The effect of gastric secretagogues on gastric mucosal histamine and histidine decarboxylase activity in rats

Life Sciences Vol. 8, Part 1, pp . 783-789, 1989 . Printed in Great Britain

Pergamon Press

THE EFFECT OF GASTRIC SECRETAGOGUES OA GASTRIC MQCOSAL HISTAMINE AND HISTAMINE DECARBOXYLASE ACTIVITY IN RATS K,-Fr, Sewing

74 Tübingen, Germar{y . (Received . l8 December 1988 ; in final form 29 April 1969)

Department of Pharmacology, University of Tübingen,

Introduction Pretreatment of rats with different gastric secretagogues has bees reported to result in a decrease of gastric histamine content (1-3), Furthermore it has been shown that a feedback mechanism exists between the gastric histamine content and histidine decarbozylase activity (4) and that pretreatment of rata with gastrin stimulates the histidine decarboxylase in vitro (5), The present paper describes the effect of various gastric aecretagoguea on gastric histamine content and histidine decarboxylase of rats in vitro . Preliminary reports have been made (6,7) . Methods 1, Gastric histamine content, The experiments were performed on female rats (FW

49

Biberach) starved for

16 hours, The animals were killed by ether asphyxia, The stomachs were removed at

4°

C and opened along the greater curvature, The mucosa was scraped off from

the muscular layer, The mucosas of two stomachs weighing together about

550

m8

were made up to a final volume of 10 ml with Krebs' solution containing aminoguanidine sulphate

(10 3 N),

In all experiments with the gastric

secretagogues a small volume of Krebs' be studied . For each ezperiment usually one serving as control and 2 incubation period of

30

3

solution was replaced by the compound to

3-4

incubation vessels were prepared,

containing the different compounds, After an

respectively 120 min at

783

37 °

C in a shaking water bath

784

GASTRIC SECRETAGOGUES

Vol. 8, No . 18

the incubation mixture wen oeatrifuged for 10 min at 2000 rpm, the supernatant removed and the histamine content in the sediment was measured fluorometric7y (8) . The results are expressed in nMolea histamine per g mucosa . 2, Gastric hiatidine decarbo~rlase, For each ezperimeat 10 rats xere killed by ether asphyxia and the stomachs were removed sad opened, The mucosas were scraped off and homogenized in 3 volumes of distilled crater. After centrifugation for 30 min at 1800 x g 0,8 ml of the supernatant vsa added to the mixtures of table 2. TABLE I, Without exogenous Irhistidine control

With exogenous L-hiatidine

+substance

control

+substance

A

B

C

D

supernatant

o.e

o.s

o,e

o,e

Phosphate buffer

1 .4

1,4

1,4

1,4

Aminoguanidine sulphate Aicotinemide

0,1

0,1

0,1

0,1

0,1

0,1

0,1

0,1

-

-

0,5

0,5

0,5

0,5

-

-

-

0,1

-

0,1

0,1

-

C,1

-

L-H3stidine Distilled water Compound Solvent for oompound

Incubation mixtures (in ml) for the estimation of histamine decarbozylase activity, The final concentrations of the different components used were as follows : phosphate buffer (pH 7,0) aminoguanidine sulphate nicotinamide

2 x 101 M 2,5 z 104 M 1 x 1Ô

2 2

M

Ithistidine

1 x 1Ô

carbacholchloride

1 z 10~ M

M

Vol. 8, No. 13

GASTRIC SECRETAGOGUES

reaerpine

785

2 .5 z 10~ M

3

Trp .Met .Aep .Phe-NH2 (gsatrintetrapeptide) insulin

z 10~ M

1 unit/ml

compound 48/80

20 Pg/ml

The miztures were incubated for 1 hour at

37°

C in a Warburg apparatus under

nitrogen atmosphere, The reaction was stopped with perchloric acid . Subsequently the histamine content was estimated fluorometricly (8) . The figures in column A and B (Table III) represent the amounts of histamine present in the tissue sad/or formed by decarboxylation of endogenous hiatidine ezpressed in nMoles histamine per g mucosa, the differences C - A and D - B show the amounts of histamine newly formed from 150 pMoles Irhietidine by 1 g mucosa within 1 hour ezpressed in nMolea . For statistical aaalysis all results were treated with the t-teat for pairs, Compounds: aminoguanidine sulphate (Fluka, Bucha), nicotinamide (Schuchardt, Munich), L-histamine (Schuchardt, Munich), carbacholchloride (Merck, Darmstadt), reaerpine (Ciba, Hods), gastrin-tetrapeptide (Thomas, Hiberach), compou~ 48/80 (Thomas, Biberach), insulin (Hoechst, Frankfurt), Results The results of the type 1 experiments are summarized in table II, The histamine content of the gastric mucosa was significantly higher than in the controls after incubation for

30

min with compound 48/80 (20 ~g/ml), reserpine

(all doses) and carbachol (20 pg/ml) . The gastrin-tetrapeptide and insulin were ineffective . An incubation for 120 min caused an increased histamine content of the gastric mucosa with reserpine (10 pg'/ml) and oa.rbachol (10 ~g/ml), but a decreased histamine level with t}ie gastrin-tetrapeptide (20 ~g/ml) and insulin (1 unit/ml) by comparison with the controls . The hietidine decarbozylase activity (type 2 experimenta) in the presence of ',:he same gastric secretagogues as used above is s++~rized in table III,

786

Vol. 8, No . 13

GASTRIC SECRETAGOGUES TABLE II .

Histamine (nMoles/g tisaue + sx) 120 mis incubation

30 min incubation Substance compound 48/80

Reserpine

Carbachol

Gastrantetrapeptide Insulin

1

dose

(~a/~)

control

+substance

control

+ substance

10

139 _+ 40

164 _+ 42

66 ± 10

69 + 13

20

107 + 20

174 + 26 1

87 + 13

87 +

10

95 + 20

113 + 21 1

78 + 16

98 + 151

20

95 + 20

117 + 18 1

71 + 10

47 +

40

95 + 20

136 + 18 1

85 + 12

53 + 121

10

107 + 20

123 + 21

65 + 14

87 + 19

20

107 + 20

173 + 31 1

66 + 1Ô

87 + 13

10

139 + 40

138 + 29

85 + 15

75 +

20

115 + 27

130 + 27

73 + 10

1+

115 + 27

116 + 18

65 +

2+

115 + 27

123 + 30

93 + 15

35 +

14

8

6

7 71

53 + 12 68

+ 14

significantly different from the controls (P < 0,05)

+ units/ml Effect of gastric secretagoguea on the gastric mucosal histamine content.

TABLE III. A

H

C - A

D - B

control

+substance

control

+substance

compound g8/80

234 + 22

268 + 28

423 + 104

460 +

Carbachol

201 + 28

196 + 28

275 + 105

510 + 1121

Beserpine

223 + 25

241 + 27

386 + 11

513 +

Gastrantetrapeptide Inaulin

166 + 25 266 + 20

242 + 61 365 + 29 1

516 + 87 330 + 86

629 + 143 363 + 31

77

511

aigaificantly different from the controls (P < 0,05) Effect of gastric secretagogues on gastric histamine decarbozylase activity (for ezplanation see tezt) .

Vol . 8, No . 13

GASTRIC SECRETAGOGUES

787

On],y in the incubation mixture with insulin the decarbozylation rate of endogenous histidine was found to be significantly higher than is the control experiments . All other compounds were ineffective, The decarbazylation rate of exogenous L-histidine wsa significantly increased by carbachol and reaerpine, but not by compound 48/80, the gastrin-tetrapeptide and insulin. Macussion The failure of compound 48/80 to release histamine from the isolated gastric mucosa confirmed the resistance of gastric histamine to pretreatment of rate with this agent (9), Reaerpine, however, in doses from 1 - 5 mg/kg has been reported to reduce gastric histamine in rats within 2 - 5 hours by

60 - 80 y6 (3,10),

In the latter experiments (10) the histamine depletion was

accompanied by a rapid reduction in the gastric histidine decarbozylase . In the present experiments reserpine has a histamine depleting action which can be demonstrated only with e concentration of 40 }ig/ml and after as incubation time of 2 hours . The increase in histidine decarboxylase activity by reaerpine is in an unexplained contrast to the results by Isaac et al . (10), Cholinergic stimuli reduce gastric histamine in rats by 19 yn (urecholine) (3), by 44 ~ (insulin) and by 2~ jô (2-deoxyglucoae)

(11), It must be

emphasized that in the last mentioned experiments (11) the control values for histamine were very high (125 - 2155 nMoles/g) . An increase in gastric histidine decarboxylase activity as obtained with carbachol in vitro can be observed in vivo with other cholinergic stimuli like insulin and 2~ieoxyglucose (11) . The ineffectiveness of insulin to affect the histamine content and histidine decarboxylase activity of the rat stomach agrees with the general view that insulin exerts its action on the stomach only in vi vo via the vague nerve, A gastric histamine release by gastrin in rata has been described frequently (3,4,11,12), No corresponding experiments have been performed with the gsstrin-tetrapeptide, In the experiments described in this paper only an extremely high dose rf the gastrin tetrapeptide resulted in a histamine

788

GASTRIC SECRETAGOGUES

Vol. 8, No. 13

depletion within 2 hours . The histidine decarborylase, however, was not stimulated by the gastrin-tetrapeptide, which is in agreement with the studies by Sz~yder and Epps (5) who failed to demonstrate a stimulating effect of gastrin on the histidine decarborylase activity when the agent was added to the incubation medium . Pretreatment of rats with gastrin increased the histidine decarboxylase activity in subsequent in vitr o, measurements . This effect of gastrin and pentagastrin in rats has also been described by other authors (11,13) . ICahlson et al . (4) au~gested a "feedback relaticn between histamine and histidine decarborylase where by lessening of end-product repression results in increased enzyme activity" in the stomach . If such a mechanism is the effective principle for the local control of gastric acid secretion no decrease in the histamine content should be observed in vivo provided teat enough endogenous histidine is available. If the histidine supply is insufficient arty gastric acid stimulant - except histamine - must undergo fatigue . Our present experiments have shown that among the gastric secretagogues ezamined only reserpine e.nd the gastrin-tetrapeptide had a histamine releasing activity and that only carbachol and reserpine stimulated the gastric histidine decarborylase activity in vitro. Whether the increased capacity of histidine decarborylation is utilized in vi vo needs further investigation . S»< The effect of different gastric secretagogues on the histamine content and the histidine decarborylase activity of a scraped off gastric mucosa has been ezamined in rata . A histamine release has been observed after 2 hours of incubation with reserpine (40 Pg/ml) and the gastrin-tetrapeptide

(20 }zg/ml) . Compound 48/80,

carbachol and insulin had no histamine releasing effect . i The histidine ~decarborylase activity was enhanced by carbachol and reserpine, but was not affected by compound 48/80, the gastrin-tetrapeptide

vol. 8, No . 13

789

GASTRIC SECRETAGOGUES

and insulin. The results demonstrated no strict connection between histamine release and stimulation of hietidine decarbozylase activity in the rat gastric mucosa . Achowledgementa. Supported by a grant from the Deutsche Forschungegemeia-

schaft . The technical assistance of Mr, W, Beer is gratefully acknowledged . References 1 . Haverback, B,J, and S,K, Wirtschafter, Adv. Phsrmacol,

1, 309 (1962),

2, Haverback, B,J L,B, Tecimer, B,J, Dyce, M, Cihen, M.I, Stubrin and A,D, Santa Ana, Life Sci . ~, 637 (1964) .

3 . Stubrin, M,I H. Dyce, T, Brem, L,B. Tecimer and B,J. Haverback, Amer . J, dig, Dis, 10, 901

(1965) .

4 . Kahlson, G., E, Rosengren, D, Svahn and R, Thunberg, J, Phyeiol, (Load,) ~~~, 4~ (1964) . 5, Sx~rder, S,H, and L, Epps, Mol, Pharmacol . ~, 187 (1968) . 6 . Sewing, K,-Fr ., Naun~vn-Schmiedebergs Arch, Pharmak . ezp. Path, 260, 200 (1968), 7, Albinus, M, and K,-Fr. Sewing, Nauayn-Schmiedebergs Arch, ~aati, ezp. Path . (in press), B, Shore, P,A., A, Burkhalter and V,H, Cohn, J, Pharmac . ezDtl. Ther, 12~, 1x2 (1959) .

9, Johnson, H,L M,A, Beaven, F, Erjavec sad B,B, Brodie, Life Sci . ~, 115 (1966) . 10, Isaac, L,, M.A, Beaven, A,K, Cho and B,B, Brodie, Pharmacologist 10, 185

(196x) . 11 . Kahlson, G E, Rosengren and R, Thunberg, J, Phyaiol. (tond,) 190, 455

(1967) . 12 . Brodie, B,B ., M,A . Heaven, F, Erjavec and H,L, Johnson. In : U,S, v. Eider and B, Uvnäs (Ed .) . 4lenner Gren Symposium Pergamon Press, Oxford p. 401 1j, Lin, T,M, and T, Evans, Fed, Proc, 26, 273 (1967),

(1966),