- Email: [email protected]
IV. Receptor regulation of ion transport in the intestinal epithelium
Life Sciences, Vol. 43, pp. 2193-2201 Printed in the U.S.A.
Pergamon Press
IV. RECEPTOR REGULATION OF ION TRANSPORT IN THE INTESTINAL E P I T H E L I ~
David R. Brown 1 ' 3 , R i t u Chandan 1 ' 3 , F r a n c i s L. Q u i t o 1 and V i r g i n i a S. Seybold 2'3
U n i v e r s i t y of Minnesota 1Department of V e t e r i n a r y B i o l o g y , C o l l e g e of V e t e r i n a r y M e d i c i n e , 1988 F i t c h Avenue, S t . P a u l , Minnesota 55108 2Department of C e l l B i o l o g y and Neuroanatomy, and 3 N e u r o s c i e n c e Graduate Program, 4-135 J a c k s o n H a l l , 321 Church S t r e e t , M i n n e a p o l i s , Minnesota 55455
Summary The a c t i v e t r a n s p o r t of ions by t h e intestinal e p i t h e l i u m is regulated by a number of e n t e r i c n e u r o t r a n s m i t t e r s , hormones and o t h e r s u b s t a n c e s . Our knowledge of t h e r e c e p t o r s m e d i a t i n g t h e actions o f t h e s e s u b s t a n c e s is g e n e r a l l y f r a g m e n t a r y . This r e v i e w summarizes c u r r e n t knowledge on t h e l o c a t i o n and f u n c t i o n a l characteristics of t r a n s m i t t e r receptors regulating transport f u n c t i o n in t h e small i n t e s t i n e , h i g h l i g h t i n g r e c e n t r e s e a r c h on c h o l i n e r g i c and b r a d y k i n i n r e c e p t o r s .
The i n t e s t i n a l mucosa h o l d s p r e c i s e l y in b a l a n c e t h e a b s o r p t i o n and s e c r e t i o n o f w a t e r and ions so as t o m a i n t a i n h o m e o s t a s i s in body f l u i d volume and c o m p o s i t i o n , p r o v i d e an aqueous medium f o r t h e b i o t r a n s f o r m a t i o n of n u t r i e n t s and o t h e r s u b s t a n c e s , and r i n s e t h e lumen of h o s t i l e m i c r o o r g a n i s m s . The f u n c t i o n of e p i t h e l i a l c e l l s i n v o l v e d in t h e s e t r a n s p o r t processes is regulated by a complex a r r a y of n e r v e s o r i g i n a t i n g from t h e two major g a n g l i o n a t e d p l e x u s e s of t h e gut and from neurons l y i n g o u t s i d e t h e d i g e s t i v e system. This n e u r a l c i r c u i t r y s e r v e s t o c o o r d i n a t e t h e a b s o r p t i v e and motor functions of the intestines. In a d d i t i o n , v a r i o u s hormonal substances synthesized and r e l e a s e d from e n t e r o e n d o c r i n e c e l l s can m o d u l a t e e p i t h e l i a l function e i t h e r through d i r e c t i n t e r a c t i o n s with t r a n s p o r t i n g enterocytes or by a l t e r i n g e n t e r i c n e u r a l a c t i v i t y . The m o r p h o l o g i c and f u n c t i o n a l a s p e c t s of t h e neurohormonal c o n t r o l o f t h e i n t e s t i n a l e p i t h e l i u m have been s u b j e c t s of r e c e n t r e v i e w a r t i c l e s (1 - 3). In t h i s a r t i c l e , we s h a l l r e v i e w b r i e f l y t h e proposed mechanisms u n d e r l y i n g ion t r a n s p o r t in t h e i n t e s t i n a l e p i t h e l i u m and d i s c u s s t h e p h a r m a c o l o g i c a l characteristics of r e c e p t o r s f o r s e l e c t e d t r a n s m i t t e r substances thought to r e g u l a t e t h i s system.
0024-3025/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
2194
Receptors and Intestinal Ion Transport
Intestinal
Vol. 43, No. 26, 1988
Ion Transport
In the mammalian small intestine, villus and c r y p t cells lining the epithelium are believed to subserve ion transporting functions. It has been p r o p o s e d t h a t t h e a c t i v e a b s o r p t i o n o f Na t , a n i o n s (Cl', HCO3" e t c . ) and organic substrates i s a c c o m p l i s h e d by c e l l s l i n i n g t h e i n t e s t i n a l villi (Fig. 1 ) . C h l o r i d e a b s o r p t i o n ( m o v e m e n t f r o m lumen t o blood) may o r may n o t be linked t o Na + t r a n s p o r t depending upon t h e i n t e s t i n a l s e g m e n t and s p e c i e s e x a m i n e d . Sodium a b s o r p t i o n i s d e p e n d e n t upon t h e operation of a ouabainsensitive Na÷/K÷-ATPase situated on t h e basolateral c e l l m e m b r a n e . On t h e other hand, ceils located in the intestinal glands or c~ypts of Lieberkuehn appear t o s e c r e t e a c t i v e l y CI" ( a n d i n some c a s e s , HCO3 ) . C h l o r i d e s e c r e t i o n ( m o v e m e n t f r o m b l o o d t o lumen) a p p e a r s t o be a t w o - s t e p process: first, a basolateral coupled influx o f Na t and CI" a l l o w s [ C l ' ] t o r i s e w i t h i n t h e crypt cell to levels above its electrochemical equilibrium and s e c o n d , alterations in the permeability o f t h e a p i c a l c e l l membrane p r o m o t e CI" e x i t into the intestinal lumen ( F i g . 1 ) .
.......
+umo~
bloo~
FIG. 1. Hodel f o r i o n t r a n s p o r t in mammalian small intestine.
WLLI
LOOP DlUnETICS lug,.
blood
; ~
B~*
K÷
The i n t e s t i n a l epithelium is relatively "leaky" relative to other epithelia, s u c h as t h o s e o f t h e c o l o n o r u r i n a r y b l a d d e r , and b o t h t h e b u l k movement o f water in response to o s m o t i c g r a d i e n t s and p a s s i v e i o n f l u x e s o c c u r i n i t s paracellular spaces. The s p o n t a n e o u s potential difference (p.d.) which develops across the epithelium is the bioelectrical result of ongoing active and p a s s i v e i o n t r a n s p o r t processes. It is possible to nullify this transmural p . d . i n Vi~Fo by v o l t a g e - c l a m p i n g the gut epithelium, thereby greatly reducing (or theoretically, eliminating) the passive electrodiffusion of ions. The resulting short-circuit c u r r e n t ( I s c ) i s t h e sum o f a l l a c t i v e i o n t r a n s p o r t by t h e t i s s u e and i s e l e c t r o c h e m i c a l l y related to the net transepithelial f l u x e s o f Na t and C l ' .
Vol. 43, No. 26, 1988
Receptors and Intestinal Ion Transport
2195
A number of intracellular second messengers have been implicated in the regulation of intestinal ion transport. These intracellular regulators include the cyclic nucleotides (cAMP, cGHP), free Ca 2+ , and probably phosphoinositides, p r o t e i n k i n a s e C and e i c o s a n o i d s . Their potential roles in ion transport processes h a v e b e e n r e v i e w e d ( 4 ) and t h e a c t i o n s o f some o f these substances are illustrated below in relation to their coupling to gut transmitter receptors (Fig. 2). Localization
of Transmitter
Receptors
Transmitter receptors have been localized within the gut through the use of physiological, biochemical, and anatomical methodologies. In general, receptors for transmitter substances affecting intestinal ion transport are localized primarily on t h e s e r o s a l a s p e c t o f t h e e p i t h e l i u m ; exposure of the luminal aspect to concentrations of transmitters far exceeding effective serosal concentrations is often not associated with alterations in transport. On t h e s e r o s a l a s p e c t o f t h e i n t e s t i n a l mucosa, receptors for transmitters have been tentatively l o c a l i z e d on e p i t h e l i a l c e l l s o r on n e r v o u s e l e m e n t s i n the submucosa examining agonist activity in denervated tissue preparations (5) o r t h e a b s e n c e and p r e s e n c e of neuronal conduction blockade produced by tetrodotoxin (TTX). Changes in ion transport occurring after transmitter application to these "denervated" preparations h a v e b e e n t a k e n as e v i d e n c e f o r a d i r e c t a c t i o n on b a s o l a t e r a l epithelial cell membranes; it is assumed that the actions of transmitters a c t i n g a t r e c e p t o r s on e n t e r i c n e u r o n s w i l l be eliminated by t h e s e maneuvers. These experiments do n o t r u l e out the possibility that a given transmitter acts secondary to the release of other bioactive substances from endocrine or paracrine-type cells in the gut epithelium-subepithelium. Information on t h e location and p h a r m a c o l o g i c a l characteristics of transmitter r e c e p t o r s on i d e n t i f i e d e n t e r i c n e u r o n s c a n be obtained through the use of electrophysiological techniques in which the action of the transmitter s u b s t a n c e on n e u r o n a l a c t i v i t y c a n be d e t e r m i n e d . Unfortunately, it has not been possible to link specific transmitter-induced changes in neural activity with ion transport. Electrophysiological techniques c o u l d be a p p l i e d t o t h e s t u d y o f r e c e p t o r - m e d i a t e d changes in ion fluxes at the level of individual epithelial cells or intestinal cell cultures; such strategies have been deployed successfully in other preparations, s u c h as the tracheal epithelium (6), but they have not been extensively utilized in the study of intestinal epithelial function. Biochemical assays of specific transmitter binding to high-affinity sites in the intestinal mucosa have been reported in mucosal homogenates, slide-mounted t i s s u e s e c t i o n s and e n t e r o c y t e membrane f r a c t i o n s . Ligand binding t o mucus secreted by c e l l homogenate preparations spontaneously or in response to transmitter exposure represents a potential artifact in receptor characterization (7). The u s e o f s l i d e - m o u n t e d t i s s u e s e c t i o n s i s a p r o c e d u r e first d e v e l o p e d by Young and K u h a r ( 8 ) w h i c h a f f o r d s a b e t t e r a p p r o x i m a t i o n o f receptor distribution i_nn s i t u , a h i g h e r d e g r e e o f s p e c i f i c b i n d i n g and no mucus a r t i f a c t s because it involves less tissue disruption and e x t e n s i v e washing steps. Both procedures suffer from the disadvantage that transmitter b i n d i n g s i t e s may n o t be d i r e c t l y related to functional receptors mediating ion transport if binding occurs to neurons or other non-transporting cells present in the preparation. Transmitter binding sites c a n be l o c a l i z e d and quantified within subregions of the intestinal mucosa t h r o u g h t h e u s e o f quantitative autoradiographic procedures in tissue sections. Binding studies using cell membrane fractions have been conducted frequently and g e n e r a l l y
2196
Receptors and Intestinal Ion Transport
allow for level.
optimum b i o c h e m i c a l
Localization
TTX A c t i on
Acetyl choline
( gp, p : - )
[9151 Angiotensin [ 1 6 , 17]
(rt:-)
II
[20 - 2 2 ] Bradykinin [ 1 6 , 231 Neuropeptide Y [24 - 2 7 , 7 6 ]
Norepinephrine
(gp:-
(rb,
Mediating
at the epithelial
(rt:
? Yes ( r t ,
rt:-;gp:÷
?
?
; p:±)
rb:-;
cell
Ion Transport i n S m a l l
Yes ( r t )
(gp:-) (rt,
sites
S p e c i f i c B i n d i n g S i t e s D e t e c t e d i n Mucosa: Biochemically Autoradiographically Yes ( r t ) ( g p : SP)
(fl:-) 19; S.M O ' G r a d y , s u b m i t t e d ]
Bombesin/GRP
of binding
TABLE 1 Receptors Intestine
o f Some T r a n s m i t t e r
T r ansmi t t e r
[18,
analysis
Vol. 43, No. 26, 1988
p:,) )
possibly
E)
( d g : MP) gp)
(gp:
SP, E)
Yes ( r t )
?
Yes ( r b )
?
[28 - 3 2 ]
Opioids
(p, gp, rb:+) No ( r t , rb) ( r t , gp: SP, E) F.L. Q u i t o and D.R. Brown, u n p u b l i s h e d o b s e r v a t i o n s ) (gp:±; p:÷) Yes ( p : m e m b r a n e ; rodent: cytosolic) ? [ 3 1 , 39 - 4 2 ; H.F. O v e r e n d and D.R. Brown, s u b m i t t e d ] Substance P (gp, r b : ± ; p : - ) ? ( r t : n o n e ; gp: SP, E) [43 - 48] VIP (gp, rb:-) Yes ( r t , gp, r b ) ( h : E) [49 - 54] [ 2 8 , 33 - 38; Somatostatin
(+) = f u l l i n h i b i t i o n of ion transport effects; (±) = p a r t i a l inhibition; ( - ) = no i n h i b i t i o n ; h= human; p = p i g ; dg = dog; r b = r a b b i t ; rt = rat; gp = g u i n e a p i g ; f l = w i n t e r f l o u n d e r ; l ~ = m y e n t e r i c p l e x u s ; SP = submucous plexus; E = intestinal epithelium; ? = not determined. Bracketed numbers indicate references.
Selected Acetylcholine
Mediators
Regulating
Ion Transport
(ACh)
Cholinergic receptors a r e l o c a l i z e d on b o t h n e u r o n a l and e p i t h e l i a l cells of the small intestine. The r e c e p t o r s m e d i a t i n g t h e a c t i o n s o f ACh t o depolarize submucous p l e x u s n e u r o n s and t o p r e s y n a p t i c a l l y i n h i b i t t h e r e l e a s e o f ACh and norepinephrine a p p e a r t o be o f t h e M1- and M 2 - m u s c a r i n i c c h o l i n e r g i c subtype, respectively (55). In addition, muscarinic cholinergic agonists rapidly evoke a serosa-positive increase in Isc in the small intestinal mucosa from most s p e c i e s . When e x a m i n e d i n more d e t a i l , this tissue Isc response represents a stimulation o f a c t i v e a n i o n s e c r e t i o n and i s r e s i s t a n t t o TTX ( T a b l e 1 ) . H i g h concentrations of the non-selective agonist carbachol have been observed to produce, in addition to an initial Isc elevation, a long latency decrease in Isc below baseline levels in the rabbit ileum. This effect seems t o involve
Vol. 43, No. 26, 1988
Receptors and Intestinal Ion Transport
t h e c a r b a c h o l - i n d u c e d a c t i v a t i o n of g a n g l i o n i c n i c o t i n i c w i t h e n t e r i c s y m p a t h e t i c neurons (56).
receptors
2197
associated
Ligand b i n d i n g a s s a y s r e v e a l t h a t c h o l i n e r g i c r e c e p t o r s on c r y p t and v i l l u s cells possess similar binding characteristics t o c h o l i n e r g i c a g o n i s t s and a n t a g o n i s t s (14) and d i f f e r from t h o s e on i n t e s t i n a l smooth muscle ( 5 7 ) . In displacing the potent muscarinic antagonist [3H]-quinuclidinyl benzilate (QNB), c h o l i n e r g i c l i g a n d s d i s p l a y o r d e r s of p o t e n c y of a t r o p i n e > s c o p o l a m i n e >> p i r e n z e p i n e > o x o t r e m o r i n e >> c a r b a c h o l in i l e a l v i l l u s and c r y p t c e l l s of t h e r a t ileum ( 1 4 ) , 4-DAMP > p i r e n z e p i n e > HeN A-343 > c a r b a c h o l > b e t h a n e c h o l in mucosal scrapings o f t h e guinea p i g i l e a l mucosa ( 1 2 ) , and a t r o p i n e > 4I~ >> p i r e n z e p i n e in s l i d e - m o u n t e d s e c t i o n s of t h e p o r c i n e j e j u n a l mucosa (V.S. S e y b o l d , R. Chandan, X.-F. Sun and D.R. Brown, s u b m i t t e d ) . T r i t i a t e d QNB binds t o t h e s e sites w i t h l ~ s r a n g i n g from 60 - 400 pM, depending upon t h e p r e p a r a t i o n employed. S p e c i f i c , - h i g h - a f f i n i t y [3H]-QNB b i n d i n g s i t e s with similar affinities for cholinergic agonists and a n t a g o n i s t s have been c h a r a c t e r i z e d in r a t and guinea p ig c o l o n i c mucosa (58, 59). In t h e guinea p i g i l e a l and c o l o n i c mucosa, t h e rank o r d e r s of b i n d i n g a f f i n i t i e s of m u s c a r i n i c cholinergic antagonists a g r e e s w e l l w i t h t h e i r o r d e r s of p o t e n c y in a l t e r i n g ion t r a n s p o r t induced by e l e c t r i c a l f i e l d s t i m u l a t i o n (EFS) of t h e s e t i s s u e s ; EFS ap p ear s t o r e l e a s e endogenous ACh from submucosal neurons (12, 59). There a r e no p u b l i s h e d data on t h e a b i l i t y of t h e s e a n t a g o n i s t s t o s h i f t c h o l i n e r g i c agonist dose-response curves for a l t e r i n g transport. The a v a i l a b l e data suggest that the muscarinic r e c e p t o r mediating the t r a n s p o r t - r e l a t e d actions of ACh is o f t h e M2-subtype, but f u r t h e r d e f i n i t i o n of t h i s receptor awaits additional investigation u s i n g a g r e a t e r v a r i e t y of s e l e c t i v e l i g a n d s . The e f f e c t o r ( s ) c o u p l i n g a g o n i s t * r e c e p t o r b i n d i n g to an i o n s e c r e t i o n have not been c h a r a c t e r i z e d f u l l y in mammalian i n t e s t i n a l p r e p a r a t i o n s ( F i g . 2). In i s o l a t e d villus cells from t h e c h i c k e n ileum however, a c t i v a t i o n of m u s c a r i n i c receptors l e a d s t o an i n c r e a s e in p h o s p h o i n o s i t i d e t u r n o v e r w i t h a c o n s e q u e n t r i s e in f r e e i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n . These b i o c h e m i c a l e v e n t s ar e temporally correlated with cholinergic agonist-induced changes in cell membrane ion t r a n s p o r t p r o c e s s e s (60; E.B. Chang and M.W. Musch, s u b m i t t e d ) .
Bradykinin
(BK)
K i n i n s a r e l i b e r a t e d from damaged i n t e s t i n a l t i s s u e and may m e d i a t e a c t i v e an i o n s e c r e t i o n in i n f l a m m a t o r y s t a t e s of t h e bowel. BK, k a l l i d i n (Lys-BK) and t h e i r a n a l o g s s t i m u l a t e n e t C1 s e c r e t i o n and t r a n s i e n t l y i n c r e a s e I s c in both t h e small intestine and c o l o n (67, 68). Moreover, s p e c i f i c b i n d i n g s i t e s h a v i n g h i g h a f f i n i t y f o r t h e s e p e p t i d e s have been d e t e c t e d on small and l a r g e intestinal epithelial cells by b i o c h e m i c a l and a u t o r a d i o g r a p h i c t e c h n i q u e s (16, 23). In t h e guinea pig i l e a l mucosa, k i n i n s have a r e l a t i v e p o t e n c y o r d e r f o r i n c r e a s i n g I s c of BK > k a l l i d i n > Met-Lys-BK > Hydroxyproline3-BK > ~ - ( 2 T h i e n y l ) - A l a S ' a - B K > Des-Argg-BK which c o r r e l a t e s w e l l w i t h t h e i r a f f i n i t i e s f o r s p e c i f i c BK b i n d i n g s i t e s in t h e t i s s u e (23). The o r d e r of a f f i n i t i e s for these substances in d i s p l a c i n g [12sI]-BK from s p e c i f i c b i n d i n g s i t e s in r a t j e j u n a l e p i t h e l i a l membranes is s i m i l a r : BK ~ Tyra-BK > k a l l i d i n >> Des-Arg 1BK (1 6 ) . Using BK a n t a g o n i s t s s p e c i f i c f o r k i n i n r e c e p t o r s u b t y p e s , Kachur e t e l . (61) have d e t e r m i n e d in t h e guinea p i g ileum t h a t t h e a c t i o n s of k i n i n s in c o n t r a c t i n g l o n g i t u d i n a l smooth muscle and e l i c i t i n g anion s e c r e t i o n a r e both m e d i a t e d by BK2-type r e c e p t o r s . The i n t r a c e l l u l a r m e d i a t o r s of k i n i n - i n d u c e d s e c+ +r e t i o n appear t o be both a r a c h i d o n a t e m e t a b o l i t e s and f r e e intracellular Ca . The p e p t i d e s appear t o induce p r o s t a n o i d and l e u k o t r i e n e f o r m a t i o n by a c t i n g upon s u b e p i t h e l i a l c e l l s ; however, c y c l o o x y g e n a s e i n h i b i t o r s such as
2198
Receptors and Intestinal Ion Transport
Vol. 43, No. 26, 1988
indomethacin reduce, but do not e l i m i n a t e t i s s u e Isc responses to kinin e x p o s u r e (62, 68 - 70)• The d i r e c t e f f e c t s of k i n i n s on ion t r a n s p o r t in intestinal epithelial cells occur independent of s u b e p i t h e l i a l prostanoid s y n t h e s i s and a p p e a r t o r e q u i r e e x t r a c e l l u l a r Ca ++ (62, 71 73)• Further research i n t h i s a r e a should a t t e m p t t o r e l a t e p e p t i d e - r e c e p t o r b i n d i n g w i t h changes in i n t r a c e l l u l a r Ca ++ and ion t r a n s p o r t function in isolated epithelial cells.
A C! -
,
|ATP
VIP
C
B CI-
cAMP
Na+
CI¢
I
L. '"~ ':
;
SP
Na÷
,
K* CF Na*
Cl-
~
....
Ca2÷ %. A( LOPERAMIDE
~ . C F x ATROPINE ANP
FIG. 2
Some Proposed Receptors and Second Nossengers Nediating I n t e s t i n a l
Ion
Transport.
(A) V a s o a c t i v e i n t e s t i n a l p e p t i d e (VIP) b i n d i n g t o r e c e p t o r s on enterocytes has been directly correlated w i t h c y c l i c AMP p r o d u c t i o n and a c t i v e anion s e c r e t i o n • The p e p t i d e may a l s o inhibit NaCl a b s o r p t i o n [ 5 3 ] . (B) A number of s u b s t a n c e s a r e b e l i e v e d t o a c t t h r o u g h Ca+÷-dependent i n t r a c e l l u l a r mechanisms• These i n c l u d e +÷ s u b s t a n c e . P (SP), which may • ÷÷ i n c r e a s e f r e e ] n t r a c e l l u l a r Ca by promoting e++x t r a c e l l u l a r Ca entry through l o p e r a m i d e - s e n s i t i v e membrane Ca c h a n n e l s [60] and a c e t y l c h o l i n e (ACh) which acts on muscarinic receptors to increase p h o s p h o i n o s i t i d e ( P I ) t u r n o v e r and i n o s i t o l 1,4,5-trisphosphate production (IP3). IP 3 in t u r n m o b i l i z e s Ca t÷ s e q u e s t e r e d in t h e e n d o p l a s m i c r e t i c u l u m (ER). E p i t h e l i a l B z - b r a d y k i n i n and 5-HT 2serotoninergic r e c e p t o r s may a l s o be co u p l ed t o PI breakdown t h r o u g h p h o s p h o l i p a s e C (PLC) [61 - 64; E.B. Chang and M.W. Musch, submitted]. (C) A l t h o u g h unknown in mammalian enterocytes, basolateral receptors c o u p l e d t o c y c l i c Gt~ p r o d u c t i o n have been d e t e c t e d in t h e t e l e o s t ( w i n t e r f l o u n d e r ) i n t e s t i n e and s e r v e t o mediate the i n h i b i t o r y e f f e c t s o f a t r i a l n a t r i u r e t i c p e p t i d e s on Na-K-C1 c o t r a n s p o r t in t h i s t i s s u e [65, 66].
Conclusions The s t u d y o f t h e r e g u l a t i o n and pharmacology of i n t e s t i n a l ion t r a n s p o r t is in its infancy. D e s p i t e numerous i n v e s t i g a t i o n s of t h e n e u r o c h e m i c a l morphology o f t h e ~,,t. the electrophysiology of e n t e r i c neurons, the
Vol. 43, No. 26, 1988
Receptors and Intestinal Ion Transport
2199
e p i t h e l i a l mechanisms u n d e r l y i n g ion t r a n s p o r t and c u r s o r y e x a m i n a t i o n s o f t h e effects of a v a r i e t y o f s u b s t a n c e s on e l e c t r o l y t e f l u x e s in t h e i n t e s t i n a l epithelium, surprisingly little i s known about t h e r e c e p t o r s m e d i a t i n g t i s s u e responses to neural and hormonal t r a n s m i t t e r s in t h i s t i s s u e . T h i s s t a t e of a f f a i r s i s in p a r t due t o t h e c e l l u l a r heterogeneity and f r a g i l i t y of t h e intestinal e p i t h e l i u m which impose d i f f i c u l t i e s in d e s i g n i n g and e x e c u t i n g p h a r m a c o l o g i c a l s t u d i e s . N e v e r t h e l e s s i n t e s t i n a l ion t r a n s p o r t , c o n t r o l l e d by a wide v a r i e t y o f hormones, n e u r o t r a n s m i t t e r s and o t h e r m e d i a t o r s , p r o v i d e s a novel b i o l o g i c a l t e m p l a t e on which can be d e v e l o p e d f u t u r e i n v e s t i g a t i o n s i n t o t h e o p e r a t i o n s of t h e e n t e r i c and autonomic n er v o u s systems and on t h e properties of g a s t r o i n t e s t i n a l drug r e c e p t o r s and t h e e f f e c t o r s c o u p l e d t o them. F u r t h e r m o r e , i t r e p r e s e n t s an i m p o r t a n t avenue f o r t h e d i s c o v e r y and evaluation o f new a n t i d i a r r h e a l drugs (74), diuretics (75) and o t h e r pharmacotherapeutic agents. Pharmacological investigations in t h i s untapped and f a s c i n a t i n g area are c l e a r l y e s s e n t i a l ; all t h a t is r e q u i r e d is a l i t t l e n e r v e and a l o t of g u t s .
~cknowledgements The a u t h o r s thank Drs. Eugene B. Chang (Dept. of M e d i c i n e , U n i v e r s i t y of Chicago), S c o t t M. O'Grady (Dept. of V e t e r i n a r y B i o l o g y , U n i v e r s i t y of M i n n e s o t a ) and Timothy S. G a g i n e l l a (Dept. of M e d i c i n e , Ohio S t a t e U n i v e r s i t y ) f o r p r o v i d i n g i n f o r m a t i o n on t h e i r r e s e a r c h work and Ms. M i c h e l l e Mere f o r excellent illustrations. This work was s u p p o r t e d in p a r t by N . I . H . g r a n t s DK35260 and DK-37497 and Minnesota A g r i c u l t u r a l E x p e r i m e n t S t a t i o n g r a n t s 1~1061-37 and !Iq-061-63.
References
1.
2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
M. COSTA, J . B . FURNESS, and I . J . LLEWELLYN-SMITH, In: P h y s i o l o g y o f t h e G a s t r o i n t e s t i n a l T r a c t , 2nd E d . , L.R. J o h n s o n , e d . , pp. 1-40, Raven P r e s s , New York (1987). H . J . COOKE, I b i d . , pp. 1307-1350 (1987). D.R. BROWN and R . J . MILLER, In: Handbook of P h y s i o l o g y : G a s t r o i n t e s t i n a l P h y s i o l o g y ; A b s o r p t i v e and S e c r e t o r y P r o c e s s e s of t h e I n t e s t i n e s , M. F i e l d and R.A, F r i z z e l l , s e c t i o n e d s . , Am. P h y s i o l o g i c a l S o c . , B e t h e s d a , Maryland. In p r e s s . H.R. DE JONGE and S.M. LOHHANN, In: M i c r o b i a l Toxins and D i a v r h o e a l D i s e a s e , Ciba Fndn. Symp. 112, pp. 116-138, Pitman, London (19865. H.V. CAREY, H . J . COOKE and M. ZAFIROVA, J . P h y s i o l . (London) ~64 69-79 (1985). M.J. WELSH, P h y s i o l . Rev. 67 1143-1184 (1987). T . J . RIMELE and T.S. GAGINELLA, Biochem. Pharmacol. 31 515-520 (1982). M.J. KUHAR, In: N e u r o t r a n s m i t t e r R e c e p t o r B i n d i n g , 2nd E d . , H . I . Yamamura, S . J . Enna and M.J. Kuhar, e d s . , pp. 153-176, Raven P r e s s , New York (1985). K.A. HUBEL, J . C l i n . I n v e s t . 62 1039-1047 (1978). R. CHANDAN, S.M. NEWELL, B.G. T1LEDER, G. SOLDANI and D.R. BROWN, Fedn. P r e c . 46 1453 (1987). H . J . COOKE, Am. J . P h y s i o l . 246 G263-G267 (1984). H.V. CAREY, X.-Y. TIEN, L . J . WALLACE and H.J. COOKE, Am. J . P h y s i o l . 253 G323-G329 (1987). P.E.T. ISAACS, J . S . WHITEHEAD and Y.S. KIM, C l i n . S c i . 62 203-207 (1982). R. WAI-IAWISAN, L . J . WALLACE and T.S. GAGINELLA, J. Pharm. Pharmacol. 38 150-153 (1986).
2200
Receptors and Intestinal Ion Transport
Vol. 43, No. 26, 1988
15. N. BUCKLEY and G. BURNSTOCK, B r a i n Res. 2 9 4 1 5 . 2 2 (1984). 16. H.M. COX, K.A. HUNDAYand J . A . POAT, B r i t . J. Pharmacol. 8__7201-209 (1986). 17. H.M. COX, A.W. CUTHBERT and K.A. HUNDAY, B r i t . J . Pharmacol. 913393-401 (1987). 18. C. BIANCHI, J. GUTKOWSKA, G. THIBAULT, R. GARCIA, J. GENEST and M. CAb/TIN, H i s t o c h e m i s t r y 8 2 4 4 1 - 4 5 2 (1985). 19. H.P. VON SCHROEDER, E. NISHIHURA, C.H.S. MclNTOSH, A . H . J . BUCHAN, N. WILSON and J . R . LEDSOHE, Can. J. P h y s i o l . Pharmacol. 6 3 1 3 7 3 - 1 3 7 7 (1985). 20. J . F . KACHUR, R . J . MILLER, M. FIELD and J. RIVIER, J . Pharmacol. Exp. Thor. 2 2 0 4 4 9 - 4 5 5 (1982). 21. R. CHANDAN, S.M. NEWELL and D.R. BROWN, Regul. P e p t i d e s , in p r e s s . 22. S.R. VIGNA, C.R. HANTYH, A.S. GIRAUD, A.H. SOLL, J.H. WALSH and P.W. HANTYH, G a s t r o e n t e r o l o g y 9 3 1 2 8 7 - 1 2 9 5 (1987). 23. D.C. MANNING, S.H. SNYDER, J . F . KACHUR, R . J . HILLER and M. FIELD, N a t u r e ~ 9 9 2 5 6 - 2 5 9 (1982). 24. K.A. HUBEL and K.S. RENQUIST, J. Pharmacol. Exp. Thor. 2 3 8 1 6 7 - 1 6 9 (1986). 25. D.R. BROWN, S.L. BOSTER and M.F. OVEREND, Faseb J. 2 A1276 (1988). 26. D.D. FRIEL, R . J . HILLER and M.W. WALKER, B r i t . J . Pharmacoi. 8_88425-431 (1986). 27. H. LABURTHE, B. CHENUT, C. ROUYER-FESSARD, K. TATEHOTO, A. COUVINEAU, A. SERVIN and B. AHIRANOFF, E n d o c r i n o l o g y 11___881910-1917 (1986). 28. J . DOBBINS, L. RACUSEN and H.J. BINDER, J. C l i n . I n v e s t . 6 6 1 9 - 2 8 (1980). 29. E.B. CHANG, M. FIELD and R . J . MILLER, Am. J. P h y s i o l . 244 G76-G82 (1983). 30. B.S. TSAI, R.G. CONWAYand R.F. BAUER, Biochem. Pharmacol. 3 4 3 8 6 7 - 3 8 7 3 (1985). 31. J. KEAST, J . B . FLrI~SS and M. COSTA, N a u n y n - S c h m i e d e b o r g ' s Arch. Pharmacol. 3 3 3 3 9 3 - 3 9 9 (1986). 32. P. BUTCHER, M. HEHLIN and H. SJOVALL, Acta P h y s i o l . Scand. 1 3 1 2 3 5 - 2 4 1 (1987). 33. T.S. GAGINELLA, T . J . RIHELE and M. WIETECHA, J . P h y s i o l . (London) 3 3 5 1 0 1 111 (1983). 34. H . J . BINDER, J . P . LAURENSON and J.W. DOBBINS, Am. J. P h y s i o l . 247 G432G436 (1984). 35. E. NISHIMORA, A.M.J. BUCHAN and C.H.S. MclNTOSH, N e u r o s c i . L e t t . 5 0 7 3 - 7 8 (1984). 36. M.R. DASHWOOD, E.S. DEBNAM, J. BAGNALL and C.S. THOMPSON, Eur. J. Pharmacol. 107 267-269 (1985). 37. E. N I S H I I ~ , A.M.J. BUCHAN and C.H.S. McINTOSH, G a s t r o e n t e r o l o g y 91 10841094 (1986). 38. P . J . BIRCH, A.G. HAYES and C . J . SPRAGGS, B r i t . J. Pharmacol. 89 833P (1986). 39. M. WEBER, T. COLE and J.M. CONLON, Am. J. P h y s i o l . 250 6679-6685 (1986). 40. E. ARILLA, M.P. LOPEZ-RUIZ, L. GONZALEZ-GUIJARRO, J . C . PRIETO, A. GOMEZPAN and B.H. HIRST, Biochim. Biophys. Acta 8 0 2 2 0 3 - 2 0 8 (1984). 41. M.P. LOPEZ-RUIZ, E. ARILLA, L. GONZALEZ-GUIJARROand J . C . PRIETO, Comp. Biochom. P h y s i o l . 81B 1041-1044 (1985). 42. M.P. LOPEZ-RUIZ, L.G. GUIJARRO and E. ARILLA, Comp. Biochem. P h y s i o l . 89A 237-241 (1988). 43. J . F . KACHUR, R . J . HILLER, M. FIELD and J. RIVIER, J . Pharmacol. Exp. Thor. 2 2 0 4 5 6 - 4 6 3 (1982). 44. K.A. HUBEL, K.S. RENQUIST and S. SHIRAZI, G a s t r o e n t e r o l o g y 8__661118 (1984). 45. E. BURCHER, C.W. SHULTS, S.H. BUCK, T.N. CHASE and T.L. O'DONOHUE, Eur. J. Pharmacol. 1 0 2 5 6 1 - 5 6 2 (1984). 46. J . R . KEAST, J . B . FURNESS and M. COSTA, N a u n y n - S c h m i e d e b o r g ' s A r c h . Pharmacol. 3 2 9 3 8 2 - 3 8 7 (1985).
Vol. 43, No. 26, 1988
Receptors and Intestinal Ion Transport
2201
47. E. BURCHER, S.H. BUCK, W. LOVENBERG and T.L. O'DONOHUE, J. Pharmacol. Exp. Ther. 2 3 6 8 1 9 - 8 3 1 (1986). 48. M.H. PERDUE, R. GALBRAITH and J . S . DAVISON, Regul. P e p t i d e s 1__8863-74 (1987). 49. H . J . COOKE, M. ZAFIROVA, H.V. CAREY, J.H. WALSH and J . GRIDER, G a s t r o e n t e r o l o g y 9/2361-370 (1987). 50. J . C . PRIETO, M. LABURTHE and G. ROSSELIN, Eur. J. Biochem. 9 6 2 2 9 - 2 3 7 (1979). 51. H . J . BINDER, G.F. LEMP and J.D. GARDNER, Am. J. P h y s i o l . 238 G190-G196 (1980). 52. J . C . PRIETO, M. LABURTHE, D. HUI BON HOA and G. ROSSELIN, Acta E n d o c r i n o l . 9 6 1 0 0 - 1 0 6 (1981). 53. K. DHARMSATHAPHORN,V. HARMS, D.J. YAMASHIRO, R . J . HUGHES, H.J. BINDER and E.M. WRIGHT, J . C l i n . I n v e s t . 7 1 2 7 - 3 5 (1983). 54. H. SAYADI, B. BASS, T.W. MOODY, J.W. HARMON,and L.Y. KORMAN G a s t r o e n t e r o l o g y 9 4 A 4 0 2 (1988). 55. R.A. NORTH, B.E. SLACK and A. SURPRENANT, J . P h y s i o l . (London) 3 6 8 4 3 5 - 4 5 2 (1985). 56. E . J . TAPPER, D.W. POWELL and S.M. MORRIS, Am. J . P h y s i o l . 23___55E402-E409 (1978). 57. X-Y. TIEN, R. WAHAWISAN, L . J . WALLACE and T.S. GAGINELLA, L i f e S c i . 36 1949-1955 (1985). 58. T . J . RIMELE, M.S. O'DORISIO and T.S. GAGINELLA, J . Pharmacol. Exp. Ther. 2 1 8 4 2 6 - 4 3 4 (1981). 59. A. KUWAHARA, X.-Y. TIEN, L . J . WALLACE and H.J. COOKE, J. Pharmacol. Exp. Ther. 2 4 2 6 0 0 - 6 0 6 (1987). 60. E.B. CHANG, D.R. BROWN, N . - S . WANG and H. FIELD, J . C l i n . I n v e s t . 7 8 2 8 1 287 (1986). 61. J . F . KACHUR, W. ALLBEE, W. DANHO and T.S. GAGINELLA, Regul. P e p t i d e s 17 63-70 (1987). 62. A.W. CUTHBERT, P.V. HALUSHKA, H.S. MARGOLIUS and J.A. SPAYNE, B r i t . J. Pharmacol. 82 597-607 (1984). 63. R. HIROSE and E.B. CHANG, Am. J. P h y s i o l . 254 G891-G897 (1988). 64. K.J. MORIARTY, N.B. HIGGS, M. WOODFORD, G. WARHURST and L.A. TURNBERG, Gut 28 844-848 (1987). 65. S.M. O'GRADY, M. FIELD, N.T. NASH and M.C. RAO, Am. J. Physiol. 249 C531C534 (1985). 66. S.M. O'GRADY, H.R. DeJONGE, A.B. VAANDRAGER and M. FIELD, Am. J. Physiol. 254 Cl15-C121 (1988). 67. A.W. CUTHBERT and H.S. MARGOLIUS, B r i t . J . Pharmacol. 75 587-598 (1982). 68. M.W. MUSCH, J . F . KACHUR, R . J . MILLER, M. FIELD and J . S . STOFF, J. C l i n . I n v e s t . 71 1073-1083 (1983). 69. G. WARHURST, M. LEES, N.B. HIGGS and L.A. TURNBERG, Am. J. P h y s i o l . 252 G293-G300 (1987). 70. L.D. LAWSON and D.W. POWELL, Am. J. P h y s i o l . 252 G783-G790 (1987). 71. A.W. CUTHBERT, P.V. HALUSKA, H.S. MARGOLIUS and J.A. SPAYNE, Brit. J. Pharmacol. 8-2587-595 (1984). 72. A.W. CUTHBERT, P.V. HALUSKA, D. KESSEL, H.S. MARGOLIUS and W.C. WISE, B r i t . J . Pharmacol. 8 3 5 4 9 - 5 5 4 (1984). 73. A.W. CUTHBERT, S.C. KIRKLAND and L . J . MacVINISH, B r i t . J . Pharmacol. 8 6 3 5 (1985). 74. J . D . FONDACARO, Am. J . P h y s i o l . 250 G1-G8 (1986). 75. S.M. O'GRADY, H.C. PALFREY and M. FIELD, J. Membr. B i o l . 9 6 1 1 - 1 8 (1987). 76. H.M. COX, A.W. CUTHBERT, R. HAKANSONand C. WAHLESTEDT, J . P h y s i o l . (London) ~98 65-80 (1988).
Pergamon Press
IV. RECEPTOR REGULATION OF ION TRANSPORT IN THE INTESTINAL E P I T H E L I ~
David R. Brown 1 ' 3 , R i t u Chandan 1 ' 3 , F r a n c i s L. Q u i t o 1 and V i r g i n i a S. Seybold 2'3
U n i v e r s i t y of Minnesota 1Department of V e t e r i n a r y B i o l o g y , C o l l e g e of V e t e r i n a r y M e d i c i n e , 1988 F i t c h Avenue, S t . P a u l , Minnesota 55108 2Department of C e l l B i o l o g y and Neuroanatomy, and 3 N e u r o s c i e n c e Graduate Program, 4-135 J a c k s o n H a l l , 321 Church S t r e e t , M i n n e a p o l i s , Minnesota 55455
Summary The a c t i v e t r a n s p o r t of ions by t h e intestinal e p i t h e l i u m is regulated by a number of e n t e r i c n e u r o t r a n s m i t t e r s , hormones and o t h e r s u b s t a n c e s . Our knowledge of t h e r e c e p t o r s m e d i a t i n g t h e actions o f t h e s e s u b s t a n c e s is g e n e r a l l y f r a g m e n t a r y . This r e v i e w summarizes c u r r e n t knowledge on t h e l o c a t i o n and f u n c t i o n a l characteristics of t r a n s m i t t e r receptors regulating transport f u n c t i o n in t h e small i n t e s t i n e , h i g h l i g h t i n g r e c e n t r e s e a r c h on c h o l i n e r g i c and b r a d y k i n i n r e c e p t o r s .
The i n t e s t i n a l mucosa h o l d s p r e c i s e l y in b a l a n c e t h e a b s o r p t i o n and s e c r e t i o n o f w a t e r and ions so as t o m a i n t a i n h o m e o s t a s i s in body f l u i d volume and c o m p o s i t i o n , p r o v i d e an aqueous medium f o r t h e b i o t r a n s f o r m a t i o n of n u t r i e n t s and o t h e r s u b s t a n c e s , and r i n s e t h e lumen of h o s t i l e m i c r o o r g a n i s m s . The f u n c t i o n of e p i t h e l i a l c e l l s i n v o l v e d in t h e s e t r a n s p o r t processes is regulated by a complex a r r a y of n e r v e s o r i g i n a t i n g from t h e two major g a n g l i o n a t e d p l e x u s e s of t h e gut and from neurons l y i n g o u t s i d e t h e d i g e s t i v e system. This n e u r a l c i r c u i t r y s e r v e s t o c o o r d i n a t e t h e a b s o r p t i v e and motor functions of the intestines. In a d d i t i o n , v a r i o u s hormonal substances synthesized and r e l e a s e d from e n t e r o e n d o c r i n e c e l l s can m o d u l a t e e p i t h e l i a l function e i t h e r through d i r e c t i n t e r a c t i o n s with t r a n s p o r t i n g enterocytes or by a l t e r i n g e n t e r i c n e u r a l a c t i v i t y . The m o r p h o l o g i c and f u n c t i o n a l a s p e c t s of t h e neurohormonal c o n t r o l o f t h e i n t e s t i n a l e p i t h e l i u m have been s u b j e c t s of r e c e n t r e v i e w a r t i c l e s (1 - 3). In t h i s a r t i c l e , we s h a l l r e v i e w b r i e f l y t h e proposed mechanisms u n d e r l y i n g ion t r a n s p o r t in t h e i n t e s t i n a l e p i t h e l i u m and d i s c u s s t h e p h a r m a c o l o g i c a l characteristics of r e c e p t o r s f o r s e l e c t e d t r a n s m i t t e r substances thought to r e g u l a t e t h i s system.
0024-3025/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
2194
Receptors and Intestinal Ion Transport
Intestinal
Vol. 43, No. 26, 1988
Ion Transport
In the mammalian small intestine, villus and c r y p t cells lining the epithelium are believed to subserve ion transporting functions. It has been p r o p o s e d t h a t t h e a c t i v e a b s o r p t i o n o f Na t , a n i o n s (Cl', HCO3" e t c . ) and organic substrates i s a c c o m p l i s h e d by c e l l s l i n i n g t h e i n t e s t i n a l villi (Fig. 1 ) . C h l o r i d e a b s o r p t i o n ( m o v e m e n t f r o m lumen t o blood) may o r may n o t be linked t o Na + t r a n s p o r t depending upon t h e i n t e s t i n a l s e g m e n t and s p e c i e s e x a m i n e d . Sodium a b s o r p t i o n i s d e p e n d e n t upon t h e operation of a ouabainsensitive Na÷/K÷-ATPase situated on t h e basolateral c e l l m e m b r a n e . On t h e other hand, ceils located in the intestinal glands or c~ypts of Lieberkuehn appear t o s e c r e t e a c t i v e l y CI" ( a n d i n some c a s e s , HCO3 ) . C h l o r i d e s e c r e t i o n ( m o v e m e n t f r o m b l o o d t o lumen) a p p e a r s t o be a t w o - s t e p process: first, a basolateral coupled influx o f Na t and CI" a l l o w s [ C l ' ] t o r i s e w i t h i n t h e crypt cell to levels above its electrochemical equilibrium and s e c o n d , alterations in the permeability o f t h e a p i c a l c e l l membrane p r o m o t e CI" e x i t into the intestinal lumen ( F i g . 1 ) .
.......
+umo~
bloo~
FIG. 1. Hodel f o r i o n t r a n s p o r t in mammalian small intestine.
WLLI
LOOP DlUnETICS lug,.
blood
; ~
B~*
K÷
The i n t e s t i n a l epithelium is relatively "leaky" relative to other epithelia, s u c h as t h o s e o f t h e c o l o n o r u r i n a r y b l a d d e r , and b o t h t h e b u l k movement o f water in response to o s m o t i c g r a d i e n t s and p a s s i v e i o n f l u x e s o c c u r i n i t s paracellular spaces. The s p o n t a n e o u s potential difference (p.d.) which develops across the epithelium is the bioelectrical result of ongoing active and p a s s i v e i o n t r a n s p o r t processes. It is possible to nullify this transmural p . d . i n Vi~Fo by v o l t a g e - c l a m p i n g the gut epithelium, thereby greatly reducing (or theoretically, eliminating) the passive electrodiffusion of ions. The resulting short-circuit c u r r e n t ( I s c ) i s t h e sum o f a l l a c t i v e i o n t r a n s p o r t by t h e t i s s u e and i s e l e c t r o c h e m i c a l l y related to the net transepithelial f l u x e s o f Na t and C l ' .
Vol. 43, No. 26, 1988
Receptors and Intestinal Ion Transport
2195
A number of intracellular second messengers have been implicated in the regulation of intestinal ion transport. These intracellular regulators include the cyclic nucleotides (cAMP, cGHP), free Ca 2+ , and probably phosphoinositides, p r o t e i n k i n a s e C and e i c o s a n o i d s . Their potential roles in ion transport processes h a v e b e e n r e v i e w e d ( 4 ) and t h e a c t i o n s o f some o f these substances are illustrated below in relation to their coupling to gut transmitter receptors (Fig. 2). Localization
of Transmitter
Receptors
Transmitter receptors have been localized within the gut through the use of physiological, biochemical, and anatomical methodologies. In general, receptors for transmitter substances affecting intestinal ion transport are localized primarily on t h e s e r o s a l a s p e c t o f t h e e p i t h e l i u m ; exposure of the luminal aspect to concentrations of transmitters far exceeding effective serosal concentrations is often not associated with alterations in transport. On t h e s e r o s a l a s p e c t o f t h e i n t e s t i n a l mucosa, receptors for transmitters have been tentatively l o c a l i z e d on e p i t h e l i a l c e l l s o r on n e r v o u s e l e m e n t s i n the submucosa examining agonist activity in denervated tissue preparations (5) o r t h e a b s e n c e and p r e s e n c e of neuronal conduction blockade produced by tetrodotoxin (TTX). Changes in ion transport occurring after transmitter application to these "denervated" preparations h a v e b e e n t a k e n as e v i d e n c e f o r a d i r e c t a c t i o n on b a s o l a t e r a l epithelial cell membranes; it is assumed that the actions of transmitters a c t i n g a t r e c e p t o r s on e n t e r i c n e u r o n s w i l l be eliminated by t h e s e maneuvers. These experiments do n o t r u l e out the possibility that a given transmitter acts secondary to the release of other bioactive substances from endocrine or paracrine-type cells in the gut epithelium-subepithelium. Information on t h e location and p h a r m a c o l o g i c a l characteristics of transmitter r e c e p t o r s on i d e n t i f i e d e n t e r i c n e u r o n s c a n be obtained through the use of electrophysiological techniques in which the action of the transmitter s u b s t a n c e on n e u r o n a l a c t i v i t y c a n be d e t e r m i n e d . Unfortunately, it has not been possible to link specific transmitter-induced changes in neural activity with ion transport. Electrophysiological techniques c o u l d be a p p l i e d t o t h e s t u d y o f r e c e p t o r - m e d i a t e d changes in ion fluxes at the level of individual epithelial cells or intestinal cell cultures; such strategies have been deployed successfully in other preparations, s u c h as the tracheal epithelium (6), but they have not been extensively utilized in the study of intestinal epithelial function. Biochemical assays of specific transmitter binding to high-affinity sites in the intestinal mucosa have been reported in mucosal homogenates, slide-mounted t i s s u e s e c t i o n s and e n t e r o c y t e membrane f r a c t i o n s . Ligand binding t o mucus secreted by c e l l homogenate preparations spontaneously or in response to transmitter exposure represents a potential artifact in receptor characterization (7). The u s e o f s l i d e - m o u n t e d t i s s u e s e c t i o n s i s a p r o c e d u r e first d e v e l o p e d by Young and K u h a r ( 8 ) w h i c h a f f o r d s a b e t t e r a p p r o x i m a t i o n o f receptor distribution i_nn s i t u , a h i g h e r d e g r e e o f s p e c i f i c b i n d i n g and no mucus a r t i f a c t s because it involves less tissue disruption and e x t e n s i v e washing steps. Both procedures suffer from the disadvantage that transmitter b i n d i n g s i t e s may n o t be d i r e c t l y related to functional receptors mediating ion transport if binding occurs to neurons or other non-transporting cells present in the preparation. Transmitter binding sites c a n be l o c a l i z e d and quantified within subregions of the intestinal mucosa t h r o u g h t h e u s e o f quantitative autoradiographic procedures in tissue sections. Binding studies using cell membrane fractions have been conducted frequently and g e n e r a l l y
2196
Receptors and Intestinal Ion Transport
allow for level.
optimum b i o c h e m i c a l
Localization
TTX A c t i on
Acetyl choline
( gp, p : - )
[9151 Angiotensin [ 1 6 , 17]
(rt:-)
II
[20 - 2 2 ] Bradykinin [ 1 6 , 231 Neuropeptide Y [24 - 2 7 , 7 6 ]
Norepinephrine
(gp:-
(rb,
Mediating
at the epithelial
(rt:
? Yes ( r t ,
rt:-;gp:÷
?
?
; p:±)
rb:-;
cell
Ion Transport i n S m a l l
Yes ( r t )
(gp:-) (rt,
sites
S p e c i f i c B i n d i n g S i t e s D e t e c t e d i n Mucosa: Biochemically Autoradiographically Yes ( r t ) ( g p : SP)
(fl:-) 19; S.M O ' G r a d y , s u b m i t t e d ]
Bombesin/GRP
of binding
TABLE 1 Receptors Intestine
o f Some T r a n s m i t t e r
T r ansmi t t e r
[18,
analysis
Vol. 43, No. 26, 1988
p:,) )
possibly
E)
( d g : MP) gp)
(gp:
SP, E)
Yes ( r t )
?
Yes ( r b )
?
[28 - 3 2 ]
Opioids
(p, gp, rb:+) No ( r t , rb) ( r t , gp: SP, E) F.L. Q u i t o and D.R. Brown, u n p u b l i s h e d o b s e r v a t i o n s ) (gp:±; p:÷) Yes ( p : m e m b r a n e ; rodent: cytosolic) ? [ 3 1 , 39 - 4 2 ; H.F. O v e r e n d and D.R. Brown, s u b m i t t e d ] Substance P (gp, r b : ± ; p : - ) ? ( r t : n o n e ; gp: SP, E) [43 - 48] VIP (gp, rb:-) Yes ( r t , gp, r b ) ( h : E) [49 - 54] [ 2 8 , 33 - 38; Somatostatin
(+) = f u l l i n h i b i t i o n of ion transport effects; (±) = p a r t i a l inhibition; ( - ) = no i n h i b i t i o n ; h= human; p = p i g ; dg = dog; r b = r a b b i t ; rt = rat; gp = g u i n e a p i g ; f l = w i n t e r f l o u n d e r ; l ~ = m y e n t e r i c p l e x u s ; SP = submucous plexus; E = intestinal epithelium; ? = not determined. Bracketed numbers indicate references.
Selected Acetylcholine
Mediators
Regulating
Ion Transport
(ACh)
Cholinergic receptors a r e l o c a l i z e d on b o t h n e u r o n a l and e p i t h e l i a l cells of the small intestine. The r e c e p t o r s m e d i a t i n g t h e a c t i o n s o f ACh t o depolarize submucous p l e x u s n e u r o n s and t o p r e s y n a p t i c a l l y i n h i b i t t h e r e l e a s e o f ACh and norepinephrine a p p e a r t o be o f t h e M1- and M 2 - m u s c a r i n i c c h o l i n e r g i c subtype, respectively (55). In addition, muscarinic cholinergic agonists rapidly evoke a serosa-positive increase in Isc in the small intestinal mucosa from most s p e c i e s . When e x a m i n e d i n more d e t a i l , this tissue Isc response represents a stimulation o f a c t i v e a n i o n s e c r e t i o n and i s r e s i s t a n t t o TTX ( T a b l e 1 ) . H i g h concentrations of the non-selective agonist carbachol have been observed to produce, in addition to an initial Isc elevation, a long latency decrease in Isc below baseline levels in the rabbit ileum. This effect seems t o involve
Vol. 43, No. 26, 1988
Receptors and Intestinal Ion Transport
t h e c a r b a c h o l - i n d u c e d a c t i v a t i o n of g a n g l i o n i c n i c o t i n i c w i t h e n t e r i c s y m p a t h e t i c neurons (56).
receptors
2197
associated
Ligand b i n d i n g a s s a y s r e v e a l t h a t c h o l i n e r g i c r e c e p t o r s on c r y p t and v i l l u s cells possess similar binding characteristics t o c h o l i n e r g i c a g o n i s t s and a n t a g o n i s t s (14) and d i f f e r from t h o s e on i n t e s t i n a l smooth muscle ( 5 7 ) . In displacing the potent muscarinic antagonist [3H]-quinuclidinyl benzilate (QNB), c h o l i n e r g i c l i g a n d s d i s p l a y o r d e r s of p o t e n c y of a t r o p i n e > s c o p o l a m i n e >> p i r e n z e p i n e > o x o t r e m o r i n e >> c a r b a c h o l in i l e a l v i l l u s and c r y p t c e l l s of t h e r a t ileum ( 1 4 ) , 4-DAMP > p i r e n z e p i n e > HeN A-343 > c a r b a c h o l > b e t h a n e c h o l in mucosal scrapings o f t h e guinea p i g i l e a l mucosa ( 1 2 ) , and a t r o p i n e > 4I~ >> p i r e n z e p i n e in s l i d e - m o u n t e d s e c t i o n s of t h e p o r c i n e j e j u n a l mucosa (V.S. S e y b o l d , R. Chandan, X.-F. Sun and D.R. Brown, s u b m i t t e d ) . T r i t i a t e d QNB binds t o t h e s e sites w i t h l ~ s r a n g i n g from 60 - 400 pM, depending upon t h e p r e p a r a t i o n employed. S p e c i f i c , - h i g h - a f f i n i t y [3H]-QNB b i n d i n g s i t e s with similar affinities for cholinergic agonists and a n t a g o n i s t s have been c h a r a c t e r i z e d in r a t and guinea p ig c o l o n i c mucosa (58, 59). In t h e guinea p i g i l e a l and c o l o n i c mucosa, t h e rank o r d e r s of b i n d i n g a f f i n i t i e s of m u s c a r i n i c cholinergic antagonists a g r e e s w e l l w i t h t h e i r o r d e r s of p o t e n c y in a l t e r i n g ion t r a n s p o r t induced by e l e c t r i c a l f i e l d s t i m u l a t i o n (EFS) of t h e s e t i s s u e s ; EFS ap p ear s t o r e l e a s e endogenous ACh from submucosal neurons (12, 59). There a r e no p u b l i s h e d data on t h e a b i l i t y of t h e s e a n t a g o n i s t s t o s h i f t c h o l i n e r g i c agonist dose-response curves for a l t e r i n g transport. The a v a i l a b l e data suggest that the muscarinic r e c e p t o r mediating the t r a n s p o r t - r e l a t e d actions of ACh is o f t h e M2-subtype, but f u r t h e r d e f i n i t i o n of t h i s receptor awaits additional investigation u s i n g a g r e a t e r v a r i e t y of s e l e c t i v e l i g a n d s . The e f f e c t o r ( s ) c o u p l i n g a g o n i s t * r e c e p t o r b i n d i n g to an i o n s e c r e t i o n have not been c h a r a c t e r i z e d f u l l y in mammalian i n t e s t i n a l p r e p a r a t i o n s ( F i g . 2). In i s o l a t e d villus cells from t h e c h i c k e n ileum however, a c t i v a t i o n of m u s c a r i n i c receptors l e a d s t o an i n c r e a s e in p h o s p h o i n o s i t i d e t u r n o v e r w i t h a c o n s e q u e n t r i s e in f r e e i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n . These b i o c h e m i c a l e v e n t s ar e temporally correlated with cholinergic agonist-induced changes in cell membrane ion t r a n s p o r t p r o c e s s e s (60; E.B. Chang and M.W. Musch, s u b m i t t e d ) .
Bradykinin
(BK)
K i n i n s a r e l i b e r a t e d from damaged i n t e s t i n a l t i s s u e and may m e d i a t e a c t i v e an i o n s e c r e t i o n in i n f l a m m a t o r y s t a t e s of t h e bowel. BK, k a l l i d i n (Lys-BK) and t h e i r a n a l o g s s t i m u l a t e n e t C1 s e c r e t i o n and t r a n s i e n t l y i n c r e a s e I s c in both t h e small intestine and c o l o n (67, 68). Moreover, s p e c i f i c b i n d i n g s i t e s h a v i n g h i g h a f f i n i t y f o r t h e s e p e p t i d e s have been d e t e c t e d on small and l a r g e intestinal epithelial cells by b i o c h e m i c a l and a u t o r a d i o g r a p h i c t e c h n i q u e s (16, 23). In t h e guinea pig i l e a l mucosa, k i n i n s have a r e l a t i v e p o t e n c y o r d e r f o r i n c r e a s i n g I s c of BK > k a l l i d i n > Met-Lys-BK > Hydroxyproline3-BK > ~ - ( 2 T h i e n y l ) - A l a S ' a - B K > Des-Argg-BK which c o r r e l a t e s w e l l w i t h t h e i r a f f i n i t i e s f o r s p e c i f i c BK b i n d i n g s i t e s in t h e t i s s u e (23). The o r d e r of a f f i n i t i e s for these substances in d i s p l a c i n g [12sI]-BK from s p e c i f i c b i n d i n g s i t e s in r a t j e j u n a l e p i t h e l i a l membranes is s i m i l a r : BK ~ Tyra-BK > k a l l i d i n >> Des-Arg 1BK (1 6 ) . Using BK a n t a g o n i s t s s p e c i f i c f o r k i n i n r e c e p t o r s u b t y p e s , Kachur e t e l . (61) have d e t e r m i n e d in t h e guinea p i g ileum t h a t t h e a c t i o n s of k i n i n s in c o n t r a c t i n g l o n g i t u d i n a l smooth muscle and e l i c i t i n g anion s e c r e t i o n a r e both m e d i a t e d by BK2-type r e c e p t o r s . The i n t r a c e l l u l a r m e d i a t o r s of k i n i n - i n d u c e d s e c+ +r e t i o n appear t o be both a r a c h i d o n a t e m e t a b o l i t e s and f r e e intracellular Ca . The p e p t i d e s appear t o induce p r o s t a n o i d and l e u k o t r i e n e f o r m a t i o n by a c t i n g upon s u b e p i t h e l i a l c e l l s ; however, c y c l o o x y g e n a s e i n h i b i t o r s such as
2198
Receptors and Intestinal Ion Transport
Vol. 43, No. 26, 1988
indomethacin reduce, but do not e l i m i n a t e t i s s u e Isc responses to kinin e x p o s u r e (62, 68 - 70)• The d i r e c t e f f e c t s of k i n i n s on ion t r a n s p o r t in intestinal epithelial cells occur independent of s u b e p i t h e l i a l prostanoid s y n t h e s i s and a p p e a r t o r e q u i r e e x t r a c e l l u l a r Ca ++ (62, 71 73)• Further research i n t h i s a r e a should a t t e m p t t o r e l a t e p e p t i d e - r e c e p t o r b i n d i n g w i t h changes in i n t r a c e l l u l a r Ca ++ and ion t r a n s p o r t function in isolated epithelial cells.
A C! -
,
|ATP
VIP
C
B CI-
cAMP
Na+
CI¢
I
L. '"~ ':
;
SP
Na÷
,
K* CF Na*
Cl-
~
....
Ca2÷ %. A( LOPERAMIDE
~ . C F x ATROPINE ANP
FIG. 2
Some Proposed Receptors and Second Nossengers Nediating I n t e s t i n a l
Ion
Transport.
(A) V a s o a c t i v e i n t e s t i n a l p e p t i d e (VIP) b i n d i n g t o r e c e p t o r s on enterocytes has been directly correlated w i t h c y c l i c AMP p r o d u c t i o n and a c t i v e anion s e c r e t i o n • The p e p t i d e may a l s o inhibit NaCl a b s o r p t i o n [ 5 3 ] . (B) A number of s u b s t a n c e s a r e b e l i e v e d t o a c t t h r o u g h Ca+÷-dependent i n t r a c e l l u l a r mechanisms• These i n c l u d e +÷ s u b s t a n c e . P (SP), which may • ÷÷ i n c r e a s e f r e e ] n t r a c e l l u l a r Ca by promoting e++x t r a c e l l u l a r Ca entry through l o p e r a m i d e - s e n s i t i v e membrane Ca c h a n n e l s [60] and a c e t y l c h o l i n e (ACh) which acts on muscarinic receptors to increase p h o s p h o i n o s i t i d e ( P I ) t u r n o v e r and i n o s i t o l 1,4,5-trisphosphate production (IP3). IP 3 in t u r n m o b i l i z e s Ca t÷ s e q u e s t e r e d in t h e e n d o p l a s m i c r e t i c u l u m (ER). E p i t h e l i a l B z - b r a d y k i n i n and 5-HT 2serotoninergic r e c e p t o r s may a l s o be co u p l ed t o PI breakdown t h r o u g h p h o s p h o l i p a s e C (PLC) [61 - 64; E.B. Chang and M.W. Musch, submitted]. (C) A l t h o u g h unknown in mammalian enterocytes, basolateral receptors c o u p l e d t o c y c l i c Gt~ p r o d u c t i o n have been d e t e c t e d in t h e t e l e o s t ( w i n t e r f l o u n d e r ) i n t e s t i n e and s e r v e t o mediate the i n h i b i t o r y e f f e c t s o f a t r i a l n a t r i u r e t i c p e p t i d e s on Na-K-C1 c o t r a n s p o r t in t h i s t i s s u e [65, 66].
Conclusions The s t u d y o f t h e r e g u l a t i o n and pharmacology of i n t e s t i n a l ion t r a n s p o r t is in its infancy. D e s p i t e numerous i n v e s t i g a t i o n s of t h e n e u r o c h e m i c a l morphology o f t h e ~,,t. the electrophysiology of e n t e r i c neurons, the
Vol. 43, No. 26, 1988
Receptors and Intestinal Ion Transport
2199
e p i t h e l i a l mechanisms u n d e r l y i n g ion t r a n s p o r t and c u r s o r y e x a m i n a t i o n s o f t h e effects of a v a r i e t y o f s u b s t a n c e s on e l e c t r o l y t e f l u x e s in t h e i n t e s t i n a l epithelium, surprisingly little i s known about t h e r e c e p t o r s m e d i a t i n g t i s s u e responses to neural and hormonal t r a n s m i t t e r s in t h i s t i s s u e . T h i s s t a t e of a f f a i r s i s in p a r t due t o t h e c e l l u l a r heterogeneity and f r a g i l i t y of t h e intestinal e p i t h e l i u m which impose d i f f i c u l t i e s in d e s i g n i n g and e x e c u t i n g p h a r m a c o l o g i c a l s t u d i e s . N e v e r t h e l e s s i n t e s t i n a l ion t r a n s p o r t , c o n t r o l l e d by a wide v a r i e t y o f hormones, n e u r o t r a n s m i t t e r s and o t h e r m e d i a t o r s , p r o v i d e s a novel b i o l o g i c a l t e m p l a t e on which can be d e v e l o p e d f u t u r e i n v e s t i g a t i o n s i n t o t h e o p e r a t i o n s of t h e e n t e r i c and autonomic n er v o u s systems and on t h e properties of g a s t r o i n t e s t i n a l drug r e c e p t o r s and t h e e f f e c t o r s c o u p l e d t o them. F u r t h e r m o r e , i t r e p r e s e n t s an i m p o r t a n t avenue f o r t h e d i s c o v e r y and evaluation o f new a n t i d i a r r h e a l drugs (74), diuretics (75) and o t h e r pharmacotherapeutic agents. Pharmacological investigations in t h i s untapped and f a s c i n a t i n g area are c l e a r l y e s s e n t i a l ; all t h a t is r e q u i r e d is a l i t t l e n e r v e and a l o t of g u t s .
~cknowledgements The a u t h o r s thank Drs. Eugene B. Chang (Dept. of M e d i c i n e , U n i v e r s i t y of Chicago), S c o t t M. O'Grady (Dept. of V e t e r i n a r y B i o l o g y , U n i v e r s i t y of M i n n e s o t a ) and Timothy S. G a g i n e l l a (Dept. of M e d i c i n e , Ohio S t a t e U n i v e r s i t y ) f o r p r o v i d i n g i n f o r m a t i o n on t h e i r r e s e a r c h work and Ms. M i c h e l l e Mere f o r excellent illustrations. This work was s u p p o r t e d in p a r t by N . I . H . g r a n t s DK35260 and DK-37497 and Minnesota A g r i c u l t u r a l E x p e r i m e n t S t a t i o n g r a n t s 1~1061-37 and !Iq-061-63.
References
1.
2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
M. COSTA, J . B . FURNESS, and I . J . LLEWELLYN-SMITH, In: P h y s i o l o g y o f t h e G a s t r o i n t e s t i n a l T r a c t , 2nd E d . , L.R. J o h n s o n , e d . , pp. 1-40, Raven P r e s s , New York (1987). H . J . COOKE, I b i d . , pp. 1307-1350 (1987). D.R. BROWN and R . J . MILLER, In: Handbook of P h y s i o l o g y : G a s t r o i n t e s t i n a l P h y s i o l o g y ; A b s o r p t i v e and S e c r e t o r y P r o c e s s e s of t h e I n t e s t i n e s , M. F i e l d and R.A, F r i z z e l l , s e c t i o n e d s . , Am. P h y s i o l o g i c a l S o c . , B e t h e s d a , Maryland. In p r e s s . H.R. DE JONGE and S.M. LOHHANN, In: M i c r o b i a l Toxins and D i a v r h o e a l D i s e a s e , Ciba Fndn. Symp. 112, pp. 116-138, Pitman, London (19865. H.V. CAREY, H . J . COOKE and M. ZAFIROVA, J . P h y s i o l . (London) ~64 69-79 (1985). M.J. WELSH, P h y s i o l . Rev. 67 1143-1184 (1987). T . J . RIMELE and T.S. GAGINELLA, Biochem. Pharmacol. 31 515-520 (1982). M.J. KUHAR, In: N e u r o t r a n s m i t t e r R e c e p t o r B i n d i n g , 2nd E d . , H . I . Yamamura, S . J . Enna and M.J. Kuhar, e d s . , pp. 153-176, Raven P r e s s , New York (1985). K.A. HUBEL, J . C l i n . I n v e s t . 62 1039-1047 (1978). R. CHANDAN, S.M. NEWELL, B.G. T1LEDER, G. SOLDANI and D.R. BROWN, Fedn. P r e c . 46 1453 (1987). H . J . COOKE, Am. J . P h y s i o l . 246 G263-G267 (1984). H.V. CAREY, X.-Y. TIEN, L . J . WALLACE and H.J. COOKE, Am. J . P h y s i o l . 253 G323-G329 (1987). P.E.T. ISAACS, J . S . WHITEHEAD and Y.S. KIM, C l i n . S c i . 62 203-207 (1982). R. WAI-IAWISAN, L . J . WALLACE and T.S. GAGINELLA, J. Pharm. Pharmacol. 38 150-153 (1986).
2200
Receptors and Intestinal Ion Transport
Vol. 43, No. 26, 1988
15. N. BUCKLEY and G. BURNSTOCK, B r a i n Res. 2 9 4 1 5 . 2 2 (1984). 16. H.M. COX, K.A. HUNDAYand J . A . POAT, B r i t . J. Pharmacol. 8__7201-209 (1986). 17. H.M. COX, A.W. CUTHBERT and K.A. HUNDAY, B r i t . J . Pharmacol. 913393-401 (1987). 18. C. BIANCHI, J. GUTKOWSKA, G. THIBAULT, R. GARCIA, J. GENEST and M. CAb/TIN, H i s t o c h e m i s t r y 8 2 4 4 1 - 4 5 2 (1985). 19. H.P. VON SCHROEDER, E. NISHIHURA, C.H.S. MclNTOSH, A . H . J . BUCHAN, N. WILSON and J . R . LEDSOHE, Can. J. P h y s i o l . Pharmacol. 6 3 1 3 7 3 - 1 3 7 7 (1985). 20. J . F . KACHUR, R . J . MILLER, M. FIELD and J. RIVIER, J . Pharmacol. Exp. Thor. 2 2 0 4 4 9 - 4 5 5 (1982). 21. R. CHANDAN, S.M. NEWELL and D.R. BROWN, Regul. P e p t i d e s , in p r e s s . 22. S.R. VIGNA, C.R. HANTYH, A.S. GIRAUD, A.H. SOLL, J.H. WALSH and P.W. HANTYH, G a s t r o e n t e r o l o g y 9 3 1 2 8 7 - 1 2 9 5 (1987). 23. D.C. MANNING, S.H. SNYDER, J . F . KACHUR, R . J . HILLER and M. FIELD, N a t u r e ~ 9 9 2 5 6 - 2 5 9 (1982). 24. K.A. HUBEL and K.S. RENQUIST, J. Pharmacol. Exp. Thor. 2 3 8 1 6 7 - 1 6 9 (1986). 25. D.R. BROWN, S.L. BOSTER and M.F. OVEREND, Faseb J. 2 A1276 (1988). 26. D.D. FRIEL, R . J . HILLER and M.W. WALKER, B r i t . J . Pharmacoi. 8_88425-431 (1986). 27. H. LABURTHE, B. CHENUT, C. ROUYER-FESSARD, K. TATEHOTO, A. COUVINEAU, A. SERVIN and B. AHIRANOFF, E n d o c r i n o l o g y 11___881910-1917 (1986). 28. J . DOBBINS, L. RACUSEN and H.J. BINDER, J. C l i n . I n v e s t . 6 6 1 9 - 2 8 (1980). 29. E.B. CHANG, M. FIELD and R . J . MILLER, Am. J. P h y s i o l . 244 G76-G82 (1983). 30. B.S. TSAI, R.G. CONWAYand R.F. BAUER, Biochem. Pharmacol. 3 4 3 8 6 7 - 3 8 7 3 (1985). 31. J. KEAST, J . B . FLrI~SS and M. COSTA, N a u n y n - S c h m i e d e b o r g ' s Arch. Pharmacol. 3 3 3 3 9 3 - 3 9 9 (1986). 32. P. BUTCHER, M. HEHLIN and H. SJOVALL, Acta P h y s i o l . Scand. 1 3 1 2 3 5 - 2 4 1 (1987). 33. T.S. GAGINELLA, T . J . RIHELE and M. WIETECHA, J . P h y s i o l . (London) 3 3 5 1 0 1 111 (1983). 34. H . J . BINDER, J . P . LAURENSON and J.W. DOBBINS, Am. J. P h y s i o l . 247 G432G436 (1984). 35. E. NISHIMORA, A.M.J. BUCHAN and C.H.S. MclNTOSH, N e u r o s c i . L e t t . 5 0 7 3 - 7 8 (1984). 36. M.R. DASHWOOD, E.S. DEBNAM, J. BAGNALL and C.S. THOMPSON, Eur. J. Pharmacol. 107 267-269 (1985). 37. E. N I S H I I ~ , A.M.J. BUCHAN and C.H.S. McINTOSH, G a s t r o e n t e r o l o g y 91 10841094 (1986). 38. P . J . BIRCH, A.G. HAYES and C . J . SPRAGGS, B r i t . J. Pharmacol. 89 833P (1986). 39. M. WEBER, T. COLE and J.M. CONLON, Am. J. P h y s i o l . 250 6679-6685 (1986). 40. E. ARILLA, M.P. LOPEZ-RUIZ, L. GONZALEZ-GUIJARRO, J . C . PRIETO, A. GOMEZPAN and B.H. HIRST, Biochim. Biophys. Acta 8 0 2 2 0 3 - 2 0 8 (1984). 41. M.P. LOPEZ-RUIZ, E. ARILLA, L. GONZALEZ-GUIJARROand J . C . PRIETO, Comp. Biochom. P h y s i o l . 81B 1041-1044 (1985). 42. M.P. LOPEZ-RUIZ, L.G. GUIJARRO and E. ARILLA, Comp. Biochem. P h y s i o l . 89A 237-241 (1988). 43. J . F . KACHUR, R . J . HILLER, M. FIELD and J. RIVIER, J . Pharmacol. Exp. Thor. 2 2 0 4 5 6 - 4 6 3 (1982). 44. K.A. HUBEL, K.S. RENQUIST and S. SHIRAZI, G a s t r o e n t e r o l o g y 8__661118 (1984). 45. E. BURCHER, C.W. SHULTS, S.H. BUCK, T.N. CHASE and T.L. O'DONOHUE, Eur. J. Pharmacol. 1 0 2 5 6 1 - 5 6 2 (1984). 46. J . R . KEAST, J . B . FURNESS and M. COSTA, N a u n y n - S c h m i e d e b o r g ' s A r c h . Pharmacol. 3 2 9 3 8 2 - 3 8 7 (1985).
Vol. 43, No. 26, 1988
Receptors and Intestinal Ion Transport
2201
47. E. BURCHER, S.H. BUCK, W. LOVENBERG and T.L. O'DONOHUE, J. Pharmacol. Exp. Ther. 2 3 6 8 1 9 - 8 3 1 (1986). 48. M.H. PERDUE, R. GALBRAITH and J . S . DAVISON, Regul. P e p t i d e s 1__8863-74 (1987). 49. H . J . COOKE, M. ZAFIROVA, H.V. CAREY, J.H. WALSH and J . GRIDER, G a s t r o e n t e r o l o g y 9/2361-370 (1987). 50. J . C . PRIETO, M. LABURTHE and G. ROSSELIN, Eur. J. Biochem. 9 6 2 2 9 - 2 3 7 (1979). 51. H . J . BINDER, G.F. LEMP and J.D. GARDNER, Am. J. P h y s i o l . 238 G190-G196 (1980). 52. J . C . PRIETO, M. LABURTHE, D. HUI BON HOA and G. ROSSELIN, Acta E n d o c r i n o l . 9 6 1 0 0 - 1 0 6 (1981). 53. K. DHARMSATHAPHORN,V. HARMS, D.J. YAMASHIRO, R . J . HUGHES, H.J. BINDER and E.M. WRIGHT, J . C l i n . I n v e s t . 7 1 2 7 - 3 5 (1983). 54. H. SAYADI, B. BASS, T.W. MOODY, J.W. HARMON,and L.Y. KORMAN G a s t r o e n t e r o l o g y 9 4 A 4 0 2 (1988). 55. R.A. NORTH, B.E. SLACK and A. SURPRENANT, J . P h y s i o l . (London) 3 6 8 4 3 5 - 4 5 2 (1985). 56. E . J . TAPPER, D.W. POWELL and S.M. MORRIS, Am. J . P h y s i o l . 23___55E402-E409 (1978). 57. X-Y. TIEN, R. WAHAWISAN, L . J . WALLACE and T.S. GAGINELLA, L i f e S c i . 36 1949-1955 (1985). 58. T . J . RIMELE, M.S. O'DORISIO and T.S. GAGINELLA, J . Pharmacol. Exp. Ther. 2 1 8 4 2 6 - 4 3 4 (1981). 59. A. KUWAHARA, X.-Y. TIEN, L . J . WALLACE and H.J. COOKE, J. Pharmacol. Exp. Ther. 2 4 2 6 0 0 - 6 0 6 (1987). 60. E.B. CHANG, D.R. BROWN, N . - S . WANG and H. FIELD, J . C l i n . I n v e s t . 7 8 2 8 1 287 (1986). 61. J . F . KACHUR, W. ALLBEE, W. DANHO and T.S. GAGINELLA, Regul. P e p t i d e s 17 63-70 (1987). 62. A.W. CUTHBERT, P.V. HALUSHKA, H.S. MARGOLIUS and J.A. SPAYNE, B r i t . J. Pharmacol. 82 597-607 (1984). 63. R. HIROSE and E.B. CHANG, Am. J. P h y s i o l . 254 G891-G897 (1988). 64. K.J. MORIARTY, N.B. HIGGS, M. WOODFORD, G. WARHURST and L.A. TURNBERG, Gut 28 844-848 (1987). 65. S.M. O'GRADY, M. FIELD, N.T. NASH and M.C. RAO, Am. J. Physiol. 249 C531C534 (1985). 66. S.M. O'GRADY, H.R. DeJONGE, A.B. VAANDRAGER and M. FIELD, Am. J. Physiol. 254 Cl15-C121 (1988). 67. A.W. CUTHBERT and H.S. MARGOLIUS, B r i t . J . Pharmacol. 75 587-598 (1982). 68. M.W. MUSCH, J . F . KACHUR, R . J . MILLER, M. FIELD and J . S . STOFF, J. C l i n . I n v e s t . 71 1073-1083 (1983). 69. G. WARHURST, M. LEES, N.B. HIGGS and L.A. TURNBERG, Am. J. P h y s i o l . 252 G293-G300 (1987). 70. L.D. LAWSON and D.W. POWELL, Am. J. P h y s i o l . 252 G783-G790 (1987). 71. A.W. CUTHBERT, P.V. HALUSKA, H.S. MARGOLIUS and J.A. SPAYNE, Brit. J. Pharmacol. 8-2587-595 (1984). 72. A.W. CUTHBERT, P.V. HALUSKA, D. KESSEL, H.S. MARGOLIUS and W.C. WISE, B r i t . J . Pharmacol. 8 3 5 4 9 - 5 5 4 (1984). 73. A.W. CUTHBERT, S.C. KIRKLAND and L . J . MacVINISH, B r i t . J . Pharmacol. 8 6 3 5 (1985). 74. J . D . FONDACARO, Am. J . P h y s i o l . 250 G1-G8 (1986). 75. S.M. O'GRADY, H.C. PALFREY and M. FIELD, J. Membr. B i o l . 9 6 1 1 - 1 8 (1987). 76. H.M. COX, A.W. CUTHBERT, R. HAKANSONand C. WAHLESTEDT, J . P h y s i o l . (London) ~98 65-80 (1988).