Diazepam-induced decrease in histamine turnover in mouse brain

European Journal of Pharmacology, 124 (1986) 337-342

337

Elsevier

D I A Z E P A M - I N D U C E D D E C R E A S E IN H I S T A M I N E T U R N O V E R IN M O U S E BRAIN R Y O Z O OISHI, M A S A H I R O NISHIBORI, Y O S H I N O R I ITOH and K1YOMI SAEKI *

Department of Pharmacology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan Received 26 September 1985, revised MS received 31 December 1985, accepted 4 March 1986

R. OISHI, M. NISHIBORI, Y. ITOH and K. SAEKI, Diazepam-induced decrease in histamine turnover in mouse brain, European J. Pharmacol. 124 (1986) 337-342. The effect of diazepam on brain histamine turnover was examined in mice. The steady state levels of histamine and tele-methylhistamine remained unchanged following the i.p. administration of 0.2-20 mg/kg of diazepam. However, diazepam in doses over 5 mg/kg significantly decreased histamine turnover, as estimated from the accumulation of tele-methylhistamine after pargyline treatment. Other benzodiazepines such as chlordiazepoxide, nitrazepam and estazolam in high doses also decreased histamine turnover. The inhibitory effect of diazepam on histamine turnover was antagonized by the pretreatment with a benzodiazepine antagonist Ro 15-1788. The histamine turnover was significantly inhibited by 2 mg/kg of muscimol. Diazepam (0.2-1 mg/kg) markedly and dose-dependently potentiated the inhibitory effect of 1 mg/kg of muscimol from non-significant to highly significant levels. The potentiation by diazepam was also antagonized by Ro 15-1788. Therefore, diazepam probably decreases histamine turnover in the brain via the benzodiazepine-GABA receptor complex. tele-Methylhistamine

Histamine turnover

Benzodiazepines

1. Introduction

The GABAergic synapse is now thought to be the primary site of action of the benzodiazepines in the central nervous system. Various pharmacological actions of the benzodiazepines may be the result of changes in a complex neuronal network, as affected by GABAergic input (Haefely et al., 1983). It has been reported that the benzodiazepines alter the turnover of brain acetylcholine, catecholamines and serotonin (Haefely, 1978). Histamine (HA) is a putative neurotransmitter in the mammalian brain (Schwartz et al., 1980). Since intraventricularly injected H A induces arousal in conscious animals (Kalivas, 1982) and most H A Hi-antagonists are sedative (Faingold, 1978), HA is assumed to play a role in modulating the level of sleep-wakefulness,. However, little is known of the effects of benzodiazepines on brain

* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.

Ro 15-1788

Diazepam

Muscimol

HAergic activity. We now report that diazepam (DZP) decreases the turnover of H A in the mouse brain.

2. Materials and methods 2.1. Animals

Male ddY mice aged 6-7 weeks and weighing 25-30 g (Kyudo Co., Kumamoto, Japan) were housed in a room with controlled temperature (22 + 2°C) and given free access to pellet food and tap water. All experiments were performed from 13 : 00 to 17 : 00 h. 2.2. Drugs

Pargyline hydrochloride and muscimol were obtained from Sigma Chemical Co. (St. Louis, MO); carboxymethyl cellulose (CMC) was from Wako Pure Chemical Industries (Osaka, Japan).

338

DZP, chlordiazepoxide and estazolam were donated by Takeda Chemical Industries (Osaka), nitrazepam by Japan Upjohn Co. (Tokyo, Japan) and Ro 15-1788 by Nippon Roche Research Center (Kamakura, Japan). All benzodiazepines and Ro 15-1788 were suspended in 0.5% CMC and pargyline was dissolved in 0.9% saline solution.

HA ng/g 60

T

20

2.3. Estimation of HA turnover

The HA turnover was estimated form the accumulation of tele-methylhistamine (t-MH) after pargyline treatment (Hough et al., 1982; Oishi et al., 1984). Mice were killed 90 min after the i.p. treatment with 80 m g / k g of pargyline hydrochloride and the whole brain excluding the cerebellum was rapidly removed. The tissue was homogenized in 0.4 M perchloric acid with 40 ng of pros-methylhistamine as the internal standard. The HA and t-MH contents were determined using high-performance liquid chromatography with fluorescence detection, as described by Tsuruta et al. (1981) with modifications (Oishi et al., 1985).

0

t-MH ng/g 150t _=.

/ 1 O0

J

0

2 CMC

3. Results

5 10 20 DZP

* SALINE

2.4. Statistical analysis

Statistical significance was evaluated by the two-tailed Student's t-test.

64 52

_ _

CMC

37

5 10 20 mg/kg DZP * PARGYLINE

Fig. 1. Effects of DZP on the HA and t-MH contents in the brain of saline- and pargyline-treated mice. CMC or DZP was administered i.p. 15 min before the treatment with pargyline hydrochloride (80 m g / k g i.p.). The mice were killed 90 min after pargyline treatment. The results are the means_+S.E.M. from 5-9 animals. The figures in the columns represent the t-MH accumulation expressed as a percentage of the control (CMC-treated group). Significantly different from the control:

3.1. Effects of D Z P on the brain levels of HA and t - M H and HA turnover

* P < 0.001.

Figure 1 shows the effect of DZP on the HA and t-MH contents in the brain of saline- and pargyline-treated mice. DZP was injected 15 rain before the treatment with pargyline. The steady state levels of HA and t-MH were unchanged by 2-20 m g / k g of DZP. In the CMC-treated control, 73 n g / g of t-MH accumulated in 90 min after pargyline treatment. The t-MH accumulation was significantly and dose-dependently inhibited by 5-20 m g / k g of DZP. No significant change was observed in the HA content after the treatment of pargyline-treated mice with DZP. In small doses (0.2-1 mg/kg), DZP had no influence on any level

of these amines or on HA turnover (data not shown). 3.2. Effect of other benzodiazepines on HA turnover

Figure 2 shows the effects of chlordiazepoxide, nitrazepam and estazolam on the pargyline-induced t-MH accumulation. These benzodiazepines, in high doses, significantly decreased the t-MH accumulation but had no influence on the steady state levels of HA and t-MH and the HA levels in the pargyline-treated mice (data not shown).

339 t-Mt"

nglg

antagonized by 20 m g / k g of Ro 15-1788 administered 5 min before DZP. Ro 15-1788 given alone had no significant effect on the steady state levels of HA and t-MH and the pargyline-induced t-MH accumulation.

._T_ 3-

T

200"

I0C

150.

3.4. Effect of the combination of D Z P and muscimol on HA turnover

100-

50.

l

CMC

20 40 CDP

2

5

10

NZP

2

5

10

ETZ

* PARGYLINE Fig. 2. Effects of chlordiazepoxide (CDP), nitrazepam (NZP) and estazolam (ETZ) on the pargyline-induced t-MH accumulation. Each drug was administered i.p. 15 rain before the treatment with pargyline hydrochloride (80 m g / k g i.p.). The mice were killed 90 rain after pargyline treatment. The results are the means_+ S,E.M. from 5 or 6 animals. The figures in the columns represent the t-MH accumulation expressed as a percentage of the control (CMC-treated group). Significantly different from the control: * P < 0.01.

3.3. Antagonism by Ro 15-1788 of the action of D Z P on HA turnover

As shown in table 1, the treatment with 10 m g / k g of DZP decreased the pargyline-induced t-MH accumulation to 54% of the control value. This inhibitory effect of DZP was significantly

The s.c. administration of 1 and 2 m g / k g of muscimol 15 rain before pargyline treatment decreased the pargyline-induced t-MH accumulation to 80 and 65% of the control value, respectively (table 2). DZP (0.2-2 mg/kg) given 15 rain before muscimol markedly enhanced the weak inhibitory effect of muscimol, although DZP administered alone in these low doses had no effect on t-MH accumulation. The combined treatment with 2 m g / k g of DZP and the same dose of muscimol decreased the t-MH accumulation to as low as 15% of the control value. 3.5. Effect of Ro 15-1788 on the inhibition by D Z P and muscimol of HA turnover

When DZP (1 m g / k g i.p.) and muscimol (1 m g / k g s.c.) were administered 30 and 15 rain before pargyline treatment, respectively, the t-MH accumulation was inhibited to 51% of the control value, although the effect of muscimol given alone was only marginal (table 3). Ro 15-1788 (20 m g / k g i.p.) given 5 min before DZP treatment all but completely abolished the enhancement by DZP of the muscimol effect on t-MH accumulation.

TABLE 1 Antagonism by Ro 15-1788 of the action of DZP on HA turnover in the mouse brain. Ro 15-1788 (20 m g / k g i.p.) and DZP (10 m g / k g i.p.) were administered 20 and 15 min before pargyline hydrochloride (80 m g / k g i.p.), respectively. The mice were killed 90 rain after pargyline treatment. Each value is the mean +_S.E.M. from 5-8 animals. At-MH represents the t-MH accumulation after pargyline treatment. The figures in parentheses represent a percentage of the control value. Treatments CMC Ro 15-1788 CMC CMC Ro 15-1788 Ro 15-1788

CMC CMC CMC DZP CMC DZP

Saline Saline Pargyline Pargyline Pargyline Pargyline

HA ( n g / g )

t-MH ( n g / g )

At-MH ( n g / g )

37.5 +_2.5 38.4 + 3.9 40.7 ± 2.8 38.9 ± 3.3 36.4 + 3.9 39.7 + 3.5

76,3 ± 1.7 73,8 _+4.9 163,5 ± 6.8 123.2 _+7.6 ~ 162.2_+ 8.5 154.4_+ 8.5 b

87.2 46.9 85.9 78.1

a p < 0.01 compared with CMC-CMC-Pargyline group; b p < 0.05 compared with CMC-DZP-Pargyline group.

(100) (54) (99) (90)

340 TABLE 2 Effect of the combination of DZP and muscimol (Mus) on HA turnover in the mouse brain. DZP (i.p.) and muse±tool (s.c.) were administered 30 and 15 min before pargyline hydrochloride (80 m g / k g i.p.) respectively. The mice were killed 90 min after pargyline treatment. Each value is the mean -+ S.E.M. from 5 or 6 animals. At-MH represents the t-MH accumulation after pargyline treatment. The figures in parentheses in the At-MH column represent a percentage of the control value. Treatments CMC CMC CMC CMC CMC DZP (0.2) DZP (0.5) DZP (1) DZP (2) CMC DZP (2)

Saline Mus (1) '~ Mus (2) Saline Mus (1) Mus (1) Mus (1) Mus (1) Mus (1) Mus (2) Mus (2)

Saline Saline Saline Pargyline Pargyline Pargyline Pargyline Pargyline Pargyline Pargyline Pargyline

HA ( n g / g )

t-MH ( n g / )

At-MH ( n g / g )

36.5 _+3.5 40.5 + 3.8 38.4 + 2.9 38.2 _+2.0 43.0±3.9 37.6 + 2.8 42.7 _+6.3 45.5 _+3.5 41.2 _+ 1.9 45.4-+ 5.5 39.9 _+2.8

72.9 -+ 3.4 71.3 ± 5.3 72.4 + 4.3 178.8 -+ 4.3 158.0-+9.8 144.6 + 8.5 116.6 + 8.9 93.6 + 5.0 94.3 -+ 4.9 141.6 _+9.0 88.9 ± 4.7

105.9 (100) 85.1 (80) 71.7 (68) 43.7 (41) 20.7 (20) 21.4 (20) 68.7 (65) 16.0 (15)

h ,.a ~'~ ~.e h ~'~

a Dose in mg/kg; h p < 0.01; ~ P < 0.001 compared with CMC-Saline-Pargyline group: a p < 0.01: ~ P < 0.001 compared with CMC-Mus(1)-Pargyline group.

4. Discussion

HA released from nerve terminals is metabolized almost exclusively by methylation to t-MH (Schwartz et al., 1971; Schayer and Reilly, 1973) and is further metabolized to tele-methylimidazoleacetic acid by type B monoamine oxidase (Hough and Domino, 1979). The HA turnover in the brain was therefore estimated from the t-MH accumulation following the administration of pargyline (Hough et al., 1982; Oishi et al., 1984). Since t-MH given to mice accumulates almost

linearly up to 2 h after pargyline treatment (Nishibori et al., 1984), the mice were killed 90 min after the treatment in the present study. As discussed in a previous paper (Nishibori et al., 1985), we have observed a very large variation in the steady state brain t-MH level in the ddY mice in our laboratory. In these present study, the steady state t-MH level ranged from 72-110 ng/g. Such a variation is unlikely to relate to the method of t-MH determination. Although the factors that control the steady state brain t-MH level are not yet clear a preliminary study showed that there

TABLE 3 Effect of Ro 15-1788 on the inhibition of the HA turnover induced by the combination of DZP and muscimol. DZP (1 m g / k g i.p.) and muscimol (l m g / k g s.c.) were administered 30 and 15 rain before pargyline hydrochloride (80 m g / k g i.p.), respectively. Ro 15-1788 (20 m g / k g i.p.) was administered 5 rain before DZP. The mice were killed 90 min after pargyline treatment. Each value is the mean-+ S.E.M. from 5-9 animals. At-MH represents the t-MH accumulation after pargyline treatment. The figures in parentheses in the At-MH column represent a percentage of the control value. Treatments CMC CMC CMC DZP DZP

Saline Saline Muscimol Muscimol Muscimol +Ro15-1788

Saline Pargyline Pargyline Pargyline Pargyline

HA ( n g / g )

t-MH (ng/g)

t-MH ( n g / g )

41.1 +4.8 47.0 -+ 2.3 50.0-+ 2.8 41.1-+ 3.3

109.4_+ 5.2 209.0 + 10.6 197.7_+ 8.5 159.7_+ 6.7 ~

99.6 (100) 88.3 (89) 50.3 (51)

38.6 -+4.4

191.0_+ 12.3b

81.6 (82)

P < 0.01 compared with CMC-Saline-Pargyline group; h p < 0.05 compared with DZP-Muscimol-Pargyline group.

341

was a diurnal variation in the t-MH level. Such a factor may be partly responsible for the different control values obtained in the present experiments. In the experiments described here pargyline induced 73-106 n g / g of t-MH accumulation during 90 min after its administration, as already reported (Nishibori et al., 1984; Oishi et al, 1985). DZP in doses over 5 m g / k g inhibited t-MH accumulation in a dose-dependent manner. Other benzodiazepines such as chlordiazepoxide, nitrazepam and estazolam at high doses also inhibited t-MH accumulation. The inhibitory effect of 10 m g / k g of DZP on HA turnover was significantly antagonized by pretreatment with Ro 15-1788, a selective benzodiazepine antagonist (Hunkeler et al., 1981), thereby suggesting that DZP decreases brain HA turnover via benzodiazepine receptors. The benzodiazepines facilitate the effects of an inhibitory neurotransmitter, GABA (Costa et al., 1975; Haefely et al., 1975). Therefore, the interaction between DZP and muscimol was examined in terms of the influence on HA turnover. Although muscimol (1 mg/kg) and DZP (0.2-2 mg/kg) given separately had no significant inhibitory effect on the pargyline-induced t-MH accumulation, DZP produced a dose-dependent inhibition in muscitool-treated mice. Furthermore, the enhancement by DZP of the muscimol inhibition of the t-MH accumulation was antagonized by Ro 15-1788. These results suggest that DZP produces an inhibitory effect on HA turnover by acting on the benzodiazepine-GABA receptor complex. Recently, Takeda et al. (1984) suggested the coexistence of glutamate decarboxylase and histidine decarboxylase in neurons in the rat hypothalamus. Thus, it is possible that the GABA released from these neurons may regulate the HAergic activity of the same neurons. Although the degree of t-MH accumulation in different pargyline control groups was fairly constant, large discrepancies in the t-MH level and the t-MH accumulation were observed in mice treated with the combination of DZP (1 mg/kg), muscimol (1 mg/kg) and pargyline in different sets of experiments (see tables 2 and 3). Such discrepancies may have been due to the differences in the steady state t-MH level and the

sensitivity of mice to DZP a n d / o r muscimol but no definite conclusion can be drawn at the present time. The HA turnover in the brain is affected by drugs acting on the central nervous system: it is inhibited by sedative drugs such as barbiturates (Pollard et al., 1974), ethanol (Itoh et al., 1985) and A%tetrahydrocannabinol (Oishi et al., 1985), whereas it is enhanced by morphine (Nishibori et al., 1985) and phencyclidine (Itoh et al., 1985). The inhibitory action of the benzodiazepines on HA turnover may contribute to their sedative effect. The benzodiazepines may produce a wide variety of actions through changes in the activity of neurotransmitter systems, such as the catecholaminergic and serotonergic, which receive a GABAergic input (Haefely et al., 1983). The inhibition of HAergic activity by the benzodiazepines should be given attention with regard to possible involvement in pharmacological actions.

Acknowledgements This work was supported by a Grant-in-Aid for Encouragement of Young Scientists, 60770165, from the Ministry of Education, Science and Culture, Japan. We thank M. Ohara of Kyushu University for comments on the manuscript.

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