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Cellular stores of gastric histamine in the developing rat
LIFE SCIENCES V o l . 6, p p . Printed in Great Britain.
2535-2543,
1967.
Pergamon Press
Ltd.
CELLULAR STORESOF GASTRIC HISTAMINE IN THE DEVELOPINGRAT R. HAkanson, Ch. Ow,mn and N.-O. SjSber~ Departments of Pharwecology, Anatomy, and Obstetrics and Gynecology at MalmS, University of Lund, Lund, Sweden.
(Received
12 S e p t e m b e r
1967)
A histochemical method sensitive enough for the demonstration of histamine at the cellular level has recently been devised (I-3). According to this method, freeze-dried tissue sections are exposed to o-phthsldialdehyde vapor, which converts histamine into a fluorophore emitting an intense blue or, under certain conditions, yellow light. In the normal adult rat, gastric histamine has been found to be stored in three cell systems (1,3): a) ~atypical ~ mast cells in the superficial layer of the mucosa (4,5), b) mast cells in the submucose and the muscular layers, and c) snterx~hromaffin-like cells, which are also capable of producing and storing monoamines (5,6). Chemical and histochemical analyses have shown that in the rat this latter system of enter~chromaffin-like cells is the major store of gastric histamine (1,8). The high activity of histidine decarboxylase in the gastric mucosa of the adult rat (6-9) indicates that the histamine present is synthesized locally, presumably within the enter~chromeffin-like cells (3,6). The enzyme is adaptive, and can be activated by, for example, feeding or injection of gastrin (9}. The role of histamine in gastric function has been a matter of some controversy (c_ff_~.10,11). Recent accumulated evidence suggests that, in the rat, histamine acts as e physiological stimulant of gastric secretion. However, the conspicuous species variation in the concentration and distribution of gastric histamine and histidine decarboxylase (6) makes it questionable whether the
25:55
2536
GASTRIC HISTAMINE
Vol. 6, No. 23
postulated role of histamine in gastric function applies to all species. The high DOPA decazboxylase activity observed in gastric mucose of some species, including the rat (6), may indicate that other amines besides histamine have a control function in gastric secretion. Certain monosmines, for exen~ole, affect the volume and composition o~ gastric juice (12). Since the gastric muoosa has been found to contain a system of histamine-storing
cells capable o~ producing
and storing also monosmines (1,5,8), it seems reasonable to assume that amine mechanisms in this particular system of entecochconeffin-like cells plays a mere intricate role in gastric function than that reflected by the storage and release of histamine alone. The'development and cellular localization of gastric histamine and the appearance of histidine decarboxylase in the stomach wall of the young rat were studied in an attempt to obtain a cytochemical basis for further investigation of the physiological significance of the enterochcomaffin-liks cell system in the gastric mucosa.
Material and Methods Animals. All together 54 adult albino rats of either sex, fed ad lib., wsi~ used for histochsmical and chemical studies of gastric histamine. A fur~chsr 55 adult female rats were mated: fetuses were collected 17 - 22 days after matin K, as verified by the presence of sperms in stained vaginal smears. Young rats wer~ obtained at 0-40 days after delivery. Histochsmical demonstration of histamine. Pisces of the stomach were taken from the glandular portion adjacent to the corllified region (cumsn) in 24 adult animals and in 69 young animals aged 0-30 days. For comparison, specimens from the duodenum from 5 of the adult animals were also used for analysis. The preparations were immediately quenched to the temperature of liquid nitr~Ken and then sectioned (20 U thickness) in a Harris cryostat at -30°C. The sections were collected on cold slides and placed in a P205-containing desiccator kept in the cryostat. After vacuum-dryin K overnight the desiccator was removed from the
Vol. 6, No. 23
GASTRIC HISTAMINE
2537
cryostat and evacuation was continued for a further 6 hours at room ten~erature. Meanwhile, 2-phthaldialdehyde was re-crystallized from heptane on the bottom of a glass jar, which was sealed and placed for 5-10 min. in an oven at +100°C to allow vaporization. The jar was then transferred to room ten~ereture0 and the dried tissue sections were removed from the desiccator and placed in the jar for treatment during 90 sec. The sections were afte~ards exposed to the steam of boiling water for 5-10 sec., dried for 5 min. in an oven at +lOO°C, and then mounted in xylene for fluomescence microscopic analysis {1,2). With the excitation filter UG I, two fluorephores could be demonstrated: one blue-fluorescent and one yellow-fluorescent,
depending at least pertly on the time
of water exposure. GG9 or OG4 were used as barrier filters. DeterTnination of histamine. The stomach tissue was weighed and homo£enized in 5 volumes of 5~ trichloraoetic acid. After centrifugation, aliquots of the supernatant were extracted with a mixture of n-butanol and chloroform (15), and histamine was determined by the conventional fluorimetricteohnlque
C13). Detel~nination of hietidine decarboxylase.
The whole stomach was
taken out, cut open along the greater curvature, washed in 0.9~ saline, dried on a filtem paper and weighed. The tissue was then homogenized in 0 . 1 M phosphate buffer (pH 7.0} to a final concentration of 100 n~/ml. The homogenate was centrifuged at 20,000 g at 0° for 20 min. The eupernatant was used either directly as enzyme source, or dialyzed over-night against re-distilled water to remove endogenous histamine. Dialysis was usually performed with extracts from the stomachs of adult rate and of young rate more than three weeks old. A fluorimetrio and a rediometric technique was used for the determination of enzyme activity in 38 animals from various stages of development. All incubations were performed under nitrogen at 37°C in a metabolic incubator equipped with a shaking device. Incubation was stopped by the addition of trichlorecetic acid. For the fluorimetric assays, aliquots (0.I-0.5 ,tl) of the dialysed or nondialysed extract were incubated with I ~
L-histidine
2538
GASTRIC HISTAMINE
Vol.
6~ No.
23
and I uS pyridoxal-5-phosphate in 0 . 1 M phosphate buffer (pH 6.4) in a total volume of I ml. After one hour, incubation was stopped and histamine was extracted with o~anic solvents and determined fluorimetrically (13). In the radiometric assays, 0.05-0.1 ml aliquots were incubated with 2 ~g C14-L -histidine (21.9 mC/mM; Radiochemical Centre, Amereham) and 0.1 p~ pyridoxal-5-phosphate in 0 . 1 M phosphate buffer (pH 7.0) in a total volume of 0.1 ml. After one hour the C14-histamine formed was extracted with organic solvents and determined radiometrically (14).
Results Chemical analysis showed the concentration of gastric histamine in the fetal and neonatal rat to be low (Fig. I). About two weeks agter birth the concentration started to increase ,~rkedly: within
a
fur~cher 2 weeks it reached
a level corTespending to about 50 per cent of the normal adult concentration.
22- Pg Ig
GASTRIC HISTAMINE
18,
14'
10'
6• 2"
|
•
,s
• e eeu
io
DAYS
AFTER MATING
ll
; T
~:
m e-
;
,; DAYS
,;
2s AFTER
3'o BIRTH
3's
, M"'
,.o
ADULT
m
FIG. I Development of histamine in whole stomach. The points up to 15 days after birth are the msans of 2-5 detezTninations, the remainder being based on Im2 determinations. The aduIt value is obtained from 30 determinations (mean + S.E.M.).
Vol.
6~ No.
25
GASTRIC HISTAMINE
2539
FIG. 2
Fluorescence photomicrographs of transversaliy sectioned wall from the glandular portion of the stomach. 115X. The number of entsrochromaffin-like csiis exhibiting an _o-phthaIdialdehyde-induced fluorescence in the mucosaI epitheIium of a 3-week old animai (a) is almost as ia~e as that of an aduit animai [b). Note fIuorescent, histamine-containing mast ceIis in the submucosa (beIow) in both pictures.
The adult level was not reached until the animals were about 40 days old. Within the first few days after birth, histochemicai anaiysis revealed the presence only of a few fIuorescent mast ceiis in the stomach waIi. Scattered ceils, mozlohoiogicaily resembiing enterochcomaffin celis and emitting a fiuocescence of low intensity, were demonstrated in the epithsiium at the bottom of the deveioping gastric glands at 5 - 6 days of age. This probabIy reflects the first histochemicaily demonstrable appearance of histamine in the specific entecochromaffin-like celi system previousiy described (1). The nunber of fiuocescent enterochcomaffin-iike ceiIs increased durinf, the foilewing stages. This was true aiso for the fiuorescent mast cells which, later on, were chacacteristicaiiy located in the superficiai region of the mucose as wail as in the submucosa and the muscuiar iayers. By about 3 weeks after birth the number of fiuorescent mast ceiIs and enterochron~fin-Iike
celis had increased to
almost aduit ieveis (Fig. 2), although the intensity of the fluorescence in the
2540
GASTRIC HISTAMINE
Vol.
6~ No.
23
FIG. 3 Fluorescence photomicmograph of duodenum from adult animal. 160X. o-Phthaldialdehyde-induced histamine fluorescence only in mast cells located in lamina propria of the mucosa.
latter cell system wes still distinctly lower than that in the adult animal. In the duodenum, histamine was histochemically demonstrable only in mast cells (Fig. 3). A significant activity of histidine decarboxylase was found in the stomach of the fetal rat, but it fell to unmeasurable level immediately after birth. The activity then increased progressively; the adult level of gastric histidine decarboxylase was established at I-2 months of age.
Discussion ~-Phthaldialdehyde forms fluorophores with a number of compounds both in solution (16) and under dry conditions (17,16). However, there is strong reason to believe that the blue (or, under certain conditions, yellow) fluorophore observed in certain cell systems after exposure to ~-phthaldialdehyde is due to the presence of histamine: a) Mast cells and b) rabbit trembocytes, known to contain histamine, become fluorescent with ~-phthaldialdehyde (I-3,19). c) Addition of histamine to the peritoneal fluid selectively increases the fluorescence of mast cells (19), which are known to take up and concentrate histamine (20). d) Quantitatively, histidine decarboxylase and histamine vary
Vol.
6,
No.
23
GASTRIC HISTAMINE
2541
characteristically in different regions of the gastric mucosa of the rat according to the distribution of the fiuorescent enterochromeffin-like ceils (1,3.5,6). e) The histamine "profiIe" obtained from serial sectioning of the gastric mucosa paraiiel to its surface (3) coincides with the distribution of fluorescent mast ceiis near the surface and of enterochromeffin-like ceiis basaIly in the mucosa. However, until the chemicaI reactions underiying the formation of the fiuorophora are fuIiy understood it is apparently necessary to combine the histochemical anaIyses with concomitant chemicai determinations of the histamine content. In the newborn and young rat the concentration of histamine in the stomach waii is low and does not reach
adult level until the animal is about 40
days of age. Gastric histidine decarboxyiase foIiows a simiIar course of development,
which supports the assumption that this enzyme is responsibie for
the local formation of histamine. The paraIIei deveIopment of a blue (or yeiiow) o-phthaldiaidehyde-induced fiuorescence in enterechromeffin,-like celIs and in mast ceils offers further strong evidence for the fIuorescence observed being due to the presence of histamine. The storage of histamine in the enterochromaffin-Iike ceils can be estabIished first at about I week after birth. Initiaily, the cells are scanty and oniy weakly fiuorascent,
but there is a progressive increase in n u ~ e r and
fluorescence intensity during subsequent stages. It has recentiy been demonstrated that the histamine-storing enterochromeffin-Iike ceils are capable of taking up certain amino acids and to convert them into their respective monoamines, which are retained in high concentration in the cytoplasm (5,5). In current experiments it has been shown that the enterochx~,,~ffin-like cells have this capacity to produce and store monoamines airsady at birth (21), whereas histamine can not be demonstrated histochemicaliy until I week iatar in these ceils. Why these ceiIs do not deveIop their full capacity to produce and store histamine untii 3-6 weeks after birth is obscure. Changes in food habits, e.~. weaning, may trigger the enzymatic mechanism responsibie for the formation of
2542
GASTRIC H I S T A M I N E
gastric histamine. It should be noted that th
Vol.
(;,
No.
23
appearance of histamine-storing
ceils in the gastric muoosa of the young rat coincides fairly well with the time of onset of hydrochloric acid secretion [22].
SunT~ry With the use of a recently developed fluorescence microscopic technique for the cellular localization of histamine in combination with chemical methods for the assay of histamine and histidine decadooxylase,the appearance of histamine-storin~
cells in the gastric mueosa was followed in the
developing rat. Histamine could be demonstrated histochemically in mast cells soon after birth. At the age of one week histamine appeared in a system of enterochromaffin-like
cells. The histochemical findings agree well with the low
concentration of histamine found in the stomach wall. The subsequent increase in nun~er and fluorescence intensity of the histamine-storing cells coincided with the increase in the content of histamine and the activity of histidine decarboxylase in the stomach. The adult level of gastric histamine was reached at about 40 days after birth.
Acknowlndgement. Supported by grant from the Association for the Aid of Crippled Children, New York; the Swedish Medical Research Council [No. K67-12X-1007-02); Albert P&hlsson's Foundation.
References I.
H&kanson, R. and Ch. Owman, Life Sci. 6, 75g (1967).
2.
Ehinger, B. and R. Thunbe~, Exp. Cell Res, In press. (1967).
3.
Thunber~, R., Exp. Cell Res. In press. [1967].
4.
Enerb~ck, L., Acta path. microbiol, stand. 67, 365 [1966).
5.
H~kanson, R., B. Lilja and Ch. Owman, Eurep. J. PharTnacol. ~, 188 [1967).
5.
H~kanson, R. and Ch. E ~ n ,
7.
Sehayer, R.W., Amer. J. Physiol. I~, 533 (1957).
Biochem. PhazTnacol. 15, 469 (1966).
Vol.
8.
6~ No.
23
GASTRIC HISTAMINE
2543
Weissbach, H., W. Lovenberg and S. Uden~riend, Biochim. Biophys. Acre 50, 177 (1961).
9.
Kahlson, G., E. Rosengren, E. Svshn and R, Thunberg, J. Physioi. (Lend.) 174, 400 (1964).
10. Code, C.F., Histamine and gastric secretion. In: G.E.W. Wolstenhoin13 and C.M. O'Connor [Ed.), Ciba Foundation S~nposium on Histamine. Churchill, London. p. 169 (1956). 11. Code, C.F., Fed. Prec. 24, 1311 (1965). 12. H&kanson, R., B. L i I j a , Ch. Owman and S. Thunell, Europ. J. Pharmacol. In press. (1967). 13. Shore, P.A., A. Burkhalter and V.H. Cohn, J. Pharmacoi. Exp. Ther. 127, 182 (1959). 14. H~kanson, R., Acta phern~coI, et t o x i c o i . 2~, 217 (1966). 15. Burkhalter, A., Biochem. Pharmacoi. 11, 315 (1962). 16. Uden{riend, S., Fiuorescence Assay in Biology and Medicine. Academic Press, New York, London. (1962). 17. SheIiey, W.B. and L. 3uhiin, J. Chromates, 22, 130 (1966). 18. Aures, D., R. Fleming and R. H~kanson, Unpubiished observations. (1967). 19. Juhlin, L. and W.B. Sheiiey, J. Histochem. Cytochem. 1~4, 525 (1966). 20, Furano, A.V. and 3.P. Green, J. Physiol. (Lend.) 170, 263 (1964). 21. H&kanson, R., Ch. Owman and N.-O. SjSbez~,, Unpublished observations. (1967) 22. HiI1, K.J., Quart. J. exp. Physiol. 41, 421 (1956).
2535-2543,
1967.
Pergamon Press
Ltd.
CELLULAR STORESOF GASTRIC HISTAMINE IN THE DEVELOPINGRAT R. HAkanson, Ch. Ow,mn and N.-O. SjSber~ Departments of Pharwecology, Anatomy, and Obstetrics and Gynecology at MalmS, University of Lund, Lund, Sweden.
(Received
12 S e p t e m b e r
1967)
A histochemical method sensitive enough for the demonstration of histamine at the cellular level has recently been devised (I-3). According to this method, freeze-dried tissue sections are exposed to o-phthsldialdehyde vapor, which converts histamine into a fluorophore emitting an intense blue or, under certain conditions, yellow light. In the normal adult rat, gastric histamine has been found to be stored in three cell systems (1,3): a) ~atypical ~ mast cells in the superficial layer of the mucosa (4,5), b) mast cells in the submucose and the muscular layers, and c) snterx~hromaffin-like cells, which are also capable of producing and storing monoamines (5,6). Chemical and histochemical analyses have shown that in the rat this latter system of enter~chromaffin-like cells is the major store of gastric histamine (1,8). The high activity of histidine decarboxylase in the gastric mucosa of the adult rat (6-9) indicates that the histamine present is synthesized locally, presumably within the enter~chromeffin-like cells (3,6). The enzyme is adaptive, and can be activated by, for example, feeding or injection of gastrin (9}. The role of histamine in gastric function has been a matter of some controversy (c_ff_~.10,11). Recent accumulated evidence suggests that, in the rat, histamine acts as e physiological stimulant of gastric secretion. However, the conspicuous species variation in the concentration and distribution of gastric histamine and histidine decarboxylase (6) makes it questionable whether the
25:55
2536
GASTRIC HISTAMINE
Vol. 6, No. 23
postulated role of histamine in gastric function applies to all species. The high DOPA decazboxylase activity observed in gastric mucose of some species, including the rat (6), may indicate that other amines besides histamine have a control function in gastric secretion. Certain monosmines, for exen~ole, affect the volume and composition o~ gastric juice (12). Since the gastric muoosa has been found to contain a system of histamine-storing
cells capable o~ producing
and storing also monosmines (1,5,8), it seems reasonable to assume that amine mechanisms in this particular system of entecochconeffin-like cells plays a mere intricate role in gastric function than that reflected by the storage and release of histamine alone. The'development and cellular localization of gastric histamine and the appearance of histidine decarboxylase in the stomach wall of the young rat were studied in an attempt to obtain a cytochemical basis for further investigation of the physiological significance of the enterochcomaffin-liks cell system in the gastric mucosa.
Material and Methods Animals. All together 54 adult albino rats of either sex, fed ad lib., wsi~ used for histochsmical and chemical studies of gastric histamine. A fur~chsr 55 adult female rats were mated: fetuses were collected 17 - 22 days after matin K, as verified by the presence of sperms in stained vaginal smears. Young rats wer~ obtained at 0-40 days after delivery. Histochsmical demonstration of histamine. Pisces of the stomach were taken from the glandular portion adjacent to the corllified region (cumsn) in 24 adult animals and in 69 young animals aged 0-30 days. For comparison, specimens from the duodenum from 5 of the adult animals were also used for analysis. The preparations were immediately quenched to the temperature of liquid nitr~Ken and then sectioned (20 U thickness) in a Harris cryostat at -30°C. The sections were collected on cold slides and placed in a P205-containing desiccator kept in the cryostat. After vacuum-dryin K overnight the desiccator was removed from the
Vol. 6, No. 23
GASTRIC HISTAMINE
2537
cryostat and evacuation was continued for a further 6 hours at room ten~erature. Meanwhile, 2-phthaldialdehyde was re-crystallized from heptane on the bottom of a glass jar, which was sealed and placed for 5-10 min. in an oven at +100°C to allow vaporization. The jar was then transferred to room ten~ereture0 and the dried tissue sections were removed from the desiccator and placed in the jar for treatment during 90 sec. The sections were afte~ards exposed to the steam of boiling water for 5-10 sec., dried for 5 min. in an oven at +lOO°C, and then mounted in xylene for fluomescence microscopic analysis {1,2). With the excitation filter UG I, two fluorephores could be demonstrated: one blue-fluorescent and one yellow-fluorescent,
depending at least pertly on the time
of water exposure. GG9 or OG4 were used as barrier filters. DeterTnination of histamine. The stomach tissue was weighed and homo£enized in 5 volumes of 5~ trichloraoetic acid. After centrifugation, aliquots of the supernatant were extracted with a mixture of n-butanol and chloroform (15), and histamine was determined by the conventional fluorimetricteohnlque
C13). Detel~nination of hietidine decarboxylase.
The whole stomach was
taken out, cut open along the greater curvature, washed in 0.9~ saline, dried on a filtem paper and weighed. The tissue was then homogenized in 0 . 1 M phosphate buffer (pH 7.0} to a final concentration of 100 n~/ml. The homogenate was centrifuged at 20,000 g at 0° for 20 min. The eupernatant was used either directly as enzyme source, or dialyzed over-night against re-distilled water to remove endogenous histamine. Dialysis was usually performed with extracts from the stomachs of adult rate and of young rate more than three weeks old. A fluorimetrio and a rediometric technique was used for the determination of enzyme activity in 38 animals from various stages of development. All incubations were performed under nitrogen at 37°C in a metabolic incubator equipped with a shaking device. Incubation was stopped by the addition of trichlorecetic acid. For the fluorimetric assays, aliquots (0.I-0.5 ,tl) of the dialysed or nondialysed extract were incubated with I ~
L-histidine
2538
GASTRIC HISTAMINE
Vol.
6~ No.
23
and I uS pyridoxal-5-phosphate in 0 . 1 M phosphate buffer (pH 6.4) in a total volume of I ml. After one hour, incubation was stopped and histamine was extracted with o~anic solvents and determined fluorimetrically (13). In the radiometric assays, 0.05-0.1 ml aliquots were incubated with 2 ~g C14-L -histidine (21.9 mC/mM; Radiochemical Centre, Amereham) and 0.1 p~ pyridoxal-5-phosphate in 0 . 1 M phosphate buffer (pH 7.0) in a total volume of 0.1 ml. After one hour the C14-histamine formed was extracted with organic solvents and determined radiometrically (14).
Results Chemical analysis showed the concentration of gastric histamine in the fetal and neonatal rat to be low (Fig. I). About two weeks agter birth the concentration started to increase ,~rkedly: within
a
fur~cher 2 weeks it reached
a level corTespending to about 50 per cent of the normal adult concentration.
22- Pg Ig
GASTRIC HISTAMINE
18,
14'
10'
6• 2"
|
•
,s
• e eeu
io
DAYS
AFTER MATING
ll
; T
~:
m e-
;
,; DAYS
,;
2s AFTER
3'o BIRTH
3's
, M"'
,.o
ADULT
m
FIG. I Development of histamine in whole stomach. The points up to 15 days after birth are the msans of 2-5 detezTninations, the remainder being based on Im2 determinations. The aduIt value is obtained from 30 determinations (mean + S.E.M.).
Vol.
6~ No.
25
GASTRIC HISTAMINE
2539
FIG. 2
Fluorescence photomicrographs of transversaliy sectioned wall from the glandular portion of the stomach. 115X. The number of entsrochromaffin-like csiis exhibiting an _o-phthaIdialdehyde-induced fluorescence in the mucosaI epitheIium of a 3-week old animai (a) is almost as ia~e as that of an aduit animai [b). Note fIuorescent, histamine-containing mast ceIis in the submucosa (beIow) in both pictures.
The adult level was not reached until the animals were about 40 days old. Within the first few days after birth, histochemicai anaiysis revealed the presence only of a few fIuorescent mast ceiis in the stomach waIi. Scattered ceils, mozlohoiogicaily resembiing enterochcomaffin celis and emitting a fiuocescence of low intensity, were demonstrated in the epithsiium at the bottom of the deveioping gastric glands at 5 - 6 days of age. This probabIy reflects the first histochemicaily demonstrable appearance of histamine in the specific entecochromaffin-like celi system previousiy described (1). The nunber of fiuocescent enterochcomaffin-iike ceiIs increased durinf, the foilewing stages. This was true aiso for the fiuorescent mast cells which, later on, were chacacteristicaiiy located in the superficiai region of the mucose as wail as in the submucosa and the muscuiar iayers. By about 3 weeks after birth the number of fiuorescent mast ceiIs and enterochron~fin-Iike
celis had increased to
almost aduit ieveis (Fig. 2), although the intensity of the fluorescence in the
2540
GASTRIC HISTAMINE
Vol.
6~ No.
23
FIG. 3 Fluorescence photomicmograph of duodenum from adult animal. 160X. o-Phthaldialdehyde-induced histamine fluorescence only in mast cells located in lamina propria of the mucosa.
latter cell system wes still distinctly lower than that in the adult animal. In the duodenum, histamine was histochemically demonstrable only in mast cells (Fig. 3). A significant activity of histidine decarboxylase was found in the stomach of the fetal rat, but it fell to unmeasurable level immediately after birth. The activity then increased progressively; the adult level of gastric histidine decarboxylase was established at I-2 months of age.
Discussion ~-Phthaldialdehyde forms fluorophores with a number of compounds both in solution (16) and under dry conditions (17,16). However, there is strong reason to believe that the blue (or, under certain conditions, yellow) fluorophore observed in certain cell systems after exposure to ~-phthaldialdehyde is due to the presence of histamine: a) Mast cells and b) rabbit trembocytes, known to contain histamine, become fluorescent with ~-phthaldialdehyde (I-3,19). c) Addition of histamine to the peritoneal fluid selectively increases the fluorescence of mast cells (19), which are known to take up and concentrate histamine (20). d) Quantitatively, histidine decarboxylase and histamine vary
Vol.
6,
No.
23
GASTRIC HISTAMINE
2541
characteristically in different regions of the gastric mucosa of the rat according to the distribution of the fiuorescent enterochromeffin-like ceils (1,3.5,6). e) The histamine "profiIe" obtained from serial sectioning of the gastric mucosa paraiiel to its surface (3) coincides with the distribution of fluorescent mast ceiis near the surface and of enterochromeffin-like ceiis basaIly in the mucosa. However, until the chemicaI reactions underiying the formation of the fiuorophora are fuIiy understood it is apparently necessary to combine the histochemical anaIyses with concomitant chemicai determinations of the histamine content. In the newborn and young rat the concentration of histamine in the stomach waii is low and does not reach
adult level until the animal is about 40
days of age. Gastric histidine decarboxyiase foIiows a simiIar course of development,
which supports the assumption that this enzyme is responsibie for
the local formation of histamine. The paraIIei deveIopment of a blue (or yeiiow) o-phthaldiaidehyde-induced fiuorescence in enterechromeffin,-like celIs and in mast ceils offers further strong evidence for the fIuorescence observed being due to the presence of histamine. The storage of histamine in the enterochromaffin-Iike ceils can be estabIished first at about I week after birth. Initiaily, the cells are scanty and oniy weakly fiuorascent,
but there is a progressive increase in n u ~ e r and
fluorescence intensity during subsequent stages. It has recentiy been demonstrated that the histamine-storing enterochromeffin-Iike ceils are capable of taking up certain amino acids and to convert them into their respective monoamines, which are retained in high concentration in the cytoplasm (5,5). In current experiments it has been shown that the enterochx~,,~ffin-like cells have this capacity to produce and store monoamines airsady at birth (21), whereas histamine can not be demonstrated histochemicaliy until I week iatar in these ceils. Why these ceiIs do not deveIop their full capacity to produce and store histamine untii 3-6 weeks after birth is obscure. Changes in food habits, e.~. weaning, may trigger the enzymatic mechanism responsibie for the formation of
2542
GASTRIC H I S T A M I N E
gastric histamine. It should be noted that th
Vol.
(;,
No.
23
appearance of histamine-storing
ceils in the gastric muoosa of the young rat coincides fairly well with the time of onset of hydrochloric acid secretion [22].
SunT~ry With the use of a recently developed fluorescence microscopic technique for the cellular localization of histamine in combination with chemical methods for the assay of histamine and histidine decadooxylase,the appearance of histamine-storin~
cells in the gastric mueosa was followed in the
developing rat. Histamine could be demonstrated histochemically in mast cells soon after birth. At the age of one week histamine appeared in a system of enterochromaffin-like
cells. The histochemical findings agree well with the low
concentration of histamine found in the stomach wall. The subsequent increase in nun~er and fluorescence intensity of the histamine-storing cells coincided with the increase in the content of histamine and the activity of histidine decarboxylase in the stomach. The adult level of gastric histamine was reached at about 40 days after birth.
Acknowlndgement. Supported by grant from the Association for the Aid of Crippled Children, New York; the Swedish Medical Research Council [No. K67-12X-1007-02); Albert P&hlsson's Foundation.
References I.
H&kanson, R. and Ch. Owman, Life Sci. 6, 75g (1967).
2.
Ehinger, B. and R. Thunbe~, Exp. Cell Res, In press. (1967).
3.
Thunber~, R., Exp. Cell Res. In press. [1967].
4.
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