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Histamine formation in rat stomach: Study of regulation mechanisms
EUROPEAN JOURNAl. OF PtIARMACOLOGY 5 (1969) 272-278. NORTII-HOLLANI) PUBLISHING COMP., AMSTt'RDAM
HISTAMINE FORMATION
IN R A T S T O M A C H :
STUDY OF REGULATION
MECHANISMS
Jean-Charles SCHWARTZ, Anna-Lisa RONNBERG, Yves COHEN and Guillaume VALETTE Laboratoire de Pharmacodynamie. Facultb de Pharmacic. Paris
Received 10 April 1968
Accepted l0 October 1968
J.C.SCIIWARTZ, A.L.RONNBERG, Y.COHEN and G.VALETFE, Histamine format#m in rat stomach: study o f rcglllation mechanisms, European J. Pharmacol. 5 (1969) 272-278. The mechanism by which the activity of the histamine-forming enzyme (histidine decarboxylase) in rat glandular stomach is elevated during ,secretion periods has been studied. It has been found that the food-induced elevation was blocked when protein synthesis was inhibited by cycloheximide but actinomycin D had no effect. Substrate (L-histidine) administration induced only a feeble enzyme activation in fasting animals while D-histidine, histamine or histamine metabolites were ineffective. On the other hand end-product (histamine) administration prevented histidine decarboxylase increase provoked either by food, insulin or gastrin. It is concluded that the modifications of the activity of histidine decarboxylase are related to the synthesis of new enzyme molecules and seem to be regulated by the level of histamine in the tissue, i.e. by repression process.
Histamine Cyclohcximide (;astrin
Histidine decarboxylase Actinomycin I)
1. INTRODUCTION There is much evidence to suggest that mucosal histamine is physiologically involved as a chemostimulator in acid gastric secretion (Code, 19(~5; Ivy and Bachrach, 1966). Schayer and Ivy (1958) demonstrated that some 14C histamine synthesized in rat stomach from injected I4C histidine can be released into the blood stream after food intake. Further, Kahlson et al. (1964) showed in in vitro experiments that this mobilization of gastric histamine during digestive periods is also accompanied by a decrease of mucosal histamine and an increase of histidine decarboxylase activity in the tissue. The same results were obtained upon vagal stimulation which causes a lowering of histamine content (Kim and Shore, 1963) and an elevation of both histamine turnover rate, measured after injection of 31-t histamine (Beaver et al., 1967) and histidine
Rat glandular stomach Insulin
decarboxylase levels (Schwartz et al., to be published) in rat stomach. This inverse relationship between histamine content and histidine decarboxylase activity ("histamine forming capacity") was observed by Kahlson et al. (1964) during gastrin-induced secretion and led these authors to propose a theory of a feed-back regulation of histamine formation: gastric histamine is maintained at constant level by adjustments of the activity of the forming enzyme. The purpose of the present work was to clarify the mechanism by which the increase of histamine formation in the stomach of the rat (measured by elevation of tissue histidine decarboxylase activity) is set up during physiological secretion states. In a first group of experiments an attempt was made by means of a drug-induced blockade of protein synthesis, to determine whether synthesis of new enzyme molecules was involved in the apparent increase of histidine decarboxylase activity. In further experiments the links
HISTAMINEFORMATIONIN RAT STOMACIt between the amount of histamine and the levels of the histamine-forming enzyme in the gastric wall were investigated.
2. MATERIALS AND METHODS
2.1. Animals Adult male rats of the Wistar strain weighing 140160 g were used. They were left without food (water ad libitum) for 24 hr before the experiments. The animals were kept in cages, the bottom of which consisted of a sparse lattice-work. This precaution was taken to avoid coprophagy, which was found to cause a pronounced increase in the level of gastric histidine decarboxylase. 2.2. Determination of enLvmatic activity The rats were killed by decapitation. The stomach was immediately removed, cut along the greater curvature and rinsed in running water. The glandular part (entire wall) of the organ between the pylorus and the end of the forestomach was separated and preserved in iced saline. The tissues were homogenized in 5 volumes of ice-cold distilled water with an Ultra-Turrax. The homogenate was centrifuged at 20,000 g for 20 min at 0°C and the supernatant used as the enzyme source (Schwartz et al., 1966). The purification of the enzyme extract by elution from a column of Sephadex G-25, recommended by kevine and Watts (1966) was tried in some preliminary experiments and it was found that free histidine could be efficiently removed by this procedure (this was checked by adding labelled histidine to the raw extract before passing it through the colunm). In spite of this treatment, there was no increase in the activity of the enzyme and so this purification was not followed in further experiments. The incubations were made in 15 ml Warburg flasks. Usually 1.5 ml of the crude supernatant, corresponding to 300 mg of tissue, was incubated with 0.7 ml of a solution containing 0.12 mmole pH 7 sodium phosphate buffer, 40 nmoles pyridoxal 5'phosphate, 30 nmoles L-histidine and 21.5 nmoles DL-histidine-carboxyle-laC (Calbiochem) (10 mC/ mmole). A small glass tube was filled with 0.2 ml of a solu-
273
tion of hyamine hydroxide (0.5 M in 50% methanol) and was placed in the center well of the Warburg flask. After incubation for I hr in air, at 37°C with agitation, the reaction was stopped by passing 0.4 ml M citric acid from the side arm of the flask into the main compartment. The flasks were shaken for one additional hour to ensure quantitative absorption of released carbon dioxide into the hyamine hydroxide. The small glass tube was then transferred to a scintillation vial containing 15 ml of toluene phosphor. The value of a blank solution obtained by stopping the reaction, by the addition of citric acid, before incubation, was substracted from the experimental value. Similar blank values could be obtained on incubation for 1 hr in the presence of 10--4 M hydrazino-histidine (MK 785, Merck, Sharp and Dohme) a potent histidine decarboxylase inhibitor (Levine et al., 1965). The results have been expressed in nanomoles of Lhistidine decarboxylated per hour and per gram of fresh tissue, taking into account the specific activity of the L-histidine added as substrate (Kobayashi, 1963). Under these conditions, a linear relation between the quantity of 14CO2 formed and the volume of enzyme extract added has been observed. This finding has been checked in every type of experiment and confirms the conclusion that endogenous histidine extracted from the stomach does not interfere with the assay. 2.3. Drug treatment of animals Actinomycin D, an inhibitor of protein synthesis (Reich et al., 1962) was injected intraperitoneally at 1 mg/kg, I br before feeding. Cycloheximide, an inhibitor of protein synthesis (Wettstein et al., 1964) was injected subcutaneously in two doses (2 X 20 mg/kg), one 30 rain before and one 75 rain after feeding. tlistidine in different doses and an amino acid solution "'Trophysan" (1 ml/100 g), were injected intraperitoneally to fasted animals 4 hr before they were killed. Histamine, methylhistamine or imidazolacetic acid were given subcutaneously at a dose of 20 mg/kg. In other experiments (indicated in the text) histamine was administered in two doses (2 X 20 mg/kg) at the start and in the middle of the experimental periods.
274
J.-C.SCHWARTZ et al.
Insulin (10 U/kg) gastrin (10 Leo Units/kg) and gastrin-like pentapeptide (20 /ag/kg) were injected subcutaneously to fasted rats. The animals were killed alter 4 hr for insulin and after 2.5 hr for the two gastrin preparations. Aminoguanidine, an inhibitor o f diamine oxidase (Buffoni, 1966) was administered subcutaneously in two 30 mg/kg doses, one dose 15 min before and one in the middle o f the experimental period. 2.4. Drugs Actinomycin D, Merck Sharp and Dohme; amino acid solution for injection " T r o p h y s a n " Egic; gastrin Leo; gastrin-pentapeptide "Peptavlon" I.C.I. 50.123; L-histidine monohydrochloride B.D.H.; histamine dihydrochloride Prolabo; insulin Endopancrine; methylhistamine and imidazolacetic acid Calbiochem.
3. RF~SULTS
3.1. Effects o f injection of actinomycin D and cycloheximide on fi)od-induced activation o f histidine decarboxylase The activity of histidine decarboxylase in the glandular part o f the stomach was low in 24 hr fasted rats but increased markedly within a few hours when the animals were again allowed food. Actinomycin D and cycloheximide, potent inhibitots o f RNA and protein synthesis were used to find out if blockade of protein synthesis would prevent this activation of the enzyme produced by feeding. In some preliminary experiments it was found that the intraperitoneal injection of actinomycin 1 hr betbre feeding did reduce the potentiation o f the enzyme. However, it was noticed that the injection of this antimitotic drug considerably diminished the voluntary intake of food and this might explain the observed effect of the drug. Thus in all further experiments voluntary feeding was replaced by the forced feeding, by stomach tube of 6 ml o f a thick paste, obtained by homogenizing standard dietary pellets (U.A.R., France) with water. Under these conditions, administration of actinoinycin D had no effect on the increased enzymatic activity of glandular stomach tissue observed 4 hr after feeding (table 1). In contrast, in rats treated with cycloheximide
and killed 2.5 hr after feeding, the activation of the gastric enzyme has not only been completely blocked but the level of activity was significantly lower (P < 0.02) than that o f the fasting animals (table 1). Table 1 Effects of actinomycin D (1 mg/kg; i.p.) and cycloheximide (2 X 20 mg/kg; s.c.) on the activation of gastric histidinc decarboxylase in rats caused by feeding. Actinomycin D was injected l hr before oral administration of dietary paste; cycloheximide injections were made 30 min before and 75 min after feeding. The time between feeding and death is indicated in brackets. Results are expressed in nanomoles per g per hr and are the means of the specified number of experiments +-S.I-.M. Number of experiments
Histidine decarboxylase (nmoles/g/hr)
Fasting
5
0.42 + 0.06
I:ed (4 hx) saline injected
5
1.64 &0.29
Fed (4 hr) actinomycin D injected
5
1.85 -+0.24
Fed (2.5 hr) saline injected
5
1.46 -+0.45
Fed (2.5 hr) cycloheximide injected
6
0.14 _+0.01
Animals .
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3.2. Effect of in/ectkm of histidine to fasting rats An attempt was. made to determine whether activation of histidine decarboxylase could be provoked by administration of the substrate as this is the case for several inducible enzymes (Knox, 1964; Conney, 1965). lntraperitoneal injection o f 10 mg L-histidine per animal 4 hr before death did not produce any effect (table 2) while with a dose of 50 mg per animal a slight but significant increase (P < 0.05) was observed. Such an activation did not take place after administration of 50 mg D-histidine, nor after the injection of an equivalent quantity of nitrogen in a mixture of amino acids (solution for injection "Trophysan" containing glycine and essential amino acids but no histidine). 3.3. l'~ffect o f injectMn o f histamh~e and its metabol-
ites on fasting rats Gastric histamine mobilization during intake of
ItISTAMINE FORMATION IN RAT STOMACH
Table 3 Effect of the subcutaneous administration (20 mg/kg) of histamine and two of its rnetabolites, methyl histamine and imidazole acetic acid, 4 hr before death on gastric histidine decarboxylase activity in fasting rats. Results are expressed as mean + S.E.M.
Table 2 Gastric histidine decarboxylase in fasting rats, 4 hr after intraperitoneal administration of saline, histidine or a mixture of amino acids. Results are given as mean of the number of experiments + S.I-.M. Treatments
Number of Histidine decarboxylase experiments (nmoles/g/hr)
Saline
9
0.61 -+ 0.08
L-histidine (10 mg)
5
0.60 + 0.09
L-histidine (50 mg)
7
1.00 + 0,16 *)
D-histidine (50 mg)
7
0.69 + 0.13
Amino acid mixture (Trophysan)
5
0.64 + 0.12
275
Treatments
Number of Itistidine decarboxylase experiments (nmoles/g/hr)
Saline Histamine Methylhistamine Imidazolacetic acid
8 7 5 8
0.49 0.82 0.63 0.59
+ 0.07 -+ 0.15 *) -+ 0.09 *) -+ 0.09 *)
*) P >0.05 using Student's t-test. ministration o f two doses (20 mg/kg subcutaneously) o f histamine before and during the experimental period, c o m p l e t e l y stopped the activation which was normally p r o v o k e d either by food intake or by the administration o f natural gastrin or a gastrin-like
*) P < 0.05 using Student's t-test. food is associated with a release o f this amine and its metabolites into the blood stream (Schayer and Ivy, 1958). It seemed o f interest to d e t e r m i n e w h e t h e r the increase o f one o f these p r o d u c t s in the b l o o d could be responsible for the e n z y m e activation. However, no significant change was observed 4 hr after the s u b c u t a n e o u s administration o f histamine, methylhistamine or imidazolacetic acid (table 3). The slight but not statistically significant (P > 0.05) increase observed on histamine administration may tentatively be attributed to secretion o f medullary h o r m o n e s f r o m the suprarenal glands p r o v o k e d by this t r e a t m e n t , as adrenaline can induce elevation of histamine forming capacity in several tissues (Perlman and Waton, 1966). 3.4. Effect of injection of histamine on the activation
o f histidine decarboxylase induced by various agents A c c o r d i n g to Kahlson et al. (1964), the histidine decarboxylase activation is due to a process of negative feedback, i.e. it is a c o n s e q u e n c e o f a decrease o f histamine c o n t e n t o f the gastric mucosa. In order to c o n f i r m this hypothesis, a large dose o f histamine was injected during the period o f e n z y m e activation to see if this w o u l d block the increase o f the activity o f the e n z y m e . This a t t e m p t seemed to be justified by the fact that injected histamine enters the endogenous amine pool o f the gastric mucosa (Beaven et •,d., 1967). The results, illustrated in fig. 1, indicate that ad-
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sohne I
I
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PJ°'I°Og!~ P
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P
0,5
r,3st,,,w~ 2 h 3 0
4h
otte," fooo ~mto;~e
ins~flm
gostrir~
penPopeptlOe ( 1CI 50.123)
Fig. 1. Effect of histamine administration (2 X 20 mg/kg, s.c.) on the activation of rat glandular stomach histidine decarboxylase provoked by several agents. Ilistamine injections were made at the start and in the middle of the experimental periods. Rats were killed 2% hr or 4 hr after feeding 6 ml of dietary paste, 4 hr after injections of insulin (10 U/kg, s.c.), and 2% hr after gastrin (10 I_.eo Units/ kg, s.c.) or gastrin-pentapeptide I.C.I. 50123 (20 ~g/kg, s.c.) Results are given as mean -+ S.E.M. Number of animals in brackets.
276
J.-C.SCIIWARTZ et al.
pentapeptide "Peptavlon". The effect of insulin was significantly decreased under the same conditions. 3.5. Affect of the injection of aminoguanMine on
fi,od- and insulin-induced activations of histidine decarboxylase The main, if not the sole, histaminolytic enzyme of the gastric mucosa of the rat is diamine oxidase (Kobayashi and Ivy, 1959. Brown et al., 1959). Inhibition of this enzyme by two doses of aminoguanidine (30 mg/kg: subcutaneously) one administered 15 min before the inducing agent and the other in tile middle of the experimental period weakly inhibited the increase of the enzyme activity (fig. 2). The animals were killed 2% hr after feeding (6 ml of paste) and 4 hr after the administration of insulin (I Unit/100 g). These negative results do not, however, exclude the fact that the gastric histamine level may interfere with histidine decarboxylase activation: in spite of the fact that aminoguanidine potentiates gastric secretory stimulants in tile rat (Code, 1965), it has not been demonstrated tlmt diamine oxidase action is a rate-limiting step in histamine inactivation in the Stolnach. ., ,,: ,. ,.-:./~qe.~.
) o,o5< p < o , f
-t-
|l
i
2 L't tel" f o o u
39 ;,';tClkO
Fig. 2. Effect of aminoguanidine administration 1,2 X 30 mg/ kg, s.c.) on the level o f rat glandular stomach histidinc dccarboxylase evaluated 4 hr after insulin injection (10 U/kg, s.c.) or 2'k hr after feeding dietary paste.
4. DISCUSSION As a general rule variations in enzyme activity depend on different factors. The main ones are: (1) variations in the amount of coenzyme in the tissue, (2) modifications of the intrinsic activity of the enzyme protein (especially in relation to the presence of an enzyme inhibitor), (3) variations of the concentration of enzyme proteins. In the case of the activation of gastric histidine decarboxylase which is observed in rats after food intake, the first hypothesis can be rejected because the enzyme activities in the present work were measured in tile presence of an excess of pyridoxal phosphate. The second hypothesis, concerning the presence of an inhibitor of histidine decarboxylase of a small molecular weight in the stomach extract, suggested by Levine and Watts (1966), has not been confirmed during our experiments. In addition the linear relation between tile enzyme activity and the wllume of the extract used in the determinations observed under the different experimental circumstances, excludes interference by any histidine in the stomach extract. On the other hand, the complete blockade by cycloheximide of the increase of enzyme activity, which is normally provoked by food intake, suggests that the activation is related to tile synthesis of new molecules of enzyme proteins. Furthermore, the reduction of enzyme activity below fasting levels within a few hours after cycloheximide treatment suggests a high turnover rate for stomach histidine decarboxylase, which may be paralleled by the histamine turnover rate in the same organ {Beaven et al., 1967). During ttle course of these experiments a similar blockade by cycloheximide of gastrin-induced activation of histidine decarboxylase together with the lack of effect of actinomycin D has been described in a preliminary communication (Snyder and Epps, 1967). Thus it appears that histidine decarboxylase activation is more readily explained as a true enzyme induction, similar to those which are observed in drug metabolizing enzymes, other than by "allosteric" modification. The absence of such an inhibitory action in animals treated with actinomycin D may appear to contradict this conclusion. However, the mechanisms
HISTAMINE FORMATION IN RAT STOMACH of action of the two compounds at the cellular level seem to be different. Cycloheximide inhibits protein synthesis mainly in the ribosomes, preventing the transport of soluble RNA to the newly formed polypeptide chains (Wettstein et al., 1964), while actinomycin D interferes with a previous step by inhibition of DNA directed RNA biosynthesis (Reich et al., 1962). There are other similar observations of discrepancies between the effects of actinomycin D and those of cycloheximide towards the induction of various enzymes (Knox, 1964). These observations suggest that messenger RNA synthesis is not ratelimiting and enzyme formation is controlled at the ribosome level. Another characteristic of several inducible enzymes is the increase in their biosynthesis which may be produced by the administration of the corresponding substrates (Knox, 1964; Conney, 1965). ltistidine decarboxylase seems to obey this rule but the phenomenon is produced only to a small extent and it requires the administration of a high dose of histidine. This fact excludes the hypothesis that histidine may play an important physiological role in gastric histamine formation during digestion. Nevertheless it is possible that overloading with histidine may, even in the absence of increased histidine decarboxylase activity, result in increased gastric histamine formation. This could, in turn, explain the hypersecretion observed in rats by R~is~nen (1956) after administration of the precursor amino acid, histidine. Kahlson et al. (1964) have proposed a feed-back theory for the regulation of histamine synthesis in rat stomach. The first experimental evidence advanced by these authors was that lowering of the histamine content was associated with a concurrent elevation of histidine decarboxylase in the mucosa. In fact as gastric histamine depletion was associated with an increased blood level of the amine and its metabolites, it was possible that one of these blood products was the true activating agent. The present work, showing that administration of histamine, methyl histamine or imidazoleacetic acid to fasting rats do not change significantly the activity of the synthesizing enzyme, rules out this last interpretation. The second experimental evidence put forward by Kahlson et al. (1964), was that administration of the end-product (histamine) to fasted rats or fed mice resulted in both cases in a decrease of enzyme level.
277
In the experiments described here it was also found that histamine injections prevented the food-induced and markedly diminished the insulin-induced enzyme activations. Since it is well known that the secretory effect of both these agents is, at least in part, mediated by gastrin release (Uvn~is, 1963) and as gastrin is a potent histidine decarboxylase activator, the inhibitory effect of histamine administration could be due to an inhibition of gastrin release due to the high antral acidity after this administration. Thus a direct regulation of histidine decarboxylase level by histamine can only be demonstrated if the blockade is still apparent when gastrin release is not involved. Such is the case, because histamine administration completely prevents the enzyme activation provoked by injections of natural gastrin or of synthetic gastrin pentapeptide (fig. I). In conclusion, Kahlson's hypothesis of a "'feedback" regulation of histidine decarboxylase activity in rat stomach is confirmed by our experiments. Moreover, this regulation seems to take place in the protein synthesizing part of tile cell (at an actinomycin-insensitive step) and should, therefore, be better described as a "repression" process. Further experiments will show if other substances besides histamine, are able to act in this repression process.
ACKNOWLEDGEMENTS The authorswish to thank Dr. B.Larsen (I.aboratoires L6o, France) for a gift of gastrin, Dr. Fitzgerald 0.C.I. ltd., Great-Britain) and Dr. Augusseau (Laboratoire Avlon, I"rance) for a gift of peptavlon.
REFERENCES Beaven, M.A., Z.Horakova, It.L.Johnson, t'i.Erjavec and B.B. Brodie, 1967, Selective labelling of histamine in rat gastric mucosa, Fed. Proc. 26, 233. Brown, D.D., R.Tomchick and J.Axelrod, 1959, The distribution and properties of a histamine methylating enzyme, I. Biol. Chem. 234, 2948. Buffoni, F., 1966, ttistaminasc and related amine oxida~s, Pharmacol. Rev..18, 1163. Code, C.F., 1965, Histamine and gastric secretion: a later look, 1955 1965, I'ed. Proc. 24, 1311. ('onney, A.II., 1965, Enzyme induction and drug toxicity, in: Proceedings of the second International Pharmacological Meeting, Vol. 4, ed. H.Raskova (Pergamon, OxfordL
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ivy, A.C. and W.H.Bachrach, 1966, Physiological significance of the effect of histamine on gastric secretion, Handb. Expcr. Phaxmacol. 18, 810. Kahlson, G., E.Rosengren, D.Swann and R.Thunberg, 1964, Mobilization and formation of histamine in the gastric mucosa as related to acid secretion, J. Physiol. (London) 174,400. Kim, K.S. and P.A.Shore, 1963, Mechanism of action of reserpine and insulin on gastric amines and gastric acid secretion and the effect of monoaminoxidase inhibition, .I. PhaImacol. 141,321. Knox, I.i., 1964, Substratc-type induction of tyrosine transaminase, illustrating a general adaptative mechanism in animals, Advan. l-nzyme Regulation 2, 311. Kobayashi, Y., 1963, Determination of histidine decarboxylase activity by liquid scintillation counting of ('140 2, Anal. Biochem. 5, 284. Kobayashi, Y. and A.C.lvy, 1959. Histamine metabolizing activity of the stomach and intestine of the rat, Am. J. Physiol. 196, 835. l.evine, R.J., I.L.Sato and A.Sjoerdsma, 1965, Inhibition of histamine synthesis in the rat by a hydrazino analog of histidine and 4-bromo-3-hydroxy benzyloxyamine, Biochem. Pharnaacol. 14, 139.
Pearlman, D.S. and N.(;.Waton, 1966, Observations on the histamine forming capacity of mouse tissues and of its potentiation after adrenaline. J. Physiol. (l.ondon) 183, 257.
Reich, t-., 1.11.Goldberg and M.Rabinowitz, 1962, Structureactivity correlations of actinomycins and their derivatives, Nature 196,743. R/isanen, T.. 1966, llistidine induced secretion in the pyloric ligated stomach, Acta Physiol. Stand. 66, 481. Schayer, R.W. and A.('.Ivy, 1958, Release of (?,14 histamine from stomach and intestine on feeding, Am. J. Physiol. 193, 40O. Schwartz. J.('., Y.('ohen and (;.Valcttc, 1966, Histidinc dccarboxylasc gastrique et ulc~re~ exp4rimentaux chez le rat, Biochem. Parmacol. 15, 2122. Snydcr, S.tt. and l,.t'?pps. 1967. Histidinc decarboxylasc in rat stomach: mechanism of its activation by gastrin, l.cd. Proc. 26. 2956. U,vn~is, B.. 1963. Gastrin rclease, in: Pathophysiology of Peptic Ulccr. cd. S.('.Skoryna (McGill Univcrsity Press. Montreal) p. 87. Wettstcin. I..O., H.Noll and S.Penman, 1964, I-ffcct of cycloheximide on ribosomal aggregates engaged in protein synthesis ht vitro, Biochim. Biophys. Acta 87, 525.
HISTAMINE FORMATION
IN R A T S T O M A C H :
STUDY OF REGULATION
MECHANISMS
Jean-Charles SCHWARTZ, Anna-Lisa RONNBERG, Yves COHEN and Guillaume VALETTE Laboratoire de Pharmacodynamie. Facultb de Pharmacic. Paris
Received 10 April 1968
Accepted l0 October 1968
J.C.SCIIWARTZ, A.L.RONNBERG, Y.COHEN and G.VALETFE, Histamine format#m in rat stomach: study o f rcglllation mechanisms, European J. Pharmacol. 5 (1969) 272-278. The mechanism by which the activity of the histamine-forming enzyme (histidine decarboxylase) in rat glandular stomach is elevated during ,secretion periods has been studied. It has been found that the food-induced elevation was blocked when protein synthesis was inhibited by cycloheximide but actinomycin D had no effect. Substrate (L-histidine) administration induced only a feeble enzyme activation in fasting animals while D-histidine, histamine or histamine metabolites were ineffective. On the other hand end-product (histamine) administration prevented histidine decarboxylase increase provoked either by food, insulin or gastrin. It is concluded that the modifications of the activity of histidine decarboxylase are related to the synthesis of new enzyme molecules and seem to be regulated by the level of histamine in the tissue, i.e. by repression process.
Histamine Cyclohcximide (;astrin
Histidine decarboxylase Actinomycin I)
1. INTRODUCTION There is much evidence to suggest that mucosal histamine is physiologically involved as a chemostimulator in acid gastric secretion (Code, 19(~5; Ivy and Bachrach, 1966). Schayer and Ivy (1958) demonstrated that some 14C histamine synthesized in rat stomach from injected I4C histidine can be released into the blood stream after food intake. Further, Kahlson et al. (1964) showed in in vitro experiments that this mobilization of gastric histamine during digestive periods is also accompanied by a decrease of mucosal histamine and an increase of histidine decarboxylase activity in the tissue. The same results were obtained upon vagal stimulation which causes a lowering of histamine content (Kim and Shore, 1963) and an elevation of both histamine turnover rate, measured after injection of 31-t histamine (Beaver et al., 1967) and histidine
Rat glandular stomach Insulin
decarboxylase levels (Schwartz et al., to be published) in rat stomach. This inverse relationship between histamine content and histidine decarboxylase activity ("histamine forming capacity") was observed by Kahlson et al. (1964) during gastrin-induced secretion and led these authors to propose a theory of a feed-back regulation of histamine formation: gastric histamine is maintained at constant level by adjustments of the activity of the forming enzyme. The purpose of the present work was to clarify the mechanism by which the increase of histamine formation in the stomach of the rat (measured by elevation of tissue histidine decarboxylase activity) is set up during physiological secretion states. In a first group of experiments an attempt was made by means of a drug-induced blockade of protein synthesis, to determine whether synthesis of new enzyme molecules was involved in the apparent increase of histidine decarboxylase activity. In further experiments the links
HISTAMINEFORMATIONIN RAT STOMACIt between the amount of histamine and the levels of the histamine-forming enzyme in the gastric wall were investigated.
2. MATERIALS AND METHODS
2.1. Animals Adult male rats of the Wistar strain weighing 140160 g were used. They were left without food (water ad libitum) for 24 hr before the experiments. The animals were kept in cages, the bottom of which consisted of a sparse lattice-work. This precaution was taken to avoid coprophagy, which was found to cause a pronounced increase in the level of gastric histidine decarboxylase. 2.2. Determination of enLvmatic activity The rats were killed by decapitation. The stomach was immediately removed, cut along the greater curvature and rinsed in running water. The glandular part (entire wall) of the organ between the pylorus and the end of the forestomach was separated and preserved in iced saline. The tissues were homogenized in 5 volumes of ice-cold distilled water with an Ultra-Turrax. The homogenate was centrifuged at 20,000 g for 20 min at 0°C and the supernatant used as the enzyme source (Schwartz et al., 1966). The purification of the enzyme extract by elution from a column of Sephadex G-25, recommended by kevine and Watts (1966) was tried in some preliminary experiments and it was found that free histidine could be efficiently removed by this procedure (this was checked by adding labelled histidine to the raw extract before passing it through the colunm). In spite of this treatment, there was no increase in the activity of the enzyme and so this purification was not followed in further experiments. The incubations were made in 15 ml Warburg flasks. Usually 1.5 ml of the crude supernatant, corresponding to 300 mg of tissue, was incubated with 0.7 ml of a solution containing 0.12 mmole pH 7 sodium phosphate buffer, 40 nmoles pyridoxal 5'phosphate, 30 nmoles L-histidine and 21.5 nmoles DL-histidine-carboxyle-laC (Calbiochem) (10 mC/ mmole). A small glass tube was filled with 0.2 ml of a solu-
273
tion of hyamine hydroxide (0.5 M in 50% methanol) and was placed in the center well of the Warburg flask. After incubation for I hr in air, at 37°C with agitation, the reaction was stopped by passing 0.4 ml M citric acid from the side arm of the flask into the main compartment. The flasks were shaken for one additional hour to ensure quantitative absorption of released carbon dioxide into the hyamine hydroxide. The small glass tube was then transferred to a scintillation vial containing 15 ml of toluene phosphor. The value of a blank solution obtained by stopping the reaction, by the addition of citric acid, before incubation, was substracted from the experimental value. Similar blank values could be obtained on incubation for 1 hr in the presence of 10--4 M hydrazino-histidine (MK 785, Merck, Sharp and Dohme) a potent histidine decarboxylase inhibitor (Levine et al., 1965). The results have been expressed in nanomoles of Lhistidine decarboxylated per hour and per gram of fresh tissue, taking into account the specific activity of the L-histidine added as substrate (Kobayashi, 1963). Under these conditions, a linear relation between the quantity of 14CO2 formed and the volume of enzyme extract added has been observed. This finding has been checked in every type of experiment and confirms the conclusion that endogenous histidine extracted from the stomach does not interfere with the assay. 2.3. Drug treatment of animals Actinomycin D, an inhibitor of protein synthesis (Reich et al., 1962) was injected intraperitoneally at 1 mg/kg, I br before feeding. Cycloheximide, an inhibitor of protein synthesis (Wettstein et al., 1964) was injected subcutaneously in two doses (2 X 20 mg/kg), one 30 rain before and one 75 rain after feeding. tlistidine in different doses and an amino acid solution "'Trophysan" (1 ml/100 g), were injected intraperitoneally to fasted animals 4 hr before they were killed. Histamine, methylhistamine or imidazolacetic acid were given subcutaneously at a dose of 20 mg/kg. In other experiments (indicated in the text) histamine was administered in two doses (2 X 20 mg/kg) at the start and in the middle of the experimental periods.
274
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Insulin (10 U/kg) gastrin (10 Leo Units/kg) and gastrin-like pentapeptide (20 /ag/kg) were injected subcutaneously to fasted rats. The animals were killed alter 4 hr for insulin and after 2.5 hr for the two gastrin preparations. Aminoguanidine, an inhibitor o f diamine oxidase (Buffoni, 1966) was administered subcutaneously in two 30 mg/kg doses, one dose 15 min before and one in the middle o f the experimental period. 2.4. Drugs Actinomycin D, Merck Sharp and Dohme; amino acid solution for injection " T r o p h y s a n " Egic; gastrin Leo; gastrin-pentapeptide "Peptavlon" I.C.I. 50.123; L-histidine monohydrochloride B.D.H.; histamine dihydrochloride Prolabo; insulin Endopancrine; methylhistamine and imidazolacetic acid Calbiochem.
3. RF~SULTS
3.1. Effects o f injection of actinomycin D and cycloheximide on fi)od-induced activation o f histidine decarboxylase The activity of histidine decarboxylase in the glandular part o f the stomach was low in 24 hr fasted rats but increased markedly within a few hours when the animals were again allowed food. Actinomycin D and cycloheximide, potent inhibitots o f RNA and protein synthesis were used to find out if blockade of protein synthesis would prevent this activation of the enzyme produced by feeding. In some preliminary experiments it was found that the intraperitoneal injection of actinomycin 1 hr betbre feeding did reduce the potentiation o f the enzyme. However, it was noticed that the injection of this antimitotic drug considerably diminished the voluntary intake of food and this might explain the observed effect of the drug. Thus in all further experiments voluntary feeding was replaced by the forced feeding, by stomach tube of 6 ml o f a thick paste, obtained by homogenizing standard dietary pellets (U.A.R., France) with water. Under these conditions, administration of actinoinycin D had no effect on the increased enzymatic activity of glandular stomach tissue observed 4 hr after feeding (table 1). In contrast, in rats treated with cycloheximide
and killed 2.5 hr after feeding, the activation of the gastric enzyme has not only been completely blocked but the level of activity was significantly lower (P < 0.02) than that o f the fasting animals (table 1). Table 1 Effects of actinomycin D (1 mg/kg; i.p.) and cycloheximide (2 X 20 mg/kg; s.c.) on the activation of gastric histidinc decarboxylase in rats caused by feeding. Actinomycin D was injected l hr before oral administration of dietary paste; cycloheximide injections were made 30 min before and 75 min after feeding. The time between feeding and death is indicated in brackets. Results are expressed in nanomoles per g per hr and are the means of the specified number of experiments +-S.I-.M. Number of experiments
Histidine decarboxylase (nmoles/g/hr)
Fasting
5
0.42 + 0.06
I:ed (4 hx) saline injected
5
1.64 &0.29
Fed (4 hr) actinomycin D injected
5
1.85 -+0.24
Fed (2.5 hr) saline injected
5
1.46 -+0.45
Fed (2.5 hr) cycloheximide injected
6
0.14 _+0.01
Animals .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
3.2. Effect of in/ectkm of histidine to fasting rats An attempt was. made to determine whether activation of histidine decarboxylase could be provoked by administration of the substrate as this is the case for several inducible enzymes (Knox, 1964; Conney, 1965). lntraperitoneal injection o f 10 mg L-histidine per animal 4 hr before death did not produce any effect (table 2) while with a dose of 50 mg per animal a slight but significant increase (P < 0.05) was observed. Such an activation did not take place after administration of 50 mg D-histidine, nor after the injection of an equivalent quantity of nitrogen in a mixture of amino acids (solution for injection "Trophysan" containing glycine and essential amino acids but no histidine). 3.3. l'~ffect o f injectMn o f histamh~e and its metabol-
ites on fasting rats Gastric histamine mobilization during intake of
ItISTAMINE FORMATION IN RAT STOMACH
Table 3 Effect of the subcutaneous administration (20 mg/kg) of histamine and two of its rnetabolites, methyl histamine and imidazole acetic acid, 4 hr before death on gastric histidine decarboxylase activity in fasting rats. Results are expressed as mean + S.E.M.
Table 2 Gastric histidine decarboxylase in fasting rats, 4 hr after intraperitoneal administration of saline, histidine or a mixture of amino acids. Results are given as mean of the number of experiments + S.I-.M. Treatments
Number of Histidine decarboxylase experiments (nmoles/g/hr)
Saline
9
0.61 -+ 0.08
L-histidine (10 mg)
5
0.60 + 0.09
L-histidine (50 mg)
7
1.00 + 0,16 *)
D-histidine (50 mg)
7
0.69 + 0.13
Amino acid mixture (Trophysan)
5
0.64 + 0.12
275
Treatments
Number of Itistidine decarboxylase experiments (nmoles/g/hr)
Saline Histamine Methylhistamine Imidazolacetic acid
8 7 5 8
0.49 0.82 0.63 0.59
+ 0.07 -+ 0.15 *) -+ 0.09 *) -+ 0.09 *)
*) P >0.05 using Student's t-test. ministration o f two doses (20 mg/kg subcutaneously) o f histamine before and during the experimental period, c o m p l e t e l y stopped the activation which was normally p r o v o k e d either by food intake or by the administration o f natural gastrin or a gastrin-like
*) P < 0.05 using Student's t-test. food is associated with a release o f this amine and its metabolites into the blood stream (Schayer and Ivy, 1958). It seemed o f interest to d e t e r m i n e w h e t h e r the increase o f one o f these p r o d u c t s in the b l o o d could be responsible for the e n z y m e activation. However, no significant change was observed 4 hr after the s u b c u t a n e o u s administration o f histamine, methylhistamine or imidazolacetic acid (table 3). The slight but not statistically significant (P > 0.05) increase observed on histamine administration may tentatively be attributed to secretion o f medullary h o r m o n e s f r o m the suprarenal glands p r o v o k e d by this t r e a t m e n t , as adrenaline can induce elevation of histamine forming capacity in several tissues (Perlman and Waton, 1966). 3.4. Effect of injection of histamine on the activation
o f histidine decarboxylase induced by various agents A c c o r d i n g to Kahlson et al. (1964), the histidine decarboxylase activation is due to a process of negative feedback, i.e. it is a c o n s e q u e n c e o f a decrease o f histamine c o n t e n t o f the gastric mucosa. In order to c o n f i r m this hypothesis, a large dose o f histamine was injected during the period o f e n z y m e activation to see if this w o u l d block the increase o f the activity o f the e n z y m e . This a t t e m p t seemed to be justified by the fact that injected histamine enters the endogenous amine pool o f the gastric mucosa (Beaven et •,d., 1967). The results, illustrated in fig. 1, indicate that ad-
,~
,'.0%/., 3,C;.
sohne I
I
,9,5.
2
1,5 u
?
PJ°'I°Og!~ P
P
P
0,5
r,3st,,,w~ 2 h 3 0
4h
otte," fooo ~mto;~e
ins~flm
gostrir~
penPopeptlOe ( 1CI 50.123)
Fig. 1. Effect of histamine administration (2 X 20 mg/kg, s.c.) on the activation of rat glandular stomach histidine decarboxylase provoked by several agents. Ilistamine injections were made at the start and in the middle of the experimental periods. Rats were killed 2% hr or 4 hr after feeding 6 ml of dietary paste, 4 hr after injections of insulin (10 U/kg, s.c.), and 2% hr after gastrin (10 I_.eo Units/ kg, s.c.) or gastrin-pentapeptide I.C.I. 50123 (20 ~g/kg, s.c.) Results are given as mean -+ S.E.M. Number of animals in brackets.
276
J.-C.SCIIWARTZ et al.
pentapeptide "Peptavlon". The effect of insulin was significantly decreased under the same conditions. 3.5. Affect of the injection of aminoguanMine on
fi,od- and insulin-induced activations of histidine decarboxylase The main, if not the sole, histaminolytic enzyme of the gastric mucosa of the rat is diamine oxidase (Kobayashi and Ivy, 1959. Brown et al., 1959). Inhibition of this enzyme by two doses of aminoguanidine (30 mg/kg: subcutaneously) one administered 15 min before the inducing agent and the other in tile middle of the experimental period weakly inhibited the increase of the enzyme activity (fig. 2). The animals were killed 2% hr after feeding (6 ml of paste) and 4 hr after the administration of insulin (I Unit/100 g). These negative results do not, however, exclude the fact that the gastric histamine level may interfere with histidine decarboxylase activation: in spite of the fact that aminoguanidine potentiates gastric secretory stimulants in tile rat (Code, 1965), it has not been demonstrated tlmt diamine oxidase action is a rate-limiting step in histamine inactivation in the Stolnach. ., ,,: ,. ,.-:./~qe.~.
) o,o5< p < o , f
-t-
|l
i
2 L't tel" f o o u
39 ;,';tClkO
Fig. 2. Effect of aminoguanidine administration 1,2 X 30 mg/ kg, s.c.) on the level o f rat glandular stomach histidinc dccarboxylase evaluated 4 hr after insulin injection (10 U/kg, s.c.) or 2'k hr after feeding dietary paste.
4. DISCUSSION As a general rule variations in enzyme activity depend on different factors. The main ones are: (1) variations in the amount of coenzyme in the tissue, (2) modifications of the intrinsic activity of the enzyme protein (especially in relation to the presence of an enzyme inhibitor), (3) variations of the concentration of enzyme proteins. In the case of the activation of gastric histidine decarboxylase which is observed in rats after food intake, the first hypothesis can be rejected because the enzyme activities in the present work were measured in tile presence of an excess of pyridoxal phosphate. The second hypothesis, concerning the presence of an inhibitor of histidine decarboxylase of a small molecular weight in the stomach extract, suggested by Levine and Watts (1966), has not been confirmed during our experiments. In addition the linear relation between tile enzyme activity and the wllume of the extract used in the determinations observed under the different experimental circumstances, excludes interference by any histidine in the stomach extract. On the other hand, the complete blockade by cycloheximide of the increase of enzyme activity, which is normally provoked by food intake, suggests that the activation is related to tile synthesis of new molecules of enzyme proteins. Furthermore, the reduction of enzyme activity below fasting levels within a few hours after cycloheximide treatment suggests a high turnover rate for stomach histidine decarboxylase, which may be paralleled by the histamine turnover rate in the same organ {Beaven et al., 1967). During ttle course of these experiments a similar blockade by cycloheximide of gastrin-induced activation of histidine decarboxylase together with the lack of effect of actinomycin D has been described in a preliminary communication (Snyder and Epps, 1967). Thus it appears that histidine decarboxylase activation is more readily explained as a true enzyme induction, similar to those which are observed in drug metabolizing enzymes, other than by "allosteric" modification. The absence of such an inhibitory action in animals treated with actinomycin D may appear to contradict this conclusion. However, the mechanisms
HISTAMINE FORMATION IN RAT STOMACH of action of the two compounds at the cellular level seem to be different. Cycloheximide inhibits protein synthesis mainly in the ribosomes, preventing the transport of soluble RNA to the newly formed polypeptide chains (Wettstein et al., 1964), while actinomycin D interferes with a previous step by inhibition of DNA directed RNA biosynthesis (Reich et al., 1962). There are other similar observations of discrepancies between the effects of actinomycin D and those of cycloheximide towards the induction of various enzymes (Knox, 1964). These observations suggest that messenger RNA synthesis is not ratelimiting and enzyme formation is controlled at the ribosome level. Another characteristic of several inducible enzymes is the increase in their biosynthesis which may be produced by the administration of the corresponding substrates (Knox, 1964; Conney, 1965). ltistidine decarboxylase seems to obey this rule but the phenomenon is produced only to a small extent and it requires the administration of a high dose of histidine. This fact excludes the hypothesis that histidine may play an important physiological role in gastric histamine formation during digestion. Nevertheless it is possible that overloading with histidine may, even in the absence of increased histidine decarboxylase activity, result in increased gastric histamine formation. This could, in turn, explain the hypersecretion observed in rats by R~is~nen (1956) after administration of the precursor amino acid, histidine. Kahlson et al. (1964) have proposed a feed-back theory for the regulation of histamine synthesis in rat stomach. The first experimental evidence advanced by these authors was that lowering of the histamine content was associated with a concurrent elevation of histidine decarboxylase in the mucosa. In fact as gastric histamine depletion was associated with an increased blood level of the amine and its metabolites, it was possible that one of these blood products was the true activating agent. The present work, showing that administration of histamine, methyl histamine or imidazoleacetic acid to fasting rats do not change significantly the activity of the synthesizing enzyme, rules out this last interpretation. The second experimental evidence put forward by Kahlson et al. (1964), was that administration of the end-product (histamine) to fasted rats or fed mice resulted in both cases in a decrease of enzyme level.
277
In the experiments described here it was also found that histamine injections prevented the food-induced and markedly diminished the insulin-induced enzyme activations. Since it is well known that the secretory effect of both these agents is, at least in part, mediated by gastrin release (Uvn~is, 1963) and as gastrin is a potent histidine decarboxylase activator, the inhibitory effect of histamine administration could be due to an inhibition of gastrin release due to the high antral acidity after this administration. Thus a direct regulation of histidine decarboxylase level by histamine can only be demonstrated if the blockade is still apparent when gastrin release is not involved. Such is the case, because histamine administration completely prevents the enzyme activation provoked by injections of natural gastrin or of synthetic gastrin pentapeptide (fig. I). In conclusion, Kahlson's hypothesis of a "'feedback" regulation of histidine decarboxylase activity in rat stomach is confirmed by our experiments. Moreover, this regulation seems to take place in the protein synthesizing part of tile cell (at an actinomycin-insensitive step) and should, therefore, be better described as a "repression" process. Further experiments will show if other substances besides histamine, are able to act in this repression process.
ACKNOWLEDGEMENTS The authorswish to thank Dr. B.Larsen (I.aboratoires L6o, France) for a gift of gastrin, Dr. Fitzgerald 0.C.I. ltd., Great-Britain) and Dr. Augusseau (Laboratoire Avlon, I"rance) for a gift of peptavlon.
REFERENCES Beaven, M.A., Z.Horakova, It.L.Johnson, t'i.Erjavec and B.B. Brodie, 1967, Selective labelling of histamine in rat gastric mucosa, Fed. Proc. 26, 233. Brown, D.D., R.Tomchick and J.Axelrod, 1959, The distribution and properties of a histamine methylating enzyme, I. Biol. Chem. 234, 2948. Buffoni, F., 1966, ttistaminasc and related amine oxida~s, Pharmacol. Rev..18, 1163. Code, C.F., 1965, Histamine and gastric secretion: a later look, 1955 1965, I'ed. Proc. 24, 1311. ('onney, A.II., 1965, Enzyme induction and drug toxicity, in: Proceedings of the second International Pharmacological Meeting, Vol. 4, ed. H.Raskova (Pergamon, OxfordL
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ivy, A.C. and W.H.Bachrach, 1966, Physiological significance of the effect of histamine on gastric secretion, Handb. Expcr. Phaxmacol. 18, 810. Kahlson, G., E.Rosengren, D.Swann and R.Thunberg, 1964, Mobilization and formation of histamine in the gastric mucosa as related to acid secretion, J. Physiol. (London) 174,400. Kim, K.S. and P.A.Shore, 1963, Mechanism of action of reserpine and insulin on gastric amines and gastric acid secretion and the effect of monoaminoxidase inhibition, .I. PhaImacol. 141,321. Knox, I.i., 1964, Substratc-type induction of tyrosine transaminase, illustrating a general adaptative mechanism in animals, Advan. l-nzyme Regulation 2, 311. Kobayashi, Y., 1963, Determination of histidine decarboxylase activity by liquid scintillation counting of ('140 2, Anal. Biochem. 5, 284. Kobayashi, Y. and A.C.lvy, 1959. Histamine metabolizing activity of the stomach and intestine of the rat, Am. J. Physiol. 196, 835. l.evine, R.J., I.L.Sato and A.Sjoerdsma, 1965, Inhibition of histamine synthesis in the rat by a hydrazino analog of histidine and 4-bromo-3-hydroxy benzyloxyamine, Biochem. Pharnaacol. 14, 139.
Pearlman, D.S. and N.(;.Waton, 1966, Observations on the histamine forming capacity of mouse tissues and of its potentiation after adrenaline. J. Physiol. (l.ondon) 183, 257.
Reich, t-., 1.11.Goldberg and M.Rabinowitz, 1962, Structureactivity correlations of actinomycins and their derivatives, Nature 196,743. R/isanen, T.. 1966, llistidine induced secretion in the pyloric ligated stomach, Acta Physiol. Stand. 66, 481. Schayer, R.W. and A.('.Ivy, 1958, Release of (?,14 histamine from stomach and intestine on feeding, Am. J. Physiol. 193, 40O. Schwartz. J.('., Y.('ohen and (;.Valcttc, 1966, Histidinc dccarboxylasc gastrique et ulc~re~ exp4rimentaux chez le rat, Biochem. Parmacol. 15, 2122. Snydcr, S.tt. and l,.t'?pps. 1967. Histidinc decarboxylasc in rat stomach: mechanism of its activation by gastrin, l.cd. Proc. 26. 2956. U,vn~is, B.. 1963. Gastrin rclease, in: Pathophysiology of Peptic Ulccr. cd. S.('.Skoryna (McGill Univcrsity Press. Montreal) p. 87. Wettstcin. I..O., H.Noll and S.Penman, 1964, I-ffcct of cycloheximide on ribosomal aggregates engaged in protein synthesis ht vitro, Biochim. Biophys. Acta 87, 525.