Nutrition, Digestion, Metabolism Proceedings of the 28th

International Congress of Physiological Sciences, Budapest,

1980

Visit the link below to download the full version of this book:

https://medipdf.com/product/nutrition-digestion-metabolism-proceedings-of-the-28
th-international-congress-of-physiological-sciences-budapest-1980/

Click Download Now

 FOREWORD

This volume is one of the series published by Akadémiai Kiado, the

Publishing House of the Hungarian Academy of Sciences in coédition with
Pergamon Press, containing the proceedings of the symposia of the 28th
International Congress of Physiology held in Budapest between 13 and 19
July, 1980. In view of the diversity of the material and the "taxonomic"
difficulties encountered whenever an attempt is made to put the various
subdisciplines and major themes of modern physiology into the semblance of
some systematic order, the organizers of the Congress had to settle for 14
sections and for 127 symposia, with a considerable number of free communi-
cations presented either orally or as posters.
The Congress could boast of an unusually bright galaxy of top names
among the invited lecturers and participants and, naturally, the ideal would
have been to include all the invited lectures and symposia papers into the vol-
umes. We are most grateful for all the material received and truly regret that
a fraction of the manuscripts were not submitted in time. We were forced
to set rigid deadlines, and top priority was given to speedy publication even
at the price of sacrifices and compromises. It will be for the readers to judge
whether or not such an editorial policy is justifiable, for we strongly believe
that the value of congress proceedings declines proportionally with the gap
between the time of the meeting and the date of publication. For the same
reason, instead of giving exact transcriptions of the discussions, we had to
rely on the introductions of the Symposia Chairmen who knew the material
beforehand and on their concluding remarks summing up the highlights of
the discussions.
Evidently, such publications cannot and should not be compared with
papers that have gone tlirougli the ordinary scrupulous editorial process of
the international periodicals with their strict reviewing policy and high
rejection rates or suggestions for major changes. However, it may be refresh-
ing to read these more spontaneous presentations written without having to
watch the "shibboleths" of the scientific establishment.

September 1, 1980 J. Szentâgothai

President of the
Hungarian Academy of Sciences

v
 PREFACE

The 28th Congress of Physiological Sciences was held in Budapest amid

a greater interest than ever on previous similar occasions.
In the Nutrition-Digestion-Metabolism section alone 268 papers were
presented including two invited lectures.
In the frames of nine symposiums 58 invited and 30 free presentations
were delivered. In the free communication part 69 oral communications and
109 posters were presented.
From all this material this volume contains the two invited lectures and
the subject matter of the following eight symposiums:

I. Vitaminsand trace elements

II. Role of cyclic nucleotides in stimulus—secretion coupling of exocrine
glands
III. Physiological components of the gastric mucosal barrier and their
role in mucosal defense
IV. Motility in control of gastric emptying
V. Intestinal polypeptides and peptidergic nerves
VI. Molecular changes during metabolic processes of gastrointestinal
peptide hormones
VII. Factors involved in the integrated mechanism of intestinal absorption
VIII. Lipoprotein metabolism, apolipoproteins, lipid constituents

The Lecture Halls were filled to capacity. In general there was a lively
discussion and a useful exchange of experiences after the lectures.
The presentations of the Poster Section were also followed with great
interest. Unfortunately — because of technical reasons — we are not able to
publish them.
We would like to thank to all the authors of the manuscripts, especially
to Prof. Bonfils and Prof. Ugolev invited lecturers as well as to Prof.
R. Buzina, Prof. J. Christophe, Prof. K. J. öbrink, Prof. E. E. Daniel, Prof.
B. Uvnäs, Prof. V. Varro, Prof. T. Z. Csaky, and Prof. P. S. Roheim;
furthermore to Dr. E. Morava, Dr. Gy. Mozsik, Prof. E. Atanassova, Dr.
J. Szolcsânyi, Dr. L. Varga, Dr. T. Vârkonyi and Dr. L. G. Szollar for the
excellent organization of the symposia.

xv
 We also thank to Professors J. A. Young and R. M. Case for organizing
the 9th Symposium titled: "Mechanism of Gastrointestinal Exocrine Sec-
retion" the material of which will appear elsewhere.
We would like to express our gratitude to the Publishing House of the
Hungarian Academy of Sciences for its valuable work.

Budapest, October 1980

Prof. T. Gâti
Chairman of the Nutrition-
Digestion-Metabolism section

xvi
 Adv. Physiol. Sei. Vol. 12. Nutrition, Digestion, Metabolism
T. Gàti, L. G. Szollàr, Gy. Ungvâry (eds)

HORMONAL RECEPTORS IN THE CELL

REGULATION OF DIGESTIVE FUNCTIONS
Serge Bonfils
Unité de Recherches de Gastroentérologie, INSERM U. 10, Hôpital Bichat, F-75877 PARIS CEDEX 18,
France

The main d i g e s t i v e f u n c t i o n s c a n be a c t i v a t e d or i n h i b i t e d by
hormones s e c r e t e d by t h e GEP s y s t e m . Strong e v i d e n c e h a s been p r e -
s e n t e d for a c t i o n of t h e s e hormones o n : 1) e x o c r i n e secretions
( w a t e r a n d e l e c t r o l y t e s , p r o t e i n s a n d g l y c o p r o t e i n s ) ; 2) motor a c t i -
v i t i e s (tonic a n d p r o p u l s i v e ) , 3) function of " s u r v i v a l " , i . e . t i s s u e
metabolism a n d cell r e n e w a l . Less documented a n d s t i l l c o n t r o -
v e r s i a l a r e t h e p o s s i b l e effects on o t h e r e n d o c r i n e s e c r e t i o n s a n d on
absorption, mainly by t r a n s c e l l u l a r p a t h w a y .
But a s i n d i c a t e d in t a b l e 1, t h e r e is so f a r no s t r i c t i d e n t i t i e s

Table I
HORMONAL DEPENDENCE OF DIGESTIVE FUNCTIONS

DIGESTIVE FUNCTIONS HORMONAL HORM . STIMUL.

RECEPTOR &/or INHIB.

Exocrine Water, electrolytes + +

secretions Proteins (enzymes) + +
Glycoproteins + +

Endocrine
secretions - ?

Motor Tonic (sphincter) + +

activity Propulsive - +

Absorption &
intestinal transepithelial
transport + ?

Survival of Metabolism ?. +
cells Renewal + +

Comparison between e v i d e n c e s for hormonal r e c e p t o r a n d

for p h y s i o l o g i c a l a c t i o n s a t t h e o r g a n l e v e l ( - : no
evidence so f a r ; ?: no d i r e c t e v i d e n c e ) .

1
between t i s s u e s u n d e r g o i n g hormonal i n f l u e n c e s and tissues (or
c e l l s ) evide i c i n g r e c e p t o r a c t i v a t i o n a n d / o r i n h i b i t i o n by the same
hormones.
The concept of h o r m o n a l r e c e p t o r is however h i g h l y s t i m u l a -
ting; it p a r t i c u l a r l y implies the c e l l u l a r e v e n t s r e s u l t i n g from
h o r m o n e - t i s s u e i n t e r a c t i o n s , t h a t could be c o n s i d e r e d a s r e p r e s e n t -
a t i v e of f u n c t i o n ( s ) ( 3 ) . This could l e a d to c o n t r o v e r s i a l r e s u l t s
when compared with o r g a n p h y s i o l o g y , a l l t h e more t h a t in e x p e r i -
mental a p p r o a c h e s c o n c e r n e d with t h e s e effects, p r o b l e m s a r i s e from
the m u l t i p l i c i t y of t h e GEP hormones t h a t may i n t e r f e r e on a same
t a r g e t o r g a n a n d the c a p a b i l i t y for one hormone of t r i g g e r i n g
simultaneously various physiological activities. A p r e r e q u i s i t for
o p t i m i z i n g r e l i a b i l i t y a n d r e p r o d u c i b i l i t y of s t u d i e s a p p e a r s t h u s to
minimize or suppress the o t h e r p a r a m e t e r s of r e g u l a t i o n , i.e.
n e r v o u s i n f l u x , f e e d - b a c k phenomena d r i v e n by the d i g e s t i v e s e c r e -
t i o n s , blood s u p p l y .

1. FUNCTIONAL MODELS AT THE CELL LEVELS

I d e a l l y , r e c e p t o r s t u d i e s s h o u l d not only encompass b i n d i n g

p a r a m e t e r s , but e x t e n d to specific f u n c t i o n a l a c t i v i t i e s : t e c h n i c a l l y
the methods used t a k e n a l o n e , a r e well defined a n d r a t h e r s o p h i s t i -
c a t e d ; however, for a n i d e a l d e m o n s t r a t i o n , four c r i t e r i a should be
f u l f i l l e d . 1) Homogeneity of the t i s s u e , i . e . c o n s t i t u t e d of one cell
type (eventually equipped with more t h a n one r e c e p t o r type).
2/ Specificity of hormone b i n d i n g to r e c e p t o r ; t h i s t e c h n i c a l l y
needs r a d i o l a b e d hormones with full b i o l o g i c a l a c t i v i t i e s . 3) C h a -
r a c t e r i z a t i o n a n d measurement of i n t r a c e l l u l a r m e s s e n g e r a c t i v i t i e s ,
in connection with 2 a n d 4 . 4) Determination of t h e most r e l e v a n t
a c t i v i t y of t h e c e l l for a s s e s s i n g the f u n c t i o n a l r e s p o n s e . These
c r i t e r i a which c o v e r the s u c c e s s i o n of e v e n t s i n t e r v e n i n g in the cell
r e g u l a t i o n of d i g e s t i v e functions a r e not often s i m u l t a n e o u s l y o b t a i -
ned ( t a b l e I I ) .
Table II. CRITERIA FOR RECEPTOR STUDIES

CRITERIA REQUIREMENTS

1. Homogeneity of the tissue Isolated cells

Enrichment in one cell type
Group of cells with a predominant
functional activity

2. Specificity of the binding Radiolabeled hormones with full

biological activity
Distinction of various types of
sites

3. Intracellular messenger cAMP, cGMP, Ca , protein

activities kinases

^. Functional responses Specific and/or relevant for

organ physiology

5. Competence within the E.Ds. obtained from 2., 3., 4.

2
I s o l a t e d Cell Models

They a r e l a r g e l y u s e d a s i n t a c t a n d l i v i n g c e l l s d e r i v e d from
v a r i o u s t i s s u e s : stomach fundic mucosa with i s o l a t i o n a n d s e p a r a -
tion of p a r i e t a l a n d n o n - p a r i e t a l c e l l s ( 8 , 13); p a n c r e a s a c i n a r
c e l l s ( 3 ) ; e n t e r o c y t e s ( 5 ) . Two of t h e four a b o v e c r i t e r i a may be
p a r a l l e l y o b t a i n e d , i . e . homogeneity of t h e t i s s u e a n d s p e c i f i c i t y of
hormone b i n d i n g ; t h i s s p e c i f i c i t y , not only d e r i v e s from the t i s s u e
homogeneity b u t a l s o from t h e p r e s e n c e of t h e hormone in i t s n a t i v e
c o n d i t i o n , a t a p r e c i s e l y known c o n c e n t r a t i o n a n d w i t h o u t i n t e r f e r e n -
ces of blood s u p p l y a n d / o r n e r v o u s i n f l u x .
On the o t h e r h a n d , a s s e s s i n g r e l e v a n t a c t i v i t y ( i e s ) of t h e cell
is often d i f f i c u l t due to t h e d e c r e a s e a n d / o r to the c h a n g e in cell
a c t i v i t y when s e p a r a t e d from n o r m a l t i s s u e s t r u c t u r e s . F u r t h e r m o r e ,
often the b i o l o g i c a l s i g n a l is not e a s i l y c h a r a c t e r i z e d in the
s u r v i v a l medium w h e r e t h e c e l l s a r e t e s t e d : for i n s t a n c e a c i d
s e c r e t i o n of t h e p a r i e t a l c e l l s c a n be only i n d i r e c t l y e v i d e n c e d with
the t r a p p i n g of t h e C l a b e l l e d weak b a s i s a m i n o p y r i n e . P a n c r e a t i c
a c i n a r cell a c t i v i t y is e a s i e r to a s s e s s by m e a s u r i n g amylase
o u t p u t . 0~ consumption (12) is a u s e f u l l tool when t h e specific
functions of the c e l l s h a v e close r e l a t i o n with o x y d a t i v e m e t a b o l i s m .
On the whole, the s e n s i t i v i t y of t h e s e models is poor ( t a b l e 111).
Table I I I
ISOLATED CELL MODELS

ADVANTAGES DISADVANTAGES

Receptors: d i r e c t access Loss of c e l l polarisation

Stimulant: not metabolised Weak \

local concentration Marginal? B i o l . response
p r e c i s e l y known Abnormal ;

No i n t e r f e r e n c e from: Inadequacy t o organ

bloocl supply physiology
nervous i n f l u x

Membrane models, such a s v e s i c l e s , c a n n o t be s t u d i e d by r e f e r -

ence to a specific b i o l o g i c a l r e s p o n s e . Measurement of i n t r a c e l l u l a r
m e s s e n g e r a c t i v i t i e s is u s u a l l y c o n s i d e r e d a s r e p r e s e n t a t i v e of the
b i o l o g i c a l r e s p o n s e , b u t c a n n o t a s c e r t a i n the p h y s i o l o g i c a l i n t e r e s t
of t h e r e s u l t s . Even if some k i n d s of b i o l o g i c a l a c t i v i t y a r e c h a -
r a c t e r i z e d ( e . g . c h a n g e in i n t r a v e s i c u l a r pH or ion t r a n s p o r t s ) ,
their r e l e v a n c e to p h y s i o l o g y may r e m a i n d i s p u t a b l e .
From t i s s u e or c e l l s one c a n o b t a i n r o u g h h o m o g e n a t e s a n d f u r -
t h e r p u r i f i c a t i o n s i n c r e a s e the s p e c i f i c i t y of b i n d i n g a n d / o r m e s s e n -
g e r s t u d i e s . However, the homogeneity of the o r i g i n a l material
r e m a i n s the most d e t e r m i n a n t f a c t o r for specific hormone r e c e p t o r s t u -
dies.

3
 I s o l a t e d c e l l s models h a v e e v i d e n c e d a n u m b e r of c e l l - h o r m o n e
r e l a t i o n s h i p s t h a t could be i n t e r p r e t e d in r e s p e c t to r e c e p t o r :
- a f f i n i t y of one hormone for one c e l l t y p e , c o n s i d e r i n g
t h e p r e s e n c e of one or m u l t i p l e r e c e p t o r t y p e ( s ) ( e . g .
h i g h a n d low a f f i n i t y ) ;
- v a r i e t y of hormone r e c e p t o r s for one cell t y p e ; site to
site interactions;
- d e t e r m i n a t i o n of t h e n u m b e r of s i t e s p e r c e l l ;
- a b i l i t y for one r e c e p t o r - t y p e to r e c o g n i z e v a r i o u s molecu-
l a r forms of one hormone or v a r i o u s hormones;
- i n t e r n a l i z a t i o n of r e c e p t o r s s u g g e s t i n g i n t r a c e l l u l a r h o r -
mone t r a n s p o r t .
I s o l a t e d cell models h a v e been p a r t i c u l a r l y fruitful in the fol-
lowing s t u d i e s :
- p a r i e t a l cell r e c e p t o r s ( 7 ) : b i n d i n g a n d s t i m u l a t i o n with
g a s t r i n (9, 13) a n d t h e i r i n h i b i t i o n by g a s t r i n a n a l o g u e s (NPS
g a s t r i n a n d p e n t a g a s t r i n ) ( 2 ) ; b i n d i n g of s o m a t o s t a t i n a n d s u b s e -
quent i n t r a c e l l u l a r events (6);
- p a n c r e a t i c a c i n a r cell receptors: comparison and interferen-
ces between s e c r e t i n a n d VIP; c a e r u l e i n a c t i v a t i o n a n d i n h i b i t i o n ;
c o m p a r a t i v e a c t i v i t y of the v a r i o u s m o l e c u l a r forms of CCK;
- e n t e r o c y t e s : VIP r e c e p t o r a n d a d e n y l a t e c y c l a s e s t i m u l a t i o n
(5);
- colonie c a r c i n o m a c e l l s : p r i v i l e d g e d a c t i v i t y of VIP on a d e n -
n y l a t e c y c l a s e with p e r s i s t e n c e of f u n c t i o n a l r e c e p t o r s ( 1 1 ) .
Supra cellular models

They a r e made of g r o u p of c e l l s s e p a r a t e d from the o r i g i n a l

t i s s u e ; b u t t h e s e c e l l s a r e k e e p i n g c o n n e c t i o n s between them t h a t
could favor p e r m a n e n c e of specific f u n c t i o n a l a c t i v i t i e s , such as ion
t r a n s p o r t ( e . g . g a s t r i c H s e c r e t i o n ) or enzyme s e c r e t i o n ( e . g . p a n -
c r e a t i c a m y l a s e ) . C u r r e n t works h a v e u s e d i s o l a t e d g a s t r i c g l a n d s
( 1 ) , p a n c r e a t i c a c i n i , p a n c r e a t i c d u c t f r a g m e n t s , c l u s t e r s of e n t e r o -
c y t e s or colonie mucosal c e l l s .
On t h e s e m o d e l s , c h a r a c t e r i z a t i o n a n d measurement of i n t r a c e l -
l u l a r messenger a c t i v i t i e s c a n be s i m u l t a n e o u s l y o b t a i n e d , b u t s p e c i -
ficity of hormone b i n d i n g to r e c e p t o r could be more d i s p u t a b l e in
r e s p e c t to cell h e t e r o g e n e i t y . For the p h y s i o l o g i s t , e v i d e n c e for m e s -
s e n g e r - m e t a b o l i s m c o u p l i n g is more s i g n i f i c a n t t h a t for b i n d i n g - m e s -
senger (recognition-activation) coupling.
C o n c l u s i v e l y , one c a n s t a t e d t h a t i s o l a t e d cell models a n d s u -
p r a c e l l u l a r models a r e c o m p l e m e n t a r y : i n c r e a s i n g the number of
c e l l s , i . e . the size of t h e sample from the o r i g i n a l t i s s u e r e s u l t s in
a n a p p r o a c h more a n d more a p p r o x i m a t i n g o r g a n p h y s i o l o g y . On the
o t h e r h a n d , p u r e homogeneous cell p r e p a r a t i o n a p p e a r s to be m a i n l y
an e x t e n s i o n of b i o c h e m i c a l r e s e a r c h on m e m b r a n e s . On both e x t r e m i -
t i e s of t h i s r a n g e , improvement s h o u l d be o b t a i n e d from a ) c h e m i c a l
i s o l a t i o n of r e c e p t o r s a n d d e t e r m i n a t i o n of t h e i r l o c a t i o n on or in
t h e c e l l s , b ) i s o l a t e d o r g a n p r e p a r a t i o n s a l l o w i n g to c o n t r o l p a r a m e -
t e r s such a s blood flow, n e r v o u s i n f l u x , v e i n o u s a r t e r i a l g r a d i e n t s
of hormones or n u t r i e n t s .

II. FUNCTIONAL MODELS AT THE ORGAN LEVEL

These models h a v e been l a r g e l y u s e d , in t h e p a s t in "in v i v o "

or "ex vivo'' s t u d i e s , without r e f e r i n g to r e c e p t o r t h e o r y .

4
 More r e c e n t l y , a n d p a r t i c u l a r l y u n d e r M. 1. Grossman's influence
functional r e s p o n s e s to GEP hormones h a v e been a n a l y z e d a c c o r d -
ing to the Michaelis-Menten e n z y m a t i c model. It is g e n e r a l l y c o n s i d e -
red t h a t t h i s model may c o r r e c t l y d e s c r i b e the s t i m u l u s - s e c r e t i o n
coupling.
However, k i n e t i c s of o r g a n r e s p o n s e s h a v e been sometimes a b u -
s i v e l y l i n e a r i z e d by m a t h e m a t i c a l t r a n s f o r m a t i o n s . C o n t r a r i w i s e to
the c a s e of r e c e p t o r models, t h e hormone c o n c e n t r a t i o n s a t the cell
level is not a c t u a l l y known, blood hormone c o n c e n t r a t i o n b e i n g not
n e c e s s a r i l y r e p r e s e n t a t i v e for t h i s p a r a m e t e r . But, in r e s p e c t to p h y -
s i o l o g i c a l m e c h a n i s m s , o r g a n s t u d i e s on the one h a n d a n d b i o c h e m i -
c a l s t u d i e s of r e c e p t o r s on the o t h e r h a n d h a v e p r o v i d e d r a t h e r com-
plementary than controversial results (4).

111. CONCLUSIONS

The c o n t r i b u t i o n of r e c e p t o r s t u d i e s to the knowledge of d i g e s -

t i v e p h y s i o l o g y is i m p r e s s i v e . This c a n be e x a m p l i f i e d in the field
of g e n e r a l c o n c e p t s : 1) the a b i l i t y to compete for the same r e c e p t o r
makes it p o s s i b l e to e x p l a i n the common b e h a v i o u r of s t r u c t u r a l l y
r e l a t e d hormones ( g a s t r i n f a m i l y , s e c r e t i n family) ( 1 0 ) . F u r t h e r m o -
r e , the knowledge of r e c e p t o r s t r u c t u r a l r e q u i r e m e n t s is a c r u c i a l
clue in the s y n t h e s i s of c o m p e t i t i v e i n h i b i t o r such a s modified
( n i t r o p h e n y l s u l f e n y l t r y p t o p h a n ) g a s t r i n ; 2) p h a r m a c o l o g i c a l a n d
p h y s i o l o g i c a l i n t e r a c t i o n s of hormones a r e b e t t e r u n d e r s t o o d t a k i n g
into a c c o u n t r e c e p t o r e v e n t s s u c h a s c o o p e r a t i v i t y a n d s p a r e n e s s
phenomena ( 3 ) ; 3) s p e c u l a t i o n on a b n o r m a l d i g e s t i v e f u n c t i o n s in
pathological conditions could be more f r u i t f u l in hypothesizing
p o s s i b l e c h a n g e in the n u m b e r or t h e s e n s i t i v i t y of t h e r e c e p t o r s .
Basic works h a v e documented t h i s h y p o t h e s i s in colonie c a r c i n o m a
(11); 4) i n t e r f e r e n c e s of systemic hormone s u p p l y may be overcome
in d i r e c t r e c e p t o r s t u d i e s a n d t h e s e could moreover inform on the
hormone c a t a b o l i s m a t the t a r g e t c e l l l e v e l . These b e n e f i t s are
especially valuable for hormone analogues; 5) a cell response
different in n a t u r e from t h a t a n t i c i p a t e d from o r g a n s t u d i e s is
sometimes o b s e r v e d : e . g . a m y l a s e s e c r e t i o n u n d e r s e c r e t i n s t i m u l a -
tion of p a n c r e a t i c a c i n a r c e l l s . A new a p p r o a c h to the p o t e n t i a l
f u n c t i o n a l a c t i v i t i e s of the c e l l is t h u s s u g g e s t e d .
However, for the p h y s i o l o g i s t a s well a s for the g a s t r o e n t e r o -
l o g i s t g a t h e r i n g p h y s i o l o g i c a l or p a t h o p h y s i o l o g i c a l i n f o r m a t i o n a t
the o r g a n level, transposition or adaptation of b a s i c science
p r o g r e s s e s to t h e i r own field is a h a r d w o r k . Many f a c t o r s a r e
hindering this inavoidable extension. Without n u m b e r i n g a l l of
them, one could s t r e s s on 3 major p o i n t s . 1) There is no common
l a n g u a g e between men i n t e r e s t e d in o r g a n functions a n d those w o r k -
ing a t the cell l e v e l : even more, t h e same words a r e often used
with a d i f f e r e n t meaning by both. 2) P a t h o p h y s i o l o g y is often
p o o r l y u n d e r s t o o d by the b a s i c s c i e n t i s t , b e c a u s e it is a s s o c i a t e d
with a l a r g e n u m b e r of n o n - c o n t r o l l e d p a r a m e t e r s : t h i s f e a t u r e is
s t r o n g l y r e j e c t e d w h a t e v e r the v a l u e of t h e r e m a i n i n g i n f o r m a t i o n .
3) An o r g a n is not a simple c l u s t e r of c e l l s b u t an a s s o c i a t i o n of
c e l l s monitored by v e s s e l s , n e r v e s , e n e r g e t i c s u p p l y , e t c . ; this
a s s o c i a t i o n s i m u l t a n e o u s l y a s s u m s specific f u n c t i o n s ( i . e . s e c r e t i o n ,
t r a n s p o r t , e c t . ) a n d c o n t r o l s i t s own c e l l r e n e w a l .
Cell p h y s i o l o g y does not a c c o u n t for a l l t h e s e i n t e r f e r e n c e s

5
whicb a r e included in (and not easily substracted from) organ
responses.

REFERENCES
1. B e r g l i n d h , T. a n d O b r i n k , K . J . : A method for p r e p a r i n g i s o l a -
ted g l a n d s f r o m the r a b b i t g a s t r i c m u c o s a . Acta P h y s i o l
S c a n d , 1976, 96: 150-159

2. D u b r a s q u e t , M., O r y - L a v o l l é e , L . , Pham Van C h u o n g , P . , Mor-

g a t , J . L . , Fromageot, P . , a n d Bonfils, S. : Effet i n h i b i t e u r
d ' u n a n a l o g u e de la p e n t a g a s t r i n e s u r la s é c r é t i o n g a s t r i q u e
a c i d e du r a t . G a s t r o e n t e r o l Clin Biol, 1980 (in p r e s s ) .

3. G a r d n e r , J . D . : Receptors for g a s t r o i n t e s t i n a l hormones. Gastro-

e n t e r o l o g y , 1979, 76: 2 0 2 - 2 H

4. Grossman, M . I . : Neural a n d hormonal r e g u l a t i o n of g a s t r o i n t e s -

t i n a l f u n c t i o n : an o v e r v i e w . Annu Rev P h y s i o l , 1979, 4T_: 27-33

5. L a b u r t h e , M., Besson, J . , Hui Bon Hoa, D . , a n d R o s s e l i n , G . :

Récepteurs du p e p t i d e i n t e s t i n a l v a s o a c t i f (VIP) d a n s les
e n t é r o c y t e s : l i a i s o n s p é c i f i q u e et s t i m u l a t i o n de l'AMP c y c l i -
q u e . C R Acad Sei ( P a r i s ) , 1977, 2M: D2139-D2U2

6. Lewin, M . J . M . : Hormonal r e c e p t o r c o n t r o l of e l e c t r o l y t e s e c r e t i o n
in the g a s t r o i n t e s t i n a l t r a c t . I n : G a s t r o i n t e s t i n a l Hormones,
e d i t e d by George B. Jerzy G l a s s , Raven P r e s s , New York,
1980, 477-50Λ

7. Lewin, M . J . M . : P a r i e t a l cell r e c e p t o r s . I n : P a t h o g e n e s i s a n d
T h e r a p y of Ulcer D i s e a s e , e d i t e d by K.H. Holtermiiller, E x c e r -
p t a Medica, Amsterdam, 1980 (in p r e s s ) .

8. Lewin, M . J . M . , C h e r e t , A.M., Soumarmon, A . , a n d G i r o d e t , J . :

Méthode p o u r l ' i s o l e m e n t et le t r i des c e l l u l e s de l a m u q u e u s e
f u n d i q u e de r a t . Biol G a s t r o e n t e r o l ( P a r i s ) , 1974, ]_: 139-H4

9. Lewin, M . ] . M . , Soumarmon, A . , a n d Bonfils, S . : G a s t r i n r e c e p -

t o r s i t e s in r a t g a s t r i c mucosa. I n : Hormonal Receptors in
Digestive T r a c t P h y s i o l o g y , e d i t e d by S. Bonfils, P . Fromageot
and G. R o s s e l i n . Elsevier/North Holland, Amsterdam, 1977,
379-387

10. Morley, J . S . : Information a b o u t p e p t i d e hormone r e c e p t o r s from

s t r u c t u r e a c t i v i t y s t u d i e s . I n : Hormonal Receptors in Digestive
T r a c t P h y s i o l o g y , e d i t e d by S. Bonfils, P . F r o m a g e o t , a n d G.
R o s s e l i n . E l s e v i e r / N o r t h H o l l a n d , Amsterdam, 1977, 3-12

11. René, E . , V i s s u z a i n e , C , Dupont, C , Lewin, M.J.M. a n d Bon-

f i l s , S. : VIP s t i m u l a t i o n of a d e n y l a t e c y c l a s e in a p o p u l a t i o n
of 11 human c o l o r e c t a l a d e n o c a r c i n o m a e : e v i d e n c e for two
c l a s s e s of r e s p o n s i v e n e s s . G a s t r o e n t e r o l o g y , 1980, J8_: 1243
(abst).

12. Soll, A.H.: The actions of secretagogues on oxygen uptake by

6
 isolated mammalian parietal cells. ] Clin Invest, 1978, 61:
—
370-380
13. Soumarmon, A., Cheret, A.M., and Lewin, M.J.M.-: Localization
of gastrin receptors in intact isolated and separated rat
fundic cells. Gastroenterology, 1977, 73 : 900-903

7
 Adv. Physio/. Sei. Vol. 12. Nutrition, Digestion, Metabolism
T. Gàti, L. G. Szollâr, Gy. Ungvâry (eds)

REVISED CONCEPT OF FUNCTIONAL AND

STRUCTURAL ORGANIZATION OF THE
ENZYME-TRANSPORT SYSTEMS OF THE
APICAL MEMBRANE OF THE ENTEROCYTES
A. M. Ugolev
/. P. Pavlov Institute of Physiology, The Academy of Sciences of the USSR, Leningrad, USSR

Introduction
The mechanism of coupling the enzyme and transport sys-
tems is a central problem not only of gastroenterology, but
also of the biology of the cells and membranes. Indeed, comp-
lex metabolic chains may ultimately be represented as a com-
bination of the (1) transformational and (2) transport links.
The means of integration of these links is a third fundamen-
tal element of metabolism in the living systems. This problem
will be discussed below and several fundamental and accepted
concepts (see Fig.1) will be revised in the light of the new
evidence.
The glycocalyx (7, 20, 22)
The external surface of the plasmic membrane in the maj-
ority of the cells including enterocytes is covered by the
glycocalyx. It is a component of the membrane forming on its
surface a layer with a thickness up to 1000 A · The glycoca-
lyx performs some vital functions such as adhesion, recogni-
tion, intercellular interactions, defense, separation of dif-
ferent types of molecules according to their sizes and charge
etc.
Until very recently ideas on this mechanism remained un-
certain since the glycocalyx was not preparatively separated
from the other cellular structures. However, we have recently
succeeded in a separation of the apical glycocalyx by the
technique of agar replica, proposed by us and schematically
represented in Fig.2. As can be seen, the apical glycocalyx
is separated from the enterocyte plasmic membrane without
disturbing the latter. This fact was confirmed not only elec-
tronmicroscopically, but also by means of membrane markers.
The results of our work were quite unexpected. It turned
out that the apical glycocalyx is practically free of inver-
tase, but contains about 60% of pancreatic amylase adsorbed
onto the structures of the intestinal mucosa. Besides, some
85% trypsin and less than 20% chymotrypsin activities were
found in the glycocalyx zone. Finally, it has been shown
that intrinsic intestinal enzymes, such as Ï -amylase, amino-
peptidase, a some dipeptidases are largely bound with the li~

9
 Fig.1. Enterocyte structure and functions. A - structure,
B - substance fluxes, C - water flows.

Fig.2. A scheme of the separation of the apical glyco-

calyx from the lipoprotein membrane.

10
poprotein membrane fraction.
The analysis of the results obtained suggests that a
passage of nutrients from the small intestinal lumen to the
surface of the lipoprotein membrane of the enterocytes thro-
ugh the glycocalyx space is under the control and selectively
facilitated for those substrates, for which in the glycocalyx
layer there are corresponding enzymes and, probably, binding
proteins. This mechanism effectively prevents the entry of
many molecules retaining antigenic and toxic properties to
the plasmic membrane.
We have made an attempt to characterize the role of the
glycocalyx enzymes and found out that for soluble starch, a
desorption of pancreatic amylase from the intestinal mucosal
surface causes a 8-10 fold decrease in the uptake of this
substrate.
N.N.Iezuitova, P.De Laey and myself have demonstrated
that in every segment of the rat small intestine there exists
a certain correlation between adsorbed enzymes and the acti-
vity of the intrinsic enzyme and transport systems of the
lipoprotein membrane. This circumstance gives reasons to sug-
gest a definite relationship between various links of the
certain enzyme-transport chains, in particular, between the
functional spectrum of the enzymes and binding proteins of
the glycocalyx, on the one hand, and between enzymes, binding
proteins and the transport systems of the lipoprotein membra-
ne, on the other hand. This rule must be taken into account
to evaluate the effective work of intestinal uptake system
and other spatially distributed systems.
The characteristics of the enzyme-transport interactions.
Phenomenology (11, 16-19, 21)
Membrane digestion was discovered in 1958· It is accomp-
lished both by the intrinsic intestinal enzymes being an in-
tegral part of the lipoprotein membrane and the enzymes ad-
sorbed onto the intestinal mucosal structures.
With a discovery of membrane digestion it became clear
that the apical membrane of the enterocytes is a structure
where the final stages of hydrolysis and initial stages of
transport occurs. The proximity of the final enzymes and the
entrances into the transport systems should enhance efficien-
cy of a coupling of these two processes. However, as can be
seen from Fig.3t for monomers (for example, glucose and amino
acids) formed during the hydrolysis of di- and oligomers the
possibility of a loss of substances due to its diffusion
through the water phase markedly increases in the region of
transition from the enzyme onto the entrance to the transport
system. Besides, the transport of enzyme-released monomers
and that of free monomers should be identical in many proper-
ties. Finally, the transport of free monomers should be a mo-
re rapid process than that of monomers formed during the hyd-
rolysis of oligomers, since in the latter case a dissipation
of the reaction products due to diffusion takes place.
In reality, the enzyme-dependent transport has many, for
a long time remained unexplicable> advantages over the trans-
port of free monomers:
(1) Monomers formed during oligomer hydrolysis are ab-

11
 LUMINAL HYDROLYSIS MEMBRANE HYDROLYSIS
AND SUBSEQUENT TRANSPORT AND SUBSEQUENT TRANSPORT

SUBSTRATE

ENZYME
MEMBRANE-BOUND
ENZYME

MEMBRANE

free monomer f
transport O
system
TRANSPORT FOLLOWED BY
system
INTRACELLUIAR HYDROLYSIS

dimer trans-X
port system φ ,

ΙΙΙΙΙΙΙΙΙ<ΙΙΙΙΙΙΙΙΙΙΙΙΐζ I SlIIIIIIIIIIMlS I JTTTTTT

iiiiim^iiiiiniiiiii; ι '.llllllllllllll! liiiilL
4 free monomer
transport
system
THE ENZYME ACTIVE
CENTER PACED THE CYTOSOL
CYTOPLASM O ENZYME

MEMBRANE HYDROLYSIS AND TRANSPORT

IN ENZYME-TRANSPORT COMPLEX

llllllllM-f-^-inilMMIMIIIIJ ι III I III II11II

Mill iimiiiiij/ \siiiiiiiiiiimi{ ι jniiinmii
ENZYME-TRANSPORT / 4 f free monomer
COMPLEX o O O transport
system

Fig.3· Different types of enzyme-transport interactions.

sorbed more rapidly than free monomers; (2) In contrast to
free monomers, the monomers released from membrane hydrolysis
do not compete for a common entrance to the transport system;
(3) A competition between oligomers and monomers is absent or
reduced. At the same time, within each group, a marked compe-
tition between different monomers and di- and oligomers takes
place; (4) During the transport of monomers formed by hydroly-
sis of oligomers Κ^ is often less, and V is higher than du-

12
ring the transport of free monomers; (5) The active transport
of glucose and amino acids (at least in vitro) is Na+-?depen-
dent process, while the transport of the same substances re-
leased during membrane hydrolysis of respective oligomers, in
many cases (although not always) is independent on the pre-
sence of Na+.
In order to explain all the characteristics of the enzy-
me-dependent transport, it is necessary to assume that it is
accomplished by another way distinguished from the transport
of free monomers. Prom this point of view, the hypothesis on
the existence of specific transport systems for di- and oli-
gomers seems especially attractive. Our alternative hypothe-
sis postulating the presence of the enzyme-transport ensemb-
les or complexes on the membrane surface of the intestinal
cells appear* to be less probable, since such a complex sho-
uld possess a number of properties seemed, at least initially,
unlikely.
However, at present it is known that the catalytic part
of membrane enzymes, as shown on the example of carbohydra-
ses, aminopeptidase etc., is projected over the apical memb-
rane and therefore faces the small intestinal lumen.
The molecular structure of membrane enzymes (2, 3, 5, 12)
Under natural conditions, membrane enzymes appear to be
oligomers consisting of two, three and even more subunits.
Most intestinal membrane enzymes are transmembrane in-
tegral proteins (namely, glycoproteins) and possess amphipa-
tic structure, composed of a hydrophobic and hydrophilic
parts. The catalytic sites of the enzymes are concentrated
in the hydrophilic head making up more than 90% of the total
mass of the enzymes. The remaining part is attributed to the
hydrophobic one (molecular mass between 8000-10000 daltons)
spanning the phospholipid bilayer of the membrane. In some
cases, the hydrophobic part of the enzyme is terminated by a
small hydrophilic peptide, exposed on the internal membrane
surface.
It has been demonstrated that the hydrophobic part of
the enzyme is necessary for realization of the anchor functi-
ons. A suggestion has been made that the hydrophobic part of
the enzyme may be involved into the transport processes. How-
ever, this hypothesis has not yet been experimentally confir-
med. We have demonstrated such a vital function of the hydro-
phobic part of enzymes as a maintenance of the optimal con-
formation of the hydrophilic catalytic part. This function
has been found when comparing the kinetic characteristics of
the triton and trypsin forms of carbohydrases and alkaline
phosphatase pf the enterocytes. The hydrophobic part tends
also to stabilize structure of the enzyme under the action
of different factors. Finally, it has been established that
the hydrophobic part is of importance for a manifestation
of allosteric functions and regulation of the enzyme activi-
ty.

13
 Cooperative interactions between the enzyme and transport
parts of the complex (8, 9, 12, 21)
The final stages of hydrolysis of most nutrients are ac-
complished on the external membrane surface and hence, a
transmembrane transfer follows hydrolysis rather than preceds
it. This conclusion is consistent with data of the kinetic
analysis of the transport of free monomers and ones formed by
di- and oligomer hydrolysis as well as with the fine locali-
zation of final enzymes.
In order to describe the evidence obtained by the pro-
perties of a single model, an assumption should be made on
the »existence of the enzyme-transport complex characterized
by the following properties: (1) the hydrolysis products sho-
uld be transferred directly from the active site of the enzy-
me onto the entrance to the transport system without dissipa-
tion into water phase; (2) the transport system should not
interact with the monomers present in the water medium. The
only way to explain this, is to postulate that a carrier and
the final enzyme form a cooperative system. In this case the-
re should be kinetic signs of such cooperativety. It is true
for an exact structure of the enzyme-transport system irres-
pective of whether it is (1) the enzyme-transport system is
a constant heterological quaternary complex consisting of
particular periodically dissociating subunits, (2) one comp-
lex molecule where the hydrophilic part carries out cataly-
tic functions and the hydrophobic part - transport ones, or
(3) the transport part represents a channel in an oligomer.
It is known that the allosteric enzymes and cooperative
systems are characterized by sigmoid kinetics. On the contra-
ry, the functioning of non-cooperative systems, including en-
zymes and carriers, may be described by Michaelis kinetics.
The typical hyperbolic curves have been drawn in our and
other laboratories when studying the accumulation of free
glucose in the rat jejunal mucosa. The behaviour of highly
purified preparations of maltase, $ -amylase and invertase of
the rat intestinal mucosa also fits well Michaelis kinetics.
We have recently suggested and later demonstrated that a
pronounced cooperativity of the enzyme-transport systems is
possible only under the conditions of undisturbed interacti-
ons between the catalytic and transport parts of the complex.
Indeed, in co-author with E.G.Gurman, E.È.Nurks and G.G.Kol-
tushkina it has been shown that the tissue accumulation of
glucose formed during maltose, sucrose and starch hydrolysis
in actively functioning intestinal preparations is described
by a sigmoid curve. Furthemore, it is important that not on-
ly the kinetics of transport but also that of sugar hydroly-
sis is described by a sigmoid curve if an interaction between
the enzyme and transport systems takes place.
An exclusion of the active transport of glucose by a
substitution of oxygen for nitrogen in the experiment, the
sigmoid curves of carbohydrate hydrolysis are transformed in-
to hyperbolic ones. Of greater interest is a possibility to
transform sigmoid curves into hyperbolic ones by a specific
blockage of the glucose transport system by phlorizin (Fig.4).
These results may serve as a direct proo'f of the exis-
tence in the apical membrane of the enterocytes of the enzyme-i

14