15.7. 1975

Speeialia

T h e I n h i b i t i o n of G a s t r i c A c i d S e c r e t i o n b y E p i d e r m a l E p i d e r m a l g r o w t h f a c t o r (EGF) is a p o l y p e p t i d e i s o l a t e d f r o m t h e s u b m a x i l l a r y glands o f male mice w h i c h h a s t h e s t r i k i n g p r o p e r t y of c a u s i n g p r e m a t u r e eye o p e n i n g a n d incisor e r u p t i o n w h e n i n j e c t e d into n e w b o r n mice 1. This h a s been s h o w n to be t h e result of i n c r e a s e d k e r a t i n i zation of t h e epidermis2. The p r i m a r y s t r u c t u r e of E G F has b e e n d e t e r m i n e d r e c e n t l y ; it is c o m p o s e d of 53 a m i n o acid residues w i t h 3 d i s u l p h i d e b o n d s a l t h o u g h a fully active p e p t i d e of 51 residues has also b e e n isolated 3. E G F will s t i m u l a t e cell p r o l i f e r a t i o n in a v a r i e t y of o r g a n c u l t u r e s 4. I t h a s similar effects to insulin in m o u s e m a m m a r y epithelial cells 5, a n d it will cause r a p i d regenera t i o n of r a b b i t corneal e p i t h e l i u m n. The full r a n g e of a c t i o n s h a s b e e n r e v i e w e d r e c e n t l y 7. I t is n o t y e t a p p a r e n t w h e t h e r this p a r t i c u l a r p e p t i d e fulfils a n y physiological role b u t it m a y p o s s i b l y be i n v o l v e d in t h e c o n t r o l of g r o w t h of c e r t a i n tissues. R e l a t i v e l y small a m o u n t s will s t i m u l a t e D N A a n d R N A s y n t h e s i s in c u l t u r e d h u m a n f i b r o b l a s t s s a n d a v a r i e t y of m a m m a l i a n tissues will b i n d e p i d e r m a l g r o w t h f a c t o r a t sites d i s t i n c t f r o m t h o s e o c c u p i e d b y o t h e r a n a b o l i c p e p t i d e s such as insulin 9. H o w e v e r , in a d d i t i o n t o t h e i r effects on tissue g r o w t h , p e p t i d e s such as insulin or g a s t r i n also h a v e a well d e f i n e d ' s h o r t t e r m ' a c t i o n a n d t h i s r e p o r t describes a d i r e c t effect of E G F u p o n g a s t r i c acid secretion w h i c h is unlikely to be t h e result of cell proliferation. Materials and methods. E p i d e r m a l g r o w t h f a c t o r was isolated f r o m t h e s u b m a x i l l a r y glands of m a l e a n d female mice a c c o r d i n g t o t h e m e t h o d of SAVAGE and COHEN a~

8

{

6 4

~

100

Histamine 300 pcj s.c.

Histamine 300 lJg s.c.

Histamine Histamine 500 IJg s.c. 300 lJg s.c. +EGF lOpg i.v.

Fig. 1. Inhibition of histamine induced acid secretion in a perfused rat stomach preparation by epidermal growth factor.

:1.15

w i t h m i n o r m o d i f i c a t i o n s . The p r o p e r t i e s of t h e p r o d u c t were fully in accord w i t h t h o s e p u b l i s h e d a n d in p a r t i c u l a r a m i n o - a c i d analysis s h o w e d a c o m p l e t e lack of alanine, p h e n y l a l a n i n e or lysine residues. T h e p o t e n c y was conf i r m e d b y t h e a c t i o n on n e w b o r n mice (Alderley P a r k Strain). S u b c u t a n e o u s i n j e c t i o n s of 5 txg p e p t i d e in 25 ~1 saline solution p e r d a y i n d u c e d eye o p e n i n g on d a y 10 c o m p a r e d t o d a y 13 in controls. The a c t i o n of t h i s p r o d u c t on gastric secretion was e x a m i n e d in b o t h r a t s a n d dogs ~. R a t t e s t i n g was carried out in a n a e s t h e t i z e d a n i m a l s (Alderley P a r k Strain) w i t h t h e s t o m a c h p e r f u s e d a n d t h e acid c o n t e n t of t h e p e r f u s a t e was d e t e r m i n e d b y t i t r a t i o n . Acid secretion was i n d u c e d b y s.c. i n j e c t i o n of h i s t a m i n e (300 ~.g) a t 1 h i n t e r v a l s a n d a f t e r 2 c o n t r o l responses, E G F was given b y i.v. i n j e c t i o n a t t h e t i m e of t h e n e x t h i s t a m i n e i n j e c t i o n (Figure 1). A l t e r n a t i v e l y , t h e action of E G F was e x a m i n e d in r a t s a g a i n s t a p l a t e a u of secretion e v o k e d b y r e p e a t e d h i s t a m i n e (20 vg) i n j e c t i o n s a t 5 m i n i n t e r v a l s 12. I n b o t h cases n e a r m a x i m a l i n h i b i t i o n was o b t a i n e d w i t h doses of 1050 Fg p e r kg. The effect of t h e g r o w t h f a c t o r was also s t u d i e d in Beagle dogs w i t h a d e n e r v a t e d gastric pouch, w i t h denerv a t e d p o u c h a n d fistula in t h e gastric r e m n a n t , or w i t h a n i n n e r v a t e d gastric pouch. T h e dogs were f a s t e d o v e r n i g h t a n d t h e n gastric secretion was s t i m u l a t e d b y a n i.v. infusion of e i t h e r h i s t a m i n e , p e n t a g a s t r i n or m e t h a c h o l i n e t o a b o u t 70% of t h e m a x i m u m o u t p u t . W h e n t h e secretion was s t e a d y , E G F was g i v e n as i.v. or s.c. i n j e c t i o n s at doses as low as 0.1 ~tg/kg (Figure 2). The d u r a t i o n of t h e i n h i b i t i o n was a b o u t 90 rain b u t w i t h t h e larger doses it t o o k longer for t h e secretion to r e t u r n to t h e original p l a t e a u level. H o w e v e r , using a sensitive r a d i o - i m m u n o a s say q u a n t i t a t i o n process, it w a s f o u n d t h a t t h e half t i m e of d i s a p p e a r a n c e of t h e p e p t i d e f r o m blood was a b o u t 4 rain ~3. Discussion. The original effect of E G F u p o n eye o p e n i n g in n e w - b o r n mice requires doses of a p p r o x i m a t e l y 200 btg/ kg s.c. p e r d a y w h e r e a s doses of 10 ~zg/kg in r a t s a n d 0.5 btg/kg in dogs p r o d u c e s a p r o f o u n d effect u p o n gastric acid secretion. This effect a p p e a r s to be specific for gastric secretion a n d doses of p e p t i d e up to 15 ~g/kg s.c. in dogs, p r o d u c e no u n t o w a r d effects u p o n respiration, h e a r t r a t e or t e m p e r a t u r e . A l t h o u g h e p i d e r m a l g r o w t h f a c t o r is f o u n d in l a r g , a m o u n t s in t h e s a l i v a r y g l a n d it also a p p e a r s in o t h e r b o d i l y fluids of t h e m o u s e i n c l u d i n g t h e saliva 14. I n t e r -

g

g

9 E. O'KEEFE, M. D. HOLLENBERG a n d P. CUATRIs

0.10 >

E 0.05

Growth Factor

1 S. COHEN, J .biol. Chem, 237, 1555 (1962). 2 S. COHEN and G. A. ELLIOT, J. Invest. Derm. 40, 1 (1963). 3 C. R. SAVAGEJR., T. INAGAMIand S. COHEN, J. biol. Chem. 247, 76]2 (1972). R. O. JoNEs, Expl. Cell Res. 43, 645 (1966). 5 IX. W. TURKINGTON, Expl. Cell Res. 57, 79 (1969). 6 C. P.. SAVAGEJR., and S. COIIEN, Expl. Eye IXes. 75, 361 (1973). 7 S. COHEN and J. M. TAYLOR, Recent Progr. Horm. Res. 533, 30 (1974).

9 9

T

825

8 M. I). t{OLLENBERG a n d P. CUATRECASAS, Proc. n a t n . A e a d . Sci.,

USA 70, 2964 (1973). Archs.

Biochem. Biophys. 164, 518 (1974). 10 C. R. SAV.~.GRand S. COHEN, J. biol. Chem. 247, 7609 (1972).

o

o

1'5

30

45

60

i5 90 105 189 Time

135 150rain

Fig. 2. The effect of a single i.v. injection of epidermal growth factor in a Heidenhain pouch dog upon volume and acid concentration secretion induced by an infusion of histamine at 30 btg/kg/h.

11 1~. CAMBLE, E. E. L. GERRING a n d H . GREGORY, G e r m a n P a t e n t

Publication No. 2415101. 12 j . D. t~'ITZGERALD, A. M. BARRETT a n d H . GREGORY, in The Physiology o/ Gastric Secretion (Eds. L. S. SE~tB and J. MYREN;

Oslo Universitets Forlaget 1968), p. 408. 13 j. BOWER, H. GREGORYand I. R. WlLI.SmRI~, unpublished work. 14 IX, L. BYYNY, D. N. ORTH, S. COHEN and E. S. 1)OYNE, Endocrinology 95, 776 (1974).

826

Specialia

e s t i n g l y t h e r e is a n a s s o c i a t i o n b e t w e e n h u m a n s a l i v a a n d t h e i n h i b i t i o n of acid secretion. T h u s in 1949 i t was f o u n d t h a t t h e i n j e c t i o n of h u m a n saliva i n t o a n i m a l s w o u l d i n h i b i t g a s t r i c acid secretion ~5 a n d i t was f o u n d later that the sublingual gland provided the greatest a m o u n t of i n h i b i t o r y m a t e r i a N 6. T h e e x t r a c t called s i a l o g a s t r o n e c a u s e d t h e i n h i b i t i o n of s e c r e t i o n in p y l o r u s l i g a t e d r a t s b y o v e r 5 0 % a t doses of 30 m g / k g . F u r t h e r purification gave material with antisecretory activity at 50 t~g/kg in r a t s b u t t h e p r o d u c t b e h a v e d as a c o m p o u n d w i t h a m o l e c u l a r w e i g h t of a b o u t 150,000~L A t t h e s a m e t i m e i n h i b i t o r y m a t e r i a l ( M W 50,000) was also o b t a i n e d f r o m m o u s e s u b m a x i l l a r y g l a n d s w i t h a n a c t i v i t y of 25 ~g/kg b u t t h e s e w o r k e r s f o u n d n o n e of t h e s e x u a l d i m o r p h i s m a s s o c i a t e d w i t h e p i d e r m a l g r o w t h f a c t o r ~s. T h i s sex difference is q u i t e s t r i k i n g ; t h e m a l e m o u s e c o n t a i n s ca. 1000 n g / m g w e t tissue a n d t h e f e m a l e o n l y ca. 70 n g / m U 9, b u t t r e a t m e n t of t h e f e m a l e w i t h testost e r o n e causes a d r a m a t i c rise in t h e levels of E G F ~4. S m a l l e r a m o u n t s were also o b s e r v e d in r a t s a l i v a r y g l a n d s ~, b u t i n h i b i t i o n of acid secretion c a n b e effected in rats, a l b e i t a t h i g h e r doses t h a n are n e c e s s a r y in t h e dog. A r e c e n t p u b l i c a t i o n described t h e i d e n t i f i c a t i o n of i m m u n o r e a c t i v e E G F in t h e p l a s m a of p r e g n a n t h u m a n females b u t n o t in n o n - p r e g n a n t females or m a l e s 2~ a n d levels u p t o 6 n g / m l were r e p o r t e d d u r i n g e a r l y p r e g n a n c y . H o w e v e r , if t h e doses a d m i n i s t e r e d t o dogs --- 0.5 ~ g / k g i.v., were c a l c u l a t e d as b l o o d levels t h e n t h e m a x i m u m briefly a t t a i n a b l e w o u l d b e a b o u t 6 n g / m l a n d t h i s rep r e s e n t s n e a r m a x i m a l i n h i b i t i o n of gastric acid secretion. If s i m i l a r doses a p p l i e d t o t h e h u m a n t h e n a p e r s i s t e n t b l o o d level of 6 n g / m l w o u l d p r e s u m a b l y b e a s s o c i a t e d w i t h g r e a t l y r e d u c e d acid secretion unless, of course, s e n s i t i v i t y was r e d u c e d d u r i n g p r e g n a n c y . T h i s m a y r e l a t e t o t h e k n o w n low i n c i d e n c e of p e p t i c u l c e r a t i o n d u r i n g pregnancy. A m o n g t h e k n o w n p r o p e r t i e s of E G F in c a u s i n g cell p r o l i f e r a t i o n is t h e s t i m u l a t i o n of o r n i t h i n e d e c a r b o x y l ase 2~. More r e c e n t l y it was r e p o r t e d t h a t h i s t i d i n e decarb o x y l a s e was also s t i m u l a t e d b y E G F in skin a n d o t h e r tissues a l t h o u g h g a s t r i c m u c o s a was n o t a m o n g s t t h o s e studied~2. N e v e r t h e l e s s a n u m b e r of Other i n h i b i t o r s of

EXPERIENTIA 31/7

gastric s e c r e t i o n are also a s s o c i a t e d w i t h i n c r e a s e d h i s t i d i n e d e c a r b o x y l a s e a c t i v i t y in t h e gastric m u c o s a e v e n t h o u g h h i s t a m i n e itself is a s t i m u l a n t of acid secretion 23 ! P o s s i b l y E G F e x e r t s a s i m i l a r effect u p o n g a s t r i c mucosa. T h e doses of E G F r e q u i r e d t o affect gastric s e c r e t i o n in dogs are in t h e s a m e region as t h o s e for t h e h o r m o n e gastrin24, a n d are low c o m p a r e d to t h o s e r e q u i r e d for t h e o t h e r o b s e r v a b l e effects, so t h a t it is t e m p t i n g to s p e c u l a t e t h a t it is a t r u l y p h y s i o l o g i c a l effect. I n d e e d it m a y well b e t h a t c o m p a r a b l e p e p t i d e s f r o m d i f f e r e n t species are i n v o l v e d in t h e c o n t r o l of g a s t r i c acid secretion.

Rdsumd. Le f a c t e u r de croissance 6 p i d e r m i q u e ( E G F ) a 6t6 isol6 A p a r t i r des g l a n d e s s o u s - m a x i l l a i r e s des souris. O n a t r o u v 6 que ce f a c t e u r est u n i n h i b i t e u r de la sCcrCtion g a s t r i q u e . Chez les chiens des doses aussi faibles que 0,1 ~g/ kg f o n t baisser la sCcrCtion g a s t r i q u e provoquCe p a r d i v e r s stimulants. J. M. BOWER, R. CAMBLE, H. GREGORY, E. L. GERRING a n d I. R. WILLSHIRE Imperial Chemical Industries Ltd., Pharmaceutical Division, Mereside A lderley Park, Maccles/ield (Cheshire SKIO d TG, England) 4 March 1975.

15 C. F. CODE, H. V. RATKE, G. R. LIVERMORE and W. LUNDBERG, Fedn. Prec. 8, 26 (1949). le R. MENGUY and M. BERLINSKI, Amer. J. dig. Dis. 12, 1 (1967). 17 1V[.KOBAYASHIand M. YAMAMOTO, J. pharm. Soc. Japan 89, 222 (1969), 18 M. KOBAYASHIand M. YAMAMOTO,J. pharm. Soc. Japan 92, 226 (1972). 19 R. L. BYVNY, D. N. ORTH and S. COHEN, Endocrinology 90, 1261

(1972). 20 I. G. ANCES, Am. J. Obstet. Gynec. 115, 357 (1973). 21 M. STASTRY and S. COHEN, Biochim. biophys. Aeta. 204, 578 (1970). 22 p. T. BLOSSE, E. L. FENTON, S. HENNINGSSON,G. KAHLSONand

E. ROSENGREN, Experientia 30, 23 (1974). 23 D. V. MAUDSLEY, Y. KOBAYASHI,E. WILLIAMSONand L. BOVAIRD,

Nature New Biol. 245, 148 (1973). 24 R. A. GREGORYand H. J. TRACu Gut 5, 103 (1964).

Modification b y C a l c i u m D o b e s i l a t e of H i s t a m i n e Effects on Capillary U l t r a s t r u c t u r e As is well k n o w n , t h e e x i s t e n c e of close c o n t a c t s b e t w e e n cells is t h e m o s t p e c u l i a r f e a t u r e of e p i t h e l i a l s t r u c t u r e s . In the vascular endothelium, and particularly at the b l o o d c a p i l l a r y level, i n t e r c e l l u l a r b o n d s t h r o u g h i n t e r d i g i t a t i o n c r e a t e a selective b a r r i e r b e t w e e n t h e v a s c u l a r c o m p a r t m e n t a n d t h e i n t e r s t i t i a l fluid ~. T h e s t a b i l i t y of t h e s e i n t e r c e l l u l a r c o n t a c t s d e p e n d s on t h e c h e m i c a l c o m p o s i t i o n of t h e cell m e m b r a n e , a n d m o r e precisely of t h e cell coat, as h a s b e e n s h o w n in several e x p e r i m e n t a l studies 2. O n t h e o t h e r h a n d , e n d o t h e l i a l cells, a n d b o n d s b e t w e e n t h e m , c o n s t i t u e t h e m a j o r s t r u c t u r a l b a s e of c a p i l l a r y p e r m e a b i l i t y a n d c a p i l l a r y resistence. Several e n d o g e n o u s chemicals, a n d a m o n g t h e m p a r t i c u l a r l y h i s t a m i n e , are c a p a b l e of d i s t u r b i n g c a p i l l a r y f u n c t i o n , l e a d i n g t o a n i n c r e a s e in c a p i l l a r y p e r m e a b i l i t y a n d a fall in c a p i l l a r y resistence. H i s t a m i n e p r o d u c e d a n d s t o r e d in m a s t cells is released in r e s p o n s e t o a local i n j u r y 3, following a n a n t i g e n - a n t i b o d y r e a c t i o n , or e v e n t h r o u g h t h e effects of p h y s i o l o g i c a l or p h a r m a c o l o g i c a l substances~ I n fact, a role for h i s t a m i n e h a s b e e n p o s t u l a t e d in conn e c t i o n w i t h t h e e s t r o g e n - i n d u c e d increase in c a p i l l a r y p e r m e a b i l i t y a t t h e e n d o m e t r i M leveD.

Since t h e classical s t u d i e s of MAINe a n d PALADE s, i t is g e n e r a l l y a c c e p t e d t h a t h i s t a m i n e effects on c a p i l l a r y p e r m e a b i l i t y m a i n l y r e s u l t f r o m a n a c t i o n of h i s t a m i n e o n p o s t c a p i l l a r y venules. T h e increase in c a p i l l a r y p e r m e a b i l i t y would be t h e r e s u l t of s e p a r a t i o n on e n d o t h e l i a l cells a t t h e i r b o u n d a r i e s , d u e t o t h e c o n t r a c t i o n a n d s h r i n k age of t h e s e cells w i t h f o r m a t i o n of s t o m a t a e . I t m u s t b e stressed, h o w e v e r , t h a t t h e e v i d e n c e in f a v o u r of a t r u e c o n t r a c t i o n of t h e e n d o t h e l i a l cell u n d e r t h e effects of h i s t a m i n e is o n l y weak, a n d consists m a i n l y in t h e similit u d e w i t h t h e effects of h i s t a m i n e o n s m o o t h muscle.

1 W. BLOOM and D. W. FAWCETT, in Textbook o/Histology (W. B. Saundcrs, Philadelphia 1968), p. 358. 2 j. Z. BORYSENKO and J. P. REVEL, Am. J. Anat. 737, 403 (1973). 3 W. G. SPECTOR, Pharmac. Rev. 10, 475 (1958). 4 C. D. TURNER, in GeneralEndocrinology (W. B. Saunders, Philadelphia 1966), p. 439. s G. MAJNO and G. E. PALADE, J. biophys. Biocheln. Cyt. 11, 571 (1961). e G. MAJNO, M. S. SHEA and M. LEVENTHAG J. Cell Biol. 42, 648 (1969).