Biochimica et Biophysica Acta 928 (1987) 227-234 227 Elsevier

BBA 12005

Induction of histidine decarboxylase by dexamethasone

in mastocytoma P-815 cells

N o r i a k i Imanish i , T a k a h i r o N a k a y a m a , M a m i Asano , K i m i o Y a t s u n a m i ,

K c n k i c h i T o m i t a a n d Atsush i I ch ikawa

Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto (Japan)

(Received 13 November 1986)

Key words: Histidine decarboxylase; Enzyme induction; Dexamethasone; (Mastocytoma P-815 cell)

Dexamethasone at a concentration as low as 10 nM significantly increased both the histamine content and histidine decarboxylase activity of cultured mastocytoma P-815 cells. Both effects were clearly seen using several glucocorticoids, which were as effective as dexamethasone. In contrast to that of histamine, the serotonin level of mastocytoma P-815 cells was decreased by treatment with dexamethasone. The de- xamethasone-induced increases in histamine content and histidine decarboxylase activity were completely suppressed by the addition of cycloheximide and actinomycin D. Mastocytoma P-815 cells were found to possess binding sites for [3Hldexamethasone in the cytosol (K d -- 15.7 nM) and the nuclei (K d -- 1.26 nM). These results show that glucocorticoids significantly stimulate de novo synthesis of histidine decarboxylase.

Introduction

Glucocorticoids, which are among the most ef- fective antiallergic drugs, also induce some specific enzymes in mammalian tissues [1]. The activity level of histidine decarboxylase (EC 4.1.1.22), which synthesizes histamine in mast cells, is known to vary with treatment of animals with gluco- corticoids. However, the few results that have been published are inconsistent. ReiUy and Schayer [2] detected a 60% increase in this enzyme activity in the stomach, but not in other tissues, after subcutaneous injection of hydrocortisone into adrenalectomized mice. Sandberg and Steinhardt [3] reported the suppression of this enzyme activ- ity in injured rat skin after cortisone administra- tion. These results suggest that the effect of gluco-

Correspondence: A. Ichikawa, Department of Health Chem- istry, Faculty of Pharmaceutical Sciences, Kyoto Univeristy, Yoshida, Sakyo-ku, Kyoto 606, Japan.

corticoids on histidine decarboxylase might be dependent on the different type(s) of histamine- synthesizing cells in these tissues [4]. However, the change of the histidine decarboxylase activity by glucocorticoids has not been determined in any type of the histamine synthesizing cells.

In contrast to their antiallergic action, gluco- corticoids were reported to produce severe ulcera- tion lesion in the stomach, which were completely inhibited by treatment of the animals with an H2-antagonist [5]. The mechanism of this effect is unclear. A monoclonal antibody against histidine decarboxylase from rat stomach was reported to recognize the enzyme in mastocytoma P-815 cells [6]. Although the type of histamine-synthesizing cells responding to glucocorticoid has not yet been identified in the stomach, mastocytoma P-815 cells provide a valuable cell model for investigating the effects of glucocorticoids on histidine decarboxy- lase. This report describes an increase in the de novo synthesis of histidine decarboxylase of mastocytoma P-815 cells by dexamethasone.

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

228

Materials and Methods

Cells and cell culture Mastocytoma P-815 cells were supplied by Dr.

M. Potter and were maintained either in a suspen- sion culture or in an ascitic form in BDF 1 mice [7]. The cells were harvested from the ascitic fluid of inoculated mice by centrifugation at 280 x g for 5 min at 4°C in siliconized centrifuge tubes and washed twice with cold phosphate-buffered saline. The cell culture and viability test were performed as described previously [7].

Preparation of cytosol and nuclei All operations were performed at 0-4°C. The

cells were suspended in 15 vol. buffer A (10 mM Tris/1 mM EDTA/12 mM thioglycerol/0.5 mM antipain, pH 7.35), homogenized in a Dounce type glass homogenizer and then centrifuged at 100 000 x g for 60 min to obtain the supernatant (cytosol) fraction.

For isolation of the nucelar fraction, the packed cells were suspended for 7 min in a hypotonic buffer B (10 mM Tris/1 mM E D T A / 4 mM MgCI2, pH 7.80) at 2.106 cells/ml. The cells were then resuspended in buffer B at 7- 107 cells/ ml, homogenized by 20 strokes in a Dounce type glass homogenizer, cell rupture being monitored under a phase contrast microscope, and then centrifuged at 1000 x g for 15 min. The pellets (nuclear fraction) obtained were washed once with the same buffer.

Assay methods Histamine and serotonin were extracted by

heating cells suspended in a 2.1% HC104 solution at 80°C for 10 min. After centrifuging at 280 x g for 5 min, the clear supernatant solution was assayed for histamine and serotonin according to Shore et al. [8] and Snyder et al. [9], respectively.

Histidine decarboxylase was assayed by mea- suring 14CO2 produced from [14C]histidine at 37°C in a reaction mixture (0.5 ml) containing 100 #M DL-[carboxy-lJ4C]histidine (0.2 #Ci), 10 /~M pyridoxal phosphate and 50 #M potassium phosphate (pH 6.8). t-Tryptophan 5-hydroxylase (EC 1.14.16.4) [10], chymotrypsin-like mast cell proteinase (chymase) [11], N-acetyl-fl-gluco- saminidase (EC 3.2.1.30) [12], fl-N-acetyl-o-

hexosaminidase (EC 3.2.1.52) [13] and acid phos- phatase (EC 3.1.3.2) [14], NADPH-cytochrome-c oxidoreductase (EC 1.6.2.4) [15], succinate dehy- drogenase (EC 1.3.99.1) [16], 5'-nucleotidase (EC 3.1.3.5) [17] and tyrosine transaminase (EC 2.6.1.5) [18] were assayed as described in the literature. One unit of enzyme activity was defined as the amount of protein required to transform 1 #mol of substrate per rain under the assay conditions employed. Protein was determined either with Folin-Ciocalteu reagent [19] or absorbance at 280 nm as described by Layne [20].

Steroid binding assays Cytosol binding. A 1 ml aliquot of the cytosol

fraction was incubated with 7 nM [3H]dexametha- sone at 4 °C for 2 h. The reaction was terminated by adding a 0.4 ml dextran-coated charcoal sus- pension (1% Norit A and 0.1% dextran T-70 in buffer A), followed by a further 2 h incubation for adsorption of the unbound labeled steroid to the charcoal. After centrifugation at 10 000 X g for 10 rain, the clear supernatant fraction obtained was assayed for radioactivity of the cytosol-bound [3H]dexamethasone in 8 ml 0.5% 2,5-diphenyl- oxazole in Tri ton/toluene (1 : 3, v/v).

Nuclear binding. Nuclear binding for masto- cytoma P-815 cells was carried out essentially according to the method of Svec and Harrison [21]. Mastocytoma P-815 cells (3.10 6 cells) were suspended in 0.4 ml of Eagle's minimum essential medium without fetal calf serum, followed by incubation with 7 nM [3H]dexamethasone (0.22 /~Ci) in the presence or absence of 1 /~M various non-radioactive steroids (1 ttM) at 37°C for 30 min. The reaction was terminated by adding 4 ml of an ice-cold buffer C (6 mM MgCI 2 and 0.05% (v/v) Triton X-100). After 5 min, the reaction mixture was centrifuged at 800 x g for 5 min at 4°C. The pellet obtained was washed twice with ice-cold buffer C containing 6 mM 2-mercapto- ethanol and then counted for radioactivity in 0.5% 2,5-diphenyloxazole in 8 ml toluene scintillant containing 0.4 ml Nuclear Chicago Scintillator. The purity of the nuclear preparations was about 80%, as determined by phase microscope monitor- ing. Non-specific binding was defined as that which was not competed for with a 10a-fold excess of the unlabeled dexamethasone.

Preparation of the dexamethasone-receptor complex and its cell-free nuclear binding

Dexamethasone-receptor complex. The complex was prepared according to the method of Kon and Spelsberg [22]. 1 ml cytosol fraction of masto- cytoma P-815 cells (1 ml) was incubated with [3H]dexamethasone dissolved in 5 /~1 buffer A / ethanol (4:1, v / v ) to give a final dexamethasone concentration of approx. 10 nM. The [3H]de- xamethasone-cytosol receptor complex was then precipitated with 40% (w/w) ammonium sulfate and stored at - 8 0 ° C . Prior to use, the complex was resuspended in buffer A, dialyzed against the same buffer for 3 h at 4°C and then centrifuged at 10 000 × g for 30 rain. The clear supernatant frac- tion (260 fmol dexamethasone /mg protein) was used for the nuclear binding assay.

Nuclear binding. 1 ml reaction mixture, which contained approx. 1.107 nuclei, [3H]dexametha- sone-cytosol receptor complex and 0.18 M KC1 in buffer A, was incubated at 22°C for 90 min with intermittent mixing. The reaction was terminated by centrifugation at 800 × g for 10 min, and then the pelleted nuclei were washed twice with 1 ml buffer A and counted in toluene scintillant as described.

Materials The materials used were obtained from the

following sources: [1,2,4,6,7-3H]dexamethasone (77 C i / m m o l ) , OL-[ carboxy1-14 C]histidine (51 C i / mmol) and a tissue solubilizer, Nuclear Chicago Scintillator, Amersham-Japan (Tokyo, Japan); and dexamethasone, progesterone, 17fl-estradiol, pred- nisolone, testosterone, corticosterone and hydro- cortisone, Nakarai Pure Chemicals (Kyoto, Japan). All other chemicals were commercial products of reagent grade.

Results

Effects of dexamethasone on the levels of histamine and histidine decarboxylase activity in cultured mastocytoma P-815 cells

As shown in Fig. 1, dexamethasone, at con- centrations ranging from 10 -9 to 10 -3 M, signifi- cantly increased the histamine content and histi- dine decarboxylase activity of mastocytoma P-815 cells incubated for 24 h at 37 ° C; the maximum

229

A Q

o c E

.I-

÷

~ n ~ o

~ m "o o

Dexamethasone ( M )

Fig. 1. Effects of dexamethasone on the histamine content and the histidine decarboxylase activity of mastocytoma P-815 cells. Mastocytoma P-815 cells (8.104 cells/ml) were cultured in 100 ml of Fischer-Sartorelli's medium containing de- xamethasone at concentrations ranging from 10 9 to 10-3 M for 24 h at 37°C. The cells were harvested, washed twice with 20 ml of cold phosphate-buffered saline and then assayed for the histamine content (e) and the histidine decarboxylase activity (©). Each value is the mean+ S.E. for triplicate sam- pies. The observed values are statistically significant at P < 0.01, as compared to the vehicle value without dexamethasone.

response occurred at around at 10 - 6 M. When the cells were incubated with 10 -7 M dexamethasone for 24 h at 37°C and then transferred to a steroid-free medium, the elevated histamine con- tent and histidine decarboxylase activity levels both gradually decreased to the original levels (Fig. 2).

Among various steroids tested at 3 . 1 0 -v M, glucocorticoids such as prednisolone, hydrocorti- sone and corticosterone were about as effective as dexamethasone in increasing the histamine level of mastocytoma P-815 cells on 20 h incubation; sex steroid hormones such as progesterone, testoster- one and estradiol were, however, not effective. In contrast, serotonin levels were decreased by treat- ment with glucocorticoids and not affected by sex steroid hormones (Table I).

In addition, dexamethasone had no significant effect on the activities of enzymes other than histidine decarboxylase (Table II). Dexametha- sone showed no effect on tryptophan 5-hydroxyl- ase, the rate limiting enzyme in serotonin bio- synthesis, suggesting that the decrease in the serotonin level in the presence of dexamethasone (Table I) was not due to a direct inhibitory action of the drug on the enzyme. Furthermore, de- xamethasone had no effect on tyrosine amino-

230

6[ T ~ , . . ~ -112 ¢

-

¢ 3 16 .o .~

J o • o

0 24 48 72 Time (h)

TABLE I

EFFECTS OF VARIOUS STEROIDS ON THE HISTA- MINE AND SEROTONIN CONTENTS OF MASTOCYTO- MA P-815 CELLS

Mastocytoma P-815 cells (8.104 cells/ml) were cultured in 30 ml of Fischer-Sartorelli's medium containing various steroids at a concentration of 3.10 -7 M for 20 h at 37°C. The cells were harvested, washed and then assayed for the histamine and serotonin contents. Each value (#g / 10 s cells) is the mean 5: S.E. for triplicate samples. * P < 0.01.

Steroid Histamine Serotonin

None 2.90+0.38 1.73 +0.10 Dexamethasone 11.8 -t-0.9 * 0.4725:0.088 * Prednisolone 9.955:0.15 * 0.608+0.120 * Hydrocortisone 9.90 5:0.50 * 0.476 5:0.076 * Corticosterone 10.1 5:1.0 * 0.6085:0.120 * Progesterone 3.525:0.30 1.74 5:0.16 Testosterone 2.95 5:0.33 1.80 5:0.17 Estradiol 3.095:0.19 1.58 5:0.12

Fig. 2. Effect of the removal of dexamethasone on the hista- mine content and histidine decarboxylase activity of de- xamethasone-stimulated mastocytoma P-815 cells for 24 h. Mastocytoma P-815 cells (8-104 cells/ml) were cultured in 200 ml of Fischer-Sartorelli's medium containing 10 -7 M de- xamethasone at 37°C ( ). At 24 h after steroid treat- ment, 100 ml of the cell suspension was removed, washed and then transferred to a steroid-free medium ( . . . . . . ). The cells were incubated for a further 48 h at 37° C. Each value is the mean + S.E. for triplicate samples.

transferase which was reported to be induced by glucocorticoids in rat liver [23], but not in other tissues [24].

Effects of cycloheximide and actinomycin D on the dexamethasone responses of histamine and histidine decarboxylase

5 /~g/ml cycloheximide and 0.5 /~g/ml actinomycin D, which did not retard cell prolifera- tion for at least 20 h at these doses, suppressed the dexamethasone-induced increases of both the histamine content and the histidine decarboxylase activity (Table III). Cycloheximide was more potent than actinomycin D. Simultaneous and prior addition of actinomycin D to dexametha- sone-treated mastocytoma P-815 cells completely suppressed the increase in the histamine content during subsequent incubation. However, the addition of actinomycin D to the cells, which had

TABLE II

EFFECTS OF DEXAMETHASONE ON VARIOUS ENZYME ACTIVITIES OF MASTOCYTOMA P-815 CELLS

Mastocytoma P-815 cells (8.105 cells/ml) were incubated in Fischer-Sartorelli's medium with or without 3 .10-7 M dexamethasone for 30 h at 37 o C, and then various enzyme activities were assayed. Each value (mUni t /mg protein) is the mean + S.E. for three determinations. * P < 0.01.

Enzyme - D e x a m e t h a s o n e + Dexamethasone (3-10 -7 M)

Histidine decarboxylase N-Acetyl-fl-glucosaminidase fl-N-Acetyl-D-hexosaminidase Tryptophan 5-hydroxylase Tyrosine aminotransferase Acid phosphatase 5 '-Nucleotidase NADPH-cytochrome-c oxidoreductase Succinate dehydrogenase Chymotrypsin-like proteinase

0.0042+ 0.0015 0.063+ 0.005 * 0.24 + 0.01 0.23 + 0.02

13 + 1.2 14 + 0.8 0.158 + 0.009 0.162+ 0.025 0.22 + 0.04 0.21 + 0.027

10 + 1.1 10 + 2.0 58 5: 2.1 54 _+ 1.4

6.6 _ 0.0 6.1 + 0.1 2000 +190 2100 _+130 1300 + 260 1500 _+ 180

TABLE III

EFFECTS OF CYCLOHEXIMIDE AND ACTINOMYCIN D ON THE HISTAMINE LEVEL OF MASTOCYTOMA P-815 CELLS

Mastocytoma P-815 cells (8-104 cells/ml) were cultured in 30 ml of Fischer-Sartorelli's medium containing 3-10 -7 M de- xamethasone (5 /~g/ml) and/or actinomycin D (0.5 /lg/ml) for 20 h at 37 o C. The cells were harvested, washed and then assayed for the histamine content and histidine decarboxylase activity. Each value is the mean + S.E. for triplicate samples. * P < 0.01.

Treatment Histamine Histidine (/~g/10 s cells) decarboxylase

( × 105 U/107cells)

None 1.50 +0.43 0.533+0.100 Dexamethasone 5.93 +0.14 5.03 +0.12 Cycloheximide 0.733 + 0.120 0.533 + 0.067 Actinomycin D 0.947 + 0.120 0.600 :k 0.120 Dexamethasone

+cycloheximide 1.01 +0.11 * 0.647+0.073 * Dexamethasone

+actinonycin D 3.01 +0.12 * 2.03 +0.11 *

been pretreated for 2 h with dexamethasone, resulted in a 50% reduct ion of the increase in the h is tamine level induced by the steroid alone.

Ac t inomycin D added after 6 h to 8 h pre incuba- t ion with dexamethasone did not suppress the

accumula t ion of cellular h is tamine (Fig. 3). In contrast , cycloheximide always suppressed the in-

crease in the h is tamine level, regardless of the t ime of its addi t ion to the steroid-pretreated cells (data

no t shown).

Dexamethasone binding to cytosolic and nuclear fractions of mastocytoma P-815 cells

Since the act ion of glucocorticoids is known to

be mediated via their in teract ion with b ind ing proteins in the cytosol and nuclear fractions, the characteristics of [3H]dexamethasone b ind ing to

mas tocytoma P-815 cells were investigated. Cytosol fraction. Scatchard plot analysis of the

total [3H]dexamethasone b ind ing to the cytosol fract ion indicated the existence of one type of b ind ing site with a K d value of 1 .57 .10 -8 M and a b ind ing capacity of 66.0 f m o l / m g protein, cor- responding to 2450 b ind ing sites per cell (Fig. 4). A m o n g various steroids tested, glucocorticoids and progesterone bu t no t other sex steroids were potent

231

X(h) (24-X)(h)

100. ] myc n D '

"R Z= ~ 5 0 "

o ~; ,~ ~ X(h)

Fig. 3. Effect of actonomycin D on the histamine level of dexamethasone-stimulated mastocytoma P-815 cells. Masto- cytoma P-815 cells (1.5-105 cells/ml) were cultured at 37 °C in 30 ml of Fischer-Sartorelli's medium containing 3 "10 -7 M dexamethasone. After X h (0-8 h) incubation, 0.5 #g/ml actinomycin D was added to the culture, and the incubation was continued until a total incubation time of 24 h. Then the cells were harvested and assayed for the histamine content. Each value is the mean_+ S.E. for triplicate samples.

in replacing [3H]dexamethasone as to b ind ing to

the cytosol fraction (Table IVa). Nuclear fraction. As shown in Fig. 5, the b ind-

ing of the [3H]dexamethasone-cytosol receptor

complex to the isolated nuclear fraction increased

with increasing amount s of the complex and rea- ched a plateau level at about 2 mg protein of the

0 - , , ' , . . . . .

o 5 0 Bound DM(fmollmg)

Fig. 4. Scatchard plots of [3 H]dexamethasone (DM) binding to mastocytoma P-815 cells. The cytosol fraction (0.5 mg pro- tein/ml) of mastocytoma P-815 cells was incubated for 2 h at 4°C with 7 nM [3H]dexamethasone in the presence of various concentrations of non-labeled dexamethasone. The unbound [3H]dexamethasone was removed through adsorption on char- coal-dextran. The binding data were plotted according to Scatchard [45].

232

3 0 ¸ ¢r 0

20- =o c

% -~ 10. E

(.9

A

~ 5ool

0

GR(mg)

m 0 1'0 2'0 30 40 Bound GR(fmol/107 n u c l e i )

Fig. 5. Analysis of the saturable binding of the mastocytoma P-815 cell [3H]dexamethasone-receptor complex to ho- mologous nuclei mixture. [3H]Dexamethasone-receptor com- plex, precipitated on 40% saturation with ammonium sulfate (GR), was added to a nuclei (1 "10 7) suspension, followed by incubation for 90 min at 22°C. The nuclei were centrifuged and washed, and then the residual radioactivity in nuclei was counted. A constant amount of nuclei was titrated against varying concentrations of ammonium sulfate. Each value is the mean + S.E. for triplicate samples (inset). The data were plotted according to the method of Scatchard [45].

TABLE IV

EFFECTS OF VARIOUS STEROIDS ON [3H]DEXA- METHASONE BINDING TO CYTOSOLIC AND NUCLEAR FRACTIONS

(a) An aliquot of the cytosol fraction prepared from masto- cytoma P-815 cells was incubated with 7 nM [3H]dexametha- sone f0.22 #Ci) with 1/tM each nonradioactive steroid for 2 h at 4°C. (b) Mastocytoma P-815 cells (3.106 cells) were in- cubated with 7 nM [3H]dexamethasone (0.22 ~Ci) in the presence or absence of various non-radioactive steroids (1/tM) for 30 min at 37°C. The [3H]dexamethasone binding to the cytosolic and nuclear fractions of mastocytoma P-815 cells was assayed. Each value represents the meaniS.E, for three de- terminations. Statistical significance: * P < 0.01, ** P < 0.05.

Non-radioactive (a) (b) steroid added Cytosol fractions Nuclear fractions

(fmol/mg protein) (fmol/106 nuclei)

None 38.80 ± 0.66 0.27 ± 0.02 Dexamethasone 4.80±0.40 * 0.16 ± 0.03 ** Prednisolone 5.61 _+ 1.80 * 0.19 ± 0.01 ** Hydrocortisone 4.00±0.81 * 0.15_+0.02 ** Corticosterone 4.63_+0.20 * 0.18±0.01 ** Testosterone 30.80 ± 0.80 0.24 ± 0.01 Estradiol 29.40 ± 0.40 0.27 ± 0.01 Progesterone 6.40 _+ 0.22 * * 0.19 ± 0.01 * *

TABLE V

EFFECT OF PROGESTERONE ON DEXAMETHASONE- INDUCED HISTAMINE SYNTHESIS IN MASTO- CYTOMA P-815 CELLS

Mastocytoma P-815 cells (1.5.105 cells/ml) were incubated with dexamethasone (1 ~M) and/or progesterone (1 or 10/tM) for 20 h at 37°C, and then the histamine content was de- termined. Each value represents the mean+S.E, for three determinations. * P < 0.05.

Steroid Concentration Histamine content (/~ M) ( ~ g/109 cells)

None 5.03 ± 1.25 Progesterone 1 4.81 ± 0.42 Dexamethasone 1 31.2 ±0.7

+ progesterone 1 24.5 ± 0.3 * + progesterone 10 18.7 ± 3.0 *

c o m p l e x (Fig. 5, inset) . S c a t c h a r d analys is o f these

d a t a i n d i c a t e d the p r e sence o f o n e type o f b i n d i n g

si te w i t h a K d va lue o f 1.26 • 10 -9 M, c o r r e s p o n d -

i ng to 2400 b i n d i n g sites pe r nuc l eus (Fig. 5). T h e

p o t e n c i e s o f g lucoco r t i co id s in d i sp l ac ing [3H]de-

x a m e t h a s o n e b o u n d to the nuc l ea r f r ac t ion ( T a b l e

I V b ) were a lmos t the s a m e as those in the case o f

d i s p l a c e m e n t o f the l abe led d r u g b o u n d to the

cy toso l f r ac t ion ( T a b l e IVa) and the e l eva t ion of

the ce l lu la r h i s t a m i n e level (Tab le I).

P roges t e rone , wh ich had no ef fec t on the ac-

c u m u l a t i o n o f ce l lu la r h i s t a m i n e by i tse l f bu t sig-

n i f i can t ly i n h i b i t e d the [ 3 H ] d e x a m e t h a s o n e b ind -

ing to the cy toso l and nuc lea r f rac t ions , cons ide r -

a b l y suppressed the s t imu la t ed h i s t a m i n e synthes is

i n d u c e d by d e x a m e t h a s o n e ( T a b l e V).

Discussion

O u r resul ts s h o w e d tha t g lucoco r t i co ids specif i -

ca l ly s t imu la t e h i s t a m i n e f o r m a t i o n wi th a con-

c o m i t a n t inc rease in the de n o v o synthes is of

h i s t i d ine d e c a r b o x y l a s e in m a s t o c y t o m a P-815

cells. D i b u t y r y l c A M P [25,26] a n d n -bu ty r i c ac id

[27] are a lso k n o w n to s t imu la t e h i s t a m i n e accu-

m u l a t i o n in this cell l ine. H o w e v e r , the h i s t a m i n e

inc rease i n d u c e d by g lucocor t i co ids is un l ike ly to

be m e d i a t e d by changes in the in t r ace l lu la r c A M P

con ten t , because g lucoco r t i co ids have no ef fec t on

the c A M P synthes i s in m a s t o c y t o m a P-815 cells

(A. I c h i k a w a et al., u n p u b l i s h e d data) . I t is a lso

233

unlikely that glucocorticoids and n-butyrate act

through similar mechanisms because, in contrast to glucocorticoids, n-butyrate consistently stimu- lated the synthesis of not only histamine but also other granular components such as serotonin and

glucosaminoglycans [27], and cyclooxygenase [28]. Furthermore, glucocorticoids are known to bind first with high affinity to the specific cytosolic receptors on the target cells, and then to the

acceptor sites in the chromatin of the nucleus as a complex with the activated receptors, and to pro- mote RNA polymerase activity and the transcrip-

tion rate of a specific gene, leading to the en-

hanced synthesis of a specific protein. In contrast, n-butyrate is assumed to act by changing the chromatin conformation [29] or by inhibiting his-

tone deacetylase [30]. Our results clearly indicate that mastocytoma P-815 cells possess considerable

amounts of cytosolic and nuclear binding sites for

dexamethasone with K, and B,, values almost comparable to those reported for other animal

tissues [22,31]. Since the minimal effective con- centration of dexamethasone for the elevation of histamine synthesis was almost the same as the K,

value of the cytosolic receptors, the appearance of the dexamethasone effect is probably mediated via the activation of the glucocorticoid receptors.

Progesterone appears to act as an antagonist of glucocorticoids in histamine synthesis in masto- cytoma P-815 cells, as in the case of its antagonis-

tic effect on the glucocorticoid-induced milk pro- duction in the rat mammary gland [32]. As for the interaction of steroids with chromatin and/or

DNA, direct interaction of the progesterone recep- tor A subunit complex with the sites flanking the

5’-terminus of the ovalbumin gene [33,34], and of the progesterone-receptor B subunit complex with the non-histone chromatin protein [35], were re-

ported. The binding site of the glucocorticoid-re- ceptor complex has recently been identified in the transcription system of the steroid-sensitive mouse mammary tumor virus [36,37]. It is certainly nec- essary to demonstrate a dexamethasone-induced

increase in newly synthesized mRNA corre- sponding to histidine decarboxylase in masto- cytoma P-815 cells.

The decrease in serotonin level caused by de- xamethasone (Table I) is probably not due to inhibition of its synthesis, because dexamethasone

had no effect on tryptophan 5-hydroxylase activ-

ity. It may be due to the stimulation by gluco- corticoids of serotonin secretion and not hista-

mine release, as in the case of the preferential

release of serotonin from mastocytoma P-815 cells induced by butoctamide [38].

It is not clear whether the glucocorticoid-in-

duced synthesis of histidine decarboxylase obser- ved in neoplastic mastocytoma P-815 cells can be

reproduced in non-neoplastic mast cells. Recently, dexamethasone was reported not to induce accu-

mulation of cellular histamine in mouse serosal

mast cells [39] or in interleukin-3 (IL-3) -depen- dent mouse bone marrow-derived mast cells

[40,41]. However, it is still questionable whether glucocorticoids have absolutely no effect on the

histidine decarboxylase activity in these histamine synthesizing cells, because changing histidine de-

carboxylase activity in several histamine synthesiz- ing cells did not necessarily follow the histamine

level [42,43,44].

The present result of dexamethasone-stimu-

lated histamine synthesis in mastocytoma P-815

cells does not appear to be directly related to the antiallergic effect of glucocorticoids. Its role in a

variety of physiologic and pathologic events of glucocorticoids has not been elucidated yet. How-

ever, our result was essentially consistent with

those reported by other investigators on the change of histidine decarboxylase activity in the stomach by glucocorticoids with using in vivo experiments, showing that hydrocortisone activated the histi- dine decarboxylase activity in rat stomach [2], and that prednisolone caused severe ulceration lesion in rat stomach which was inhibited by H,

antagonist [5]. The type(s) of histamine-synthesiz-

ing cells participating in the induction of gluco- corticoid-stimulated ulceration has not been dis-

covered. However, since a monoclonal antibody

against stomach histidine decarboxylase reacted with the enzyme from mastocytoma P-815 cells

[4], and the biochemical properties of the char- acteristics of mastocytoma P-815 cells in the mast cells were almost consistent with those of the mucosal mast cells but not with the connective tissue mast cells (A. Ichikawa et al., unpublished data, and Ref. 4), the mastocytoma P-815 cells might provide the proper in vitro system to ex- plore the mechanisms of glucocorticoid-induced

234

u l ce ra t i on les ion in s t o m a c h tissue. In add i t ion ,

g lucoco r t i co id s m a y be r e spons ib l e fo r p r o m o t i n g

the r e a c c u m u l a t i o n o f b o t h the h i s t a m i n e a n d

h i s t i d ine d e c a r b o x y l a s e d u r i n g g r a n u l o p o i e s i s in

d e g r a n u l a t e d m a s t cells o r the d i f f e r e n t i a t i o n o f

m a s t cells newly f o r m e d f r o m s t e m b o n e m a r r o w

cells [40]. Th i s p o i n t c e r t a in ly requ i res fu r the r

inves t iga t ion .

Acknowledgement

This w o r k was s u p p o r t e d in pa r t by g ran t s

f r o m T h e M i n i s t r y o f E d u c a t i o n , Sc ience and Cu l -

ture o f J a p a n .

References

1 Liddle, G.W. (1961) Ciin. Pharmacol. Ther. 2, 615-635 2 Reilly, M.A. and Schayer, R.W. (1972) Br. J. Pharmacol.

45, 463-469 3 Sandberg, N. and Steinhardt, C. (1964) Acta Chir. Scand.

127, 574-577 4 Katz, H.R., Stevens, R.L. and Austen, K.F. (1985) J. Al-

lergy Clin. Immunol. 76, 250-259 5 Nobuhara, Y., Takeuchi, K. and Okabe, S. (1985) Japan. J.

Pharmacol. 38, 219-222 6 Pollard, H., Pachot, I., Legrain, P., Buttin, G. and Schwartz,

J.-C. (1985) Adv. Biosci. 51,103-117 7 Ichikawa, A., Esumi, K., Takagi, M., Yatsunami, K. and

Tomita, K. (1980) J. Pharmaco-bio. Dyn. 3, 123-135 8 Shore, P.A., Burkhalter, A. and Cohn, V.H., Jr. (1959) J.

Exp. Ther. 127, 182-186 9 Snyder, S.H., Axelrod, J. and Zweig, M. (1965) Biochem.

Pharmacol. 14, 831-835 10 Friedman, P.A., Kappelman, A.H. and Kaufman, S. (1972)

J. Biol. Chem. 247, 4165-4173 11 Okuno-Kaneda, S., Saito, T., Kawasaki, Y., Ichikawa, A.

and Tomita, K. (1980) Biochem. Pharmacol. 29, 1715-1722 12 Leaback, D.H. and Walker, P.G. (1961) Biochem. J. 78,

151-156 13 Schwartz, L.B., Austen, K.F. and Wasserman, S.I. (1979) J.

Immunol. 123, 1445-1450 14 Ostrowski, W. and Tsugita, A. (1961) Arch. Biochem. Bio-

phys. 94, 68-78 15 Omura, T. and Takesue, S. (1970) J. Biochem. (Tokyo) 67,

249-257 16 Pennington, R.J. (1961) Biochem. J. 80, 649-654 17 Heppel, L.A. and Hilmoe, R.J. (1955) Methods Enzymol. 2,

546-549

18 Hayashi, S., Grarmer, D.K. and Tomkins, G.M. (1967) J. Biol. Chem. 242, 3998-4006

19 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275

20 Layne, E. (1957) Methods Enzymol. 3, 447-454 21 Svec, F. and Harrison, R.W. (1979) Endocrinology 104,

1563-1568 22 Kon, O.L. and Spelsberg, T.C. (1982) Endocrinology 111,

1925-1935 23 Greengard, O., Smith, M.A. and Acs, G. (1963) J. Biol.

Chem. 238, 1548-1551 24 Lin, E.C.C. and Knox, W.E. (1958) J. Biol. Chem. 233,

1186-1189 25 Takagi, M., Ichikawa, A., Esumi, K., Yatsunami, K.,

Negishi, M. and Tomita, K. (1980) J. Pharamco-bio. Dyn. 3, 136-148

26 Davis, J. and Ralph, R.K. (1975) Cancer Res. 35, 1495-1504 27 Moil, Y., Akedo, H., Tanigaki, Y., Tanaka, K.M., Okada,

M. and Nakamura, N. (1980) Exp. Cell Res. 127, 465-470 28 Koshihara, Y., Senshu, T., Kawamura, M. and Murota, S.

(1980) Biochim. Biophys. Acta 617, 536-539 29 Vidali, G., Botta, C., Bradburg, E.M. and Allfrey, V.G.

(1978) Proc. Natl. Acad. Sci. USA 75, 2239-2243 30 Cousens, L.S., Gallwitz, D. and Alberts, B.M. (1979) J.

Biol. Chem. 254, 1716-1723 31 Rousseau, G.G., Baxter, J.D. and Tomkins, G.M. (1972) J.

Mol. Biol. 67, 99-115 32 Maki, M., Hirose, M. and Chiba, H. (1980) J. Biochem. 88,

1845-1854 33 Compton, J.G., Schrader, W.T. and O'Malley, B.W. (1983)

Proc. Natl. Acad. Sci. USA 80, 16-20 34 Mulvihill, E.R., LePennec, J.P. and Chambon, P. (1982)

Cell 24, 621-632 35 Pilder, G.M., Webster, R.A. and Spelsberg, T.C. (1976)

Biochem. J. 156, 399-408 36 Govindan, M.V., Spiess, E. and Majors, J. (1982) Proc.

Natl. Acad. Sci. USA 79, 5157-5161 37 Chandler, V.L., Maler, B.A. and Yamamoto, K.R. (1983)

Cell 33, 489-499 38 Ichikawa, A., Kaneko, H., Moil, Y. and Tomita, K. (1977)

Biochem. Pharmacoi. 26, 197-202 39 Daeron, M., Sterk, A.R., Hirata, F. and Ishizaka, T. (1982)

J. Immunol. 129, 1212-1218 40 Schrader, J.W., Lewis, S.J., Clark-Lewis, I. and Culvenor,

J.G. (1981) Proc. Natl. Acad. Sci. USA 78, 323-327 41 Robin, J.L., Austen, K.F. and Lewis, R.A. (1985) J. Im-

munol. 135, 2719-2726 42 Kahlson, G., Rosengren, E., Svahn, D. and Thunberg, R.

(1964) J. Physiol. 174, 400-416 43 Beaven, M.A., Aiken, D.L., Woldmussie, E. and Soil, A.H.

(1983) J. Pharmacol. Exp. Ther. 224, 620-626 44 Lemmi, C.A.E. (1984) Agents Actions 14, 185-194 45 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660