Dose-Response Curves of Denervated Autogenous and Homotransplanted Canine Fundic Pouches Stimulated with Purified Gastrin and Histamine

Vol. 51, Xo. 2 Pri11ted in U.S. A.

GASTROENTEROLOGY

Copyright © 1966 by The Williams & Wilkins Co.

DOSE-RESPONSE CURVES OF DENERVATED AUTOGENOUS AND HOMOTRANSPLANTED CANINE FUNDIC POUCHES STil\IULATED WITH PURIFIED GASTRIN AND HISTAMINE lR.-1. A.

W ARRE:\ D. DAVIDSON, M .D., JA~I ES

H.

MILLER, AND J AMES

c.

DAVES, :M.D., CARLOS

TH O~ !P SOX, M.D . ,

A.

LE~nu,

1\LA.

D epartments of Surgery and Jliedicine , H arbor General Ho spital, TorTance, California, and the Unive1·sity of Californi a Los Angeles School of Jledicine, Los .Angeles, California

In 1964 Gregory and Tracy 1 isolated from hog antral mucosa two polypeptides, gastrins I and II. These proved t o be the most potent of all known stimuli of gastric secret ion. In later communications2· 3 they and their colleagues reported the complete characterization of the two heptadecapeptides and the t otal synthesis of both. Gastrins I and II are, on a mol ar b asis, about 500 times more potent gastric stimulants than histamine in man. 4 The presence of large amounts of histamine in gastric mucosa and the ability of this compound to stimulate gastric secretion have suggested that it may also have some physiological role in the secretion of acid by the stomach. 5 However, direct experimental evidence to support t his hypothesis is lacking. The relationship, if any, of gastrin to histamine is unknown. Several studies have been designed to compare the actions of gastrin and histamine on acid secretion by gastric pouches. Previous studies utilizing relatively crude gastrin extracts have shown that the maximal rate of acid secretion of a vagally innervated fundic pouch stimulated by gastrin is apReceived J anu ary 10, 1966. Accepted April, 12 1966. Address requests for reprints to: Dr. Warren D. D avidson, H arb or General H ospital , 1124 W . Carson Street, T orr ance, California 90509. These studies have been supported in part by Grants AM-07961 and AM-07962 from the United States Public Health Service, an d by a grant from the John A. H artford F oundat ion , Inc., to the University of California L os Angeles . Dr. D a vidson is a r ecipient of a United States Public Health Service Research Career Development Award (K3-AM-13853) .

proximately equal to the maximal rnte in response to histamine. 6 Denervation of a gastric pouch depresses the maximal acid response to gastrin to ab out 50 % of t he maximal response to histamine. 6 The effects of antral denervation and antrectomy on the response of t he denervated fundic pouch to gastrin and histamine have been the subj ect of several previous studi es.7·12 Several authors7 ·10 reported that antrectomy did not alter the acid response of P avlov or H eidenhain pouches t o a single submaximal subcutaneous dose of histamine. Antrectomy had no effect on maximal gastrin response of dogs with Heidenhain pouches, but this procedure increased t he response to submaximal doses of gastrin. In contrast, antrectomy had no effect on the acid response to submaximal doses of histamine, but did reduce the maximal histamine response.12 Vagot omy failed to reduce t he maximal acid response to histamine in nonantrectomized dogs but did reduce this response in antrectomized animals. 11 Also, it was shown that the pepsin-stimulating action of high doses of gastrin in Heidenhain pouch dogs was abolished by antrectomy.U Hm-vever, in a subsequent study antrectomy was found t o have no effect on pepsin secretion by a denervated fundic pouch. 12 The present study was undertaken to determine t he characteristics of acid and pepsin secretion by denervated fundic pouches in response to graded intravenous doses of histamine and gastrin, and to determine the effect of antral denervation; antrectomy, and homotransplantation on these responses. Four types of denervated fundic pouches were studied: (1) Heidenhain pouch (HP),

180

August 1966

RESPON SES TO GASTRIN AND HISTAMINE

(2) Heidenhain pouch with denervated antral pouch (HP-DAP), (3) Heidenhain pouch with antrectomy (HP-AX), and (4) heterotopic homotransplanted fundic pouch with a homotransplanted antral pouch (TP) .

l\'laterial and Methods Surgical T echniques Thirty-eight adult mongrel dogs were used. Denervated autogenous fundic and antral pouches were prepared by our standard operative techniques .13 Animals with autogenous pouches 1wighed 17 to 28 kg. Six dogs were prepared with Heidenhain pouches, 7 with Heidenhain and denen ·ated antral pouches, and 7 dogs under\Yent antrectomy plus the formation of Heidenhain pouches. Kine gastric homografts were placed in 9 host animals, 5 of which had no autogenous pouches, 11·hile 4 had previously been provided with autogenous H eidenhain and denervated antral pouches. The donor animals weighed approximately 10 kg; the recipients weighed between 30 and 35 kg. The procedure of gastric homotransplantation is described elsewhere.l 3 Briefly, this technique allowed the preparation of a gastric graft from the distal stomach. The blood supply was dissected to the celiac artery and to the junction of the gastrosplenic vein with the superior mesenteric vein. The homograft was divided into antral and fundic pouches with an internal mucous membrane barrier. Antrum was delineated from fundu s with pH indicator paper following the subcutaneous injection of 12.5 llg of gastrin, similar to the method of Olbe.l 4 The homograft was placed in a subcutaneous pocket in the neck of the recipient dogs. With the use of a domestic vascular stapling device (Codman and Shurtleff, Boston), anastomosis was performed between the celiac artery of the homograft and the common carotid of the host, and between the gastrosplenic and external jugular vein. The mean period of ischemia was less than 8min.

Immunosuppressive Therapy The 9 host dogs received azathioprine (Burroughs-Wellcome Company, Tuckahoe, N . Y.) daily with the addition of prednisolone and actinomycin C (Farbenfabriken Bayer A. G., Leverkusen, Germany) during rejection crisis. 13 Each day the animals were weighed and had white blood cell counts and hematocrit determinations. Mucosal biopsies were obtained every 4th day in most dogs. The usual first sign of immune rejection was fundic hypochlorhydria or achlorhydria

181

following histamine. This was followed by swelling and induration of the graft. These signs were usually reversible with drug therapy.

Secretory Stimulants Histamine. Histamine was given in the fonn of the acid phosphate. However, all doses are expressed in te1ms of the weight of the free base. Gastrin. Gastrin was prepared from the antral mucosa of hog stomachs according to the technique described by Gregory and Tracy1 in 1964. The gastrin was first extracted from the mucosa with boiling water and then adsorbed onto diethylaminoethyl cellulose floc (Whatman DE-I floc, W. and R. Balston, Ltd ., England) in a batch procedure. The gastrin was eluted from the cellulose with sodium hydroxide and precipitated from solution with glacial acetic acid. The precipitate was redissolved in dilute ammonium hydroxide, and potassium phosphate and isobutanol were added. After mixing, the isobutanol layer was extracted with water and ether, and the final aqueous layer was extracted with ether. Residual ether was removed in a vacuum evaporator at room temperature. The gastrin was precipitated by the addition of glacial acetic acid. The precipitate was redissolved in dilute ammonium hydroxide and subj ected to gel filtration on a dextran (Sephadex G-50, fine bead fo1m; Pha1macia Fine Chemicals, Uppsala, Sweden) column with an ammonium bicarbonate liquid phase. Proteins and peptides were dete1mined in fractions of the eluate by measuring optical density at 280 mJ.i.. The active peak was subjected to lyophilization to remove the volatile ammonium bicarbonate. Lyophilization was repeated until a constant weight of a pure white powder was obtained. This material was dissolved in 0.9% sodium chloride solution for use in secretory studies. Since gastrins I and II have identical physiological actions,! no attempt was made to separate the two forms by gradient elution chromatography from aminoethyl cellulose. Gregory and Tracy1 have shown that this final step results in a loss of about 30 % of the material obtained from the active peak eluted from the Sephadex column. Since the specific activity of the material obtained at each of these steps is not given, it is not clear whether the loss represents inert impurity or the loss of gastrin. In this study, all doses of gastrin are expressed in terms of the weight of the final lyophilized product.

Chemical Assays Acid titration. The output of hydrochloric acid by the gastric pouches was dete1mined by titra-

182

D.A VIDSO~Y ET AL.

tion of 1 ml of gastric juice ''"ith 0.100 x l\ aOH, utilizing Topfer's reagent as an indicator. Pepsin assay. Pepsin actiYity of gastric juice "·as determined by the method of Grossman and Marks,1 5 with the single modification that the albumin substrate (Abbott Laboratories, Chicago) was labeled \Yith P 25 instead of I 131 • Samples " ·ere counted in a well scintillation counter. Sufficient counts "·ere recorded to reduce counting errors to less than 3 %. Peptic actiYity \Yas e:-..-pressecl as micrograms of pepsin per milliliter by comparison with a standard enzyme solution made up with porcine pepsin (Calbiochem, Los Angeles) which had been recrystallized three times.

Secretory Studies Dogs "·ith autogenous gastric pouches \Yere not studied until 1 month after operation, and those with transplanted pouches "·ere not studied until 2 weeks after operation. Secretory studies were perfom1ecl only on healthy animals. Dogs with transplanted pouches were used only \Yhen there was no sign of immunosuppressive drug toxicity or immune rejection. The latter was heralded by the sudden appearance of hypochlorhydria or achlorhydria. All animals were fasted for 16 hr prior to study and all tests were perfom1ecl with the dogs in Pavlov stands. Gastric juice was collected in a rubber balloon attached to a cannula inserted in the gastric pouch. Infusion of gastrin or histamine was initiated after the animal had attained a basal secretory state which we defined as a 15-min output of less than 0.5 ml of gastric juice with an acid concentration of less than 20 mEq per liter. The histamine and gastrin, dissolved in 0.9 % sodium chloride solution, were infused intravenously with a motor-driven syringe (Harvard Apparatus Company, Dover, Mass.). The same dosage schedule was used in all dogs and each dose was expressed as micrograms of gastrin or histamine per minute per kilogram of body weight. The dosage range for histamine was 0.01 to 4.0 f..Lg per kg per min, and for gastrin, 0.0005 to 0.625 J.Lg per kg per min. The volume of fluid administered varied between 3.75 and 37.5 ml per hr; the total volume in any 8-hr study did not exceed 180 ml. Each dose level was administered for 1 hr. Gastric juice was collected every 30 min and each collection was titrated for acid . Only the second collection in each hour was assayed for pepsin activity. At the end of each hour, the dose was increased 2- or 27'2-fold, depending on the gear ratio of the infusion apparatus. This procedure was continued until an increase in close produced no further increase in the rate of acid

Yol. 51, .Yo. 2

secretion or, in the case of gastrin, until clear-cut inhibition of gastric acid secretion had occurred. Histamine and gastrin infusions "·ere carried out on successive clays. If erratic responses were obsen·ecl during any test, the entire procedure m1' repeated the follo"·ing clay. If clogs Yomited, sali,·atecl excessi,·ely, or sho"·ecl other signs of toxicity to either agent, the test was tenninated and repeated the follo,Ying clay, unless such sigm appeared at least 2 hr beyond the establishment of a peak rate of secretion ..~bout one-half oi all infusions with either agent required repetition because of erratic response, eYiclence of toxicit)· or other technical difficulties.

Et•aluation of Data In constructing the acid and pepsin doseresponse cun·es, only the Yalues obtained in the second 30-min collection of each hour "·ere utilized. The output of acid in the second collection was ahrays higher than in the first 30-min collection unless the peak secretory rates had been achieved, or inhibition had occurred. In order to compare secretory rates of pouches of different sizes, all results are expressed as a percentage of the peak histamine response of acid and pepsin in each dog. By this means, the secretory rates of small pouches could be compared with those of large pouches. The data from each dog consisted of a single pair of gastrin and histamine dose-response curves. No study was repeated unless technical difficulties occurred in the initial study. No dog was included in more than one of the four groups. In each group of dogs, a mean value was calcu· lated for the rate of secretion of acid and pepsin at each dosage level. Statistical differences between the mean responses were detennined by using the t-test formula for unpaired groups of different sizes. 16 The linear ascending portions of each pair of composite dose-response curves were inspected visually and the segments which seemed to be parallel were subj ected to mathematical tests for parallelismP The segments evaluated corresponded to a dosage range of 0.005 to 0.025 f..L g of gastrin per kg per min and 0.1 to 1.0 f..L g of histamine per kg per min fo r the HP's; 0.005 to 0.025 f..Lg of gastrin per kg per min and 0.2 to 1.0 f..Lg of histamine per kg per min for the HP-AX's; 0.0025 to 0.005 f..Lg of gastrin per kg per min and 0.04 to 0.1 J.Lg of histamine per kg per min for the HP-DAP's; and 0.005 to 0.025 f.l.g of gastrin per kg per min and 0.1 to 1.0 J.Lg of histamine per kg per min for the TP's. Differences in sensitivity to gastrin and histamine were determined by measuring the lateral displacement of the parallel portions of the corre·

100

183

RESPONSES TO GASTRIN AND HISTAkiiNE

.-J.ugust 1966 HEIDENHAIN POUCH 6 DOGS

1

90

I

~ 80

100

90

\ \

I

80

/ \i

"'a.

HEIDENHAIN POUCH DENERVATED ANTRAL POUCH 7 DOGS

[

"'~

70

I

<[

>(f) X

I

60

u..

I

0

>-

z

"' (J 50

cr

"'a.1

40

1-

~

s

30

0 0

~

20

10

_.--t 0.0005 0.001

0.0025 0.005 0.01

,ug/kg/min-HISTA 1.41 NE

100

/

/

~J

I

I

I I

I I

GASTRIN

-

HISTAMINE---

0.!

0.625- Jtglk<;~/min GASTRIN

025

0~4

1

o.M

0.1

0. 2

1~0

1

1

2.0

4 .0

HEIDENHAIN POUCH, ANTRECTOMIZED 7 DO GS

90

J\

I \

I

I I

" eo

~

\

I

w

l

\

70

I

60

J

j'! Vl

~ 0 1-

z

I

w 50 u 0: w

.

":0

30

0

i3

"

20

/¥1

10

0.0005 0.001

00025 0.005 0.01

,..,,.Q/min~HISTAMINE

BASE-

0~01

1

0.02

0.25 1

0.04

0.1

0. 2

0.625-pg/kg/min-GASTRIN

1~0 2~0 4~0

!

9 DOGS

~ 80

w

a.

~

j

60

0 1-

I

I

~

")

z lj50 I

I 4o

I

1::l

~

::l 0

/\1 II

I I

Vl

I

J'

T/ 30

11 I

/{

-

0.625-pCJ/kg/min-GASTRIN

0~4

0.25

0.~ 0~4

0.1

90

HISTAMINE---

0.1

~~

HOMOTRANSPLANTED FUNDIC POUCH

1~0

Z.o ~.0

GASTRIN

-

HISTAMINE---

/I--f

/

0025 ,MS

0.025 0.05

o.(ll o.Oz o.04

cr w a.

GASTRIN

/

_J

100

HISTAMINE SASE -

~ 70 ;'!

x' j!'

~ 40

5

I

0.0025 0.005 0.01

JLQikg/min

w

I

i

/~ ~.~.: . =

u "'

"

--f

0.025 0.05

BASE-o.Or o.Oz

X

0.0005 0.001

0.0025 0.005

p.9/kq/min-HISTAWI~

opi

BASE- 0.01

?.025 I 0.05 O.OZ 0.04

0;1 0.1

0.25

o.'z

0.625-pg/kQ/min-GASTRIN 0:4

1:0

2~0

1

4.0

Fw. 1. Acid response to graded doses of gastrin and histamine. Each point is the mean secretory rate expressed as a percentage of the peak secretory response to histamine in each dog. Ve1·tical bars at each point represent the standard error of the mean. Gastrin and histamine doses are expressed as micrograms per kilogram per minute.

sponding gastrin and histamine curves. These n1easurements were performed visually, utilizing detailed graphs of the dose-response curves and using the midpoint of the gastrin segment as the reference point for drawing a horizontal line between the two parallel segments. The anti-

logarithm of the length of this horizontal line gave a comparison of potency on a weight basis.

Resu]ts

Secretion of acid. The response to graded doses of histamine and gastrin in each of the

184

DA YIDSO~l\T ET AL.

Vol. 51, 1Yo. 2

100

90 ~ 80 w a. w

~

70


f-

(f)

I

60

lL

0 f-

z w 50

t)

a:: w a.

40 f:::J

a.

~ 30

0 0

u
20

10

0.0005 0.001

0.0025 0005 0.01 I

p.g/kg i'llin

~HISTAMINE

BASE- 0.01

0.025 0.05 I

I

0.1 I

0.02 0.04

0.1

, 0.25, 0.625--;--p.g/~g/min~GASTRI N 0.2

0.4

1.0

2.0

4.0

FIG. 2. Acid response to graded doses of gastrin and histamine, providing comparison of four groups of dogs with various denervated fundic pouch preparations. Points marked with asterisks differ significantly (*, P < 0.01; **, P < 0.05) from corresponding points on each of the dose response curves of the other three groups of dogs. For statistical purposes, the final point on the gastrin curve for the transplanted pouches (0.625 pg per kg per min) was compared to the final points on the gastrin curve for the other three groups (0.25 pg per kg per min). HP, Heidenhain pouch; HP, DAP, Heidenhain pouch with denervated antral pouch; HP, AX, Heidenhain pouch with antrectomy; TP, heterotopic homotransplanted fundic pouch with a homotransplanted antral pouch.

four groups of dogs is depicted in figure 1. Each point represents the mean secretory rate expressed as a percentage of the peak secretory response to histamine. It should be noted, however, that none of the hista~ mine curves reaches 100 %, since not all of the dogs in each group attained their peak rates of secretion at the same dosage level. Vertical bars at each point represent the standard error of the mean. In order to make a more accurate comparison of the groups, all data for the four groups are plotted on a single graph (fig. 2). It can be seen that the histamine responses are similar. None of the groups showed any response to histamine infused at a rate of 0.01 J..l.g per kg per min. However, all groups showed small but measurable responses to

0.025 J..l.g per kg per min. Tllis latter figure can be taken to be the threshold dose of histamine in this study. The peak rates of secretion in response to histamine occurred at a dosage of 2 J..l.g per kg per min for the HP, HP-AX, and TP dogs and at 1 J..l.g per kg per min for the HP-DAP dogs. The only statistically significant difference between the histamine curves is the increased response of HP-DAP dogs to submaximal doses of histamine (fig. 2). Another common characteristic of the histamine-response curves is the apparent inhibition of acid secretion by higher doses of histamine. Inhibition occurred in the absence of any obvious signs of histamine toxicity, i.e., vomiting, excessive salivation, irritability, or signs of hypotension. In some

A11gust 1966

185

RESPONSES TO GASTRIN AND HISTAMINE

dogs the rate of acid secretion at higher histamine doses fell to less than 50% of the peak secretory rate. The gastrin response curves show greater differences between the groups. None of the groups secreted at a dose level of 0.0005 p.g per kg per. min. Small responses were observed at 0.001 p.g per kg per min in all of the groups. Peak rates of secretion occurred at a different dosage level for each group: 0.01 p.g per kg per min for the HP-DAP's, 0.025 p.g per kg per min for the HP-AX's, 0.05 p.g per kg per min for the HP's, and 0.1 p.g per kg per min for the TP's. The ratio of the maximal gastrin-stimulated acid response to the rl.1aximal histamine-stimulated response (Gmax: Hrnax) differed in each of the groups. The mean Gmax:Hmax ratio was 0.40 for the HP~DAP's , 0.56 for the HP-AX's, 0.68 for the HP's and 1.01 for the TP's. The latter figure for the TP's differs significantly (P < 0.001) from the Gmax:Hmax values of each group of atltogenous pouches. In addition, the Gmax: H max value for the HP's differs significantly (P < 0.02) from the value for the HP-DAP's, but not from the HP-AX's (P > 0.2). By measuring the lateral distance between the parallel portions of each pair of histamine and gastrin cu.rves, one can calculate the relative sensitivity of each type of fundic pouch to gastrin and to histamine. On a weight basis, gastrin was found to be many times more potent than histamine as a stimulant of acid secretion. In the TP's, gastrin was 35 times as potent as histamine; in the HP's, 21 times; in the HP-DAP's, 14 times; and in the HP-AX's, 42 times. Since the molecular weight of Gregory's gastrin is about 20 times that of histamine,2 the ratio of activity of gastrin compared with histamine, mole for mole, was 700 in the TP's, 420 in the HP's, 280 in the HP-DAP's, and 840 in the HP-AX's. The value for the HPDAP's differs significantly (P < 0.02) from the values for the TP's and the HP-AX's, but not from the value for the HP's (P > 0.2). Since our gastrin is not entirely pure, the relative potency of gastrin to histamine is actually greater than shown in these calculations. The apparent inability to inhibit acid se-

DOG a T-866 HOST HEIDENHAIN POUCH PLUS HOMOTRANSPLANTED FUNDIC POUCH

6

1\

I I I

L

I I

I I

I

/ •

I

I

;<6\ i~ /

u

:r:

::J

1 I \

OST

//

0.2

I

0.000 5 0.001

I

I-- HOST

l

I

I

\1.

/

I

e

1- TRANSPLANT 1

/

6-}6--;A I

I

//\\

I

0

&

'I

I

\

3

\

I .\

\ \ l rRANSPLAN'

/

~~~::~E=

/

I •/ ~.---0.0025 0.005 0.01 1

pq / kQ /min - HISTAMINE BASE - 0 .01

?.025 0.05 1

0.02 0.04

~. 1

0.1

, 0.25, 0.625 ~pq/~g /min -,GAS TR!N 0.2

0.4

1.0

2.0

4.0

FIG. 3. Typical acid response (milliequivalents of HCl per 30 min) of an autogenous Heidenhain pouch and a homotransplanted fundic pouch in a single dog . The responses of both pouches were determined simultaneously.

cretion of transplanted pouches with large doses of gastrin was an unexpected finding. In the dogs with autogenous pouches, after peak secretory rates had been achieved, a 2to 4-fold increase in gastrin dosage was always sufficient to produce marked inhibition of gastric acid secretion. Inhibition did not occur in TP's with similar increases in gastrin dosage. This phenomenon is best illustrated in dogs with both an autogenous (HP-DAP) and a transplanted (TP) fundic pouch (fig. 3). The responses of both pouches to gastrin or histamine were observed simultaneously. In this dog, the peak secretory rate to gastrin was achieved at 0.01 p.g per kg per min for the host pouch and at 0.025 p.g per kg per min for the transplanted pouch. A 10-fold increase in dosage to 0.05 p.g per kg per min produced no significant change in the rate of secretion of the transplanted pouch. Both pouches achieved peak rates of secretion in response to histamine at the same dose level, 2.0 p.g per kg per min. Both autogenous and transplanted fundic pouches showed significant inhibition when the histamine dose was increased to 4.0 p.g per kg per min. The Gmax :Hmax ratio for the trans-

186

DAVIDSON ET AL. TABLE

Vol . 51, Xo. 2

·1. Acid secretion I

Groupa

Maximal histamine response

(mean±

SE)

Dose for

Dose for

mean histamine

threshold

mean peak histamine response

0.025 0. 025 0.025 0.025

2.0 1.0 2 .0 2.0

mEq/ 30 mi"

HP (6 dogs) .. .... HP -DAP (7 dogs) . . HP-AX (7 dogs) . .. TP (9 dogs). . . ....

2 .95 2 . 93 2.62 2 . 56

± ± ± ±

0.61 0.41 0.28 0.52

Dose for mean gastrin

threshold

Dose for mean peak gastrin response

Gmax:Hm ax b

(mean±

SE)

RelatiYe gastrin potencyc

(mean)

M f kg/mi"

0.001 0 .001 0.001 0.001

0.05 0.01 0 .025 0 .1

0.68 0.40 0.56 1.01

± ± ± ±

0 . 12d 0.08 0.08 0.12' 1

420 280• 840 700

a HP, Heidenhain pouch; HP-DAP, Heidenhain pouch with denervated antral pouch; HP-AX , Heidenhain pouch with antrectomy; TP, heterotopic homotransplanted fundic pouch with a homotransplanted antral pouch. b Gmax:Hmax, ratio of maximal secretory rate to gastrin to the maximal secretory rate to histamine. • An expression showing the number of histamine molecules required to give the same acid-stimulat ing effect as 1 molecule of gastrin. d This value is significantly different (P < 0.02) from the value for the HP-DAP 's but is not significantly different (P > 0.2) from the value for the HP-AX's. • This value is significantly different (P < 0.02) from the values for the HP-AX's and TP's but not from the value for the HP's (P > 0.1). I This value is significant (P < 0.001) compared to each of the three groups of autogenous pouches.

planted pouch was 1.31, and for the host pouch, 0.69. The acid secretory data following gastrin and histamine stimulation are summarized in table 1. Pepsin secretion. The response to graded doses of histamine and gastrin in each of the four groups of dogs is depicted in figure 4. Each point represents the mean secretory rate at t hat dose expressed as a percentage of the peak secretory response to histamine. Vertical bars represent the standard error of the mean. As in the acid-response curves, none of the histamine curves reach 100% because different dogs attained their peak rate of secretion at different dosage levels. No major differences were noted between the histamine-response curves. No measurable pepsin secretion was observed at a dosage level of 0.01 J.Lg per kg per min except in the TP's, which showed a significant secretion at this dose. The threshold dose for the other three groups was 0.02 J.Lg per kg per min. Peak response to histamine occurred at a dose of 0.2 J.L g per kg per min for the HPDAP's and at 0.4 J.L g per kg per min for the other groups. The peak pepsin response to histamine occurred at 75 the dose (i.e., two dose levels earlier) of the peak acid response in all four groups. After the peak rates of secretion were

achieved, further increases in histamine dosage were associated with substantial decreases in pepsin secretion. This " ·as observed in all groups except the HP's. The responses to gastrin infusion were more variable. All groups except the HPAX's were found to be secreting pepsin at the lowest dose level, i.e., 0.0005 J.Lg per kg per min. The threshold for the HP-A.-"'C's was 0.001 J.Lg per kg per min. Peak rates of pepsin secretion occurred at 0.025 J.L g per kg per min for the HP-DAP's and the TP's, at 0.1 J.L g per kg per min for the HP's, and at 0.25 J.L g per kg per min for the HP-AX's. In contrast to the histamine-response curves, gastrin-stimulated pepsin secretion remained at maximal or near-maximal rates until the termination of the infusion. This was true in spite of inhibition of acid and volume se· cretion at the highest dosage levels. The ratio of the maximal gastrin-stimulated pepsin secretion rate to the maximal histamine· stimulated rate (Gmax :Hmax) was 1.00 for the HP-DAP's, 1.04 for the HP-AX's, 1.17 for the TP's, and 1.70 for the HP's. The latter figure differs significantly (P < 0.05) from the values for the other three groups. The pepsin secretory data following gas· trin and histamine are summarized in ta· ble 2.

August 1966 230l

RESPON SES TO GASTRIN AND HISTAMIN E

HEIDENHAIN

POUCH

j

HEIDENHAIN POUCH DENERVATED AN TRAL POU CH 7 DOGS

200

6 DOGS I SO

2101

..

187

GASTRIN

"' ;:l

160

0.

w z

ro

w

i

"'

140

.

120

0

e

1-

:

--

HISTAMINE - - -

160

GASTRIN

-

~

H I STAMINE - - -

i

120

0

gz

.

~ 100

100

ffi

w

0.

80

eo

1-

~::>

e

~ 60

,

e

0

60

0

"' ~

40

.. z

m

40

0.

~H-]

w

20

20

0 p;/k;/l!lin-HlSTA M!NE

2 00

..

B~ SIE: - 0.01

?-025 ,o.os 0.02

0.04

Oil 0.1

~

,o.zs I 0.2

00005 0.001

0. 625 T f9'~g/min,-GAST RI/i

0.4

1.0

2.0

--

H IS TAM INE -

-

-

"' ;:l

i

0.1

0.25

0.1

o'z o~4

w

0.625 --,.ug /kg/min·GASTRIN

,Q z~o 4~o

180 GAS T RIN

0.

--

HISTAMINE - - -

i"'

160

..

140

1-

120

50.

100

~:r

140

0.025 0.05

' 0.04 ' 0.01 0.02

HOMOTRANSPLANTED FUNDIC POUCH 9 DOGS

200

180 GASTRIN

o.oozs o.oos oior

,uglkg /min -HISTAMINE BASE -

4.0

HE IDENHAIN POUCH, ANTRECTOMIZED 7 DOGS

w 160 w z

>

/-!"

0

0.0005 0.001 0.0025 0.005 o.pl

0

~

0

120

~

. e

z ~ 100 ~

, ,~

e

0

z

1-

80

ii.' 0

60

"'

;;; ~

•

eo

!; 60

iii

~

40

20

40

20

0

oooos o.oo1 o.oozs coos op1

JtQikg/min·H ISTAMINE BASE -

0.01

~.o2s

0.02

.•o.os 0;1

0.04

0.1

0.25

0~2 o:4

0.625-J.Iq / k\j/min-GASTAIN

tO z:o 4~0

{t1l~t r !A H

0•+--.---.-.~.--.--.-.---.--.-----. 0.0005 0.00 1 0.0025 O.Cl9S 0.~1

nl kq /min -H ISTAMINE BASE -

0.01

?.OZS 0.05 1

O.OZ

0.0<1

Oil 0.1

0.ZS

0 .6Z57pq/~q/min.-GAS1R !N

1

1

O.Z

0.4

1.0

Z.O

<1.0

FI G. 4. P epsin response to graded doses of gastrin a nd histamine. Each point is the mean secretory rate expressed as a percentage of the peak secretory response to hista mine in each dog. V e1·tical ba1·s at each p oint represent the standard error of the mean.

Discu ssion The acid and pepsin responses of canine fundic pouches to gastrin and histamine have been the subject of several reports.l 1 • 12. 1s No previous dose-response study of gastric secretion in dogs has utilized a highly purified form of the antral hormone.! As much as 2.0 mg of gastrin are required for a single

dose-response study. The use of this material permits reference to an actu al weight of hormone rather than to the wet weight of hog antral mucosa, allowing one to make some estimate of the comparative molar potency of gastrin and histamine. S ecretion of acid. The dose-response curves to histamine were virtually identical in all

188

Fol. 51, .Yo. 2

DAVIDSON ET AL. TABLE

2. Pepsin secretion I

:Maximal Group•

response

to histamine (mean± SE)

Maximal response to gastrin

Dose for

Dose for

Dose for

mean histamine

mean gastrin

(mean±

threshold

mean peak histamine response

SE)

;.g pepsin/30 min

HP (6 dogs) . ..... . 950 HP-DAP (7 dogs) .. 611 HP-AX (7 dogs) ... 645 TP (9 dogs) ... . ... 970 a b c

± ± ± ±

372 1849 ± 875 263 656 ± 141 239 536 ± 111 210 978 ± 235

threshold

I

;

Dose for mean peak gastrin response

;.g/ kg/min

0.02 0.02 0.02
0.4 0.2 0.4 0.4

<0.0005b <0 . 0005b

0.001 <0.0005b

a

i

Gmax:Hm3 X

(mean±

SE)

! i

0.1 0.025 0.25 0.025

fl. 70

± 0.28' ).00 ± 0.18 :1.0-! ± 0.2-! '1.17 ± 0.26

Abbreviations are defined in table 1. Pepsin secretion was noted at the initial dosage level. This value differs significantly (P < 0.05) from the values for the other three groups.

four groups, with the single exception that the response curve for the HP-DAP's was shifted slightly to the left, indicating an increase in sensitivity to histamine (fig. 2). In addition, the averages of the maximal histamine responses (milliequivalents of HCl per 30 min) in all groups were very nearly equal (table 1, column 1), indicating that antral denervation, antrectomy, or homotransplantation had no apparent effect on the mean secretory response to histamine. It might be expected on the basis of previous work by Passaro et al./ 9 showing potentiation between gastrin and histamine, that the presence of the antrum would enhance the response of the parietal cell to exogenous histamine. However, antrectomized dogs in our study had a mean peak secretory rate of 2.53 mEq per 30 min, a value not significantly different from that of the HP's, i.e., 2.93 mEq per 30 min. Furthermore, the histamine dose-response curves for the two groups are nearly identical, indicating that there is also no difference in threshold dose or in responsiveness to submaximal doses of histamine. Comparisons between groups of dogs with pouches of slightly different sizes are not as reliable as serial observations made on the same animal following antral denervation or antrectomy. Our studies suggest that neither antral denervation, antrectomy, nor transplantation altered the HP maximal response to histamine. However, in sequential studies performed on the same 5 dogs before and after antrectomy, Andersson and Grossman12 reported that antrectomy caused a significant diminution in maximal acid response to histamine in 3 dogs, no essential

change in 1 dog, and a significant increase in 1 dog. The apparent increase in sensiti,·ity to histamine in dogs with a denervated antral pouch has not been described previously, and we can offer no explanation for this phenon1enon. In about 50% of all dogs studied, supra· maximal doses of histamine produced significant inhibition of acid secretion. This phenomenon occurred in each of the four groups and was observed in the absence of any obvious signs of histamine toxicity. Gillespie 20 has shown that large doses of histamine dihydrochloride (2 to 6 mg) given as a single intravenous injection will also inhibit previously established gastric secretion in the dog. However, in his study pro. found hypotension was regularly observed and may have been the cause of the inhibition of acid secretion. Inhibition by histamine was not observed in the dose-response study of Andersson and GrossmanY However, the final dose of histamine used was smaller than that in our study. Slight inhibition was observed in the 3 dogs studied by Heathcote et al./8 utilizing doses of histamine comparable to those in our study. It is impossible to stat·e unequivocally that the inhibition observed in response to supramaximal doses of histamine is a result of a true pharmacological action of histamine, since some subtle toxic manifestation of histamine overdosage may have gone unnoticed. When present, the degree of inhibition observed in response to histamine was similar in magnitude to the inhibition seen with supramaximal doses of gastrin. As ·with histamine, we have no data indicating

August 1966

RESPONSES TO GASTRIN AND HIST Alv!INE

11·hether this inhibitory effect of large amounts of gastrin is due to a physiological response or to a specific toxic action which affects the secretory mechanism. The responses of the four groups of dogs to gastrin were considerably more variable than their responses to histamine. The peak gastrin response relative to the corresponding histamine response was depressed by antral denervation and was augmented by homotransplantation (fig. 2) . The ascending limbs of the gastrin-response curves of the HP-AX's and the HP-DAP's rise more rapidly than the corresponding portion of the curve of the HP's (fig. 2). Also, the peak responses in the HP-AX's and the HPDAP's occu r at lower doses than does the peak response of the HP's. These are the only data which suggest that antrectomy or antral denervation increases the sensitivity of the parietal cell to gastrin stimulation. The threshold doses in all groups were identical The depression of the peak response to gastrin with antral denervation or antrectomy could be the result of a decrease in responsiveness to the stimulatory action of gastrin or an increase in responsiveness to the inhibitory action of gastrin. It is impossible to make this distinction with the existing data. A decreased responsiveness of a Heidenhain pouch to food stimulus after antral denervation has been demonstrated by Johnson and Koos. 21 However, this decrease in responsiveness was attributed to a decreased vagal release of endogenous gastrin from the antrum. This observation would not explain our results, since exogenous gastrin was used. The responses of the homotransplanted pouches to gastrin exhibited three major differences from the responses of the autogenous pouches. The peak response occurred later, i.e., at higher doses of gastrin; the magnitude of the peak response was nearly identical to the peak histamine response; and there was no inhibition by supramaximal doses of gastrin. None of the changes can be attributed to immunosuppressive therapy, since these phenomena were not observed in autogenous pouches in the same dogs. We have previously shown13 that chronic azathioprine administration does not

189

significantly alter pouch reactivity to stand~ ard stinmli. Therefore, the process of transplantation or the immune response of the host must account for the altered response of the transplanted pouch. The transplanted pouches showed variability of secretory re-. sponse, depending upon the threat of immune rejection. These studies were performed only on dogs showing brisk secretory responses and no histological or gross signs of rejection. Inhibition of acid secretion in autogenous pouches was invariably produced by 2- to 5-fold increase of gastrin dosage beyond the peak response. Transplanted pouches, however, were not inhibited by even greater increases (10 to 25 times) beyond the peak response dose of gastrin. The inability to inhibit the transplanted pouches may also explain the equality of the gastrin and histamine peak responses in this group of dogs. However, an equality of gastrin and histamine peak responses has also been observed in dogs with innervated autogenous pouches, which do show inhibition of secretion with high doses of gastrin. 6 In addition, the inability to inhibit transplanted pouches is not absolute, since gastrin given intravenously as a single dose will inhibit previously established secretion in these pouches. 13 Calculation of molar sensitivity (table 1) indicates that gastrin is, on the average, 570 (average of all 29 dogs) tin1es more potent than histamine as a stimulant of acid secretion. The low value of 280 for the HP-DAP's reflects an increase in sensitivity to histamine rather than a decreased sensitivity to gastrin. Pepsin secretion. Changes in pepsin secretion were much more difficult to evaluate because of the inherent variability of secretion both in a single dog and· among groups of dogs. This study was designed primarily to evaluate the dose-response of acid secretion, which could be followed during the course of the infusion. The pepsin responses, however, were not available for interpretation until after the study has ended. They were quite variable, and often a plateau of pepsin secretion was not obtained. Patterns of secretion within groups of dogs varied greatly. For example, some dogs were noted

190

D A_ Y IDSON ET A L.

to h ave significant secretion of pepsin even wit h the smallest doses of gastrin or histamine. In a few animals, this "basal" secretion of pepsin was the highest observed during the course of the infusion. As a result of t his v ariability, t he stat istical st andard d eviations of all responses at all dose levels were large. The pepsin responses t o histamine in t he four groups were qualitatively similar. The p eak response occurred in all four groups at Ys the dose (i.e., hYo doses earlier) of the corresponding peak acid response. In addition, the response in all grOUpS \YaS bi phasic; that is, with smaller amounts of hist amine, increases in dosage caused increased pepsin secretion, while at higher doses, the tot al pepsin output decreased as acid and volume output increased. This biphasic response to histamine has been previously observed in HP dogs. 6 • 11 P epsin responses to gastrin infusion in t he HP-DAP's, HP-AX's, and TP's were qualit atively similar. In these t hree groups hiphasic r esponses t o gastrin occurred which were similar to those observed with histamine. However, in t he HP's t he pepsin output continued t o rise with increasing doses of gastrin, and the greatest secretion was reached at the t ime t he infusion was terminated, i.e. , after the acid and volume output had been inhibited. This phenomenon has not been observed in dogs wit h innervated fundic pouches, but has been described by Andersson and Grossman11 in HP dogs with intact antra. These authors also state that the marked pepsigogic action of gastrin is abolished by ant rectomy. However, in a subsequent study12 t he same workers conclude that antrectomy has no effect on gastrin-stimulated pepsin secretion by denervated fundic pouches. I nspection of t heir data (fig. 5 in reference 12) reveals that at the time their infusion study was terminated the pepsin response of t he HP dogs was still rising while the response of the antrectomized HP dogs had r eached a plateau. Continuation of the infusion of gastrin at hi gher doses might have revealed significant differences between the antrectomized and nonantrectomized HP dogs. In this study, ant rectomy, antral denerva-

V ol . 51, 1Y o. 9

tion, and homotransplantation abolished the marked pepsigogic effect of gastrin obsernd in the HP's. Gregory and Tracy 1 and Konturek and Grossman22 have shmn1 that t his pepsi gogic eff ect in dogs \Yith vagally denervat ed fu ndic pouches is a property of gastrin itself and cannot be attribut ed to contaminating substances. Our data ;mgge~t that an intact antrum is required for t his effect. This is further support ed by the fact t hat in t he --! dogs with an autologous fu ndic and denervat ed ant ral pouch and a homotransplanted antral and fundi c pouch , t he mean pepsin Gmax :Hmax ratio was lmH r (0.99) than in .5 dogs with intact stom ac-hs and a homot ran splanted antral and fund ic pouch CGmax :Hmax = 1.39). This difference is not statistically significant, perhaps, because of t he small number of dogs in each group. Since t he marked pepsigogic action of gast rin has been observed only in dogs \Yith denervated pouches and at high dose levels of gastrin, t he physiological role, if any, of gastrin in stimulating pepsin secretion in t he intact stom ach is unknown. Summary

The dose-response relations of denervat ed autogenous and homotransplanted fundic pouches to gastrin and histamine " ·ere studied in 29 dogs: 6 wit h a H eidenhain pouch, 7 wit h a H eidenhain pouch and a denervated ant ral pouch, 7 with a Heidenhain pouch and antrectomy, and 9 \Yith heterotopic homotransplanted fundic and ant ral pouches. Histamine-stimulat ed acid response curves in all four groups were virtually identical except for an increased sensitivity t o submaximal histamine stimulus of the H eidenhain pouch wit h denervated antral pouch. The maximal acid r esponse t o gastrin stimulation was depressed by antral denervat ion and was apparently enhanced by homot ransplantation. Inhibition of acid secretion was noted wit h supramaximal doses of bot h gastrin and hist amine with the single exception t hat homotransplanted pouches w ere not inhibited by supramaximal doses of gastrin. The r atio of the maximal gastrinstimulated acid response t o t he m aximal

RESPONSES TO GASTRIN AND HISTAMINE

191

5. Code, C. F. 1956. Histamine and gastric secrehistamine-stimulated acid response (Gmax: tion, p. 189. In G. E. W. Wolstenholme and H max) was 0.40 for dogs with a Heidenhain C. M. O'Connor [ed .], Histamine (Ciba pouch and denervated antral pouch, 0.56 Foundation Symposium). Little, Brown and for dogs with a Heidenhain pouch and anCompany, Boston . trectomy, 0.68 for dogs with a Heidenhain 6. Passaro, E. P ., Jr., and lVI. I. Grossman. 1964. pouch, and 1.01 for dogs with a transplanted Effect of vagal innervation on aci d and pouch. On a molar basis, gastrin was, on the pepsin response to histamine and gastrin. average, 570 times as potent as histamine Amer. J . Physiol. 206: 1068-1076 . as a stimulant of acid secretion. 7. Woodward, E. R., R. R. Bigelow, and L. R. The histamine-stimulated pepsin-response Dragstedt. 1950. Effect of resection of the antrum of the stomach on gastric secretion curves were similar in all four groups of in Pavlov dog pouches. Amer. J. Physiol. dogs. The response to histamine stimulation 162 : 99-109. \\"aS biphasic; i.e., with small doses of his8. Langlois, K. J., and M. I. Grossman. 1950. tamine, increases in dosage caused increased Effect of surgical extirpation of the pyloric pepsin secretion, while at higher doses, the portion of the stomach on response of fundic total pepsin output decreased as acid and glands to histamine and urecholine in dogs. volume output continued to increase. With Amer. J. Physiol. 163: 38-4.0. gastrin stimulation, responses similar to 9. Andersson, S., C. E . Elwin, and B. Uvnas.1958. those observed with histamine were noted The effect of exclusion of the a ntrum and in all groups except in dogs with a Heidenduodenum, and subsequent resection of the antrum, on the acid secretion in Pavlov hain pouch and intact antrum. In these pouch dogs . Gastroenterology 34: 636-657. animals, pepsin output continued to increase as gastrin dosage increased. Therefore, 10. Johnson, A. N ., Jr. 1964. Acid inhibition by the gastric antrum; experiments on antral and antrectomy and antral denervation abolished fundic pouches. Ann. Surg. 159: 252- 259. the marked pepsigogic response of dener- 11. Andersson, S., and M. I. Grossman. 1965. vated fundic pouches to gastrin stimulation. Effect of vagal denervation of pouches on The ratio of the maximal gastrin-stimulated gastric secretion in dogs with intact or pepsin response to the maximal histamineresected antrum . Gastroenterology 48: 449stimulated pepsin response (Gmax : H max) was 462. 1.00 for dogs with a Heidenhain pouch and 12. Andersson, S., and M. I. Grossman. 1965. Effect of antrectomy on gastric secretion of a denervated antral pouch, 1.04 for dogs acid and pepsin in response to histamine and with a Heidenhain pouch and antrectomy, gastrin in dogs. Gastroenterology 49: 2461.17 for dogs with a transplanted pouch, 255. and 1.70 for dogs with a Heidenhain pouch. REFERENCES 1. Gregory, R. A., and H . J. Tracy. 1964. The constitution and properties of two gastrins extracted from hog a ntral mucosa. Gut 5: 103- 117 . 2. Gregory, H., P . M . Hardy, D. S. Jones, G. W. Kenner, and R. C. Sheppard. 1964. Struc ture of gastrin. Nature (London) 204: 931933. 3. Anderson, J. C., M.A. Barton, R. A. Gregory, P.M. Hardy, G. W. Kenner, J . K . MacLeod, J. Preston , R. C . Sheppard, and J . S. Morley. 1964 . Synthesis of gastrin. Nature (London) 204: 933-934 . 4. Makhlouf, G. M ., J.P. A. McManus, and W. I. Card. 1964. The action of gastrin II on gastric-acid secretion in man. Lancet 2: 485-4.89.

13. Thompson, J. C., I. A. Daves, W. D. Davidson, and J . H. Miller. 1965. Studies on the humoral control of gastric secretion in dogs with autogenous and homotransplanted antral and fundic pouches . Surgery 58: 84109. 14. Olbe, L . 1963. Significance of vagal release of gastrin during the nervous phase of gastric secretion in dogs . Gastroenterology 44: 463468. 15. Grossman, M. I., and I. N. Marks. 1960. Secretion of pepsinogen by the pyloric glands of the dog, with some observations on the histology of the gastric mucosa. Gastroenterology 38: 343-352. 16. Snedecor, G. W . 1956. Statistical methods, Ed. 5, p . 91. Iowa State College Press, Ames. 17. Emmens, C. W., and P . Hartley. 1948. Principles of biological assay, p. 88. Chapman and Hall, Ltd., London.

192

DAVIDSON ET AL.

18. Heathcote, B. V., D. W. Daly, and I. E. Gillespie . 1965. Secretory responses before and after vagal denervation of a gastric pouch. Gastroenterology 48: 463-4 71. 19. Passaro , E. P., Jr., I. E. Gillespie, and i\1. I. Grossman. 1963. Potentiation between gastrin and histamine in stimulation of gastric secretion. Proc . Soc. Exp. Bioi. :Med. 114: 5G-52. 20. Gillespie, I. E . Inhibition of acid secretion by gastrin extracts. In M. I. Grossman [ed.],

Vol.51,Xo.2

UCLA forum in medical sciences, Vol. 5. University of California Press, Los Angel es . In press . 21. Johnson, A. N., and F . A. Koos. 196-±. Signifi. cance of vagal influences on release of antral gastrin. Amer. J. Surg . 108: 31-35. 22. Konturek, S., and M. I. Grossman. 1965. Effect of large doses of gastrin I on pepsin secretion. Proc. Soc. Exp . Bioi. :i\Ied. 119: 443-444.