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Kinetics of Inhibition of Canine Gastric Secretion by Vasopressin
Vol. 61 , No. 4. Pa rt 1 Printed in U.S.A.
G ASTROENTEROLOGY Copyri ~h t
© 1971 by T he Williams & Wilk ins Co.
KINETICS OF INHffiiTION OF CANINE GASTRIC SECRETION BY VASOPRESSIN ANDRE L. BLUM, M.D.
Division of Gastroenterology, Department of Medicine , Univ ersity of Alabama in Birmingham , School of Medicine, and Veterans Administration Hospital, Birmingham, Alabama
The kinetics of inhibition of acid secretion by vasopressin were studied in gastric fistula dogs. Inhibition was dose-dependent with a threshold of 10 mU per kg in each dog. Dose-response curves with the inhibitor dose held constant showed that inhibition was of the competitive type: maximal response was unaltered but the dose required to elicit half the maximal response increased 2-fold. Kinetics of this type are postulated to arise from interference in the reaction of stimulant and receptor but can be mimicked in vivo by mechanisms restricting the delivery of the stimulant to the target cells. In each case, however, the mechanisms were consistent with either the vasoactive or water-conserving properties of vasopressin. For some inhibitors of gastric secretion such as secretin and compounds structurally related to gastrin, theories have been advanced which provide a satisfactory account of their mechanism of action. 1 In other instances, the mechanism of inhibition remains obscure as in the case of histamine-stimulated secretion by atropine. 2 Even more intriguing is the ineffectiveness in vitro of inhibitors like Diamox and vasopressin which are highly potent in vivo. 3 • 4 In this study, the kinetics of inhibition of canine acid secretion by vaso-
pressin were analyzed in an attempt to attribute the effect of vasopressin either to its vasoactive or to its water-conserving properties.
Kinetics of Inhibition According to a model proposed by Makhlouf et al., 5 • 6 the relationship between acid response and dose of stimulant may be expressed as follows: Rd r =
K
+d
(1)
where r represents the observed response, R the maximal response, d the exogenous dose of stimulant, and K the value of the dose which elicits half the maximal response or ED 50 . It is assumed in this model that the response is proportional to the number of activated secretory units or stimulant-receptor complexes and that the concentration of stimulant in proximity to the receptor sites remains a constant fraction of the exogenous dose. The formal analogy between this model and enzyme-substrate interaction is apparent and the kinetics of the two systems in respect of inhibition and acceleration are alike: as applied to exocrine secretion,
Received December 2, 1970. Accepted May 7, 1971. Address requests for reprints to: Dr. Andre L. Blum, Medizinische Klinik, Stadtspital Triemli, 8055 Zurich, Switzerland. This work was supported by National Institutes of Health Grants TIAM 2A-5286 and AM09260, National Science Foundation Grant GB8351 , and Research Funds of Veterans Administration Hospital, Birmingham, Alabama. This work was submitted as a docent thesis to the University of Zurich ; it was suggested and guided by Dr. Gabriel M. Makhlouf. Expert technical assistance by Mr. Robert Perkins and Mr. Michael Lachina is acknowledged. Synthetic vasopressin 8-lysine was a gift of Sandoz Pharmaceuticals, Hanover, New Jersey. 461
462
BLUM
competitive inhibition, which is the only type discussed here, may be stated as follows 7 : Rd
r
=
K(1
+ ilk) + d
(2)
where i and k; are respectively the dose and dissociation constant of the inhibitor. The function (1 + ilk;) is constant for a fixed dose of inhibitor and represents the factor of increase inK or the ED 5 0 • An alternative manner of stating equation 2 indicates that the inhibitor acts as if to render the dose of stimulant less effective by a factor (1 + ilk;): r
=
K
R d[ll(1 + ilk)] + d[l/(1 + ilk;)]
(3)
Equation 2 may be transformed into 1/r
=
1IR
+ K(1 + ilk;)IRd
(4)
which states that, for a constant dose of inhibitor i, the relationship between 1/r and 1/d remains linear and the intercept on the response axis is maintained equal to 1IR while the slope of the relation is increased by a factor of (1 + ilk;). For a constant dose of stimulant, the relationship between i and 1/r is linear. The latter condition applies also to noncompetitive inhibition. An alternative linear transformation more suitable for calculation 8 is: r
=
R - K(1
+ ilk) rid
(5)
which states that, for a constant i, the relationship between r and rid remains linear, the intercept on the response axis is maintained equal to R, and the slope is increased by a factor (1 + ilk;).
Material and Methods Three female mongrel dogs weighing 21-25 kg were equipped with stainless steel gastric cannulae and Komarov type esophageal fistulae for external drainage of saliva during collection of gastric juice. Experiments were started several months after operation. The dogs were fasted for 16 hr after which the stomachs were gently washed with 100 ml of lukewarm tap water and unstimulated secretion was collected for at least 30 min. Two intravenous infusions were then started into leg veins for separate administration
Vol. 61 , No . 4, Part I
of histamine acid phosphate and synthetic vasopressin 8-lysine. Two types of experiments were performed in which either inhibitor or stimulant was held constant. 1. Vasopressin dose varied, histamine dose constant. Histamine was infused intravenously at a rate of 50 f.Lg of base per kg per hr for a period of 330 min. After allowing 60 min for secretion to reach a steady rate, vasopressin was injected intravenously three times at intervals of 90 min. Three out of four dose levels (10, 20, 30, or 40 m U per kg) were employed in the course of each experiment. The order of vasopressin doses was randomized as follows: a dose, for example 10 mU per kg, was injected at 60 min in one experiment, at 150 min in a second, and at 240 min in a third, and so on for all four doses. This procedure was instituted in order to minimize differences in the response arising from repeated administration of vasopressin or continous infusion of histamine. In retrospect, this precaution proved unnecessary since no significant differences could be detected both by paired t-tests and by regression analysis between responses at 60 min and responses at 150 or 240 min. Responses were therefore averaged independent of their time of occurrence. 2. Vasopressin dose constant, histamine dose varied. In a control series, histamine was infused at rates of 10, 25, 50 or 100 f.Lg of base per kg per hr for a period of 270 min. Saline was infused concurrently at a separate site. The dose of histamine was changed every 90 min and the order of infusions randomized such that each dose was given from 0 to 90 min in one experiment, from 90 to 180 in a second, and from 180 to 270 in a third. In this manner, differences that might have resulted from the fade of secretion were minimized. In a second series, the same procedure was followed except that vasopressin was added to the saline and infused from the start at a rate of 50 mU per kg per hr. Gastric juice was collected every 10 min except during the period following prompt intravenous injection of vasopressin when it was collected every 5 min for a period of 20 min. Acid content was determined by titration with 0.1 N NaOH using an automatic titrator and pH meter (Radiometer, Copenhagen).
Results 1. Vasopressin dose varied, histamine dose constant. Intravenous injection of vasopressin resulted in a rapid tall of histamine-stimulated secretion followed by
VASOPRESSIN INHIBITION OF GASTRIC SECRETION
October 1971 -20
0
20
40
-20
0
20
40 100
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40 ~
::::>
20
i
j 10mUkg1
x~
20mUkg-1
80 60 40 20
~
[._
0 0 o-
80 60 40 20
30mUk -1 -20
0
20
40
40mUk -1 - 20
0
20
20
40
MINUTES FIG 1. Effect of single injections of lysine vasopressin on gastri c secretion during stimulation with histamine (50 p.g per kg per hr). Vasopressin injections are represented by an arrow at time zero. The dose of vasopressin is given in each graph. Acid output (mean ± SE) and acid concentration (black dots; SE smaller than symbols) of 3 dogs were normalized and expressed in percentage of the highest value obtained in each test.
a gradual return to control levels (fig. 1). The inhibitory effect was dose-dependent but was not accompanied by a change m acid concentration. The results are recorded in table 1. A convention which served the purpose of normalizing the data was utilized: the response during the 30-min period following vasopressin was expressed as a fraction of the control response during the 30-min period prior to inhibition. As shown in figure 2, a linear relation prevailed between the reciprocal of this fractional response and the dose of vasopressin. The slope of this relation was similar in the 3 dogs and indicated that a threshold of 10 m U per kg existed for the inhibitory effect of intravenous vasopressin on acid secretion. Analysis of published data 9 - 13 on inhibition of exocrine secretion by vasopressin disclosed a similar kinetic pattern including the existence of a threshold ·dose (table 2). 2. Vasopressin dose constant, histamine dose varied. Responses to increasing doses of histamine infused alone or in
463
combination with vasopressin are recorded in table 3. Vasopressin reduced acid output and flow rate, but had no detectable effect on acid concentration. The results were analyzed for each dog separately in terms of equations 2 to 5. There was a good fit of the data to equation 4 which predicts a linear relation between the reciprocals of response and dose of stimulant. Calculations of the constants R and K were based on an alternative linear transformation (equation 5) relating response and response per dose (table 4). The kinetics showed the characteristics of competitive inhibition in that the maximal response R remained unchanged while the dose which elicited half the maximal response rose by more than 2-fold from 57 J.L g per kg per hr with histamine alone to 128 J.Lg per kg per hr on addition of vasopressin. On the assumption of competitive inhibition, the effect of vasopressin was further characterized by the inhibitor constant k i calculated from equation 5 to be 40 m U per kg per hr. Discussion This study confirms earlier reports on dose-dependent inhibition of gastric acid secretion by vasopressin. 10 • 1 2 - 1 7 Kinetic analysis of published data shows a similar dose-dependent inhibition of pancreatic secretion and of splanchnic and resting gastric blood flow (table 4). Conditions in which only the inhibitor dose is changed do not, however, allow conclusions regarding the type of inhibition. By instituting a further condition in which the stimulant dose was varied while the inhibitor dose was held constant, it was possible to show that inhibition by vasopressin was of the competitive type. It is characteristic of this type of inhibition that the calculated maximal response to the stimulant alone is not altered in the presence of the inhibitor (table 4, fig. 3) while the dose of stimulant required to elicit half the maximal response is increased by a factor (1 + i /k;) which is dependent on the dose of the inhibitor. There are several ways in which inhibition in vivo may give rise to competitive kinetics. (a) In the simplest case, expressed
464 TABLE
BLUM
Vol. 61, No.4, Part 1
1. Effect of single injections of vasopressin on acid output and volume output stimulnted with 50~ per
kg per hr of histamine" Dose of vasopressin Regression of 1/r on dose of vasopressin
Dog 10
20
30
40
m U/kg
mU/kg
Acid output
Dog A
0 . 994
0.792
0. 637
Dog B
1. 009
0 . 771
0.632
Dog C
0 . 980
0 . 783
0.686
3 dogs (mean ±
Volume output
SE)
Dog A
0.994 ±0 . 020 0.956
Dog B
1.038
Dog C
0 . 974
3 dogs (mean ±
SE)
0 . 989 ±0 .031
0.782 0 . 651 ±0.035 ±0.027 0.824 0. 773 0.789 0 . 795 ±0.040
Lowest effective dose of vasopressin
0.644
0 . 552
1/r = 0. 0273i + 0. 73, p < 0.01 0. 566 1/r = 0 . 0262i + 0.76, p < 0 .01 0.623 1/r = 0.0193i + 0.86, p < 0.01 0 . 580 1/r = 0.0241i ±0.023 + 0.78, p < 0 .01 0 . 603
1/r = 0. 0218i + 0.82, p < 0.683 0 . 615 1/r = 0.0216i + 0.89, p < 0.703 0 . 650 1/r = 0. 0169i + 0.80, p < 0 . 677 0.623 1/r = 0. 0200i ±0.026 ±0.023 + 0.84, p <
9. 9 9 .3 7.6 9. 0
8.0 0.01 9 .3 0.01 6.0 0 .01 8.1 0. 01
a Response is the output during the 30-min period following injection of vasopressin and is expressed as fraction of the output in the 30-min period before injection. Regression equations were calculated for dose of vasopressin {i) and reciprocal of the response (1/r). Correlation coefficients were in all cases >0. 98. Threshold doses of vasopressin were calculated from the regression equations (see text) . Regressions for acid output are plotted in figure 2.
VASOPRESSI N DOSE
FIG 2. Dose-response curve for single vasopressin injections during a histamine infusion. The dose of histamine is 50 p.g per kg per hr. The dose of vasopressin is given in mU per kg. Acid output in the 30 min following vasopressin injection is expressed as fraction of the acid output before injection. The reciprocal of this fraction (control/response) is plotted against the dose of vasopressin. An arrow points at the intercept on the X axis which represents the lowest effective dose of vasopressin. Regression equations are given in table 1.
by equation 2, the inhibitor acts by competing for the same receptor site as the stimulant, an unlikely event in this instance in view of the structural dissimilarity between histamine and vasopressin. Allosteric modification of the receptor site for histamine appears more probable. This would interfere with the rate at which the stimulant is bound to and/or discharged from the receptors. Since the over-all constant K is determined by the ratio of the rate constants for these two opposing processes, 7 its value increases in the presence of the inhibitor and the system appears less sensitive to the influence of the stimulant in that it requires a higher dose to elicit the same response. (b) An alternative manner of stating competitive kinetics is given in equation 3 and leads to a different interpretation of inhibition by vasopressin in vivo. Equation 3 implies that the effective dose of the stimulant is reduced
Type of
Shehadeh et al., 1969 29 Bell and Battersby, 1969 26
Banks et al. , 1968 9
Perks et a!., 1964 11
Forrest et al., 1954 35
Karlmark and Obrink, 1967 10
Schapiro et al., 1968 1 3
Schapiro et al., 1966"
Author
Stim ulant
Inhibitor
Volume output
Histamine, 50 p.g sub- Pitressin, single injection cutaneously every 15 min Pitressin, single Meat, 200-400 g Volume output injection Lysine and arForcemeat, 10 g per Volume output ginine vaso10 min pressin infusion Norepinephrine Histamine, 25 p.g/ Acid output infusion kg/hr Pitressin, single Secretin, 1 U/kg Volume output injection Pitressin, single Secretin, 1 U/kg Volume output injection Norepinephrine Electromagnetic flow None infusion meter Pitressin infusion Gastric mucosal Kr •• None clearance
Met hod
1/r = 0.0124i
1/r = 0.0195i
1/r = 0.0437i
1/r = 0.0775i
= 0.025)
(P
(P
(P
= 0.01) = 0.005)
+ 0.447
(P
+ 1.31
= 0.002)
= 0.0005)
+ 1.07
(P
+ 0.603
(P
= 0.001)
= 0.0003)
+ 1.02
(P
= 0.001)
+ 0.047
(P
+ 0.077
+ 0.692
1/r = 0.0338i
1/r = 0.251i
1/r = 0.0961i
1/r = 0.0442i
Regression of 1/r and i
Effective of vasoactive drugs on canine digestive secretion and blood flow•
44 mU/kg/hr
0 p.g/kg/hr
0 mU/kg
5 mU/kg
0 p.g/kg/hr
2 mU/kg/hr
6 mU/kg
5 mU/kg
Lowest effective dose of inhibitor
• Fit of a model relating dose of a vasoactive agent and response of exocrine secretion or blood flow. Gastric secretion was studied in dogs with Heidenhain pouches. Pancreatic secretion was studied in dogs with Thomas cannulas. Blood flow was measured in anesthetized animals. For regression analysis, the response observed after administration of the inhibitor was expressed as fraction of the response observed before inhibition. The reciprocal of this fraction (1 /r) was expressed as a function of the dose i of the inhibitor. For calculation of the lowest effective dose see text.
Mesenteric blood flow Gastric blood flow
Pancreatic secretion
Gastric secretion
experiment
TABLE 2.
~
Cll
0)
..,..
:<;
a~
t>l
::tl
(J
(::3
~
CJ:J
§2
~
~
~ ~
t5
~ ~
~
~
~
CJ:J
:::::!
......
~
o-
c
ag.
466
BLUM
Vol. 61, No.4, Part 1
TABLE 3. Effect of histamine infusions (control) and of histamine plus vasopressin infusions (+VA) on acid output, volume output, and acid concentration• Dose of histami ne 10
Dog
Acid output (mEq/hr)
±SE) Dog A Dog B Dog C 3 dogs (mean
± SE) Acid concentration (mEq/liter)
+ VA
Control
Dog A Dog B Dog C 3 dogs (mean
Volume output (ml/hr)
25
3 dogs (mean ± SE)
12.44 6.78 3.42 2.23 6.64 2.99 7.50 4.00 ±1.52 ± 0.91
50
+VA
Control
23 . 75 11.14 7.44 4.58 12 .24 7.96 14.48 7.89 ±2. 49 ± 1.51
-
92.4 32.2 52.7 59.1 ± 10.2
49.7 20 .4 19.7 29 .9 ±2 .0
168 . 1 54.5 82 .7 101 .7 ± 17 .4
80.3 38.4 50.6 56.4 ±9.2
134.1 ±2. 9
130.6 ±3. 1
142 .0 ± 1.6
141.5 ± 2.0
100
+VA
Control
16. 50 12.17 11.09 13 .25 ±1. 62
29.26 15.72 20.40 21.79 ± 2.32
194 .3 116.7 108 .8 106.2 135.3 73.1 146.1 98 .7 ± 13 .6 ± 11.1 148.8 ± 1.6
146.5 ± 1.6
+ VA
Control
28.98 27.63 20.61 25.74 ± 2.23
24.92 18 .63 18.30 20 .62 ±2. 10
198 .6 157.5 186.3 126.9 130.7 116.4 171 .9 133 .5 ± 12. 7 ± 13 .0 149.7 ± 1.9
152 .5 ± 1.2
• Dose of histamine base is given in /J.g per kg per hr. Dose of vasopressin was 50 mU per kg per hr. Doseresponse curves are plotted in figure 3.
TABLE 4. Regression equations for plots of I /response versus I /dose (see figure 3) and of response versus
~esponse /dose• Histamin e plus vasopressin
Histamine a lone
Regression of
1/r and 1/d, equation 4
1/r
=
0.0281
+ 0.982 (P
r and r/d, equation 5
r
=
1/d
1/r
< 0.001)
36.62 - 56.97 r/d (P < 0.025)
=
0.0269
+ 0.242
1/d
(P
r
=
< 0.001)
36.72 - 128.80 r/d (P
< 0.05)
a Response (r) represents acid output per 60 min in milliequivalents. Dose (d) represents the dose of histamine which was either infused alone or together with 50 mU per kg per hr of vasopressin.
\)() j
002
0.02
004
006
0 00
0 10
I I HISTAMINE DOSE
FIG 3. Dose-response curves for histamine infusions (open circles) and histamine infusions given together with vasopressin infusions (closed circles) in 3 dogs. The dose of vasopressin is 50 m U per kg per hr. The dose of histamine is given in /J.g per kg per hr. The response in the upper graph is the volume output in ml per hr and in the lower graph it is the acid output in mEq per hr. Regression equations are given in table 4.
in the presence of an inhibitor to an even smaller fraction [1 /(1 + ilk)] of the exogenous dose. It should be recalled that the convention which permits the use of the exogenous dose in lieu of the local concentration of stimulant in the target organ is based on the assumption that the latter is a constant fraction of the former. This fraction could, however, be altered by a drug which restricts the delivery of the stimulant to the receptor sites. The resultant kinetics would simulate competitive inhibition without involving an effect of the inhibitor drug at receptor level. Various processes in the target organ or at more distant locations might lead to this outcome; for example, a decrease in vas-
October 1971
VASOPRESSIN INHIBITION OF GASTRIC SECRETION
cular permeability coupled with a decrease in capillary flow and surface area; a decrease in cellular permeability; or an increase in nonspecific binding or disposal of the stimulant. Mechanisms of inhibition consistent with the two alternatives listed under "a" and "b" above may be sought respectively in the water-conserving and vasoactive properties of vasopressin. 1. Consistent with its physiological action, the requirements of vasopressin for water conservation by the renal tubule are small. Antidiuretic responses in vivo may be observed with doses as low as 10 J.LU per kg per hr 18 while in vitro concentrations below 1 J.LU per ml are still active. 19 Much higher concentrations ( > 100 mU per ml) are needed in vitro to stimulate absorptive mechanisms in amphibian skin, 20 bladder, 2 1 and colon. 22 The mature stomach, like other exocrine organs, is largely secretory and only to a minor extent absorptive. Water conservation in this organ would, therefore, mainly result from restriction of secretion. In fact, the only mechanism which leads to disappearance of acid from the gastric lumen appears to be passive and without significant effect on movement of water. 23 Recent attempts to demonstrate inhibition of secretion in vitro have not met with success. A wide range of concentrations failed to inhibit spontaneous or histamine-stimulated acid secretion by the isolated frog gastric mucosa (R. L. Shoemaker, A. L. Blum, and G. M. Makhlouf, unpublished observations). No information is available on secretion by the isolated mammalian mucosa, but there is no reason to believe that it would in this respect respond differently from the amphibian mucosa. A similar lack of inhibition was observed by Buranarugsa and Moore 3 in the perfused pancreas of the rabbit which in vivo promptly responded to inhibition by vasopressin. Such lack of inhibition in vitro appears to preclude an effect of vasopressin at receptor level or through an effect on cellular permeability. Conclusions based on the absence of in vitro effects, however, are not decisive since the preparations employed are sub-
467
optimal and secrete at rates which are low and may mask inhibitory effects. 24 More pertinent, an inhibitor-like vasopressin may require an adjuvant available only in vivo for its effect to be fully manifest. Both the lack of secretory inhibition and the need for large concentrations to stimulate absorption in vitro are consistent with this view. It is known that low concentrations of vasopressin potentiate the effect of catecholamines on vascular muscle. 2 5 A similar type of cooperation in respect of its water-conserving properties would endow vasopressin with more general physiological significance than the apparent ineffectiveness in vitro would suggest. 2. Of the various possibilities listed under "b" above which might lead to inhibition by restricting delivery of the stimulant only the first is consistent with the vasoactive properties of vasopressin. Vasopressin is known to decrease splanchnic blood flow and in particular total and mucosal gastric blood flow. 1 5 • 16• 26" 29 Larger doses than were used in this study to inhibit secretion are apparently needed to bring about a reduction in resting mucosal blow flow, 26 • 30 but there are no systematic studies on dose requirements for this effect during active secretion. Following stimulation of acid secretion there prevails a high ratio of blood flow to secretion rate 3 1• 3 2 which makes it improbabk that anything but an extreme reduction in blood flow could have imposed a limit on the amount of water cleared in secretion by allowing the osmolality of the tissues to rise excessively. It is equally improbable that a limit could have been set on secretion by the supply of nutrients since this would have led to irreversible damage 3 3 inconsistent with the observed rapid recovery of function following intravenous vasopressin. Restriction to the delivery of the stimulant is possible but remains conjectural in the case of histamine. The rates at which small molecules diffuse in and out of capillaries exceed greatly the rates at which they are borne by the blood, and for these substances capillary permeability is not an essential factor in blood tissue exchange. For some substances like antipyrine cellu-
468
BLUM
lar permeability is also high; their clearance approaches blood flow and their distribution is said to be blood flow-limited. Others, like urea, have a low cellular permeability and therefore their clearance changes little despite wide variations in blood flow. It should be noted that these conclusions are based on a model which assumes uniform perfusion of widely dilated capillaries with a constant surface area. For a given blood flow during vasoconstriction even the clearance of antipyrine is reduced. 3 4 Conceivably the properties of gastric stimulants reside between the two extremes of flow- and diffusionlimited substances. By inducing a reduction in capillary flow and surface area, vasopressin might limit the extraction of a stimulant by the mucosa and lead to a reduction in secretion. A similar mechanism might apply to inhibition of secretion by noradrenaline for which an identical kinetic pattern has been observed 3 5 (table 2) . A direct study of stimulant extraction by target tissues under various conditions should yield valuable information on the mechanism of secretory inhibition by vasoactive agents in vivo. REFERENCES 1. Grossman MI: Gastrin, cholecystokinin and secretin act on one receptor. Lancet 1:1088- 1089, 1970 2. Durbin RP, Thorpe CD: Inhibition by atropine of acid secretion stimulated by acetylcholine, histamine and gastrin in isolated frog gastric mucosa. Gastroenterology 58:944, 1970 3. Buranarugsa P, Moore WW : The effect of vasopressin on secretin-stimulated pancreatic secretion in the rabbit, in vivo and in vitro. Physiologist 13: 159, 1970 4. Durbin RP, Heinz E : Electromotive chloride transport and gastric acid secretion in the frog. J Gen Physiol 41 :1035-1047, 1958 5. Makhlouf GM, McManus JPA, Card WI: Action of pentapeptide (ICI 50 123) on gastric secretion in man. Gastroenterology 51 :455- 465, 1966 6. Makhlouf GM, McManus JPA, Knill JR: Quantitative aspects of synergism and inhibition of gastric secretion. Gastroenterology 54:532-537, 1968 7. Dixon M, Webb EC: Enzymes. Longmans Green and Co, 1958, p 171- 181 8. Dowd JE, Riggs DS: A comparison of estimates of Michaelis Menten kinetic constants from var-
9.
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22.
Vol. 61, No.4, Part 1 ious linear transformations. J Bioi Chern 240:863869, 1965 Banks PA, Rudick J, Dreiling DA, et a!: Effect of antidiuretic hormone on pancreatic exocrine secretion. Amer J Physiol215:361-365, 1968 Karlmark B, Obrink KJ : The antidiuretic hormone as an inhibitor of gastric secretion. Scand J Gastroent 2:124-128, 1967 Perks AM, Schapiro H, Woodward ER: The influence of antidiuretic hormone on pancreatic exocrine secretion. Acta Endocr (Kobenhavn) 45:340-348, 1964 Schapiro H, Storer EH, Britt LG : Action of antidiuretic hormone on gastric secretion. Arch Surg (Chicago) 92 :699- 703, 1966 Schapiro H, Storer EH, Britt LG: Action of antidiuretic hormone on food-stimulated gastric secretion. Amer Surg 34:315- 316, 1968 Baisset A, Montastruc P : Action des hormones neuro-hypophysaires sur les secretions salivaire et gastrique. Arch Sci Physiol (Paris) 14:393409, 1960 Cutting WC, Dodds EC, Noble RL, et a! : Pituitary control of alimentary blood flow and secretion : The effect of posterior pituitary extract on the alimentary secretions of intact animals. Proc Roy Soc [Bioi] 123:27- 38, 1937 Jacobson ED, Linford RH, Grossman MI: Gastric secretion in relation to mucosal blood flow studied by a clearance technic. J Clin Invest 45:1- 13, 1966 Lawson LJ, Dragstedt LR II: Vasopressin and gastric secretion. Arch Surg (Chicago) 90:273278, 1965 Hollander W Jr, Williams TF, Fordham CC III, et a!: A study of the quantitative relationship between antidiuretic hormone (vasopressin) and the renal tubular reabsorption of water. J Clin Invest 36:1059-1071, 1957 Grantham JJ, Orloff J: Effect of prostaglandin E, on the permeability response of the isolated collecting tubule to vasopressin, adenosine 3', 5'monophosphate and theophylline. J Clin Invest 47 :1154- 1161, 196~ Cuthbert AW, Painter E: Independent action of antidiuretic hormone, theophylline and cyclic 3', 5' -adenosine monophosphate on cell membrane permeability in frog skin. J Physiol (London) 199:593-612, 1968 Hays RM , Leaf A: Studies on the movement of water through the isolated toad bladder and its modification by vasopressin. J Gen Physiol 45: 905, 1962 Cofre G, Crabbe J: Active sodium transport by the colon of bufo marinus: Stimulation by aldosterone and antidiuretic hormone. J Physiol (London) 188:177-190, 1967
October 1971
VASOPRESSIN INHIBITION OF GASTRIC SECRETION
23. Obrink KJ, Waller M: The transmucosal migration of water and hydrogen ions in the stomach. Acta Physiol Scand 63:175-185, 1965 24. Sachs G, Clark LC, Makhlouf GM : The use of fluorocarbon emulsion in the Ussing Chamber. Proc Soc Exp Bioi Med (in press), 1971 25. Bartelstone HJ, Nasmyth PA: Vasopressin potentiation of catecholamine actions in dog, rat, cat and rat aortic strip. Amer J Physiol 208:754-762, 1968 26. Bell PRF, Battersby C: Effect of vasopressin (Pitressin) on gastric mucosal blood flow measured by clearance of krypton. Surgery 66:510-514, 1969 27. Berde VB, Weidmann H, Cerletti A: Ueber Phenylalanin-Lysin-Vasopressin. Helv Physiol Pharmacal Acta 19:285-302, 1961 28. Delaney JP, Grim E: Influence of hormones and drugs on canine pancreatic blood flow. Amer J Physiol 211:1398- 1402, 1966 29. Shehadeh Z, Price WE, Jacobson ED: Effects of vasoactive agents on intestinal blood flow and
30.
31.
32.
33.
34.
35.
469
motility in the dog. Amer J Physiol 216:386- 392, 1969 Cowley DJ, Code CF: Effects of secretory inhibitors on mucosal blood flow in nonsecreting stomach of conscious dogs. Amer J Physiol218:270-274, 1970 Harper AA, Reed JD, Smy JR: Gastric blood flow in anesthetized cats. J Physiol (London) 194:795807, 1968 Moody FG: Gastric blood flow and acid secretion during direct intraarterial histamine administration. Gastroenterology 52:216-224, 1967 Nedzel AJ: Experimental production of gastric ulcers in dogs by inducing vascular spasm with pitressin. Amer J Dig Dis 10:283-296, 1943 Renkin EM : Effects of blood flow on diffusion kinetics in isolated perfused hindlegs of cats. Amer J Physiol183:125-136, 1955 Forrest APM, Code CF: The inhibiting effect of epinephrine and norepinephrine on secretion induced by histamine in separated pouches of dogs. Pharmacal J 110:477-450, 1954
G ASTROENTEROLOGY Copyri ~h t
© 1971 by T he Williams & Wilk ins Co.
KINETICS OF INHffiiTION OF CANINE GASTRIC SECRETION BY VASOPRESSIN ANDRE L. BLUM, M.D.
Division of Gastroenterology, Department of Medicine , Univ ersity of Alabama in Birmingham , School of Medicine, and Veterans Administration Hospital, Birmingham, Alabama
The kinetics of inhibition of acid secretion by vasopressin were studied in gastric fistula dogs. Inhibition was dose-dependent with a threshold of 10 mU per kg in each dog. Dose-response curves with the inhibitor dose held constant showed that inhibition was of the competitive type: maximal response was unaltered but the dose required to elicit half the maximal response increased 2-fold. Kinetics of this type are postulated to arise from interference in the reaction of stimulant and receptor but can be mimicked in vivo by mechanisms restricting the delivery of the stimulant to the target cells. In each case, however, the mechanisms were consistent with either the vasoactive or water-conserving properties of vasopressin. For some inhibitors of gastric secretion such as secretin and compounds structurally related to gastrin, theories have been advanced which provide a satisfactory account of their mechanism of action. 1 In other instances, the mechanism of inhibition remains obscure as in the case of histamine-stimulated secretion by atropine. 2 Even more intriguing is the ineffectiveness in vitro of inhibitors like Diamox and vasopressin which are highly potent in vivo. 3 • 4 In this study, the kinetics of inhibition of canine acid secretion by vaso-
pressin were analyzed in an attempt to attribute the effect of vasopressin either to its vasoactive or to its water-conserving properties.
Kinetics of Inhibition According to a model proposed by Makhlouf et al., 5 • 6 the relationship between acid response and dose of stimulant may be expressed as follows: Rd r =
K
+d
(1)
where r represents the observed response, R the maximal response, d the exogenous dose of stimulant, and K the value of the dose which elicits half the maximal response or ED 50 . It is assumed in this model that the response is proportional to the number of activated secretory units or stimulant-receptor complexes and that the concentration of stimulant in proximity to the receptor sites remains a constant fraction of the exogenous dose. The formal analogy between this model and enzyme-substrate interaction is apparent and the kinetics of the two systems in respect of inhibition and acceleration are alike: as applied to exocrine secretion,
Received December 2, 1970. Accepted May 7, 1971. Address requests for reprints to: Dr. Andre L. Blum, Medizinische Klinik, Stadtspital Triemli, 8055 Zurich, Switzerland. This work was supported by National Institutes of Health Grants TIAM 2A-5286 and AM09260, National Science Foundation Grant GB8351 , and Research Funds of Veterans Administration Hospital, Birmingham, Alabama. This work was submitted as a docent thesis to the University of Zurich ; it was suggested and guided by Dr. Gabriel M. Makhlouf. Expert technical assistance by Mr. Robert Perkins and Mr. Michael Lachina is acknowledged. Synthetic vasopressin 8-lysine was a gift of Sandoz Pharmaceuticals, Hanover, New Jersey. 461
462
BLUM
competitive inhibition, which is the only type discussed here, may be stated as follows 7 : Rd
r
=
K(1
+ ilk) + d
(2)
where i and k; are respectively the dose and dissociation constant of the inhibitor. The function (1 + ilk;) is constant for a fixed dose of inhibitor and represents the factor of increase inK or the ED 5 0 • An alternative manner of stating equation 2 indicates that the inhibitor acts as if to render the dose of stimulant less effective by a factor (1 + ilk;): r
=
K
R d[ll(1 + ilk)] + d[l/(1 + ilk;)]
(3)
Equation 2 may be transformed into 1/r
=
1IR
+ K(1 + ilk;)IRd
(4)
which states that, for a constant dose of inhibitor i, the relationship between 1/r and 1/d remains linear and the intercept on the response axis is maintained equal to 1IR while the slope of the relation is increased by a factor of (1 + ilk;). For a constant dose of stimulant, the relationship between i and 1/r is linear. The latter condition applies also to noncompetitive inhibition. An alternative linear transformation more suitable for calculation 8 is: r
=
R - K(1
+ ilk) rid
(5)
which states that, for a constant i, the relationship between r and rid remains linear, the intercept on the response axis is maintained equal to R, and the slope is increased by a factor (1 + ilk;).
Material and Methods Three female mongrel dogs weighing 21-25 kg were equipped with stainless steel gastric cannulae and Komarov type esophageal fistulae for external drainage of saliva during collection of gastric juice. Experiments were started several months after operation. The dogs were fasted for 16 hr after which the stomachs were gently washed with 100 ml of lukewarm tap water and unstimulated secretion was collected for at least 30 min. Two intravenous infusions were then started into leg veins for separate administration
Vol. 61 , No . 4, Part I
of histamine acid phosphate and synthetic vasopressin 8-lysine. Two types of experiments were performed in which either inhibitor or stimulant was held constant. 1. Vasopressin dose varied, histamine dose constant. Histamine was infused intravenously at a rate of 50 f.Lg of base per kg per hr for a period of 330 min. After allowing 60 min for secretion to reach a steady rate, vasopressin was injected intravenously three times at intervals of 90 min. Three out of four dose levels (10, 20, 30, or 40 m U per kg) were employed in the course of each experiment. The order of vasopressin doses was randomized as follows: a dose, for example 10 mU per kg, was injected at 60 min in one experiment, at 150 min in a second, and at 240 min in a third, and so on for all four doses. This procedure was instituted in order to minimize differences in the response arising from repeated administration of vasopressin or continous infusion of histamine. In retrospect, this precaution proved unnecessary since no significant differences could be detected both by paired t-tests and by regression analysis between responses at 60 min and responses at 150 or 240 min. Responses were therefore averaged independent of their time of occurrence. 2. Vasopressin dose constant, histamine dose varied. In a control series, histamine was infused at rates of 10, 25, 50 or 100 f.Lg of base per kg per hr for a period of 270 min. Saline was infused concurrently at a separate site. The dose of histamine was changed every 90 min and the order of infusions randomized such that each dose was given from 0 to 90 min in one experiment, from 90 to 180 in a second, and from 180 to 270 in a third. In this manner, differences that might have resulted from the fade of secretion were minimized. In a second series, the same procedure was followed except that vasopressin was added to the saline and infused from the start at a rate of 50 mU per kg per hr. Gastric juice was collected every 10 min except during the period following prompt intravenous injection of vasopressin when it was collected every 5 min for a period of 20 min. Acid content was determined by titration with 0.1 N NaOH using an automatic titrator and pH meter (Radiometer, Copenhagen).
Results 1. Vasopressin dose varied, histamine dose constant. Intravenous injection of vasopressin resulted in a rapid tall of histamine-stimulated secretion followed by
VASOPRESSIN INHIBITION OF GASTRIC SECRETION
October 1971 -20
0
20
40
-20
0
20
40 100
~
40 ~
::::>
20
i
j 10mUkg1
x~
20mUkg-1
80 60 40 20
~
[._
0 0 o-
80 60 40 20
30mUk -1 -20
0
20
40
40mUk -1 - 20
0
20
20
40
MINUTES FIG 1. Effect of single injections of lysine vasopressin on gastri c secretion during stimulation with histamine (50 p.g per kg per hr). Vasopressin injections are represented by an arrow at time zero. The dose of vasopressin is given in each graph. Acid output (mean ± SE) and acid concentration (black dots; SE smaller than symbols) of 3 dogs were normalized and expressed in percentage of the highest value obtained in each test.
a gradual return to control levels (fig. 1). The inhibitory effect was dose-dependent but was not accompanied by a change m acid concentration. The results are recorded in table 1. A convention which served the purpose of normalizing the data was utilized: the response during the 30-min period following vasopressin was expressed as a fraction of the control response during the 30-min period prior to inhibition. As shown in figure 2, a linear relation prevailed between the reciprocal of this fractional response and the dose of vasopressin. The slope of this relation was similar in the 3 dogs and indicated that a threshold of 10 m U per kg existed for the inhibitory effect of intravenous vasopressin on acid secretion. Analysis of published data 9 - 13 on inhibition of exocrine secretion by vasopressin disclosed a similar kinetic pattern including the existence of a threshold ·dose (table 2). 2. Vasopressin dose constant, histamine dose varied. Responses to increasing doses of histamine infused alone or in
463
combination with vasopressin are recorded in table 3. Vasopressin reduced acid output and flow rate, but had no detectable effect on acid concentration. The results were analyzed for each dog separately in terms of equations 2 to 5. There was a good fit of the data to equation 4 which predicts a linear relation between the reciprocals of response and dose of stimulant. Calculations of the constants R and K were based on an alternative linear transformation (equation 5) relating response and response per dose (table 4). The kinetics showed the characteristics of competitive inhibition in that the maximal response R remained unchanged while the dose which elicited half the maximal response rose by more than 2-fold from 57 J.L g per kg per hr with histamine alone to 128 J.Lg per kg per hr on addition of vasopressin. On the assumption of competitive inhibition, the effect of vasopressin was further characterized by the inhibitor constant k i calculated from equation 5 to be 40 m U per kg per hr. Discussion This study confirms earlier reports on dose-dependent inhibition of gastric acid secretion by vasopressin. 10 • 1 2 - 1 7 Kinetic analysis of published data shows a similar dose-dependent inhibition of pancreatic secretion and of splanchnic and resting gastric blood flow (table 4). Conditions in which only the inhibitor dose is changed do not, however, allow conclusions regarding the type of inhibition. By instituting a further condition in which the stimulant dose was varied while the inhibitor dose was held constant, it was possible to show that inhibition by vasopressin was of the competitive type. It is characteristic of this type of inhibition that the calculated maximal response to the stimulant alone is not altered in the presence of the inhibitor (table 4, fig. 3) while the dose of stimulant required to elicit half the maximal response is increased by a factor (1 + i /k;) which is dependent on the dose of the inhibitor. There are several ways in which inhibition in vivo may give rise to competitive kinetics. (a) In the simplest case, expressed
464 TABLE
BLUM
Vol. 61, No.4, Part 1
1. Effect of single injections of vasopressin on acid output and volume output stimulnted with 50~ per
kg per hr of histamine" Dose of vasopressin Regression of 1/r on dose of vasopressin
Dog 10
20
30
40
m U/kg
mU/kg
Acid output
Dog A
0 . 994
0.792
0. 637
Dog B
1. 009
0 . 771
0.632
Dog C
0 . 980
0 . 783
0.686
3 dogs (mean ±
Volume output
SE)
Dog A
0.994 ±0 . 020 0.956
Dog B
1.038
Dog C
0 . 974
3 dogs (mean ±
SE)
0 . 989 ±0 .031
0.782 0 . 651 ±0.035 ±0.027 0.824 0. 773 0.789 0 . 795 ±0.040
Lowest effective dose of vasopressin
0.644
0 . 552
1/r = 0. 0273i + 0. 73, p < 0.01 0. 566 1/r = 0 . 0262i + 0.76, p < 0 .01 0.623 1/r = 0.0193i + 0.86, p < 0.01 0 . 580 1/r = 0.0241i ±0.023 + 0.78, p < 0 .01 0 . 603
1/r = 0. 0218i + 0.82, p < 0.683 0 . 615 1/r = 0.0216i + 0.89, p < 0.703 0 . 650 1/r = 0. 0169i + 0.80, p < 0 . 677 0.623 1/r = 0. 0200i ±0.026 ±0.023 + 0.84, p <
9. 9 9 .3 7.6 9. 0
8.0 0.01 9 .3 0.01 6.0 0 .01 8.1 0. 01
a Response is the output during the 30-min period following injection of vasopressin and is expressed as fraction of the output in the 30-min period before injection. Regression equations were calculated for dose of vasopressin {i) and reciprocal of the response (1/r). Correlation coefficients were in all cases >0. 98. Threshold doses of vasopressin were calculated from the regression equations (see text) . Regressions for acid output are plotted in figure 2.
VASOPRESSI N DOSE
FIG 2. Dose-response curve for single vasopressin injections during a histamine infusion. The dose of histamine is 50 p.g per kg per hr. The dose of vasopressin is given in mU per kg. Acid output in the 30 min following vasopressin injection is expressed as fraction of the acid output before injection. The reciprocal of this fraction (control/response) is plotted against the dose of vasopressin. An arrow points at the intercept on the X axis which represents the lowest effective dose of vasopressin. Regression equations are given in table 1.
by equation 2, the inhibitor acts by competing for the same receptor site as the stimulant, an unlikely event in this instance in view of the structural dissimilarity between histamine and vasopressin. Allosteric modification of the receptor site for histamine appears more probable. This would interfere with the rate at which the stimulant is bound to and/or discharged from the receptors. Since the over-all constant K is determined by the ratio of the rate constants for these two opposing processes, 7 its value increases in the presence of the inhibitor and the system appears less sensitive to the influence of the stimulant in that it requires a higher dose to elicit the same response. (b) An alternative manner of stating competitive kinetics is given in equation 3 and leads to a different interpretation of inhibition by vasopressin in vivo. Equation 3 implies that the effective dose of the stimulant is reduced
Type of
Shehadeh et al., 1969 29 Bell and Battersby, 1969 26
Banks et al. , 1968 9
Perks et a!., 1964 11
Forrest et al., 1954 35
Karlmark and Obrink, 1967 10
Schapiro et al., 1968 1 3
Schapiro et al., 1966"
Author
Stim ulant
Inhibitor
Volume output
Histamine, 50 p.g sub- Pitressin, single injection cutaneously every 15 min Pitressin, single Meat, 200-400 g Volume output injection Lysine and arForcemeat, 10 g per Volume output ginine vaso10 min pressin infusion Norepinephrine Histamine, 25 p.g/ Acid output infusion kg/hr Pitressin, single Secretin, 1 U/kg Volume output injection Pitressin, single Secretin, 1 U/kg Volume output injection Norepinephrine Electromagnetic flow None infusion meter Pitressin infusion Gastric mucosal Kr •• None clearance
Met hod
1/r = 0.0124i
1/r = 0.0195i
1/r = 0.0437i
1/r = 0.0775i
= 0.025)
(P
(P
(P
= 0.01) = 0.005)
+ 0.447
(P
+ 1.31
= 0.002)
= 0.0005)
+ 1.07
(P
+ 0.603
(P
= 0.001)
= 0.0003)
+ 1.02
(P
= 0.001)
+ 0.047
(P
+ 0.077
+ 0.692
1/r = 0.0338i
1/r = 0.251i
1/r = 0.0961i
1/r = 0.0442i
Regression of 1/r and i
Effective of vasoactive drugs on canine digestive secretion and blood flow•
44 mU/kg/hr
0 p.g/kg/hr
0 mU/kg
5 mU/kg
0 p.g/kg/hr
2 mU/kg/hr
6 mU/kg
5 mU/kg
Lowest effective dose of inhibitor
• Fit of a model relating dose of a vasoactive agent and response of exocrine secretion or blood flow. Gastric secretion was studied in dogs with Heidenhain pouches. Pancreatic secretion was studied in dogs with Thomas cannulas. Blood flow was measured in anesthetized animals. For regression analysis, the response observed after administration of the inhibitor was expressed as fraction of the response observed before inhibition. The reciprocal of this fraction (1 /r) was expressed as a function of the dose i of the inhibitor. For calculation of the lowest effective dose see text.
Mesenteric blood flow Gastric blood flow
Pancreatic secretion
Gastric secretion
experiment
TABLE 2.
~
Cll
0)
..,..
:<;
a~
t>l
::tl
(J
(::3
~
CJ:J
§2
~
~
~ ~
t5
~ ~
~
~
~
CJ:J
:::::!
......
~
o-
c
ag.
466
BLUM
Vol. 61, No.4, Part 1
TABLE 3. Effect of histamine infusions (control) and of histamine plus vasopressin infusions (+VA) on acid output, volume output, and acid concentration• Dose of histami ne 10
Dog
Acid output (mEq/hr)
±SE) Dog A Dog B Dog C 3 dogs (mean
± SE) Acid concentration (mEq/liter)
+ VA
Control
Dog A Dog B Dog C 3 dogs (mean
Volume output (ml/hr)
25
3 dogs (mean ± SE)
12.44 6.78 3.42 2.23 6.64 2.99 7.50 4.00 ±1.52 ± 0.91
50
+VA
Control
23 . 75 11.14 7.44 4.58 12 .24 7.96 14.48 7.89 ±2. 49 ± 1.51
-
92.4 32.2 52.7 59.1 ± 10.2
49.7 20 .4 19.7 29 .9 ±2 .0
168 . 1 54.5 82 .7 101 .7 ± 17 .4
80.3 38.4 50.6 56.4 ±9.2
134.1 ±2. 9
130.6 ±3. 1
142 .0 ± 1.6
141.5 ± 2.0
100
+VA
Control
16. 50 12.17 11.09 13 .25 ±1. 62
29.26 15.72 20.40 21.79 ± 2.32
194 .3 116.7 108 .8 106.2 135.3 73.1 146.1 98 .7 ± 13 .6 ± 11.1 148.8 ± 1.6
146.5 ± 1.6
+ VA
Control
28.98 27.63 20.61 25.74 ± 2.23
24.92 18 .63 18.30 20 .62 ±2. 10
198 .6 157.5 186.3 126.9 130.7 116.4 171 .9 133 .5 ± 12. 7 ± 13 .0 149.7 ± 1.9
152 .5 ± 1.2
• Dose of histamine base is given in /J.g per kg per hr. Dose of vasopressin was 50 mU per kg per hr. Doseresponse curves are plotted in figure 3.
TABLE 4. Regression equations for plots of I /response versus I /dose (see figure 3) and of response versus
~esponse /dose• Histamin e plus vasopressin
Histamine a lone
Regression of
1/r and 1/d, equation 4
1/r
=
0.0281
+ 0.982 (P
r and r/d, equation 5
r
=
1/d
1/r
< 0.001)
36.62 - 56.97 r/d (P < 0.025)
=
0.0269
+ 0.242
1/d
(P
r
=
< 0.001)
36.72 - 128.80 r/d (P
< 0.05)
a Response (r) represents acid output per 60 min in milliequivalents. Dose (d) represents the dose of histamine which was either infused alone or together with 50 mU per kg per hr of vasopressin.
\)() j
002
0.02
004
006
0 00
0 10
I I HISTAMINE DOSE
FIG 3. Dose-response curves for histamine infusions (open circles) and histamine infusions given together with vasopressin infusions (closed circles) in 3 dogs. The dose of vasopressin is 50 m U per kg per hr. The dose of histamine is given in /J.g per kg per hr. The response in the upper graph is the volume output in ml per hr and in the lower graph it is the acid output in mEq per hr. Regression equations are given in table 4.
in the presence of an inhibitor to an even smaller fraction [1 /(1 + ilk)] of the exogenous dose. It should be recalled that the convention which permits the use of the exogenous dose in lieu of the local concentration of stimulant in the target organ is based on the assumption that the latter is a constant fraction of the former. This fraction could, however, be altered by a drug which restricts the delivery of the stimulant to the receptor sites. The resultant kinetics would simulate competitive inhibition without involving an effect of the inhibitor drug at receptor level. Various processes in the target organ or at more distant locations might lead to this outcome; for example, a decrease in vas-
October 1971
VASOPRESSIN INHIBITION OF GASTRIC SECRETION
cular permeability coupled with a decrease in capillary flow and surface area; a decrease in cellular permeability; or an increase in nonspecific binding or disposal of the stimulant. Mechanisms of inhibition consistent with the two alternatives listed under "a" and "b" above may be sought respectively in the water-conserving and vasoactive properties of vasopressin. 1. Consistent with its physiological action, the requirements of vasopressin for water conservation by the renal tubule are small. Antidiuretic responses in vivo may be observed with doses as low as 10 J.LU per kg per hr 18 while in vitro concentrations below 1 J.LU per ml are still active. 19 Much higher concentrations ( > 100 mU per ml) are needed in vitro to stimulate absorptive mechanisms in amphibian skin, 20 bladder, 2 1 and colon. 22 The mature stomach, like other exocrine organs, is largely secretory and only to a minor extent absorptive. Water conservation in this organ would, therefore, mainly result from restriction of secretion. In fact, the only mechanism which leads to disappearance of acid from the gastric lumen appears to be passive and without significant effect on movement of water. 23 Recent attempts to demonstrate inhibition of secretion in vitro have not met with success. A wide range of concentrations failed to inhibit spontaneous or histamine-stimulated acid secretion by the isolated frog gastric mucosa (R. L. Shoemaker, A. L. Blum, and G. M. Makhlouf, unpublished observations). No information is available on secretion by the isolated mammalian mucosa, but there is no reason to believe that it would in this respect respond differently from the amphibian mucosa. A similar lack of inhibition was observed by Buranarugsa and Moore 3 in the perfused pancreas of the rabbit which in vivo promptly responded to inhibition by vasopressin. Such lack of inhibition in vitro appears to preclude an effect of vasopressin at receptor level or through an effect on cellular permeability. Conclusions based on the absence of in vitro effects, however, are not decisive since the preparations employed are sub-
467
optimal and secrete at rates which are low and may mask inhibitory effects. 24 More pertinent, an inhibitor-like vasopressin may require an adjuvant available only in vivo for its effect to be fully manifest. Both the lack of secretory inhibition and the need for large concentrations to stimulate absorption in vitro are consistent with this view. It is known that low concentrations of vasopressin potentiate the effect of catecholamines on vascular muscle. 2 5 A similar type of cooperation in respect of its water-conserving properties would endow vasopressin with more general physiological significance than the apparent ineffectiveness in vitro would suggest. 2. Of the various possibilities listed under "b" above which might lead to inhibition by restricting delivery of the stimulant only the first is consistent with the vasoactive properties of vasopressin. Vasopressin is known to decrease splanchnic blood flow and in particular total and mucosal gastric blood flow. 1 5 • 16• 26" 29 Larger doses than were used in this study to inhibit secretion are apparently needed to bring about a reduction in resting mucosal blow flow, 26 • 30 but there are no systematic studies on dose requirements for this effect during active secretion. Following stimulation of acid secretion there prevails a high ratio of blood flow to secretion rate 3 1• 3 2 which makes it improbabk that anything but an extreme reduction in blood flow could have imposed a limit on the amount of water cleared in secretion by allowing the osmolality of the tissues to rise excessively. It is equally improbable that a limit could have been set on secretion by the supply of nutrients since this would have led to irreversible damage 3 3 inconsistent with the observed rapid recovery of function following intravenous vasopressin. Restriction to the delivery of the stimulant is possible but remains conjectural in the case of histamine. The rates at which small molecules diffuse in and out of capillaries exceed greatly the rates at which they are borne by the blood, and for these substances capillary permeability is not an essential factor in blood tissue exchange. For some substances like antipyrine cellu-
468
BLUM
lar permeability is also high; their clearance approaches blood flow and their distribution is said to be blood flow-limited. Others, like urea, have a low cellular permeability and therefore their clearance changes little despite wide variations in blood flow. It should be noted that these conclusions are based on a model which assumes uniform perfusion of widely dilated capillaries with a constant surface area. For a given blood flow during vasoconstriction even the clearance of antipyrine is reduced. 3 4 Conceivably the properties of gastric stimulants reside between the two extremes of flow- and diffusionlimited substances. By inducing a reduction in capillary flow and surface area, vasopressin might limit the extraction of a stimulant by the mucosa and lead to a reduction in secretion. A similar mechanism might apply to inhibition of secretion by noradrenaline for which an identical kinetic pattern has been observed 3 5 (table 2) . A direct study of stimulant extraction by target tissues under various conditions should yield valuable information on the mechanism of secretory inhibition by vasoactive agents in vivo. REFERENCES 1. Grossman MI: Gastrin, cholecystokinin and secretin act on one receptor. Lancet 1:1088- 1089, 1970 2. Durbin RP, Thorpe CD: Inhibition by atropine of acid secretion stimulated by acetylcholine, histamine and gastrin in isolated frog gastric mucosa. Gastroenterology 58:944, 1970 3. Buranarugsa P, Moore WW : The effect of vasopressin on secretin-stimulated pancreatic secretion in the rabbit, in vivo and in vitro. Physiologist 13: 159, 1970 4. Durbin RP, Heinz E : Electromotive chloride transport and gastric acid secretion in the frog. J Gen Physiol 41 :1035-1047, 1958 5. Makhlouf GM, McManus JPA, Card WI: Action of pentapeptide (ICI 50 123) on gastric secretion in man. Gastroenterology 51 :455- 465, 1966 6. Makhlouf GM, McManus JPA, Knill JR: Quantitative aspects of synergism and inhibition of gastric secretion. Gastroenterology 54:532-537, 1968 7. Dixon M, Webb EC: Enzymes. Longmans Green and Co, 1958, p 171- 181 8. Dowd JE, Riggs DS: A comparison of estimates of Michaelis Menten kinetic constants from var-
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Vol. 61, No.4, Part 1 ious linear transformations. J Bioi Chern 240:863869, 1965 Banks PA, Rudick J, Dreiling DA, et a!: Effect of antidiuretic hormone on pancreatic exocrine secretion. Amer J Physiol215:361-365, 1968 Karlmark B, Obrink KJ : The antidiuretic hormone as an inhibitor of gastric secretion. Scand J Gastroent 2:124-128, 1967 Perks AM, Schapiro H, Woodward ER: The influence of antidiuretic hormone on pancreatic exocrine secretion. Acta Endocr (Kobenhavn) 45:340-348, 1964 Schapiro H, Storer EH, Britt LG : Action of antidiuretic hormone on gastric secretion. Arch Surg (Chicago) 92 :699- 703, 1966 Schapiro H, Storer EH, Britt LG: Action of antidiuretic hormone on food-stimulated gastric secretion. Amer Surg 34:315- 316, 1968 Baisset A, Montastruc P : Action des hormones neuro-hypophysaires sur les secretions salivaire et gastrique. Arch Sci Physiol (Paris) 14:393409, 1960 Cutting WC, Dodds EC, Noble RL, et a! : Pituitary control of alimentary blood flow and secretion : The effect of posterior pituitary extract on the alimentary secretions of intact animals. Proc Roy Soc [Bioi] 123:27- 38, 1937 Jacobson ED, Linford RH, Grossman MI: Gastric secretion in relation to mucosal blood flow studied by a clearance technic. J Clin Invest 45:1- 13, 1966 Lawson LJ, Dragstedt LR II: Vasopressin and gastric secretion. Arch Surg (Chicago) 90:273278, 1965 Hollander W Jr, Williams TF, Fordham CC III, et a!: A study of the quantitative relationship between antidiuretic hormone (vasopressin) and the renal tubular reabsorption of water. J Clin Invest 36:1059-1071, 1957 Grantham JJ, Orloff J: Effect of prostaglandin E, on the permeability response of the isolated collecting tubule to vasopressin, adenosine 3', 5'monophosphate and theophylline. J Clin Invest 47 :1154- 1161, 196~ Cuthbert AW, Painter E: Independent action of antidiuretic hormone, theophylline and cyclic 3', 5' -adenosine monophosphate on cell membrane permeability in frog skin. J Physiol (London) 199:593-612, 1968 Hays RM , Leaf A: Studies on the movement of water through the isolated toad bladder and its modification by vasopressin. J Gen Physiol 45: 905, 1962 Cofre G, Crabbe J: Active sodium transport by the colon of bufo marinus: Stimulation by aldosterone and antidiuretic hormone. J Physiol (London) 188:177-190, 1967
October 1971
VASOPRESSIN INHIBITION OF GASTRIC SECRETION
23. Obrink KJ, Waller M: The transmucosal migration of water and hydrogen ions in the stomach. Acta Physiol Scand 63:175-185, 1965 24. Sachs G, Clark LC, Makhlouf GM : The use of fluorocarbon emulsion in the Ussing Chamber. Proc Soc Exp Bioi Med (in press), 1971 25. Bartelstone HJ, Nasmyth PA: Vasopressin potentiation of catecholamine actions in dog, rat, cat and rat aortic strip. Amer J Physiol 208:754-762, 1968 26. Bell PRF, Battersby C: Effect of vasopressin (Pitressin) on gastric mucosal blood flow measured by clearance of krypton. Surgery 66:510-514, 1969 27. Berde VB, Weidmann H, Cerletti A: Ueber Phenylalanin-Lysin-Vasopressin. Helv Physiol Pharmacal Acta 19:285-302, 1961 28. Delaney JP, Grim E: Influence of hormones and drugs on canine pancreatic blood flow. Amer J Physiol 211:1398- 1402, 1966 29. Shehadeh Z, Price WE, Jacobson ED: Effects of vasoactive agents on intestinal blood flow and
30.
31.
32.
33.
34.
35.
469
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