Inhibitory Effects of a Purified Enterogasterone, Secretin, and Cholecystokinin on Histamine-Stimulated Gastric Acid Secretion

Vol. fl9, No. 5

GASTitOENTJmOLOGY

Copyright © 1970 by 'The Williams & Wilkins Co.

Printed in ll. .S. A.

INHIBITORY EFFECTS OF A PURIFIED ENTEROGASTERONE, SECRETIN, AND CHOLECYSTOKININ ON HISTAMINE-STIMULATED GASTRIC ACID SECRETION HAROLD

w.

LUCIEN, PH.D., ZEN ITOH, M.D., PH.D., AND

A.

v. SCHALLY,

PH.D.

Gastrointestinal Hormone Laboratory, Endocrine and Polypeptide Laboratories, Veterans Administration Hospital and Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana

Inhibitory effects of a purified enterogastrone, highly purified secretin, cholecystokinin (Karolinska Institutet, Stockholm, Sweden), and Cecekin on gastric secretion stimulated with one-half the dose of histamine required for maximal stimulation were studied in dogs with Heidenhain's pouches. The enterogastrone preparation (0.5- and 2.0-mg doses) inhibited gastric acid secretion, but secretin (75 clinical units) and cholecystokinin (Karolinska) (75 Ivy dog units) did not. The 0.5-mg dose of the inhibitor decreased gastric acid secretion 18'/;; for 15 min. The 2.0-mg dose produced about 30'/i, decrease of gastric acid secretion for 30 min. Cecekin, a crude preparation of cholecystokinin, inhibited histamine-stimulated gastric secretion at a dose of 20 Ivy dog units. This paper describes the inhibitory effects of a purified substance with enterogastrone-like activity on histamine-stimulated gastric secretion and compares its action with that of secretin and cholecystokinin (CCK).

Materials and Methods Preparation of enterogastrone. The details of the concentration and properties of the en1 terogastrone were reported previously; however, a summary of the procedure is given beReceived February 17, 1969. Accepted April 24, 1970.

Address requests for reprints to: Dr. Harold W. Lucien, G. I. Hormone Research Laboratory, Veterans Administration Hospital, 1601 Perdido Street, New Orleans, Louisiana 70140. This study was supported by Veterans Administration Research Service and in part by United States Public Health Service Grant AM 07467. Dr. Itoh is a Postdoctoral Research Scholar Abroad of Education Ministry, Japan. His permanent address is: Department of Surgery (Director: Professor K. Ishihara), Gunma, University School of Medicine, Maebashi, Japan. The authors wish to acknowledge the technical assistance of Mr. Nathaniel Carlton.

low. The first 90 em of gut were removed from freshly slaughtered hogs, everted, washed in ice water, heated to 100 C to inactivate enzymes, and then extracted with 5~;, acetic acid at room temperature. The enterogastrone activity was adsorbed on alginic acid and then eluted with 0.2 M HCI. A precipitate which contained the enterogastrone activity was obtained by adjusting the pH to 2.5 and adding 30 g of sodium chloride per 100 ml of solution.' Enterogastrone activity in the salt cake was further concentrated by precipitation with !-butanol from a 60% aqueous ethanol solution at -25 C. The precipitate was dissolved in 0.2 M acetic acid and lyophilized. Final concentration of the activity was effected by gel filtration of Sephadex G-50 and ion-exchange chromatography on triethylaminoethyl cellulose. An inhibitor concentrate resulting from the second fractionation on triethylaminoethyl cellulose was used in the study. This produce inhibited gastric acid secretion 37% of the preinjection secretory rate at the 0.5-mg dose level. The enterogastrone preparation used in this study was assayed by methods described below.

Assay methods Enterogastrone. Enterogastrone activity was 707

708

LUCIEN ET AL.

assayed in 5 adult male mongrel dogs weighing 10 to 15 kg each. Heidenhain's pouches were prepared at least 1 month prior to assay. The dogs were kept in individual cages and fed once each day at a fixed time with a standard dog food containing 1 teaspoon of table salt. Before each experiment, the dogs were fasted overnight but were allowed water ad libitum. Gastric secretion was stimulated with a continuous intravenous infusion of one-half of the minimal dose of histamine that produced the maximal gastric secretion. Histamine was infused at a rate of 20 ml per hour with an infusion pump (Harvard Apparatus Company, Dover, Mass.). The dose of free histamine base required to produce the desired stimulation varied from 40 to 60 11g per kg of dog weight per hour. The volume of 15-min gastric juice samples was measured, and the acidity was titrated with 0.2 N sodium hydroxide with phenol red as the indicator and expressed as microequivalents per 15 min. After the gastric secretion reached a plateau and remained at a constant secretory level for 1 hr, a single rapid intravenous injection of the test material dissolved in 1 ml of saline was made between the seventh and eighth 15-min collection period. Inhibitory activity was expressed as percentage of the preinjection output of gastric secretion. The preinjection value was determined by taking the mean of four 15min preinjection outputs immediately before the injection of the test material. Statistical significance of the data was evaluated by Student's t- test. Secretin and cholecystokinin. The enterogastrone preparations were assayed for secretin, CCK, and vasodepressor activities. Secretin was assayed by the method of Jorpes and Mutt! CCK was assayed by the method of Ivy and Jamecek.' CCK (batch no. 26731) and highly purified secretin (batch no. 16741) were obtained from the Chemistry Department, Karolinska Institute, Stockholm, Sweden. According to a personal communication from Dr. V. Mutt, the secretin was pure and the CCK (Karolinska) contained 250 Ivy dog units of CCK and 18 clinical units of secretin per mg. Cecekin (Vitrum, Stockholm, Sweden), which contains CCK, was also used. The samples of enterogastrone did not show any detectable secretin or CCK activity when assayed in doses as high as 10 mg. Vasodepressor and pyrogen. The blood pressure of Nembutal-anesthetized dogs was recorded on a physiograph (E and M Company, Houston, Texas) after cannulating the femoral artery. Vasodepressor activity was expressed as

Vol. 59, No. 5

the percentage of the preinjection level. Rectal temperatures were taken before and after injection of an enterogastrone sample into conscious dogs. In addition to vasodepressor and pyrogen activities, careful observation was also made of other possible side effects, such as vomiting, salivation, and lip licking. The purified enterogastrone at dose levels as high as 4.0 mg induced none of these effects. These effects were not investigated with doses of the inhibitor in excess of 4.0 mg in conscious dogs. Vasodepressor and pyrogen activities were not observed in Nembutal-anesthetized cats with doses of up to 10.0 mg.

Results Gastric secretory response to histamine and the saline control. Table 1 shows that gastric acid secretory responses remained at a constant level of secretion for 2 hr after a single injection of 1.0 ml of saline. All dogs responded satisfactorily to a fixed dose of histamine, and the day-to-day flue- . tuation was very small. Effect of purified enterogastrone. Figure 1 shows the results of a single intravenous injection of purified enterogastrone in six experiment on 3 dogs. When 0.5 mg of enterogastrone was injected, gastric acid output was inhibited by 18.0 ± 2.5 % in the first 15-min collection and returned to the preinjection level during the second 15-min collection. When 2.0 mg of the enterogastrone were injected, 35.0 ± 3.3% inhibition of acid secretion occurred during the first 15-min collection, and 30.0 ± 2.8% inhibition was observed during the second 15-min collection, both being statistically significant.

Effects of CCK (Karolinska) and highly purified secretin. Figure 2 shows the effect of 75 Ivy dog units of CCK (Karolinska) on gastric acid secretion. This dose did not inhibit but in some instances actually increased the acid output to 110% (NS) of the preinjection level within 30 min after administration. Figure 3 shows that administration of 75 clinical units of highly purified secretin also resulted in slight stimulation (NS) instead of inhibition of gastric acid secretion. It was concluded that, in denervated pouch dogs, either 75 units of

November 1970

INHIBITORY EFFECTS OF ENTEROGASTRONE

709

TABLE 1. Effect of a single intravenous injection of 1.0 ml of normal saline on ga~tric ~ec retion stimulated with histamine Dog

no.

A A

B B

c c D D E E

Hnlf of

MEH

Prcinjcction sccretionu

mg/hr

"Eq/15 min

0.9 0.9 0.6 0.6 0.6 0.6 0.9 0.9 0.6 0.6

1720 1820 742 820 840 772 1952 2156 616 600

Acid outpt.tt/1!) min nft.cr injection ( 1;r, of the control)h

Mean ±SE

I

2

3

4

r,

r.

7

R

99 115 106 99 106 102 93 95 102 95

109 110 102 90 101 106 117 69 101 104

90 109 100 93 102 150 93 66 109 98

120 125 105 93 87 110 97 67 90 110

112 99 103 99 125 148 97 105 103 104

95 106 101 107 108 112 97 100 114 113

118 96 102 92 115 114 97 101

118 98 102 103 92

103 115

86 95 84 125

101 ±2.1

101 ±4.2

101 ±5.4

100 ±5.0

109 ±5.0

105 ±2.1

105 ±:l.O

100 ±4.1

99

" Control is the mean of four 15-min collections immediately before the injection. ' Consecutive 15-min collection periods after injection of test material. !Histamine di HCI,

Histamine di HCI, I.V. Infusion

I.V.l

jHistominr di HCI, I .V. Inf.l

120

A

t

110

'I t

CCK

+

100

90 80

70 60 0.5 1.0

0.5 1.0 1.5 2 .0

1.5 2.0 Hours

FIG. 1. The effect of a single intravenous injection of purified enterogastrone on histamine-stimulated gastric acid secretion. The effects of 0.5 and 2.0 mg of the inhibitor are shown at the arrow in A and B, respectively. Outputs were expressed as the mean percentage of the preinjection secretory level. The preinjection secretory level is the mean of four 15-min outputs immediately preceding the injection of the test material. The mean preinjection level of dogs A, B, and E was 995 microequivalents per 15 min. Asterisks, so from the preinjection secretory level ; •, P < 0.01, ••, P < 0.005 for six experiments on 3 dogs. Vertical bars, SE.

secretin or 75 units of CCK (Karolinska) have no inhibitory effect on gastric secretion induced by infusing one-half the dose of histamine required for maximal stimulation.

0.5

1.0

1.5

2.0

2.5

Hours

FIG. 2. The effect of a single intravenous injection of cholecystokinin (CCK (Karolinska)) (75 Ivy dog units) on histamine-stimulated gastric acid secretion . Results were obtained from six experiments on 3 dogs. Preinjection secretory level of dogs C, D, and E was 1295 microequivalents per 15 min. Vertical bars, SE.

Effect of Cecekin_ Figure 4 shows that 20 Ivy dog units of Cecekin significantly inhibited gastric acid secretion. Inhibition occurred to the extent of 20% during the first 15 min, after which gastric secretion returned to the preinjection secretory level. The inhibitory activity of Cecekin

LUCIEN ET AL.

710 Histamine di HCI, l.V. Infu•ion

120

-.... ~

~

110

~

.::

, ..,

.....

~

~:l: "" 0:::

~-~

.., ..,

100 90

~.'1>

·- sol ~ I

~


"-

""

70

~

05

1.0

1.5

20

2.5

Hours

3. The effect of a single intravenous injection of highly purified secretin (75 clinical units) on histamine-stimulated gastric acid secretion. Results were obtained from six experiments on 3 dogs. Preinjection secretory level of dogs B, C, and D was 1202 microequivalents per 15 min. FIG.

Histamine di HCI, I.V. Infusion CecttA:in

t

0.5

1.0 1.5 2.0 Hours

2.5

FIG. 4. The effect of a single intravenous injection of Cecekin (20 Ivy dog units) on histamine-stimulated gastric acid secretion. Results were obtained from six experiments on 3 dogs. The preinjection secretory level of dogs A, B, and C was ll20 microequivalents per 15 min. Asterisk, sn from the preinjection secretory level, *, P < 0.01. Vertical bars, SE.

was not attributable to CCK, since CCK (Karolinska) did not show any inhibitory effects on gastric secretion under the same experimental conditions.

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Discussion When discussing the inhibitory action of humoral substances, it is essential to indicate the kind of gastric stimulant used and the extent of gastric secretion elicited, as was reported in elaborate studies on histamine-stimulated gastric secretion by Code et al. 5 In the present experiments, gastric acid secretion was induced with onehalf of the maximal dose of histamine in each dog, and it was clearly demonstrated that of the agents tested only two exerted an inhibitory effect on gastric acid secretion. These were Cecekin and the substance isolated by us. In this experimental system, secretin and CCK (Karolinska) did not inhibit gastric secretion. Cecekin was investigated because it is a crude CCK preparation and has been reported to inhibit gastric acid secretion. Figure 4 shows that a dose of Cecekin equivalent to 20 Ivy dog units of CCK was an effective inhibitor of histamine-stimulated secretion. However, since CCK (Karolinska) did not induce any inhibition, the action of Cecekin could possibly be attributed to a contamination with an enterogastrone-like substance. This suggestion is based on the fact that recent attempts to isolate active fractions of an enterogastrone have been made on crude materials containing CCK. 1' 6 Highly purified secretin and CCK (Karolinska) actually appear to increase gastric acid secretion slightly, as shown in figures 2 and 3, but the effects were not significant. Magee and Nakamura7 reported that CCK increased gastric secretion when it was infused at the relatively low dose rate of 0.5 to 4.0 units per min. Gillespie and Grossman 8 found that secretin did not inhibit histaminestimulated gastric acid secretion and that CCK inhibited only the stimulation induced by small doses of histamine. Stening et al. 9 observed that, whereas both secretin and CCK inhibited gastrin-stimulated secretion, only CCK inhibited histamine-induced secretion, and this effect was related to the infusion dose of histamine. Thus, the degree of purification of the inhibitor of gastric secretion and the differences in doses of histamine might account

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INHIBITORY EFFECTS OF ENTEROGASTRONE

for the differences between the present results and those previously reported. The present results do not contradict the likelihood that secretin and CCK participate in the gastric inhibitory mechanism under physiological conditions. However, our experiments demonstrate the existence of a substance of duodenal origin which inhibits gastric acid secretion stimulated via infusion of intermediate doses of histamine. Highly purified secretin and CCK (Karolinska) did not inhibit acid secretion under the same experimental conditions. In view of the observation that fat in the duodenum inhibits gastric acid secretion stimulated by histamine 10 and that the enterogastrone-like substance is of duodenal origin and free of secretin and CCK contamination, this substance might be the humoral agent responsible for the inhibition of histamine-stimulated gastric secretion by fat. REFERENCES 1. Lucien HW, Sun DCH, Itoh Z, et al: The purification of enterogastrone from porcine gut. Arch Biochem 134:180-184, 1969 2. Jorpes JE, Mutt V: United States Patent 3,013,944. 1961

711

3. Jorpes JE, Mutt V: On the biological assay of secretin. The reference standard. Acta Physiol Scand 66:316-325, 1966 4. Ivy AC, Janecek HM: Assay of Jorpes-Mutt secretin and cholecystokinin. Acta Physiol Scand 45:220-230, 1959 5. Code CF, Blackburn CM, Livermore GR, et al: A method for the quantitative determination of gastric secretory inhibition. Gastroenterology 13:573-588, 1949 6. Brown JC, Pederson RA, Jorpes E, et al: Preparation of highly active enterogastrone. Canad J Physio1Pharmacol47:113-114, 1969 7. Magee DF, Nakamura M: Action ofpancreozymin preparation on gastric secretion. Nature (London) 212:1487-1488, 1966 8. Gillespie IE, Grossman MI: Inhibitory effect of secretin and cholecystokinin on Heidenhain pouch responses to gastrin extract and histamin e. Gut 5:342-345, 1964 9. Stening GF, Johnson LR, Grossman MI: Effect of cholecystokinin and caerulein on gastrin- and histamine-evoked gastric secretion. Gastroenterology 57:44-50, 1969 10. Johnson LR, Grossman MI: Effects of fat, secretin and cholecystokinin on histamine-stimulated gastric secretion. Amer J Physiol 216:1176-1179, 1969