n i s m w h i c h elevates gastric acid s e c r e t i o n m a y be a n t a g o n i z e d by a n o r a d r e n e r g i c inhibitory mechanism. The role of the central nervous s y s t e m in r e g u l a t i o n of gastric f u n c t i o n has been e x t e n s i v e l y e x a m i n e d at the level of the hypothalamus (1-4) and other regions of the brain (5,6). Nevertheless, there remains a p a u c i t y in i n f o r m a t i o n c o n c e r n i n g tentative roles of central n e u r o t r a n s m i t t e r c a n d i d a t e s such as m o n o amines, a c e t y l c h o l i n e (ACh) and GABA, in the r e g u l a t i o n of gastric functions. Osumi et al. r e p o r t e d the e x i s t e n c e of a central nora d r e n e r g i c i n h i b i t o r y m e c h a n i s m in r e g u l a t i o n of gastric functions and s u g g e s t e d that the sites of action were p r o b a b l y the lateral h y p o t h a l a m i c area (LHA) and/or the ala c i n e r e a (7,8). Recently, we noted the e x i s t e n c e of this n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m w i t h i n the ala cinerea, of w h i c h n o r a d r e n e r g i c nerve t e r m i n a l s o r i g i n a t e from the locus c o e r u l e u s (9). Thus, we a t t e m p t e d to d e t e r m i n e w h e t h e r the i n h i b i t o r y role of n o r a d r e n a l i n e (NA) in r e g u l a t i o n of gastric acid s e c r e t i o n m a y 0024-3205/83/121363-08503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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be p r e s e n t at the level of the LHA. The n o r a d r e n e r g i c - c h o l i n e r g i c i n t e r a c t i o n w i t h i n the LHA and the p o s s i b l e role of o t h e r m o n o amines and G A B A in r e g u l a t i o n of g a s t r i c acid s e c r e t i o n w e r e also examined. Materials
and M e t h o d s
M a l e W i s t a r rats w e i g h i n g 220-250 g w e r e m a i n t a i n e d at 22-24 °C u n d e r a c o n s t a n t d a y - n i g h t r h y t h m for 7 to i0 days and g i v e n food ( l a b o l a t o r y chow, CA-l, J a p a n C L E A Co.) and tap w a t e r ad libitum. P r i o r to e a c h e x p e r i m e n t , all food but n o t w a t e r w-as w i t h h e l d for 16 hr. U n d e r u r e t h a n e a n e s t h e s i a (I g/kg, i.p.), the a b d o m e n was o p e n e d by a t r a n s v e r s e i n c i s i o n and a r o u n d - t i p cannula (5 cm long, 0.5 cm o u t e r diameter) c o n n e c t e d to a p o l y e t h y lene tube was i n s e r t e d into the s t o m a c h via an i n c i s i o n into the d u o d e n u m (i cm d i s t a l from the p y l o r i c s p h i n c t e r ) . The tip of the c a n n u l a lay just above the p y l o r i c s p h i n c t e r and was h e l d in p l a c e by two l i g a t u r e s a r o u n d the duodenum, one at the p o i n t of i n c i s i o n and the o t h e r c l o s e to the pylorus, as d e s c r i b e d by M a i n and W h i t t l e (i0). To r e m o v e the solid contents, the s t o m a c h was f l u s h e d w i t h saline, t a k i n g care to a v o i d d i s t e n t i o n . A f t e r rep e a t e d w a s h i n g s , two ml of s o l u t i o n p r e w a r m e d to 38°C was p l a c e d in the s t o m a c h at the b e g i n n i n g of e a c h 15 m i n c o l l e c t i o n period. The c o m p o s i t i o n of this s o l u t i o n was a 1:5 (v/v) m i x t u r e of g l y c ine and m a n n i t o l a d j u s t e d to 300 m o s M and pH 3.5 by a d d i t i o n of 0.i N HCI, a c c o r d i n g to the m e t h o d of B l a i r et al. (ii). Determ i n a t i o n s in 2 c o n s e c u t i v e 1 5 - m i n - c o l l e c t i o n s of g a s t r i c juice w e r e then m a d e to e s t a b l i s h the basal value. In the o t h e r series of e x p e r i m e n t s , the rats had b e e n g i v e n an i n t r a p e r i t o n e a l (i.p.) a d m i n i s t r a t i o n of r e s e r p i n e 2 mg/kg, 20 hr b e f o r e the e x p e r i m e n t . The p u r p o s e of this p r o c e d u r e was to i n v e s t i g a t e the e f f e c t of A C h on g a s t r i c functions, in the a b s e n c e of a n o r a d r e n e r g i c inhibitory m e c h a n i s m . The test s u b s t a n c e to be m i c r o i n j e c t e d w a s a d j u s t e d to 300 m o s M w i t h N a C I solution. A s t a i n l e s s steel m i c r o p i p e t t e (outer d i a m e t e r 0.35 mm) was then i n s e r t e d into the L H A (AP: 5.0, L: 1.5, H: 2.0) f o l l o w i n g the B r a i n A t r a s by K o n l g and K l i p p e l (12), and 1 ~i of test s u b s t a n c e was a d m i n i s t e r e d d u r i n g 60 sec. This m i c r o p i p e t t e also s e r v e d as the anode side of the b i p o l a r electrode. The d i a m e t e r of the o t h e r c a t h o d e side of the b i p o l a r e l e c t r o d e was 0.i mm. For e l e c t r i c a l s t i m u l a t i o n of the r e s t r i c t ed region, into w h i c h the test s u b s t a n c e had b e e n m i c r o i n j e c t e d , b i p h a g i c s q u a r e - w a v e p u l s e s of 0.5 mA, 2 msec, i0 c y c l e s / s e c w e r e a p p l i e d for i0 m i n by m e a n s of a e l e c t r o n i c s t i m u l a t o r (Model 3 2 0 ~ N i h o n K o d e n LTD., Japan). rd
,
A c i d o u t p u t was d e t e r m i n e d by t i t r a t i o n of g a s t r i c samples to pH 7.0 w i t h 0.01 N NaOH, u s i n g a pH meter. At t e r m i n a t i o n of the e x p e r i m e n t , the b r a i n was r e m o v e d and f r o z e n s e c t i o n s cut at 30 ~un w e r e s t a i n e d w i t h c r e s y l v i o l e t for m i c r o s c o p i c study of the sites of m i c r o i n j e c t i o n . The e x p e r i m e n t a l p r o c e d u r e for m i c r o i n j e c t i o n of test s u b s t a n c e into the L H A is r e p r e s e n t e d in Fig. i. N i n e t y to 120 m i n after the test s u b s t a n c e (in case of this rat, N A 30 nmoles) was g i v e n into the LHA, u n i l a t e r a l e l e c t r i c a l stimulation w a s p e r f o r m e d . W h e n i n c r e a s e in the a c i d o u t p u t w i t h electrical s t i m u l a t i o n occurred, and w h e n the tip of the p i p e t t e p l a c e d w i t h i n the L H A was h i s t o l o g i c a l l y confirmed, the r e s u l t s of m i c r o i n j e c t i o n of the test s u b s t a n c e s w e r e s u m m a r i z e d for s t a t i s -
Vol. 32, No. 12, 1983
NA and ACh in Gastric Acid Secretion
tistical calculation. S t a t i s t i c a l s i g n i f i c a n c e of the data was Student's t-test for paired comparison.
calculated
1365
using
1
"~15 E uD
\ O" U.I
=.10
Fig.
O.
The e x p e r i m e n t a l p r o c e d u r e for microinjection; effect on gastric acid output. NA 30 nmoles was m i c r o i n j e c t e d into the LHA of rats in a total volume of 1 91. ES: e l e c t r i c a l stimulation given at i0 cycles/sec, 2 msec, 0.5 mA for i0 min.
=1 0
t 0
t i
i
60
i
1
!
i
120 rain Results
M e a n basal gastric acid output o b t a i n e d from rats in the control series was 6.4 ± 0.5 ~ E q / 1 5 min (n=78). In the r e s e r p i n e p r e t r e a t e d animals, this basal level was 17.2 ± 2.5 ; E q / 1 5 min (n=31). Effects of NA m i c r o i n j e c t e d into the LHA on gastric acid output. The a d m i n i s t r a t i o n of saline alone into the LHA did not slgn--iTicantly alter the basal acid output, as shown in Fig. 2. NA 30 nmoles but not 3 nmoles a p p l i e d into the LHA d e c r e a s e d gastric acid output. A c i d output in this NA 30 n m o l e s - t r e a t e d group d e c r e a s e d to the lowest level at the 45-min c o l l e c t i o n (56.5 ± 9.4 % of the basal value, n=6) and then r e t u r n e d toward the basal level. This d e c r e a s e d level was s i g n i f i c a n t l y different, as compared to the r e s p e c t i v e basal values, just before the a p p l i c a t i o n of N A (p < 0.01). In the r e s e r p i n e p r e t r e a t e d animals, NA 3 nmoles a dose w i t h o u t effect in n o n - r e s e r p i n i z e d animals, s i g n i f i c a n t l y d e c r e a s e d gastric acid output. Effect of A C h m i c r o i n j e c t e d into the LHA on gastric acid output. M i c r o i n j e c t i o n of A C h 300 nmoles into the LHA significantly i n c r e a s e d the gastric acid o u t p u t (Fig. 3). Acid output i n c r e a s e d to the h i g h e s t level at the 45-min c o l l e c t i o n p e r i o d (186.8 ± 24.3 % of the basal level, n=10), and r e t u r n e d toward the basal level. In the r e s e r p i n e p r e t r e a t e d animals, the A C h - i n d u c e d increase in acid o u t p u t was potentiated, A C h 30 nmoles a dose w i t h o u t effect in n o n - r e s e r p i n i z e d animals, s i g n i f i c a n t l y increased the gastric acid output.
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A
C:
v
'10
0
'
30 Fig.
60
min
90
2
E f f e c t of N A g i v e n into the LHA on g a s t r i c acid output. In n o n - r e s e r p i n i z e d rats: O O : s a l i n e (n=6), D D : NA 3 n m o l e s (n=4), ~ : N A 30 nmoles (n=6). In r e s e r p i n i z e d rats: O----O: NA 3 n m o l e s (n=8). ,: p < 0.05, **: p < 0.01 ( s t a t i s t i c a l l y signif i c a n t as c o m p a r e d to the r e s p e c t i v e b a s a l v a l u e s just b e f o r e the a p p l i c a t i o n of NA.
E f f e c t s of c h o l i n e r g i c agonists, d o p a m i n e , s e r o t o n i n and G A B A m i c r o i n j e c t e d into the L H A on g a s t r i c acid output. A c h o l i n e r g i c m u s c a r i n i c agonist, b e t h a n e c h o l i0 nmoles b u t not 3 n m o l e s g i v e n i n t o the LHA i n d u c e d a r e m a r k a b l e i n c r e a s e in gastric a c i d o u t p u t (Fig. 4). However, the c h o l i n e r g i c n i c o t i n i c agonist, nicotine, did not s i g n i f i c a n t l y a f f e c t g a s t r i c a c i d output, e v e n in a d o s e of 30 nmoles. D o p a m i n e 30 nmoles, s e r o t o n i n 30 n m o l e s and G A B A 300 n m o l e s g i v e n into the L H A h a d no s i g n i f i c a n t e f f e c t on g a s t r i c acid o u t p u t (Data not shown). Discussion The a d m i n i s t r a t i o n of N A into the L H A d e c r e a s e d g a s t r i c acid output, w h i l e A C h i n c r e a s e d it, a n d the r e s p o n s e s i n d u c e d by N A and A C h w e r e p o t e n t i a t e d in the r e s e r p i n e - p r e t r e a t e d animals. We h a v e a l r e a d y r e p o r t e d that a n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m seems to be p r e s e n t w i t h i n the ala cinerea, of w h i c h
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E f f e c t of A C h g i v e n into the LHA on g a s t r i c acid output. In n o n - r e s e r p i n i z e d rats: O O : A C h 30 n m o l e s (n=7), H : ACh 300 n m o l e s (n=10). In r e s e r p i n i z e d rats: O----O : A C h 30 nmoles (n=6). ,: p < 0.05, **: p < 0.01 ( s t a t i s t i c a l l y s i g n i f i c a n t as c o m p a r e d to the r e s p e c t i v e basal v a l u e s just b e f o r e the a p p l i c a tion of ACh).
n o r a d r e n e r g i c nerve t e r m i n a l s o r i g i n a t e d f r o m the locus c o e r u l e u s (9). A c c o r d i n g to d a t a o b t a i n e d in h i s t o c h e m i c a l and n e u r o c h e m ical studies, the L H A has a large n u m b e r of NA n e r v e t e r m i n a l s (~, 14) and these n o r a d r e n e r g i c t e r m i n a l s w i t h i n the L H A p r o b a b l y o r i g i n a t e from AI, A 2 cell groups t h r o u g h n o r a d r e n e r g i c v e n t r a l bundles, but not the locus c o e r u l e u s (15-17). In l i g h t of all these evidences, there m a y be a c e n t r a l n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m w h i c h r e g u l a t e s g a s t r i c functions, at the level of the L H A as w e l l as the ala cinerea, as we s u g g e s t e d e l s e w h e r e (7).
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E f f e c t s of b e t h a n e c h o l and n i c o t i n e on g a s t r i c acid o u t p u t following a d m i n i s t r a t i o n into the LHA. B----g : b e t h a n e c h o l 3 n m o l e s (n=6), D----O : b e t h a n e c h o l i0 n m o l e s (n=5), O O : n i c o t i n e 30 nmoles (n=7). ,: p < 0.05, **: p < 0.01 ( s t a t i s t i c a l l y s i g n i f i c a n t as c o m p a r e d to the r e s p e c t i v e b a s a l v a l u e s just b e f o r e the a p p l i c a t i o n of b e t h a n e c h o l ) .
As for the c e n t r a l c h o l i n e r g i c r e g u l a t i o n of g a s t r i c functions, the e f f e c t s of A C h and o t h e r c h o l i n e r g i c a g o n i s t s on gastric a c i d s e c r e t i o n have b e e n r e p o r t e d (18-21). Intracerebrov e n t r i c u l a r or i n t r a c a r o t i d i n f u s i o n of A C h i n c r e a s e d g a s t r i c sec r e t i o n (18,19) and p i l o c a r p i n e and m e t h a c h o l i n e e n h a n c e d g a s t r i c s e c r e t i o n w h e n i n j e c t e d into the i n f e r i o r c e r e b e l l a r p e d u n c l e or s u p r a o p t i c n u c l e u s (20,21). However, the e f f e c t on g a s t r i c functions of c h o l i n e r g i c a g o n i s t s a p p l i e d into the L H A w a s not d e t e r mined. The L H A is d e f i n e d as the s o - c a l l e d "feeding center", and e l e c t r i c a l s t i m u l a t i o n of this area in rats e x h i b i t s a s i g n i f i c a n t i n c r e a s e in the v o l u m e of g a s t r i c s e c r e t i o n and acid o u t p u t (3). In the p r e s e n t study, it was c l e a r l y d e m o n s t r a t e d that A C h applied into the LHA r e s u l t e d in an i n c r e a s e in g a s t r i c acid secretion,
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therefore, it is p r o b a b l y the m u s c a r i n i c r e c e p t o r s w h i c h are activated by ACh, since the same r e s p o n s e was o b t a i n e d w i t h a small dose of the pure m u s c a r i n i c r e c e p t o r agonist, bethanechol. Nicotine was w i t h o u t effect, even in a larger dose than that used in our p r e v i o u s study (22). In this previous study, we r e p o r t e d that n i c o t i n e given into the caudal p o r t i o n of the v e n t r o m e d i a l hypothalamus increases gastric acid s e c r e t i o n (22). Therefore, both c h o l i n e r g i c m u s c a r i n i c receptors w i t h i n the LHA and n i c o t i n i c receptors w i t h i n the caudal p o r t i o n of v e n t r o m e d i a l h y p o t h a l a m u s may be related to the central e x c i t a t o r y mechanism, in r e g u l a t i o n of gastric functions. In r e s e r p i n e p r e t r e a t e d animals, the N A - i n d u c e d d e c r e a s e in acid output was p o t e n t i a t e d p r o b a b l y b e c a u s e of the p o s t s y n a p t i c NA receptors s u p e r s e n s i t i v i t y w i t h i n the LHA. The levels of N A and d o p a m i n e in various brain regions of the r e s e r p i n e - t r e a t e d rats were found to be r e m a r k a b l y lower than in the r e s p e c t i v e controls (9) and the basal gastric acid o u t p u t in these rats was m u c h higher than in the n o n - t r e a t e d controls (9,23). Thus, the inhibitory effect on gastric acid s e c r e t i o n of NA given into the ala cinerea was r e a d i l y m a n i f e s t e d in these N A - s u p e r s e n s i t i v e animals (9). It is i n t e r e s t i n g that the e x c i t a t o r y e f f e c t of A C h was also p o t e n t i a t e d in these r e s e r p i n i z e d animals. A C h in a dose of 30 nmoles (which was w i t h o u t effect in the n o n - r e s e r p i n i z e d rats), induced a r e m a r k a b l e increase in acid output. This p o t e n t i a t i o n cannot be a t t r i b u t e d to a d e c r e a s e d s y m p a t h e t i c o u t f l o w at the level of gastric A u e r b a c h ' s plexus, since the increase in acid output induced by i n t r a h y p o t h a l a m i c a l l y a p p l i e d n i c o t i n e was not p o t e n t i a t e d in the r e s e r p i n i z e d animals (22). Therefore, one has to consider w h e t h e r the effect of ACh was perhaps p o t e n t i a t e d by d y s f u n c t i o n s of the central n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m in the r e s e r p i n i z e d animals. Such a functional i n t e r a c t i o n b e t w e e n n o r a d r e n e r g i c and c h o l i n e r g i c neuron systems w i t h i n the brain is well known. P r e t r e a t m e n t w i t h 6 - h y d r o x y d o p a m i n e b l o c k e d the cataleptic effects of pilocarpine, a m u s c a r i n i c c h o l i n e r g i c a g o n i s t and p o t e n t i a t e d the l o c o m o t o r s t i m u l a n t effects induced by scopolamine, a m u s c a r i n i c a n t a g o n i s t (24,25). Methacholine increased h y p o t h a l a m i c blood flow, and this increase was b l o c k e d by chemical s y m p a t h e c t o m y of the hypothalamus, using 6 - h y d r o x y d o p a m i n e (26). S t i m u l a t i o n of the i n t r a c e r e b r a l n o r a d r e n e r g i c p a t h w a y increases h y p o t h a l a m i c blood flow, and this increase was a b o l i s h e d by an i n t r a h y p o t h a l a m i c injection of the m u s c a r i n i c antagonist, atropine and by a n i c o t i n i c antagonist, m e c a m y l a m i n e (27). We t e n t a t i v e l y conclude that the central n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m in r e g u l a t i o n of gastric functions is present at the level of the LHA as well as the ala cinerea. Furthermore, the c h o l i n e r g i c m u s c a r i n i c system in the LHA has an e x i t a t o r y action on gastric acid s e c r e t i o n w h i c h is a n t a g o n i z e d by n o r a d r e n ergic i n h i b i t o r y mechanisms. Acknowledgements Thanks are due to M. Ohara for p r e p a r i n g this m a n u s c r i p t . This w o r k was s u p p o r t e d by a G r a n t - i n A i d for S c i e n t i f i c Research, No. 457060 from the M i n i s t r y of Education, Science and Culture, Japan, and by a Grant from the Japan T o b a c c o and Salt public Cor-
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poration. References 1. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
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