European Journal o f Pharmacology, 56 (1979) 217--223
217
© Elsevier/North-Holland Biomedical Press
I N H I B I T I O N O F G A S T R I C A C I D S E C R E T I O N BY A G L Y C O P R O T E I N I S O L A T E D F R O M HUMAN URINE (HUMAN URINARY GASTRIC INHIBITOR) RICCARDO NIADA *, GIUSEPPE PRINO * , GIUSEPPE LUGARO, GIACOMO CARREA, PIERO PASTA and MARIA M. CASELLATO * Crinos Biological Research Laboratories, 22079 Villa Guardia, Italy, and Laboratorio di Chimica degli Ormoni, C.N.R., 20131 Milano, Italy
Received 11 October 1978, revised MS received 23 February 1979, accepted 1 March 1979
R. NIADA, G. PRINO, G. LUGARO, G. CARREA, P. PASTA and M.M. CASELLATO, Inhibition o f gastric acid secretion by a glycoprotein isolated from human urine (human urinary gastric inhibitor), European J. Pharmacol. 56 (1979) 217--223. The gastric antisecretory activity of an inhibitor newly isolated from human urine (Human Urinary Gastric Inhibitor or HUGI) has been studied. HUGI was given intravenously and its activity determined in the following test systems: gastric secretion in the rat with pyloric ligation; gastric secretion in the dog with a Heidenhain pouch stimulated with pentagastrin, histamine and a protein meal; acid secretion by the isolated gastric mucosa of the rat; gastrointestinal motility; bile flow and gall-bladder tone and arterial and venous blood pressure and heart rate. HUGI was found to have marked activity only in the pyloric-ligated rats and in the dogs with Heidenhain pouches stimulated by a protein meal. Particularly in the dog, HUGI (0.1 to 6.4 pg/kg, i.v.) markedly inhibited gastric secretion, dose-dependently and without changing the plasma gastrin concentration. Negative results were obtained in the other tests, but these results serve to demonstrate that HUGI is an inhibitor welldifferentiated from other glycoproteins or peptides with gastric antisecretory activity, such as urogastrone and GIP. The results obtained to date are not sufficient to allow the mechanism of action of HUGI to be defined. Human urinary gastric inhibitor
Glycoproteins
1. I n t r o d u c t i o n I t has b e e n k n o w n since 1 9 3 8 t h a t h u m a n urinary extracts have a marked inhibitory e f f e c t o n gastric s e c r e t i o n (Sandweiss et al., 1 9 3 8 , 1 9 3 9 ; G r a y e t al., 1 9 4 0 ) , b u t it was o n l y in t h e last f e w y e a r s t h a t it has b e e n possible t o isolate t h e various c o m p o n e n t s r e s p o n s i b l e f o r this e f f e c t in a p u r e state a n d to study their physical and chemical properties. I n v e s t i g a t i o n s carried o u t b y G r e g o r y ( G r e g o r y , 1 9 7 5 , G r e g o r y a n d Willshire, 1 9 7 5 ; ( G r e g o r y a n d Preston, 1 9 7 7 ) a n d in o u r l a b o r a t o r i e s (Carrea et at., 1 9 7 3 ; L u g a r o et al., 1 9 7 6 ) h a v e s h o w n t h a t h u m a n u r i n e contains at least t w o s t r o n g l y a n t i s e c r e t o r y fac-
Gastric secretion
t o r s w h i c h are c o m p l e t e l y c h e m i c a l l y different from one another. T h e i n h i b i t o r isolated b y G r e g o r y is a 53aminoacid polypeptide (Gregory, 1975), while t h e o n e we h a v e p u r i f i e d ( H u m a n U r i n a r y Gastric I n h i b i t o r or H U G I ) is a high m o l e c u l a r w e i g h t g l y c o p r o t e i n (S~0,w = 8; Ve = V o o n Bio-Gel A 1.5 M), c o n t a i n i n g 37% c a r b o h y d r a t e (9% sialic acid). T h e r a t i o o f g l u c o s a m i n e / g a l a c t o s a m i n e is 4 / 1 . All t h e g a l a c t o s a m i n e is b o u n d t o t h e p o l y p e p t i d e chain as O-glycoside. T h e glycop r o t e i n is m a d e u p o f m a n y identical subunits w i t h a m o l e c u l a r w e i g h t o f a b o u t 1 4 , 0 0 0 D a l t o n s ; t h e g l y c o p r o t e i n was active in t h e p o l y m e r i c f o r m ( L u g a r o e t al., 1 9 7 6 ) . T h e active p r i n c i p l e f o u n d b y G r e g o r y
218 (Urogastrone) was obtained from urine of both men and women. HUGI is also present in the urine of both men and women. We extracted it from the urine of pregnant women since several investigators (Sandweiss et al., 1938, 1939; Gray et al., 1940) observed that during gestation there is a marked decrease in gastric secretion and a temporary remission of gastric ulcers; this would seem to indicate t h a t a larger a m o u n t of the antisecretory factor should be present during pregnancy. The aim of the present work was to study the antisecretory activity of HUGI in the dog and in the rat, to use different experimental models to study its mechanism of action and to compare its biological characteristics with those of other antisecretory factors.
2. Materials and methods
2.1. Preparation o f the gastric secretion inhibitor (HUGI) from human urine HUGI was obtained from pooled urine of women in the third or f o u r t h m o n t h of pregnancy, as previously reported (Carrea et al., 1973). The purification procedure included chromatography on an IONAC A-540 column, benzoic acid extraction, fractional precipitation with organic solvents and gel filtration on Sephadex G-200 or Bio Gel A-1.5 m.
2.2. Effect against gastric acid and pepsin secretion in pyloric-ligated rats HUGI, buffered to pH 7.2, was injected i.v. (from 2.5 to 20 pg/kg b o d y weight) into 180 + 5 g male Wistar (Morini Italy), maintained on the MIL® type R diet (Morini, Italy), but fasted for 48 h before treatment, as described by Lugaro et al. (1965). 2 h after injection and pyloric ligation, the volume of gastric fluid was measured, as well as the total acid and hydrochloric acid as H ÷ output, using an automatic Radiometer TTA-60 titrator.
R. NIADA ET AL. Pepsin o u t p u t was measured by the Prino et al. modification (1971a,b) of Hunt's method (1948). The experiments were planned according to a randomized block design (4 rats per group; 4 replications).
2.2. Effects against gastric acid secretion in dogs with Heidenhain pouches Male mongrel dogs, weighing 10--15 kg, were prepared with Heidenhain pouches and fed the McCay standard diet (Piccioni, Italy). The tests were begun 4 months after the surgical procedure. Before treatment with HUGI the dogs were conditioned to the silent, temperature,controlled room. The reproducibility of the hypersecretory responses to pentagastrin stimulation (Gastrodiagnost®, Merck, 6 pg/kg, s.c.), to histamine (histamine dihydrochloride, C. Erba, Italy, i.v. perfusion, 80 pg/ kg/h) and to a protein meal (150 ml of a 10% solution of Liebig's meat extract) was determined. The secretory stimuli induced nearmaximal responses under our experimental conditions. In the study of the effects of HUGI, each dog was used as its own control. The inhibitor was injected i.v. (from 0.1 t o 6.4 gg/kg body weight) 30 rain before ~;";~-,~.,~ the hypersecretory stimulus. The gastric secretion was collected every 15 min for 2 h, the volumes measured and the total acid and hydrochloric acid concentration titrated.
2.4. Determination o f plasma gastrin levels in Heidenhain pouch dogs Plasma gastrin concentrations were determined by a modification of the radioimmunoassay of Yalow and Berson (1970). The GASK kits (Sorin Biomedica, Italy) used contain synthetic human gastrin I 1-17 (ICI Ltd., MacClesfield, Cheshire, England) and rabbit antigastrin antiserum produced against synthetic h u m a n gastrin I 1-17. Gastrin from the same source was iodinated with ~:sI by the chloramine T m e t h o d and purified by polyacrilamide gel electrophoresis.
HUMAN URINARY GASTRIC INHIBITOR
219
2.5. Isolated rat gastric mucosa
2.7. Bile flow in the rat and gall-bladder tone in the guinea pig
T h e gastric m u c o s a f u n d u s o f t h e rat s t o m a c h was p r e p a r e d b y t h e t e c h n i q u e s described b y Wan et al. ( 1 9 7 4 ) , and F o r t e et al. ( 1 9 7 5 ) . Acid secretion was s t i m u l a t e d with h i s t a m i n e (histamine d i h y d r o c h l o r i d e , BDH, 25 p g / m l ) , with pentagastrin (Gastrodiagnost®, Merck, 20 p g / m l ) or with DBcAMP ( N 6 - 2 ' - ( O - d i b u t y r y l - a d e n o s i n e - 3 ' , 5 ' cyclic m o n o p h o s p h a t e , Boehringer, 100 pg/ml). H U G I (in a c o n c e n t r a t i o n u p t o 1 0 0 p g / m l ) was placed in c o n t a c t with t h e serosa 5 m i n b e f o r e the stimuli.
2.6. Gastrointestinal motility H U G I was given i.v. t o rats, cats and dogs in doses f r o m 10 t o 1 0 0 pg/kg. In t h e rat, t h e rate o f transit o f a c h a r c o a l suspension was m e a s u r e d b y t h e t e c h n i q u e o f Witkin et al. ( 1 9 6 1 ) . In t h e a n e s t h e t i z e d cat, m o t i l i t y was r e c o r d e d b y i n t r o d u c i n g into t h e s t o m a c h and the d u o d e n u m rubber balloons connected to HP m o d e l 1 2 8 0 pressure transducers. In the u n a n e s t h e t i z e d dog, t h e r u b b e r b a l l o o n was placed in t h e H e i d e n h a i n p o u c h .
T r a d i t i o n a l m e t h o d s for bile d u c t were used f o r t h e and gall-bladder t o n e was a t t a c h e d to t h e end o f t h e an isometric t r a n s d u c e r Ljungberg (1964).
cannulation of the rat bile flow s t u d y r e c o r d e d b y a line gall-bladder and t o as described b y
2.8. Cardiovascular system T h e a n e s t h e t i z e d cats used for o b s e r v a t i o n o f gastrointestinal m o t i l i t y were p r e p a r e d f o r simultaneous recording of carotid artery and jugular vein pressures, using t r a d i t i o n a l m e t h o d s . In a d d i t i o n , changes in h e a r t rate were o b t a i n e d f r o m t h e E C G b y an HP m o d e l 8 8 1 2 / A rate c o m p u t e r . H U G I was given i.v. in doses b e t w e e n 10 and 1 0 0 pg/kg.
3. Results
3.1. Pylorus-ligated rats In t h e pyloric-ligated rat, H U G I had a m a r k e d gastric a n t i s e c r e t o r y effect, w h i c h was
TABLE 1 Effect of urinary glycoprotein (HUGI) on gastric secretion at 120 min after pyloric ligation in rats. Mean ± S.E. from 16 rats per group. Each mean was compared to the control values with Student's t-test. Percentage inhibition in parentheses. Treatment pg/kg i.v.
Gastric juice volume (ml)
Hydrochloric acid output (pEq H÷)
Total acid output (pEq H÷)
Pepsin output (//moles L-tyrosine)
Saline 5 ml/kg HUGI 2.5 3.75 5 10 20
3.84 ± 0.24
303.71 ± 37.51
443.03 + 41.11
84.61 ± 4.05
3.05± 0.38 (20.6) 2.10 ± 0.20 3 (45.3) 1.64 ± 0.19 3 (57.3) 1.10± 0.103 (71.3) 0.89 -+ 0.09 3 (76.8)
153.91+ 44.47 139.10 ± 24.95 65.31 -+ 15.89 38.46± 7.98 32.16 +- 10.05
284.45+ 55.88 1 (35.8) 223.59 ± 28.35 3 (49.5) 132.31 ± 21.31 3 (70.1) 93.48± 11.043 (78.9) 70.77 +- 10.41 3 (84.0)
65.30± 6.91 38.81 ± 5.01 31.28 ± 4.28 21.55-+ 2.47 17.77 ± 1.63
1 p < 0.05, 2 p < 0.005, 3 p < 0.001.
1 (49.3) 2 (54.2) 3 (78.5) 3 (87.3) 3 (89.4)
1 (22.8) 3 (54.1) 3 (63.0) 3 (74.5) 3 (79.0)
220
R. NIADA ET AL.
manifested by the volume of the secretion, the acid output and the pepsin output. The inhibition started to become significant with t h e 2 . 5 p g / k g i.v. d o s e a n d w a s p r o p o r t i o n a l t o t h e d o s e s g i v e n f o r all t h e p a r a m e t e r s m e a s u r e d ( t a b l e 1).
600 ' ¢
400
i' ~
i
-
ConlrOI
/ g
C,,nT,,,i kq
~, BOO
3.2. Heidenhain pouch dogs In the dogs with Heidenhain pouches, HUGI i n j e c t e d i.v. in t h e m a x i m a l d o s e o f 6 . 4 p g / k g , a d o s e h i g h l y e f f e c t i v e in i n h i b i t i n g t h e a c i d output stimulated by a protein meal, had no effect on the gastric secretion stimulated by pentagastrin or histamine. No changes were s e e n in t h e t o t a l a c i d o u t p u t , w h i c h w a s 1503 + 196 pEq H÷/2 h after pentagastrin s t i m u l a t i o n a n d 2 1 1 4 -+ 1 2 6 laEq H * / 2 h a f t e r histamine stimulation. But HUGI did have marked dose-dependent inhibitory effects on the gastric secretion stimulated by a protein meal. For 0.1, 0.4, 1 . 6 a n d 6 . 4 p g / k g i.v. d o s e s o f t h e i n h i b i t o r , t h e r e w e r e d e c r e a s e s in v o l u m e w h i c h r a n g e d f r o m 2 8 t o 7 0 % , a n d d e c r e a s e s in t o t a l a c i d o u t p u t r a n g i n g f r o m 2 6 t o 8 2 % ( t a b l e 2). T h e concentration of acid was also influenced by the treatment, at least at the highest doses,
'"
i 400 ; !
30
Conrro~
/'\Control
/kg~
HUGI 16 pg/kg
0
30
60
T I
HUGI 109~meat extract
90
120
30
0
30
60
90
12Omin
t I
HUGI 10~meat extract
Fig. 1. Acid output from Heidenhain pouches. Inhibitory effect of i.v. injected HUGI on hypersecretion induced by a protein meal. Effect on total acid output (mean ± S.E. of four tests in 3 dogs). when the hydrogen ion concentration was decreased by about 50%. At least at the highest doses of HUGI had a n i n h i b i t o r y e f f e c t a t all t h e t i m e s o f t e s t i n g a f t e r t h e s t i m u l u s w a s g i v e n (fig. 1).
3.3. Plasma gastrin concentration of
In another series of experiments, the effects HUGI on the plasma concentration of
TABLE 2 Effect of urinary glycoprotein (HUGI) on gastric hypersecretion induced by meat extract in Heidenhain pouches. Mean values +- S.E. of four tests in 3 dogs. Each mean was compared to the control values with Student's t-test. Percentage inhibition in parentheses. Treatment (pg/kg i.v.)
Gastric juice volume/2 h (ml)
/~Eq H ÷ output/2 h Hydrochloric acid
Total acid
Saline HUGI
3 ml 0.10
12.66 -+ 1.02 9.11 ± 0.42 1 (28.04)
1312.33 ± 63.94 952.66 ± 82.39 1 (27.40)
1511.66 ± 70.89 1119.00 ± 59.25 2 (25.91)
Saline HUGI
3 ml 0.40
12.16 ± 0.99 6.46 ± 0.76 2 (46.87)
1255.33 ± 71.39 453.33 ± 41.25 a (63.88)
1478.00 -+ 47.93 642.00 ± 76.69 4 (56.56)
Saline HUGI
3 ml 1.60
12.12 ± 0.76 5.17 ± 0.18 4 (57.34)
1386.66 -+52.66 371.00 ± 93.02 4 (73.24)
1589.00 -+ 17.67 459.33 ± 96,56 4 (71.09)
Saline HUGI
3 ml 6.40
12.71 ± 1.05 3.85 ± 0.48 3 (69.70)
1466.33 -+ 39.56 241.66 ± 18.94 a (83.51)
1697.33 ± 60.45 306.66 ± 13.38 4 (81.93)
i p < 0.05, 2 p < 0.02, 3 p < 0.005, 4 p < 0.001.
221
HUMAN URINARY GASTRIC INHIBITOR - - - r - - / ~ - -
80 "~ 800p ':
10%meat [
m
,
+
"
I
exltract
The cat showed no significant changes in arterial or venous pressure or in heart rate.
60
4. Discussion 4 0 -=- 4 0 0 ~
~.
e
--~:.20 +~ 200 ~-
Control
, ,
30
0
15
30
45
60
75
90
105 120
min
I
HUGI
Fig. 2. Acid output from Heidenhain pouches. Effect
of intravenously injected HUGI (6.4 pg/kg) on the plasma gastrin levels (©) and total acid output (A) stimulated by a protein meal (mean _+S.E. of three tests in 2 dogs).
gastrin were evaluated in dogs with Heidenhain pouches. Under the experimental conditions previously described, the basal plasma levels were 28.5 + 2.92 pg/ml. After stimulation with the protein meal, the concentrations of gastrin rose to a maximal level of 73.0 + 6.02 pg/ml. The changes in the plasma gastrin concentration and in total acid o u t p u t determined simultaneously were plotted vs. time (fig. 2). When HUGI was given intravenously 30 min before the stimulus, even the maximal dose of 6.4 pg/kg was n o t able to m o d i f y the plasma gastrin concentration, but it decreased the total acid o u t p u t by 77% at the second hour of observation. 3.4. Other tests
Negative results were obtained in all the other tests. When isolated rat mucosa was incubated with HUGI, even at concentrations of 100 pg/ml, there was no change in the peak of acid secretion induced by histamine (5.56 + 0.77 pEq H+/cm2/30 min) by pentagastrin (4.63 + 0.32 pEq H*/cm2/30 min) or by DBcAMP (9.76 + 1.02 pEq H*/cm~/30 min). The bile flow in the rat, gall-bladder tone in the guinea pig and gastrointestinal motility of the rat, cat and dog were not affected by HUGI up to 100 pg/kg i.v.
HUGI is a newly found antisecretory factor from human urine and has been shown to be a high molecular weight glycoprotein (Carrea et al., 1973; Lugaro et al., 1976). The biological assay procedure used during its isolation and purification was based on its inhibition of gastric secretion in the rat with ligated pylorus (Carrea et al., 1973). The results listed in table 1 confirm the inhibitory effect of HUGI given i.v. to the rat on both the volume of secretion and the acid output. The pepsin o u t p u t was also decreased, but in this respect, one must keep in mind that this may have been due to the drastically reduced volume of gastric juice secreted. In the Heidenhain pouch dog, HUGI demonstrated a striking anti-secretory effect when gastric secretion was stimulated by a protein meal, but did not affect the hypersecretion induced by pentagastrin of histamine. This confirms the earlier results obtained in Pavlov pouch dog (Gandini et al., 1970). The inhibitory effect on hypersecretion induced by a protein meal was not accompanied by any corresponding change in the plasma gastrin levels. Therefore, it would be difficult to explain its antisecretory effect as an action at the level of the G cells where it could interfere with the synthesis or the release of gastrin, as might have been suggested by the other results. HUGI had no effect on hypersecretion from the isolated mucosa of the rat stimulated with pentagastrin, histamine or DB-cAMP. it did not change gastrointestinal motility in the rat, dog or cat and the gallbladder tone in the guinea pig was not influenced. It did not affect bile flow even at doses much higher than those which maximaUy inhibited gastric secretion. These results are specific characteristics of the biological properties of HUGI, especially as compared
222 with t h o s e already k n o w n f o r u r o g a s t r o n e and GIP. Unlike H U G I , u r o g a s t r o n e and GIP inhibit the hypersecretory response to exogenous pentagastrin and h i s t a m i n e in t h e H e i d e n h a i n p o u c h dogs (Gerring and H a w o r t h , 1 9 7 0 ; Pederson and B r o w n , 1 9 7 2 ) . Like H U G I , t h e y inhibit the s e c r e t i o n s t i m u l a t e d b y a p r o t e i n meal, b u t o n l y GIP also r e d u c e s t h e plasma gastrin levels (Villar et al., 1 9 7 5 ; Elder et al., 1975). On isolated gastric m u c o s a , urogastrone, unlike H U G I , inhibits the h y p e r secretion caused b y pentagastrin or histamine (Bower, 1 9 7 3 ) . Like H U G I , u r o g a s t r o n e does n o t a f f e c t gastric m o t i l i t y or bile flow (Getring, 1 9 7 0 ; G r o s s m a n et al., 1 9 7 4 ) , while GIP decreases t h e p o u c h c o n t r a c t i o n s in t h e d o g ( P e d e r s o n , 1 9 7 1 ) . These observations show t h a t H U G I is an i n h i b i t o r o f gastric secretion which differs f r o m u r o g a s t r o n e and GIP with respect to biological p r o p e r t i e s as well chemical n a t u r e (Carrea et al., 1 9 7 3 ; Lugaro et al., 1976). As a f u r t h e r c o n f i r m a t i o n , we can cite t h e radioimmunological trials of Gregory (personal c o m m u n i c a t i o n ) , which s h o w e d t h a t t h e r e was n o cross-reaction b e t w e e n H U G I and u r o g a s t r o n e at c o n c e n t r a t i o n s as m u c h as 2.5 × l 0 s times greater (5 pg o f H U G I against 20 pg o f u r o g a s t r o n e ) and we t h e r e f o r e reject the h y p o t h e s i s t h a t t h e polyp e p t i d e ( u r o g a s t r o n e ) is derived f r o m the glycoprotein (HUGI). Until now, the m e c h a n i s m o f a c t i o n o f H U G I has n o t been explained. H o w e v e r , we can state t h a t H U G I inhibits o n l y t h e gastric secretion i n d u c e d b y physiological stimuli and is ineffective against pharmacologically induced secretion. A n o t h e r i m p o r t a n t fact is t h a t H U G I can be held largely a c c o u n t a b l e for t h e t o t a l a n t i s e c r e t o r y activity o f h u m a n urine. In fact 2 mg o f H U G I / l i t e r in t h e pure state were o b t a i n e d f r o m urine (Carrea et al., 1 9 7 3 ) whereas the yield o f u r o g a s t r o n e was o n l y 1 #g/liter ( G r e g o r y , 1975). Even if t h e specific activity o f u r o g a s t r o n e were greater t h a n t h a t o f H U G I , its c o n c e n t r a t i o n in urine is less t h a n o n e t h o u s a n d t h the c o n c e n t r a t i o n o f HUGI.
R. NIADA ET AL. Acknowledgement The assistance of Franco Paties is gratefully acknowledged.
References Bower, J.M., 1973, The effect of urogastrone on isolated gastric mucosa, M.Sc. Thesis, University of Sheffield. Carrea, G., M.M. Caseltato, E. Manera, P. Pasta and G. Lugaro~ 1973, Piirificati6n 5~a ~u-rnanu)]na@~ glycoprotein with gastric antisecretory activity, Biochim. Biophys. Acta 295,274. Elder, J.B., P.C. Ganguli, I.E. Gillespie, E.L. Gerring and H. Gregory, 1975, Effect of urogastrone on gastric secretion and plasma gastrin levels in normal subjects, Gut 16,887. Forte, J.C., T.M. Forte and T.E. Machen, 1975, Histamine~stimulated hydrogen ion secretion by in vitro piglet gastric mucosa, J. Physiol. (London) 244, 15. Gandini, A., G. Lugaro, L. Ferrari and P. Lualdi, 1970, Influenza dell'urogastrone sugli effetti motori e secretivi del tetrapeptide terminale della gastrina, Boll. Chim. Farm. 109,118. Gerring, E.L., 1970, Actions of urogastrone. Ph.D. Thesis, University of London. Gerring, E.L. and E. Haworth, 1970, The effect of urogastrone on gastric acid secretion in dogs and cats, Rendiconti romani di Gastroenterologia 2, 167. Gray, J.S., C.U. Culmer, E. Wieczorowski and J.L. Adkinson, 1940, Preparation of pyrogen-free urogastrone Proc. Soc. Exptl. Biol. Med. 43, 225. Gregory, H., 1975, Isolation and structure of Urogastrone and its relationship to epidermal growth factor, Nature (London) 257,325. Gregory, H. and I.R. Willshire, 1975, The isolation of Urogastrones-inhibitors of gastric acid secretion from human urine, Hoppe-Seyler's Z. Physiol. Chem. 356, 1765. Gregory, H. and B.M. Preston, 1977, The primary structure of human Urogastrone, Intern. J. Pept. Protein Res. 9, 107. Grossman, M.I. and others, 1974, Candidate hormones of the gut, Gastroenterology 67,730. Hunt, J.N., 1948, A method for estimating peptic activity in gastric contents, Biochem. J. 42, 104. Ljungberg, S., 1964, Biologisk styrkebest~/mming av cholecystokinin, Sven. Farm. Tidskr., 68,351. Lugaro, G., I. Lupi and A. Corbellini, 1965, Ricerche sull'urogastrone. Nota IV. Le attivita antiulcerosa e antisecretoria dei prodotti di frazionamento dell'urogastrone, G. Biochim. 14, 126.
HUMAN URINARY GASTRIC INHIBITOR Lugaro, G., P. Pasta, M.M. Casellato, G. Mazzola and S. Carrea, 1976, Chemical and physical properties of the Human Urinary Glycoprotein with gastric antisecretory activity, Biochem. J. 1 5 3 , 6 4 1 . Pederson, R.A. 1971, The isolation and physiological actions of gastric inhibitory polypeptide. Thesis, University of Britisch Columbia, Vancouver. Pederson, R.A. and J.C. Brown, 1972, The inhibition of histamine-pentagastrin and insulin-stimulated gastric secretion by pure gastric inhibitory poly-peptide, Gastroenterology 6 2 , 3 9 3 . Prino, G., S. Paglialunga, G. Nardi and A. Lietti, 1971a, Inhibition of experimentally-induced gastric ulcers in the rat by a new sulfated glycopeptide, European J. Pharmacol. 1 5 , 1 1 9 . Prino, G., A. Lietti and S. Paglialunga, 1971b, Inhibition of gastric peptic activity by a new sulfate glycopeptide (GLPS), Arzneim. Forsch. 21, 918. Sandweiss, D.J., H.C. Saltzstein and A. Farbman, 1938, The prevention of healing of experimental peptic ulcer in Mann-Williamson dogs with the anterior pituitary-like hormone (antuitrin-S). A preliminary report, Amer. J. Dig. Dis. 5, 24.
223 Sandweiss, D.J., H.C. Saltzstein and A. Farbman, 1939, The relation of sex hormones to peptic ulcer, Amer. J. Dig. Dis. 6, 6. Villar, H.V., H.R. Fender, P.L. Rayford, S.R. Bloom, N.I. Ramus and J.C. Thompson, 1976, Suppression of gastrin release and gastric secretion by gastric inhibitory polypeptide (GIP) and vasoactive intestinal polypeptide (VIP), Ann. Surg. 184, 97. Wan, B.Y.C., E.S.K. Assem and H.O. Schild, 1974, Inhibition of in vitro stimulated gastric acid secretion by an Histamine H2-receptor antagonist, Metiamide, European J. Pharmacol. 29, 83. Witkin, L.B., C.F. Huebner, F. Galdi, E. O'Keefe, P. Spitaletta and A.J. Plummer, 1961, Pharmacology of 2-amino-indane hydrochloride (SU-8629): a potent non-narcotic analgesic, J. Pharmacol. Exptl. Therap. 1 3 3 , 4 0 0 . Yalow, R.S. and S.A. Berson, 1970, Radioimmunoassay of Gastrin, Gastroenterology 58, 1.
217
© Elsevier/North-Holland Biomedical Press
I N H I B I T I O N O F G A S T R I C A C I D S E C R E T I O N BY A G L Y C O P R O T E I N I S O L A T E D F R O M HUMAN URINE (HUMAN URINARY GASTRIC INHIBITOR) RICCARDO NIADA *, GIUSEPPE PRINO * , GIUSEPPE LUGARO, GIACOMO CARREA, PIERO PASTA and MARIA M. CASELLATO * Crinos Biological Research Laboratories, 22079 Villa Guardia, Italy, and Laboratorio di Chimica degli Ormoni, C.N.R., 20131 Milano, Italy
Received 11 October 1978, revised MS received 23 February 1979, accepted 1 March 1979
R. NIADA, G. PRINO, G. LUGARO, G. CARREA, P. PASTA and M.M. CASELLATO, Inhibition o f gastric acid secretion by a glycoprotein isolated from human urine (human urinary gastric inhibitor), European J. Pharmacol. 56 (1979) 217--223. The gastric antisecretory activity of an inhibitor newly isolated from human urine (Human Urinary Gastric Inhibitor or HUGI) has been studied. HUGI was given intravenously and its activity determined in the following test systems: gastric secretion in the rat with pyloric ligation; gastric secretion in the dog with a Heidenhain pouch stimulated with pentagastrin, histamine and a protein meal; acid secretion by the isolated gastric mucosa of the rat; gastrointestinal motility; bile flow and gall-bladder tone and arterial and venous blood pressure and heart rate. HUGI was found to have marked activity only in the pyloric-ligated rats and in the dogs with Heidenhain pouches stimulated by a protein meal. Particularly in the dog, HUGI (0.1 to 6.4 pg/kg, i.v.) markedly inhibited gastric secretion, dose-dependently and without changing the plasma gastrin concentration. Negative results were obtained in the other tests, but these results serve to demonstrate that HUGI is an inhibitor welldifferentiated from other glycoproteins or peptides with gastric antisecretory activity, such as urogastrone and GIP. The results obtained to date are not sufficient to allow the mechanism of action of HUGI to be defined. Human urinary gastric inhibitor
Glycoproteins
1. I n t r o d u c t i o n I t has b e e n k n o w n since 1 9 3 8 t h a t h u m a n urinary extracts have a marked inhibitory e f f e c t o n gastric s e c r e t i o n (Sandweiss et al., 1 9 3 8 , 1 9 3 9 ; G r a y e t al., 1 9 4 0 ) , b u t it was o n l y in t h e last f e w y e a r s t h a t it has b e e n possible t o isolate t h e various c o m p o n e n t s r e s p o n s i b l e f o r this e f f e c t in a p u r e state a n d to study their physical and chemical properties. I n v e s t i g a t i o n s carried o u t b y G r e g o r y ( G r e g o r y , 1 9 7 5 , G r e g o r y a n d Willshire, 1 9 7 5 ; ( G r e g o r y a n d Preston, 1 9 7 7 ) a n d in o u r l a b o r a t o r i e s (Carrea et at., 1 9 7 3 ; L u g a r o et al., 1 9 7 6 ) h a v e s h o w n t h a t h u m a n u r i n e contains at least t w o s t r o n g l y a n t i s e c r e t o r y fac-
Gastric secretion
t o r s w h i c h are c o m p l e t e l y c h e m i c a l l y different from one another. T h e i n h i b i t o r isolated b y G r e g o r y is a 53aminoacid polypeptide (Gregory, 1975), while t h e o n e we h a v e p u r i f i e d ( H u m a n U r i n a r y Gastric I n h i b i t o r or H U G I ) is a high m o l e c u l a r w e i g h t g l y c o p r o t e i n (S~0,w = 8; Ve = V o o n Bio-Gel A 1.5 M), c o n t a i n i n g 37% c a r b o h y d r a t e (9% sialic acid). T h e r a t i o o f g l u c o s a m i n e / g a l a c t o s a m i n e is 4 / 1 . All t h e g a l a c t o s a m i n e is b o u n d t o t h e p o l y p e p t i d e chain as O-glycoside. T h e glycop r o t e i n is m a d e u p o f m a n y identical subunits w i t h a m o l e c u l a r w e i g h t o f a b o u t 1 4 , 0 0 0 D a l t o n s ; t h e g l y c o p r o t e i n was active in t h e p o l y m e r i c f o r m ( L u g a r o e t al., 1 9 7 6 ) . T h e active p r i n c i p l e f o u n d b y G r e g o r y
218 (Urogastrone) was obtained from urine of both men and women. HUGI is also present in the urine of both men and women. We extracted it from the urine of pregnant women since several investigators (Sandweiss et al., 1938, 1939; Gray et al., 1940) observed that during gestation there is a marked decrease in gastric secretion and a temporary remission of gastric ulcers; this would seem to indicate t h a t a larger a m o u n t of the antisecretory factor should be present during pregnancy. The aim of the present work was to study the antisecretory activity of HUGI in the dog and in the rat, to use different experimental models to study its mechanism of action and to compare its biological characteristics with those of other antisecretory factors.
2. Materials and methods
2.1. Preparation o f the gastric secretion inhibitor (HUGI) from human urine HUGI was obtained from pooled urine of women in the third or f o u r t h m o n t h of pregnancy, as previously reported (Carrea et al., 1973). The purification procedure included chromatography on an IONAC A-540 column, benzoic acid extraction, fractional precipitation with organic solvents and gel filtration on Sephadex G-200 or Bio Gel A-1.5 m.
2.2. Effect against gastric acid and pepsin secretion in pyloric-ligated rats HUGI, buffered to pH 7.2, was injected i.v. (from 2.5 to 20 pg/kg b o d y weight) into 180 + 5 g male Wistar (Morini Italy), maintained on the MIL® type R diet (Morini, Italy), but fasted for 48 h before treatment, as described by Lugaro et al. (1965). 2 h after injection and pyloric ligation, the volume of gastric fluid was measured, as well as the total acid and hydrochloric acid as H ÷ output, using an automatic Radiometer TTA-60 titrator.
R. NIADA ET AL. Pepsin o u t p u t was measured by the Prino et al. modification (1971a,b) of Hunt's method (1948). The experiments were planned according to a randomized block design (4 rats per group; 4 replications).
2.2. Effects against gastric acid secretion in dogs with Heidenhain pouches Male mongrel dogs, weighing 10--15 kg, were prepared with Heidenhain pouches and fed the McCay standard diet (Piccioni, Italy). The tests were begun 4 months after the surgical procedure. Before treatment with HUGI the dogs were conditioned to the silent, temperature,controlled room. The reproducibility of the hypersecretory responses to pentagastrin stimulation (Gastrodiagnost®, Merck, 6 pg/kg, s.c.), to histamine (histamine dihydrochloride, C. Erba, Italy, i.v. perfusion, 80 pg/ kg/h) and to a protein meal (150 ml of a 10% solution of Liebig's meat extract) was determined. The secretory stimuli induced nearmaximal responses under our experimental conditions. In the study of the effects of HUGI, each dog was used as its own control. The inhibitor was injected i.v. (from 0.1 t o 6.4 gg/kg body weight) 30 rain before ~;";~-,~.,~ the hypersecretory stimulus. The gastric secretion was collected every 15 min for 2 h, the volumes measured and the total acid and hydrochloric acid concentration titrated.
2.4. Determination o f plasma gastrin levels in Heidenhain pouch dogs Plasma gastrin concentrations were determined by a modification of the radioimmunoassay of Yalow and Berson (1970). The GASK kits (Sorin Biomedica, Italy) used contain synthetic human gastrin I 1-17 (ICI Ltd., MacClesfield, Cheshire, England) and rabbit antigastrin antiserum produced against synthetic h u m a n gastrin I 1-17. Gastrin from the same source was iodinated with ~:sI by the chloramine T m e t h o d and purified by polyacrilamide gel electrophoresis.
HUMAN URINARY GASTRIC INHIBITOR
219
2.5. Isolated rat gastric mucosa
2.7. Bile flow in the rat and gall-bladder tone in the guinea pig
T h e gastric m u c o s a f u n d u s o f t h e rat s t o m a c h was p r e p a r e d b y t h e t e c h n i q u e s described b y Wan et al. ( 1 9 7 4 ) , and F o r t e et al. ( 1 9 7 5 ) . Acid secretion was s t i m u l a t e d with h i s t a m i n e (histamine d i h y d r o c h l o r i d e , BDH, 25 p g / m l ) , with pentagastrin (Gastrodiagnost®, Merck, 20 p g / m l ) or with DBcAMP ( N 6 - 2 ' - ( O - d i b u t y r y l - a d e n o s i n e - 3 ' , 5 ' cyclic m o n o p h o s p h a t e , Boehringer, 100 pg/ml). H U G I (in a c o n c e n t r a t i o n u p t o 1 0 0 p g / m l ) was placed in c o n t a c t with t h e serosa 5 m i n b e f o r e the stimuli.
2.6. Gastrointestinal motility H U G I was given i.v. t o rats, cats and dogs in doses f r o m 10 t o 1 0 0 pg/kg. In t h e rat, t h e rate o f transit o f a c h a r c o a l suspension was m e a s u r e d b y t h e t e c h n i q u e o f Witkin et al. ( 1 9 6 1 ) . In t h e a n e s t h e t i z e d cat, m o t i l i t y was r e c o r d e d b y i n t r o d u c i n g into t h e s t o m a c h and the d u o d e n u m rubber balloons connected to HP m o d e l 1 2 8 0 pressure transducers. In the u n a n e s t h e t i z e d dog, t h e r u b b e r b a l l o o n was placed in t h e H e i d e n h a i n p o u c h .
T r a d i t i o n a l m e t h o d s for bile d u c t were used f o r t h e and gall-bladder t o n e was a t t a c h e d to t h e end o f t h e an isometric t r a n s d u c e r Ljungberg (1964).
cannulation of the rat bile flow s t u d y r e c o r d e d b y a line gall-bladder and t o as described b y
2.8. Cardiovascular system T h e a n e s t h e t i z e d cats used for o b s e r v a t i o n o f gastrointestinal m o t i l i t y were p r e p a r e d f o r simultaneous recording of carotid artery and jugular vein pressures, using t r a d i t i o n a l m e t h o d s . In a d d i t i o n , changes in h e a r t rate were o b t a i n e d f r o m t h e E C G b y an HP m o d e l 8 8 1 2 / A rate c o m p u t e r . H U G I was given i.v. in doses b e t w e e n 10 and 1 0 0 pg/kg.
3. Results
3.1. Pylorus-ligated rats In t h e pyloric-ligated rat, H U G I had a m a r k e d gastric a n t i s e c r e t o r y effect, w h i c h was
TABLE 1 Effect of urinary glycoprotein (HUGI) on gastric secretion at 120 min after pyloric ligation in rats. Mean ± S.E. from 16 rats per group. Each mean was compared to the control values with Student's t-test. Percentage inhibition in parentheses. Treatment pg/kg i.v.
Gastric juice volume (ml)
Hydrochloric acid output (pEq H÷)
Total acid output (pEq H÷)
Pepsin output (//moles L-tyrosine)
Saline 5 ml/kg HUGI 2.5 3.75 5 10 20
3.84 ± 0.24
303.71 ± 37.51
443.03 + 41.11
84.61 ± 4.05
3.05± 0.38 (20.6) 2.10 ± 0.20 3 (45.3) 1.64 ± 0.19 3 (57.3) 1.10± 0.103 (71.3) 0.89 -+ 0.09 3 (76.8)
153.91+ 44.47 139.10 ± 24.95 65.31 -+ 15.89 38.46± 7.98 32.16 +- 10.05
284.45+ 55.88 1 (35.8) 223.59 ± 28.35 3 (49.5) 132.31 ± 21.31 3 (70.1) 93.48± 11.043 (78.9) 70.77 +- 10.41 3 (84.0)
65.30± 6.91 38.81 ± 5.01 31.28 ± 4.28 21.55-+ 2.47 17.77 ± 1.63
1 p < 0.05, 2 p < 0.005, 3 p < 0.001.
1 (49.3) 2 (54.2) 3 (78.5) 3 (87.3) 3 (89.4)
1 (22.8) 3 (54.1) 3 (63.0) 3 (74.5) 3 (79.0)
220
R. NIADA ET AL.
manifested by the volume of the secretion, the acid output and the pepsin output. The inhibition started to become significant with t h e 2 . 5 p g / k g i.v. d o s e a n d w a s p r o p o r t i o n a l t o t h e d o s e s g i v e n f o r all t h e p a r a m e t e r s m e a s u r e d ( t a b l e 1).
600 ' ¢
400
i' ~
i
-
ConlrOI
/ g
C,,nT,,,i kq
~, BOO
3.2. Heidenhain pouch dogs In the dogs with Heidenhain pouches, HUGI i n j e c t e d i.v. in t h e m a x i m a l d o s e o f 6 . 4 p g / k g , a d o s e h i g h l y e f f e c t i v e in i n h i b i t i n g t h e a c i d output stimulated by a protein meal, had no effect on the gastric secretion stimulated by pentagastrin or histamine. No changes were s e e n in t h e t o t a l a c i d o u t p u t , w h i c h w a s 1503 + 196 pEq H÷/2 h after pentagastrin s t i m u l a t i o n a n d 2 1 1 4 -+ 1 2 6 laEq H * / 2 h a f t e r histamine stimulation. But HUGI did have marked dose-dependent inhibitory effects on the gastric secretion stimulated by a protein meal. For 0.1, 0.4, 1 . 6 a n d 6 . 4 p g / k g i.v. d o s e s o f t h e i n h i b i t o r , t h e r e w e r e d e c r e a s e s in v o l u m e w h i c h r a n g e d f r o m 2 8 t o 7 0 % , a n d d e c r e a s e s in t o t a l a c i d o u t p u t r a n g i n g f r o m 2 6 t o 8 2 % ( t a b l e 2). T h e concentration of acid was also influenced by the treatment, at least at the highest doses,
'"
i 400 ; !
30
Conrro~
/'\Control
/kg~
HUGI 16 pg/kg
0
30
60
T I
HUGI 109~meat extract
90
120
30
0
30
60
90
12Omin
t I
HUGI 10~meat extract
Fig. 1. Acid output from Heidenhain pouches. Inhibitory effect of i.v. injected HUGI on hypersecretion induced by a protein meal. Effect on total acid output (mean ± S.E. of four tests in 3 dogs). when the hydrogen ion concentration was decreased by about 50%. At least at the highest doses of HUGI had a n i n h i b i t o r y e f f e c t a t all t h e t i m e s o f t e s t i n g a f t e r t h e s t i m u l u s w a s g i v e n (fig. 1).
3.3. Plasma gastrin concentration of
In another series of experiments, the effects HUGI on the plasma concentration of
TABLE 2 Effect of urinary glycoprotein (HUGI) on gastric hypersecretion induced by meat extract in Heidenhain pouches. Mean values +- S.E. of four tests in 3 dogs. Each mean was compared to the control values with Student's t-test. Percentage inhibition in parentheses. Treatment (pg/kg i.v.)
Gastric juice volume/2 h (ml)
/~Eq H ÷ output/2 h Hydrochloric acid
Total acid
Saline HUGI
3 ml 0.10
12.66 -+ 1.02 9.11 ± 0.42 1 (28.04)
1312.33 ± 63.94 952.66 ± 82.39 1 (27.40)
1511.66 ± 70.89 1119.00 ± 59.25 2 (25.91)
Saline HUGI
3 ml 0.40
12.16 ± 0.99 6.46 ± 0.76 2 (46.87)
1255.33 ± 71.39 453.33 ± 41.25 a (63.88)
1478.00 -+ 47.93 642.00 ± 76.69 4 (56.56)
Saline HUGI
3 ml 1.60
12.12 ± 0.76 5.17 ± 0.18 4 (57.34)
1386.66 -+52.66 371.00 ± 93.02 4 (73.24)
1589.00 -+ 17.67 459.33 ± 96,56 4 (71.09)
Saline HUGI
3 ml 6.40
12.71 ± 1.05 3.85 ± 0.48 3 (69.70)
1466.33 -+ 39.56 241.66 ± 18.94 a (83.51)
1697.33 ± 60.45 306.66 ± 13.38 4 (81.93)
i p < 0.05, 2 p < 0.02, 3 p < 0.005, 4 p < 0.001.
221
HUMAN URINARY GASTRIC INHIBITOR - - - r - - / ~ - -
80 "~ 800p ':
10%meat [
m
,
+
"
I
exltract
The cat showed no significant changes in arterial or venous pressure or in heart rate.
60
4. Discussion 4 0 -=- 4 0 0 ~
~.
e
--~:.20 +~ 200 ~-
Control
, ,
30
0
15
30
45
60
75
90
105 120
min
I
HUGI
Fig. 2. Acid output from Heidenhain pouches. Effect
of intravenously injected HUGI (6.4 pg/kg) on the plasma gastrin levels (©) and total acid output (A) stimulated by a protein meal (mean _+S.E. of three tests in 2 dogs).
gastrin were evaluated in dogs with Heidenhain pouches. Under the experimental conditions previously described, the basal plasma levels were 28.5 + 2.92 pg/ml. After stimulation with the protein meal, the concentrations of gastrin rose to a maximal level of 73.0 + 6.02 pg/ml. The changes in the plasma gastrin concentration and in total acid o u t p u t determined simultaneously were plotted vs. time (fig. 2). When HUGI was given intravenously 30 min before the stimulus, even the maximal dose of 6.4 pg/kg was n o t able to m o d i f y the plasma gastrin concentration, but it decreased the total acid o u t p u t by 77% at the second hour of observation. 3.4. Other tests
Negative results were obtained in all the other tests. When isolated rat mucosa was incubated with HUGI, even at concentrations of 100 pg/ml, there was no change in the peak of acid secretion induced by histamine (5.56 + 0.77 pEq H+/cm2/30 min) by pentagastrin (4.63 + 0.32 pEq H*/cm2/30 min) or by DBcAMP (9.76 + 1.02 pEq H*/cm~/30 min). The bile flow in the rat, gall-bladder tone in the guinea pig and gastrointestinal motility of the rat, cat and dog were not affected by HUGI up to 100 pg/kg i.v.
HUGI is a newly found antisecretory factor from human urine and has been shown to be a high molecular weight glycoprotein (Carrea et al., 1973; Lugaro et al., 1976). The biological assay procedure used during its isolation and purification was based on its inhibition of gastric secretion in the rat with ligated pylorus (Carrea et al., 1973). The results listed in table 1 confirm the inhibitory effect of HUGI given i.v. to the rat on both the volume of secretion and the acid output. The pepsin o u t p u t was also decreased, but in this respect, one must keep in mind that this may have been due to the drastically reduced volume of gastric juice secreted. In the Heidenhain pouch dog, HUGI demonstrated a striking anti-secretory effect when gastric secretion was stimulated by a protein meal, but did not affect the hypersecretion induced by pentagastrin of histamine. This confirms the earlier results obtained in Pavlov pouch dog (Gandini et al., 1970). The inhibitory effect on hypersecretion induced by a protein meal was not accompanied by any corresponding change in the plasma gastrin levels. Therefore, it would be difficult to explain its antisecretory effect as an action at the level of the G cells where it could interfere with the synthesis or the release of gastrin, as might have been suggested by the other results. HUGI had no effect on hypersecretion from the isolated mucosa of the rat stimulated with pentagastrin, histamine or DB-cAMP. it did not change gastrointestinal motility in the rat, dog or cat and the gallbladder tone in the guinea pig was not influenced. It did not affect bile flow even at doses much higher than those which maximaUy inhibited gastric secretion. These results are specific characteristics of the biological properties of HUGI, especially as compared
222 with t h o s e already k n o w n f o r u r o g a s t r o n e and GIP. Unlike H U G I , u r o g a s t r o n e and GIP inhibit the hypersecretory response to exogenous pentagastrin and h i s t a m i n e in t h e H e i d e n h a i n p o u c h dogs (Gerring and H a w o r t h , 1 9 7 0 ; Pederson and B r o w n , 1 9 7 2 ) . Like H U G I , t h e y inhibit the s e c r e t i o n s t i m u l a t e d b y a p r o t e i n meal, b u t o n l y GIP also r e d u c e s t h e plasma gastrin levels (Villar et al., 1 9 7 5 ; Elder et al., 1975). On isolated gastric m u c o s a , urogastrone, unlike H U G I , inhibits the h y p e r secretion caused b y pentagastrin or histamine (Bower, 1 9 7 3 ) . Like H U G I , u r o g a s t r o n e does n o t a f f e c t gastric m o t i l i t y or bile flow (Getring, 1 9 7 0 ; G r o s s m a n et al., 1 9 7 4 ) , while GIP decreases t h e p o u c h c o n t r a c t i o n s in t h e d o g ( P e d e r s o n , 1 9 7 1 ) . These observations show t h a t H U G I is an i n h i b i t o r o f gastric secretion which differs f r o m u r o g a s t r o n e and GIP with respect to biological p r o p e r t i e s as well chemical n a t u r e (Carrea et al., 1 9 7 3 ; Lugaro et al., 1976). As a f u r t h e r c o n f i r m a t i o n , we can cite t h e radioimmunological trials of Gregory (personal c o m m u n i c a t i o n ) , which s h o w e d t h a t t h e r e was n o cross-reaction b e t w e e n H U G I and u r o g a s t r o n e at c o n c e n t r a t i o n s as m u c h as 2.5 × l 0 s times greater (5 pg o f H U G I against 20 pg o f u r o g a s t r o n e ) and we t h e r e f o r e reject the h y p o t h e s i s t h a t t h e polyp e p t i d e ( u r o g a s t r o n e ) is derived f r o m the glycoprotein (HUGI). Until now, the m e c h a n i s m o f a c t i o n o f H U G I has n o t been explained. H o w e v e r , we can state t h a t H U G I inhibits o n l y t h e gastric secretion i n d u c e d b y physiological stimuli and is ineffective against pharmacologically induced secretion. A n o t h e r i m p o r t a n t fact is t h a t H U G I can be held largely a c c o u n t a b l e for t h e t o t a l a n t i s e c r e t o r y activity o f h u m a n urine. In fact 2 mg o f H U G I / l i t e r in t h e pure state were o b t a i n e d f r o m urine (Carrea et al., 1 9 7 3 ) whereas the yield o f u r o g a s t r o n e was o n l y 1 #g/liter ( G r e g o r y , 1975). Even if t h e specific activity o f u r o g a s t r o n e were greater t h a n t h a t o f H U G I , its c o n c e n t r a t i o n in urine is less t h a n o n e t h o u s a n d t h the c o n c e n t r a t i o n o f HUGI.
R. NIADA ET AL. Acknowledgement The assistance of Franco Paties is gratefully acknowledged.
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