Histamine-induced cyclic AMP accumulation in type-1 and type-2 astrocytes in primary culture

European Journal of Pharmacology' ~ Molecuhlr PhannacokJgy Section, 208 (1991) 249-253 ,c.t~ 1991 Elsevier Science Publishers B.V. All rights reserved 0922-41116/01/$03.50

249

EJPMOL 9¢1238

Histamine-induced cyclic AMP accumulation in type-! and type-2 astrocytes in primary culture Akiharu Kubo, Hiroyuki Fukui, Naoyuki Inagaki, Akira Kanamura and Hiroshi Wada Department o]"Pharmacolo,~' H, Faculty ~f Medicine, Osaka Unicersity, Ya:nadaoka 2-2, Suita 565, Japan Received 2 August 1991. accepted 13 August 1091

Histamine-induced cyclic AMP (cAMP) accumulation was studied in purified primary, cultures of typc-I and type-2 astrocytcs from neonatal rat brain. Histamine induced remarkable cAMP accumulation in type-I astrocytcs in a dose-dependent manner (ECs~~= 1.2 x 10 -5 M, E,,,~,x= 1100% of control). In contrast, histamine had no significant effect on cAMP accumulation in type-2 astrocytes. Famotidine, an He-antagonist, dose-dependently inhibited histamine-induced cAMP accumulation in typc-1 astrocytes ( K i = 3 × 10 s M), but mepyraminc (10 ~' M). an H~-antagonist. had no effect. Dimaprit and impromidine, Hz-agonists, stimulated cAMP accumulation, but 2-pyridylcthylaminc, an Hragonist, did not stimulate it nor augment the H,.-agonist-iindueed cAMP accumulation. These results indicate that (1) histamine induces cAMP accumulation in type-1 astrocytcs but not in type-2 astrocytes, and that (2) histamine-induced cAMP accumulation in type-I astrocytes is mediated by He-receptors without significant augmentation via H z-receptors. Histamine: Histamine H e receptor; Astrocytes (type 1); cAMP: (Rat)

1. Introduction

Histamine is one of the chemical messengers released from neurons in the brain (Prell and Green, 1986; Hough, 1988; Yamatodani et al., 1990; Schwartz et al., 1991). The cell bodies of the histaminergic neurons are localized in the posterior hypothalamic regions and send their fiber terminals to almost all parts of the brain (Watanabe et al., 1984; Inagaki et al., 1988; Panula et al., 1989). Consistent with the wideranged fiber projection, three subtypes of histamine receptors, i.e. H~-, H z- and H3-receptors, are widely distributed throughout the brain (Arrang et al., 1987; Bouthenet et al., 1988; Hill, 1990; Ruat et al., 1990). Previous studies with cultured astrocytes have indicated that astrocytes which express H t- and H_,-receptors may be one of the main targets of the central histaminergic neurons. Astrocytes in explant cultures are reported to have both H~- and He-receptors, and activation of these astrocytes changes membrane potentials (H6sli et al., 1984). Primary cultures of as~.rocytes have been shown to express high densities of H ~-receptors (Inagaki et al., 1989), which induce phosphoinositide hydrolysis (Arbon6s et al., 1988) and in-

Correspondence to: H, Fukui. Department of Pharmacology II, Faculty of Medicine. Osaka University. Yamadaoka 2-2. Suita 565. Japan. Tel. 81-6-877-5111 (Ext. 5392): Fax 81-6-878-7419.

tracellular Ca -'+ elevation (Fukui et al., 1991; Inagaki et al., 1991), and He-receptors which are coupled to cyclic AMP (cAMP) formation (Agull6 et al., 1990). Recently, Raft and his colleagues have demonstrated that cultured astrocytes consist of two distinct populations, i.e. type-I and type-2 astrocytes (Miller et al., 1989; Raft. 1989). Although both of these express a specific marker for astrocytes, i.e. glial fibrillary acid protein (GFAP), they differ in morphology, antigenic phenotypcs, developmental lineage and ion channel expression (Miller et al., 1989; Raft, 1989: Barres et al., 1990). However, little information is available regarding the distributions of histamine receptors on type-1 and type-2 astrocytes. In the present study, we examined the effects of histamine on cAMP accumulation in these two types of astrocytes and found that H z-receptors were preferentially expressed on type-1 astrocytes but not on type-2 astrocytes. Preliminary results of the present study have been presented in abstract form (Fukui et al., 1991).

2. Materials and methods

2.1. Culture preparation Primary cultures of type-1 and type-2 astrocytes were prepared as reported previously (Aloisi et al., 1988; Inagaki et al., 1991). Newborn Wistar rats were

250 sacrificed by decapitation. The cerebral hemispheres were collected in cold Joklik modified minimum essential medium (MEM) under sterile conditions and the meninges were carefully removed, The cerebral hemispheres were triturated mechanically with a pipette and then enzymatically dissociated with Dispase (neutral protease, Dispase grade II) solution (3 m g / m l ) in Joklik modified MEM (Frangakis and Kimelberg, 1984; Inagaki et al., 1989). The dissociated cells were seeded in culture flasks (75 cm 2) at a density of 1 × 1(I~ cells/cm-'. The cultures were incubated at 37°C in a humid atmosphere of 95% air and 5% CO~ and the culture medium (Eagle's MEM containing 100 U / m l penicillin G, 60 g g / m l kanamycin and 10% fetal calf serum) was changed the next day, and twice a week thereafter. After 2 weeks in vitro, the small process-bearing O-2A progenitor cells (Raft, 1989) grown on top of the type-I astrocyte monolayer were removed mechanically by shaking the culture flasks overnight, seeded onto polystyrene-treated culture wells (22 mm diameter), and subcultured in the same medium containing fetal calf serum for differentiation into type-2 astrocytes (Raft, 1989). After 7 days, they were used as type-2 astrocyte-enriched cultures and more than 70% of the cultured ceils exhibited the characteristics of type-2 astrocytes, namely, stellate shaped and immunoreactive to both G F A P and A2B5 (donated by Dr. M. Nirenberg: Eisenbarth et al., 19797 antibodies (Miller et al., 1989). The remaining cells adhering to the culture flasks were again shaken 2-3 times for complete removal of the process-bearing cells (McCarthy and de Vellis, 1980). The remaining cells were dissociated with Dispase and similarly subt ultured in the culture wells. After 7 days, they were used as type-I astrocyte-enriehed cultures and more than 96% of the cultured cells exhibited the characteristics of typed astrocytes; polygonal shaped, G F A P ( + ) and A2B5 ( - ) (Miller et al., 1989).

2.Z Measurement of cAMP Cultured astrocytes were preincubated for 10 min at 37°C in 800 /zl of incubation medium (Eagle's MEM containing 10 mM HEPES, 1 m g / m l bovine serum albumin and 0.5 mM isobutyl-l-methylxanthine (IBMX)) either with or without histamine antagonists. They were then incubated either in the prc~ence or absence of histamine or its agonists in the same medium for 5-80 min. At the end of the incubation, the medium was removed and 400 ~tl of trichloroacetic acid (TCA, 10%) was added. Cells were scraped, poured into Eppendorf tubes, sonicated and then centrifuged for 10 min at 15,000×g. To extract the TCA, 200 /zl of supernatant was collected, combined with 1 ml of water-saturated diethyl ether and vortexed for 10 rain.

Finally, the upper phase of diethyi ether was discarded. This extraction procedure was repeated 4 times, cAMP in the sample was measured using a radioimmunoassay kit (cyclic A M P kit 'Yamasa', Yamasa Shouyu, Japan). Protein in the precipitate was solubilized with 1 ml of 0.4 N NaOH and measured using a BCA protein assay kit (Pierce) (Smith et al., 1985).

2.3. Anab'sis of data The concentration of histamine required for halfmaximal accumulation (EC50) of cAMP was calculated using a Scatchard plot according to the following equation: E - 1 . . . . S ECso

Em~,x E+ - ECs~~

where Em~,x is the maximal accumulation of cAMP, E is the accumulation of cAMP and S is the concentration of histamine. The concentration of famotidine required for half-maximal inhibition (IC50) of adenylate cyclase activity was also calculated using a Scatchard plot. The inhibition c o n s t a n t (K i) was calculated according to the following equation: K~

IC50 S I+-ECso

where S is the concentration of histamine ( i 0 -4 M).

2.4. Drags The sources of drugs used were as follows: Joklik modified MEM (Gibco), Dispase (Boehringer-Mannhelm Bioproducts), Eagle's MEM (Nissui), fetal calf serum (Bioproducts), histamine (Nakarai Tesque), 2pyridylethylamine (Smith Kline & French Laboratories) dimaprit (Smith Kline & French Laboratories), impromidine (Smith Kline & French Laboratories), mepyramine (Sigma), famotidine (Yamanouchi Pharmaceutical Co.), IBMX (Aldrich).

3. Results

Fig. 1 shows cAMP accumulation induced by a 10 min incubation with 10 -4 M histamine in type-I astrocyte-enriched and type-2 astrocyte-enriched primary cultures. Histamine at 10 -4 M stimulated cAMP accumulation in type-I astrocytes (1100% of control, n = 5, P < 0.001 by Student's t-test) in the presence of 0.5 mM IBMX. On the other hand, the same concentration of histamine had no significant effect on cAMP formation in type-2 astrocytes. The identical results were obtained even if the concentration of histamine

251 c

900

Type-2

Type- 1

~lO00I

600

/

500 300

";

~

0

c

C

HA IO-4M

HA 10-4M

Fig. I. The effects of histamine (10 -4 M) on cAMP accumulation in type-] astrocytes and type-2 astrocytes. Astrocytes were incubated with histamine for ]0 min. Experiments were performed in the presence of 0.5 mM IBMX to inhibit the action of phosphodiesterase. Each column represents mean + S.E.M. of three independent experiments performed in duplicate. H A = histamine; C = control.

and the incubation time were changed (data not shown). Forskolin (10 -6 M), a direct activator of adenylate cyclase, stimulated cAMP accumulation in type-2 astrocytes (970% of control). Moreover, prostaglandin E2 was demonstrated to evoke cAMP accumulation in type-2 astrocytes (Ito et al., submitted). These results clearly indicate that the lack of cAMP accumulation in type-2 astrocytes was not due to the lack of adenylate cyclase in them. The time course of histamine-induced cAMP accumulation in type-I astrocytes is shown in fig. 2. Concentrations of cAMP increased for the first 10 min up to the maximal level and then decreased gradually thereafter. As shown in fig. 3, histamine-induced cAMP accumulation in type-1 astrocytes was dose-dependent. The ECs0 was 1.2 × 10 -5 M and the maximal response was obtained at 10 -4 M. Famotidine, a histamine H2-receptor antagonist, inhibited histamine-induced cAMP accumulation in type-

!1--~!

~

0

1 astrocytes in a dose-dependent manner (K~ = 3 x 10 -~ M); however, 10 -6 M mepyramine, an Ht-rece ptor antagonist, did not inhibit it (fig. 4). Furthermore, 10 -6 M mepyramine had no significant influence on the inhibition curve of famotidine on the histamine-induced cAMP accumulation (fig. 4). These results strongly indicate that histamine-induced cAMP accumulation in type-1 astrocytes was mediated by H2-receptors without the participation of Hi-receptors. This was further supported by the agonist studies. Dimaprit (10 -4 M) and impromidine (10 -5 M), H2-agonists, stimulated cAMP accumulation in type-1 astrocytes (table 1). However, 2-pyridylethylamine (10 -4 M), an H t-agonist, neither stimulated cAMP accumulation, nor augmented H z-agonist-induced cAMP accumulation significantly (table 1). Thioperamide (10 -6 M), an H 3-antagonist, had no effect on the histamine-induced cAMP accumulation (data not shown), suggesting that

lO0[

T

-'3

Fig. 3. Dose-response curve of histamine-induced cAMP accumulation in type-I astrocytes. Astrocytes were incubated with histamine for 10 min. Experiments were performed in the presence of 0.5 mM IBMX. Values represent means +_S.E.M. of four independent experiments performed in duplicate.

"~ 900

J4 ~/ - '7 -'6 J5 Histamine (log[M])

0

T

C)-O ~epyramine{tO 6M)

0-0

",

~epyrar~ine (-)

)

~~00

6oo

i

~

N ,2

30[

o

? 0

10

20

40

Time{min)

80

Fig. 2. Time course of cAMP accumulation in type-1 astrocytes induced by 10 -4 M histamine (e)- (©) control curve. Experiments were performed in the presence of 0.5 mM ]BMX. Values represent means + S.E.M. of five independent experiments performed in duplicate.

_

,6

4;

0 -9 J8 J7 -~6 -'5 Famotidine(log[W]) Fig. 4. Dose-dependent inhibition of histamine (10 -4 M)-induced cAMP accumulation in type-I astrocytes by famotidine in the presence (©) or absence (e) of mepyramine (10 -6 M). Astrocytes were incubated with drugs for 10 rain. Experiments were performed in the presence of 0.5 mM IBMX. Values represent means_+S.E.M, of three independent experiments performed in duplicate.

252 "]'ABLE 1 cAMP accumulationin cultured typc-I astrocytes inducedby various histaminergicH I" and H:-agonists. The experimentwas performed in the presenceof 0.5 mM IBMX to inhibit the action of phosphodiesterase. Results are means± S.E.M. obtained from four indepc.ndentexperimentsperformed in duplicate. Treatment

cAMP (pmol/ mg protein/ 10 min)

Control tiistamine ( 10 ~ M) 2-Pyridylethylamine(10 4 M) lmpromidine( 10 5 M) Dimaprit (Ill 4 M) lmpromidine(1(I s M) + 2-pyridylethylamine(10 4 Me Dimaprit (I(V4 M) + 2-pyridylethylamine( 10 4 M)

41 ± S 454 ± 52 46± 3 215+ 22 146±34 223 + 49 177+ 59

** P < 0.',Jill. * P <0.01 as compared with control by Student's tlest.

H3-receptors also did not participate in the accumulation.

4. Discussion

To investigate the functions of the central histaminergic system, identification of receptor locations in the brain is essential. Recently, Ruat et al. 11990) revealed the wide-spread distribution of histamine I-12-receptors in the guinea-pig brain using [~2Sl]iodoaminopotentidine as a marker. At the cellular level, Hz-receptors have been identified on various types of cells in the brain, including neurons (Agull6 et al., 1990), microvessels (Karnushina et al., 1980) and astrocytes (H6sli et al., 1984; Agull6 et al., 1990). In the present study, we further investigated the distribution of H,-receptors on two distinct types of astrocytes, i.e. type-1 and type-2 astrocytes, by examining histamine-induced cAMP accumulation in the two types of astrocytes. Histamine induced significant accumulation of cAMP in type-1 astrocytes via Hz-receptors but not in type-2 astrocytes. These results clearly indicate that H z-receptors a~-". preferentially expressed on type-1 astrocytes but no~. on type-2 astrocytes. It is notable that the maximal cAMP accumulation observed here in rat typeol a,~trocytes (1100% of control) was much higher than tha, !eported in rat cerebral slices (200% of control; ~,chultz and Daly, 1973), neuron-enriched primary cuhure from rat cerebrum 1120% of control; Agull6 et al., 1990) and astrocyte-enriched primary cultures from rat cerebrum (400% of control; Agull6 et al., 1990). The prominent elevation of cAMP in type-I

astrocytes suggests that type-I astrocytes may be one of the major contributors to the Hz-receptor-mediated cAMP responses in the rat cerebrum. Another finding of the present study is that histamine-indaced cAMP accumulation in type-1 astrocytes is mediated solely by Hz-receptors. This clearly contrasts with the well known observations stating that stimulation of Ht-receptors enhances Hz-receptormediated cAMP accumulation in guinea-pig and rabbit brain slices (Palacios et al., 1978; AI-Gadi and Hill, 1985; Hill, 1990; S¢:hwartz et al., 1991). Recently, we demonstrated that histamine induced inositol phosphates accumulation in type-2 astrocytes via Ht-receptors but not in type-I astrocytes (Kondou et al., 1991). Furthermore, histamine elevated the intracellular C a 2+ concentration in 73% of type-2 astrocytes via H t-receptors, but only 17% of type-I astrocytes (Fukui et al., 1991; l nagaki et al., 1991 ). These observations indicate that H rreceptors are poorly expressed on type-I astrocytes, although they are abundant in type-2 astrocytes, and that the lack of augmentation is due to the poor expression of H rreceptors on type-I astrocytes. Agull6 et al. 11990) also reported the lack of synergism between H ~- and H 2-receptors in cAMP accumulation in astrocyte-enriched cultures, i.e. mixed cultures of H 2receptor-enriched type-I astrocytes and Hrreceptorenriched type-2 astrocytes. Therefore, co-expression of H~- and H z-receptors in the same cells may be required for the synergistic interaction between these two receptors. Little information is available on the functions of H z-receptors on type-I astrocytes. It is speculated that type-I astrocytes extend their processes to neurons and blood vessels in the brain to form the blood-brain barrier (Janzer and Raft, 1987; Raft, 1989). Oishi et al. (1989) showed that activation of Hz-receptors in the brain is involved in the increase in the blood-brain barrier permeability to sodium fluorescein caused by opioid receptor agonists. Therefore, a possibility is that central histamine regulates blood-brain permeability through H2-receptors on type-1 astrocytes. Arbon6s et al. (1990) recently reported that activation of H~- and H_,-receptors triggers glycogen breakdown in mixed-astrocyte cultures. It would therefore be intriguing to investigate the effect of histamine on glycogen metabolism in type-I and type-2 astrocytes.

Acknowledgements

This work was supported by Grants-in-Aid(No. 63065004 and No. 63481}120Jfrom the Ministryof Education,Science and Culture of Japan and by a grant from the Japan Brain Foundationfor 1989. We are grateful to Smith Kline& French Laboratoriesfor the gift of histamine agonists.

253

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