Journal of Neurochemislry Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells Norimichi Nakahata, Marilene T. Abe, Isao Matsuoka, Tomoyuki Ono, and Hironori Nakanishi Department of Phurmaco/ugy, Fukushirna Medical College, Fukushima, Jupan

Abstract: The effect of adenosine on phosphoinositide hydrolysis was examined in 132IN1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5‘(N-ethy1carboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA > adenosine > NECA, a finding indicating that A,-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A,-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5’47-

thi0)triphosphate (GTPyS)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyltheophylline antagonized the inhibition. L-PIA could not inhibit GTPyS-induced accumulation of inositol phosphates in IAP-treated membranes. G;/G,, purified from rabbit brain, inhibited GTPyS-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A,class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells. Key Words: Adenosine-Gi/G,-Pertussis toxin-Islet-activating protein-Phosphoinositide hydrolysis-Cyclic AMP-Astrocytoma cells. Nakahata N. et al. Adenosine inhibits histamine-induced phosphoinositide hydrolysis mediated via pertussis toxin-sensitive G protein in human astrocytoma cells. J. Neuruchem. 57, 963-969 (1 99 1).

Adenosine has been reported to modulate the release of many neurotransmitters in the CNS and the PNS (Snyder, 1985). Adenosine in various tissues could inhibit and stimulate adenylate cyclase via A1- and A*class adenosine receptors, respectively (Van Calker et al., 1979; Londos et al., 1980). An N6-substituted adenosine analogue, L-N6-phenylisopropyladenosine (L-PIA), is a more potent agonist of Al-class adenosine receptors than adenosine and other analogues, whereas another adenosine analogue, 5’-(N-ethylcarboxamido)adenosine (NECA), is a more potent agonist of A2-classadenosine receptors than adenosine and other analogues, including L-PIA. Hughes and Harden (1986) have reported that

1321N 1 human astrocytoma cells possess Al-class adenosine receptors that interact with inhibitory GTPbinding regulatory protein (Gi) to inhibit adenylate cyclase, using pertussis toxin [islet-activating protein (IAP)], which catalyzes ADP-ribosylation and inactivates Gi, which couples inhibitory receptors to adenylate cyclase (Hazeki and Ui, 1981; Bokoch et al., 1983; Codina et al., 1983). Recent lines of evidence suggest that stimulation of Al-class adenosine receptors results in inhibition of phospholipase C (Long and Stone, 1987; Delahunty et al., 1988; Kendall and Hill, 1988), which catalyzes hydrolysis of phosphatidylinositol 1,4-bisphosphate to two second messengers, inositol 1,4,5-trisphosphate

Received August 29, 1990; revised manuscript received January 29, 1991; accepted February 13, 1991. Address correspondence and reprint requests to Dr. N. Nakahata at Department of Pharmacology, Fukushima Medical College, Fukushima 960-12, Japan. Abbreviations used: DEAE, diethylaminoethyl; G protein, GTPbinding regulatory protein; G , , inhibitory GTP-binding regulatory

protein; Go,GTP-binding regulatory protein from brain with an asubunit of 39 kDa; G,, stimulatory GTP-binding regulatory protein; GTPyS, guanosine 5’-(y-thi0)triphosphate;IAP, islet-activating protein; NECA, 5’-(Nethy1carboxamido)adenosine;L-PIA, L-N6-phenylisopropyladenosine; TEM buffer, 20 mMTris, 1 mMEDTA, and 5 mM 2-mercaptoethanol (pH 8.0) buffer.

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and diacylglycerol (Bemdge and Irvine, 1984; Nishizuka, 1984), although there is opposing evidence that stimulation of Al-class adenosine receptors results in activation or potentiation of phospholipase C (Hill and Kendall, 1988; Okajima et al., 1989). To date, the detailed mechanism of inhibition of phospholipase C by stimulation of AI-class adenosine receptors has not been established. It is also unknown whether both stimulatory and inhibitory modulations of phospholipase C exist, like the case of adenylate cyclase, which is regulated by stimulatory GTP-binding regulatory protein (G,) and Gi . The present study was undertaken to investigate the transmembrane control of adenosine receptor stimulation in 1321N 1 human astrocytoma cells, with special reference to inhibition of phospholipase C. The results obtained suggest that stimulation of Al-classadenosine receptors causes inhibition of phospholipase C as well as adenylate cyclase mediated via an IAP-sensitive GTP-binding regulatory protein (G protein).

MATERIALS AND METHODS Cell culture Human astrocytoma cells (1 32 1N I), a generous gift from Prof. T. K. Harden (Department of Pharmacology, University of North Carolina, Chapel Hill, NC, U.S.A.), were grown in Dulbecco’s modified Eagle’s medium with 5% fetal calf serum, 50 U/ml of penicillin, and 50 pg/ml of streptomycin at 37°C in an atmosphere of 5% C 0 2 in air.

3H-Inositol phosphate quantification in intact cells 3H-Inositol phosphates were quantified as previously described (Nakahata et al., 1986, 1989a,b). The cells were cultured in 12-well plates at a density of l Os/well after subculture by trypsinization and used 4-5 days after subculture. The cells in 12-well plates were labeled with 1 or 2 pCi/ml of [3H]inositolfor 48 h in Dulbecco’s modified Eagle’s medium. Before addition of drugs, the cells were incubated in Eagle’s minimal essential medium buffered to pH 7.35 with 25 m M HEPES for 10 min. Then, the drugs were added to the medium in the presence of 10 mM LiCl for 15 min, and the reactions were terminated by addition of 1 ml of ice-cold 5% trichloroacetic acid after aspiration of the medium. The trichloroacetic acid extracts were washed several times with diethyl ether to remove trichloroacetic acid, and then they were applied to a column (bed volume, 0.6 ml) of anion exchange resin (AG 1x4, 100-200 mesh, formate form; Bio-Rad). Total 3H-inositol phosphates were eluted by 6 ml of 1 M ammonium formate in 0.1 M formic acid.

3H-Inositol phosphate quantification in membrane preparations Accumulation of 3H-inositol phosphates in membrane preparations was analyzed by the method previously described (Nakahata et al., 1989a,b, 1990), with minor modifications. In brief, the cells in 150-mm-diameter dishes were labeled with 8 pCi/ml of [3H]inositolfor 2 days and lysed on ice with 2 mM EDTA and 10 m M HEPES (pH 7.0). Membranes were obtained by centrifugation at 15,000 g and washed twice with 2 mM EDTA/IO mM HEPES (pH 7.0)

J Neuroehem , Vol. 57, No 3, 1991

and 2 mM EGTA/lO mMHEPES (pH 7.0); then they were suspended in 2 mM EGTA/ 10 mMHEPES (pH 7.0). Membranes were incubated at 37°C for 10 min in 200 pl of medium of the following composition: I10 mM KC1, 10 mM NaCI, 1 mMM 2 P 0 4 , 0 . 3 mM MgC12, 10 mMLiCl, 1 mM EGTA, 0.18 mM CaC12 (free concentration, 0.1 p M ) , 0.1 mMATP, and 20 mMHEPES (pH 7.0). The reactions were terminated by addition of 1 ml of 5% trichloroacetic acid. 3H-Inositol phosphates were analyzed by the same method described above.

Cyclic AMP assay Cells were grown on 12-well plates at a density of lo5cells/ well after subculture by trypsinization. Cells were used 4 days after subculture. The incubation was started by addition of drugs in Eagle’s minimal essential medium containing 25 mMHEPES (pH 7.35) after several washes of the cells with Eagle’s minimal essential medium containing 25 mM HEPES. The reaction was terminated by addition of 1 ml of 5% trichloroacetic acid after aspiration of the medium. The trichloroacetic acid extracts were applied to a 1-ml column of aluminum oxide (neutral). Cyclic AMP was eluted by 3 ml of 0.5 M Tris-HC1 (pH 7.4) after the column was washed with 4 ml of water. Cyclic AMP was quantified by a proteinbinding method, described previously (Nakahata et al., 1977).

Purification of Gi/G, The Gi/G, fraction (Go is a G protein from brain with an a-subunit of 39 kDa) was obtained by the method described by Katada et al. (1986), with minor modifications. Membranes of rabbit brain cortex were prepared by homogenization in 10 mM HEPES/10 mM EDTA (pH 7.4) and three centrifugations at 45,000 g for 10 min. The membranes were dissolved in a small volume of 0.32 M sucrose, 5 mMMgC12, and 10 mMHEPES (pH 7.4) and were stored at -80°C until use. Brain membranes (2.5 g) from 16 rabbits were solubilized by 1% cholate in 20 mMTris, 1 mMEDTA, and 5 mM 2-mercaptoethanol (pH 8.0) (TEM buffer) for 1 h. Solubilized membranes were obtained by a 100,000-gcentrifugation for 1 h and applied to a diethylaminoethyl (DEAE)-Sephacel chromatography column (2.2 X 20 cm), which had been equilibrated by 25 mMNaCl and 1% cholate in TEM buffer. The column was then eluted at a rate of 1 ml/min with a linear gradient of NaCl(0-0.5 M ) in the same buffer, The eluate was collected in fractions of 8 ml. [35S]Guanosine5‘-(7-thio)triphosphate ([35S]GTPyS)binding (Northup et al., 1982) was used to quantify G proteins. Then, active fractions, which had been concentrated to 10 ml by ultrafiltration with Minimodule NM-3 (Asahi Kasei), were applied to an S-200 gel filtration column (1.3 X 70 cm), which had been equilibrated by 100 mMNaCl and 1% cholate in TEM buffer. The column was eluted at the rate of 1 ml/ min in the same buffer. The eluate was collected in fractions of 6 ml. The active fractions were then applied to a hydroxyapatite column ( I .6 X 20 cm), which had been equilibrated by 20 mM Tris, 0.1 mMEDTA, 5 m M 2-mercaptoethanol, 100 mM NaCI, and 1% cholate, pH 8.0. The column was then eluted at a rate of 1 ml/min with a linear gradient (0200 mM) of KH2P04.The eluate was collected in fractions of 2 ml. The active fraction, which had been diluted by 3 volumes of 0.67% Lubrol in TEM buffer, was further applied to a DEAE-Toyopearl 650(S) column (1.6 X 20 cm), which had been equilibrated by 0.5%Lubrol and 25 m M NaCl in TEM

ADENOSINE INHIBITS PHOSPHOINOSITIDE HYDROLYSIS buffer.The column was eluted with a linear gradient of NaCl (25-200 d) in 0.5% Lubrol in TEM buffer. Active fractions contained Gi/Go,which were ADP-ribosylated by IAP treatment. The Gi/G, fractions were dialyzed against a large volume of 20 mM Tns, 0.1 mM EGTA, 5 mM 2-mercaptoethanol, 25 mM NaCl, and 0.02% Lubrol (pH 8.0) for 48 h to use for analysis of phosphoinositide hydrolysis. Protein assay Protein concentrations were determined with the method described by Lowry et al. (1 95 1) or the protein-dye method (Bradford, 1976) using bovine serum albumin as the standard. Materials Adenosine, L-PIA, NECA, histamine, S-phenyltheophylline, and sodium cholate were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). GTPyS was obtained from Boehringer Mannheim (Indianapolis, IN, U.S.A.). Dulbecco’s modified Eagle’s medium and Eagle’s minimal essential medium were purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Fetal calf serum was from Cell Culture Laboratory (Cleveland, OH, U.S.A.). Aluminum oxide (neutral) was from Merck. IAP was from Funakoshi Pharmaceutical (Tokyo). rnyo-[2-3H]Inositol was purchased from American Radiolabeled Chemicals (St. Louis). Other chemicals or drugs were of reagent grade or the highest quality available.

RESULTS Adenosine and adenosine analogues (L-PIA and NECA) alone had no effect on 3H-inositol phosphate accumulation, but they inhibited histamine ( 100 pM)induced accumulation of 3H-inositol phosphates in a concentration-dependent manner (Fig. 1). The rank order in potencies of adenosine analogues to inhibit histamine-induced 3H-inositol phosphate accumulation was L-PJA > adenosine > NECA, a finding indicating that A,-class adenosine receptors could be involved in the inhibition. The inhibition by L-PIA of

L-PIA

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FIG. 1. Effects of adenosine(AD)analogueson histamine-induced phosphoinositide hydrolysis. Cells in which phosphoinositides had been labeled with [3H]inositolwere incubated with or without drugs for 15 min; without drug (A), 100 pM histamine (0),100 @ his100 pA-4 histamine plus AD tamine plus L-PIA (1 nM-100 @) ,).( (0.1-lOOpM)(m),and 100pM histamine plus NECA(O.l-100pM) (A).Data are mean SE (bars) values from three determinations, and the results were representative of three similar experiments.

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965

histamine-induced 3H-inositol phosphate accumulation was attenuated in the presence of 8-phenyltheophylline (20 p M ) , a nonselective adenosine receptor antagonist (Fig. 2). Therefore, the inhibition of 3Hinositol phosphate accumulation by adenosine analogues might be mediated via A,-class adenosine receptors. When the cells were treated with IAP (100 ng/ml) for 48 h, Gi in the cells was ADP-nbosylated and inactivated (Nakahata et al., 1989a, 1990). In the IAPtreated cells (Fig. 3), L-PIA (30 p M ) failed to inhibit histamine ( 100 pM)-induced accumulation of 3H-inositol phosphate as well as isoproterenol (10 pM)-induced accumulation of cyclic AMP. The results indicate that an IAP-sensitive G protein, which is activated by stimulation of A,-class adenosine receptors, might inhibit phospholipase C as well as adenylate cyclase. In membrane preparations, L-PIA (30 p M ) inhibited GTPyS (10 pM)-induced 3H-inositol phosphate accumulation, and 8-phenyltheophylline antagonized the inhibition (Fig. 4a). Furthermore, L-PIA failed to inhibit GTPyS-induced 3H-inositol phosphate accumulation in IAP-treated membranes (Fig. 4b). The data from membrane preparations support the results from intact cells that stimulation of A,-class adenosine receptors inhibits phospholipase C. Although L-PIA potentiated GTPyS-induced 3H-inositol phosphate accumulation in IAP-treated membranes (Fig. 4b), the potentiation was not inhibited by 8-phenyltheophylline, a result suggesting that the potentiation was not receptor mediated. The purified Gi/Go fraction from rabbit brain migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to 4 1, 39, 36, and 35 kDa, corresponding to GIs, Goa, 636, and ,& (Fig. 5). Although the ysubunit (8 kDa) could not be detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit might exist in this fraction, because the ysubunit has been reported to bind tightly to the @-subunit (Bokoch et al., 1983). The concentration of Gi/ Go was calculated using as the molecular mass of Gi/ Go 83 kDa. Gi/Go (7.5 pmol/0.2 ml) significantly decreased GTPyS (10 pM)-induced accumulation of 3Hinositol phosphates in membrane preparations (55 pg), although Gi/Goalone increased the hydrolysis (Fig. 5). Inhibition of GTPyS-induced accumulation of 3Hinositol phosphates by Gi/Go in membrane preparations was observed in a concentration-dependent manner (Fig. 6). When GJG, that had been heated for 5 min was used, the inhibition of GTPyS-induced accumulation of 3H-inositol phosphates by Gi/Go was significantly weakened (Fig. 6), a result suggesting that the native form of GJG, is necessary to inhibit GTPySinduced accumulation of 3H-inositol phosphates. Gi/ Go alone increased the accumulation of 3H-inositoJ phosphates without GTPyS in membrane preparations, but the heated GJG, also increased the accumulation with a concentration dependency similar to

J . Neurochem., Vol. 57, No. 3, 1991

N. NAKAHATA ET AL.

P < 0.05 -2

COnt

L-PIA

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20pM

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FIG. 2. Effect of 8-phenyltheophylline (8-PT)on L-PIA-induced inhibition of 3H-inositolphosphate accumulation. L-PIA (30 p M ) significantly inhibited histamine (HA; 100 pM)-induced accumulation of 3H-inositolphosphates ( p < 0.05), and 8-PT (20 pM) significantly attenuated the L-PIA-inducedinhibition( p < 0.05). Each drug was added to the medium simultaneously. Data are mean f SE (bars) values from three determinations, and the results were representative of two similar experiments.cont, control.

that of native G,/G, (Fig. 6), a finding indicating that the increase might not be due to Gi/G,. DISCUSSION A I -class adenosine receptors in various tissues have been reported to couple to GI,which inhibits adenylate cyclase, resulting in a decrease in cyclic AMP content (Hazeki and Ui, 1981; Delahunty et a]., 1988). Stimulation of A]-class adenosine receptors in human astrocytoma cells also elicited a decrease in cyclic AMP content in the present study, as has been shown by Hughes and Harden (1986). The decrease in cyclic AMP content by Al-class adenosine receptors was completely inhibited by treatment of the cells with IAP (Fig. 3 ) (Hughes and Harden, 1986). Thus, stimulation of Al-class adenosine receptors results in inhibition of adenylate cyclase via G,. In addition to inhibition of adenylate cyclase, stimulation of A,-class adenosine receptors resulted in inhibition of histamine-stimulated phospholipase C in human astrocytoma cells. The inhibition of phospholipase C by stimulation of A I-class adenosine receptors was also completely inhibited by treatment of the cells with IAP (Figs. 3 and 4), a finding indicating an involvement of an IAP-sensitive G protein in inhibiting phospholipase C by A,class adenosine receptor stimulation. The IAP-sensitive G protein in inhibiting phospholipase C might be G,, which has been accepted to be activated by Al-class adenosine receptor stirnulation. In fact, purified G,/G, from rabbit brain inhibited GTPyS-induced activation of phospholipase C in membrane preparations (Figs.

J N i w m hem.. Vo/. 5 7. No. 3. 1991

5 and 6), a result supporting the idea that stimulation of A,-class adenosine receptors activates G i, which in turn inhibits phospholipase C. The results suggest that Gi has at least two roles in human astrocytoma cells: an inhibition of adenylate cyclase and an inhibition of phospholipase C. However, stimulation of a2-adrenoceptors in rabbit platelets results in activation of Gi and inhibition of adenylate cyclase but enhances phospholipase C activity stimulated by a thromboxane A2 analogue (Matsuoka et al., 1990). The discrepancies of Gi action in different cells might be explained by the existence of subclasses of Gi (Itoh et al., 1988). Although each Gi subclass might have different physiological roles (Gierschik et al., 1989; Linder et al., 1990), no Gi subclass has been reported to inhibit phospholipase C. As purified Gi/Go from rabbit brain inhibited phospholipase C in human astrocytoma cell membranes (Figs. 5 and 6), the fraction of purified Gi/G, may contain a G protein that inhibits phospholipase C. Lapetina ( 1986) reported that thrombin-stimulated accumulation of inositol phosphates in saponin-permeabilized human platelets was enhanced by IAP treatment, an observation indicating that G , could inhibit phospholipase C. Furthermore, Litosch ( 1989) reported that an IAP-sensitive G protein could inhibit phospholipase C in cerebrocortical membranes. In all cases, including the present study, the inhibitory effect of Gi or G protein on phospholipase C was not so potent. In membranes treated with IAP (Fig. 4b), L-PIA potentiated GTPyS-induced activation of phospholipase C, and

IS0

HA

FIG. 3. Effect of IAP on L-PIA (PIA)-induced inhibition of cyclic AMP (CAMP)accumulation and 3H-inositolphosphate (IP) accumulation. Left: Effect of PIA (30 pM) on isoproterenol (ISO; 10 pM)-induced accumulation of CAMP.Right: Effect of PIA (30 pM) on histamine (HA; 100 @)-induced accumulationof IPS.Top: Cells without IAP treatment. Bottom: Cells treated with IAP (100 nglrnl, for 48 h). PIA significantly inhibited ISO-inducedcAMP accumulation as well as HA-induced IP accumulation (*p < 0.05) in the cells without IAP treatment. However, PIA failed to inhibit the accumulation of cAMP and IPS in IAP-treated cells. Data are mean k SE (bars)values from three determinations,and the results were representativeof two similar experiments. cont, control.

ADENOSINE INHIBITS PHOSPHOINOSITIDE HYDROLYSIS

b. IAP(+)

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1

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FIG. 4. Effect of L-PIA on GTPyS-induced accumu-

lation of 3H-inositolphosphates in membrane preparations treated with or without IAP. a: Control membranes (without IAP treatment). L-PIA (30 pM) significantly inhibitedGTPyS (10 pM)-inducedaccumulation of 3H-inositol phosphates ('p