Moh,cular Pharmacolo&1' Secnon, 206 {199i j 47 52 199l Elsevier Science Publishers B.V. (Biomedical Di~q>ion}0922-4106/91/503.5c5
European Journal of Pharmacolo,~"
47
ADONIS 09224!069100056Q
EJPMOL °:9131
P,-purinergic activation of phosphoinositide turnover is potentiated by A :receptor stimulation in thyroid cells Martina Nazarea. Fumikazu Okajima and Yoichi Kondo Departmem of P]~swal Bl~cheml~lrv, Institute of End:~ rl nol~)~. Gunma t "ni~'eMm. ~1acha~h~ : 71. J~l~an
Recei'~ed 4 July 1990. revised MS received 4 Seplernbe~ 1990. accepIed 25 September t990
We recently discovered that in FRTL-5 cells the Pi-purinergic agonist PIA tphenylisopropyladenosinel markedly enhanced P2-purinergic agonist-induced responses in an lAP (islet-activating protein or pertu~sis tovJnt-sensitive manner. ~n thi> study we tested PIA and other P~ agonists for their permissive effects on GTP (a P: agonistl-m,'luced inositol phosphate production and arachidonate release and found that the order of potency was PIA = CHA (cyclnhex',ladenosine} > NECA (N-ethylearboxa~ndoadenosine) = CADO (chtoradenosine). The P~ agonists also caused an inhibition of thyrotropin-induced cAMP increase in FRTL-5 cells as well as a stimulation of cAMP accumulation in lAP-treated cells. The order of potency was very :.imflar for phosphoinositide turnover, arachidonate release and cAMP inhibition, and therefore suggestive of an adenosine A1 receptor thee. As for cAMP stimulation, CADO. PIA and CHA were weaker than NECA and thus in agreement v.ith the A : receptor t;~e. The order of potency of four adenosine antagonists also revealed a similarity between arachidonate release and cAMP inhibition and a difference for arachidonate release and cAMP stimulation. These results indicate that ,Both A l- and A:-recepmr subtypes are present in FRTL-5 cells and that extracethilar adenosine enhances the P:-purinergic agonist-induced response> by >timulating an A 1 receptor which is coupled to an IAP-sensiti;e G-protein,%). Adenosine; Adenosine A 1 receptor: FRTL-5 thyroid cells: Phosphoinositide turnover: ArachiJonate release: Purine ~ receptor
1. Introduction
Purine nucleoside and nucleotide receptors are currently classified into two subclasses (Burnstock, 1987). namely the purine Pt receptor which has a high affinity for extracellular adenosine, and the purine P= receptor at which A T P binds more strongly. For the former, two further subclasses can be distinguished by a negative (A t) or positive (A z) coupling to adenytate cs~tase (Van Calker et at.. 1979; Londos et at.. 1980). T N s classification is also based on the relative potency of adenosine derivatives including phenyfisopropyladenosine (P]A) and N-ethylcarboxamidoadenosine ( N E C A ) to influence c A M P levels. The former is the strongest agonist at A~. receptors, whereas the latter is most potent at A : receptors. Both receptor types can be competifivdy antagonized by xanthine derivatives. A t~ird ( A ) ) subtype. which is characterized by its linkage to calcium metabolism rather than adenylate cydase, has been postulated by Ribeiro and Sebastiae (!986). In FRTL-5 coils purine ~ receptor stimulation caused
Correspondence to: Manina Nazm-ea. Department of Physical Biochemis,.D', institute of Endocnnology, (.~unma Univerxity~Maeb~sba 371. Japan.
phospholipase C activation which ~.~as accompanied by Ca r* mobdization, aracbJdonate release and I efftux (Okajima et al.. t988: Sho et al.. !989). A simultaneous stimulation of the adenosine receptor by' PIA. which by i~_self causes no such stimutatoQ effects, led to an enhancement of phosphotipase C and its re!ated responses (Okajima et ai.. t989a}. For cq-adrenoceptors the same potentiation was inducible by PIA ( O k ~ i m a . 1989b}. A similar potentiation induced by adenosine has been described for RBL-2H3 mast cells (All et ai., 1990) and guinea-pig cerebra! cortex (Holiingsworth et ai.. 1986: Hill and K e n d a l l !987}. Besides adenosine, other neuromoduiator> have been found to induce a similar positive cross-talk. For instance, epinephrine potentiates reactions induced by serotonin (De Chaffoy de Cource!tes el aI.. 1987L ~asopressin {Busi~field e~ aL, 1987l and thrombin (green et al.. !988: Crouch and kapetina, t988) in human platelets. In CCL39 fibroblast~ {Paris et aL. 1988) thrombin reactions have been described to become enhanced by a number of growth factors. In some cases (All et M.. t990: Okajima et ak, 1989a.b) G : p r o t e i n involvement for the perwdssive effect has been demonstrated. In FRTL-5 ceils, for instance, I A P (islet-activating protein or pertussis toxin) treatment, which inactivates IAP-st~bstrate G-proteins
by ADP ribosytation, had almost no influence on the Pz-purinergic receptor-induced phospholipase C activation and the subsequent responses, but completely abolished the permissive effect induced by PIA. The aim of the present study was to characterize the adenosine (P1)-receptor subtype which potentiates the P,-receptor-mediated phosphoinositide turnover and arachidonate release in FRTL-5 cells in an IAP-sensirive manner. Judging from agonist and antagonJ ~, orders of potency, we conclude that the Aj receptor md,Jced the above-described potentiation.
2.3. A rachidonic acid release measurement Cells labeled for 6 h with [*H]arachidonate were washed three times at t0-min intercals at 37°C with HEPES-buffered medium which consisted of (mM): HEPES 10 (pH 7.4). NaCI 134, KCI 4.7, KH2PO4 1,2, MgSO4 1.2, CaCI: 2.0. NaHCO3 2.5 and glucose 5, and bovine serum albumin (fraction V) 0,1%. Cells were incubated for I0 rain with the indicated agents in 750 #1. Radioactivity of the medium (0.5 ml) was measured in a liquid scintilhtion spectrometer (Beckman LS 7500). Data were expre,,sed as percentages of total radioactivity incorporated into the cells.
2. Materials and methods 2.1. Drugs and abbrel,iations From Sigma. adenosine. N~-(L-2-phen31isopropylladenosine (PIA), 5"-N-ethylcarboxamidoadenosine (NECA), N6-cyclohexytadenosine (CHA). 2-chloradenosine (CADO). 3-isobutyl-l-methylxanthine (IBMX) and adenosine deaminase were bought. From Research Biochemicals Inc. (Wayland, MA) we purchased 8-cyclopentyl-l,3-dipropylxanthine (CPX). 8-cyctopentyl-l.3-dimethylxanthine (CPT). and L3-diethyl-8-phenyl×anthine (DPX), GTP was obtained from Yamasa Shoyu Co. (Choshi, Japan). Bovine TSH was purified by the method of Yora and Ui (1978) and had an activity of 2'3 U / m g based on its follicle-reconstructing activity in culture. Myo-[2-3H]inositol (15.6 Ci/mmol) and [5.6,8.9Al,12,14.15-3H]arachidonic acid (100 Ci/mmol) were obtained from Du Pont-New England Nuclear. lAP and RO 20-1724 (4-(3-butoxy-4methoxybenzyl)-2-imidazolidinone) were gifts from Dr. Michio Ui (Tokyo Univer-~ity, Tokyo, Japan) and Nippon Roche Research Center (Kamakura, Japan), respectively. The reagents for the cAMP radioimmunoassay were generously provided by Yamasa Shoyu Co. (Choshi, Japan). Sources of reagents for cell culture and other purposes were those described by Okafima etal. (i98~).
2.4. cA M P measurement Cel!s were washed once with HEPES-buffered medium and incubated for 10 rain at 37°C in the same buffer in a final volume of 750 #1, A 10-min incubation was terminated by adding 10/tl of 1 N HC1 and cAMP content was measur~.~dby radioimmunoassay (Honma et al.. 1977). 2.5. Measurement of mositol phosphate production [~H]inositol-labele6 cells were washed once and proincubated for 10 miE with HEPES-buffered medium. The indicated agents were added in a final volume of 750 #1 together with i0 mM LiC1, GTP (300/zM) and adenosine deaminase (0.5 units/ml; it was omitted, however, in adenosine preparations) and incubated for 30 min. The reaction was terminated by aspirating the medium, adding 700 ul of 0.i N HC1 and overnight fieezing. The separation of [3H]inositol phosphate was performed as described in Okajima et al. (1988).
3. Results 3.1, Adenosine derit,ati~,es enhance phosphoinositide and arachidonate release J'esponses to GTP
2,2. Cell ctdture FRTL-5 ceils, a cell fine deri:ed from normal rat thyroid (Ambesi-lmpiombato et ak. 1980). were obtained from Interthyr Research Foundation (Baltimore, MD) and grown as described by Okajima et al, (1989a). When cells were 60-70% confluent in 12-welI plates, the cahure medium (Coon's F-12 containing 5% calf serum and six hormones including TSH) wa:; changed for a further 2 days to Ham's F-10 containing only 5% calf serum (TSH depletion). Where indicated, this medium was supplemented with ehher 30 ng/mf lAP or [-~Htinositol (2 #Ci/ml), or both. For arachidonate release measurement, [-~H]arachidonate (0.1 #Ci/ml) was added 6 h prior to agent addition,
GTP stimulates phosphoiipase C in FRTL-5 cells via a P2-purinergic receptor (Okajima, 1989a). This stimulation was hardly affected by IAP treatment of the cells. Here, GTP was added together with adenosine or adenosine analogues. Although these adenosine analogues alone had no effect on either inositol phosphate production or arachidonate release, they enhanced both of these GTP-induced responses in a dose-dependent manner, As for the inositol phosphate production (fig. IA). the order of potency was CHA = PIA > adenosine = CADO = NECA and was roughly the same as for arzehidonate release (fig. 1B) with P1A and CHA being the most potent agonists (see EDs0s in legend to fig. 1B) and NECA, CADO and adenosine approximately one
49 eta!., 1 9 8 9 a ) a n d this s u g g e s t s t h e i n v o l v e m e n t o f I A P - s u b s t r a t e G - p r o ' , e i n s in a d e n © s i n e - r e c e p t o r - m e d i a ted p o t e n t i a t i o n o f G T P r e c p o n s e s .
lOO
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3.2. c A M P receptors
/~e t
i
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i
reveal both A,;- and A 2 - o T e
By r a i s i n g c A M P levels w i t h T S H in p r e v i o u s t } r T S H - d e p l e t e d c e l l s (fig. 2 A ) w e c o u l d o b s e r v e a n i n h i b i t i o n b y a d e n o s i n e a n d its a n a l o g u e s . T h e o r d e r o f potency for the four adenosine derivatives was PIA > C H A > N E C A > C A D © . A d e n o s i n e itself w a s r a t h e r e f f e c t i v e at l o w e r c o n c e n l r a t i o n s a n d t h i s i n h i b i t i o n d i d
0
100
measurements
t
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{A) Controt o / "
100 I
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8 7 6 S Agonlsts (-log M) Fig. 1. The effect of adenosine agonists on inositol phosphate production (A) and arachidonate release (B). ©. PIA; e. adenosine; a. NECA; A. CAD©; [2. CHA. GTP (300 vM) had been added to all and adenosine deaminase (0.5 u.,fits/ml, to all but the adenosine preparations. Incubation times were 30 min (A) and t0 .win (BL In the absence of adenosine derivatives. GTP increased the inositol phosphate production from 3.7 x 103 dpm to 20.2 x 10 ~ dpm IA) and the arachidonate release from 73 dpm to 832 dpm (B). Adenosine derivatives alone had no effect on basal values (they were in the range of 3.6-3.9 x I03 dpm for inositol phosphate production and bet~een 70 and 90 dpm for arachidonate release), but enhanced GTP-induced actions roughly twofold in the case of inositol phosphate production and fivefold for arachidonate release. The increments of responses to GTP induced by adenosine derivatives were plotted as at pet~.~ntage of the increment induced at 10 t~M PIA (for inositol phosphate production 27.0x103 dpm and for arachidonate release 3368 dpm). Data represent the means of two (A) and three (B) experiments carried out in duplicate or triplicate. In {B) S.E. values were smaller than tot{ and EDso values+S.E. (nM) were 18,5 ±0.7 for P1A, 89.1 ±6,4 for adenosine, 50.3±4.5 for NECA. 70.85:7.0 for CAD© and 24.2+_20 for CHA.
o r d e r o f p o t e n c y w e a k e r in i n d u c i n g a p o t e n t i a t i o n o f the GTP-induce.d stimulation of phospholipase C and a r a c h i d o n a t e r elease. T h i s f i n d i n g w a s c o n s i s t e n t w i t h p r e v i o u s r e s u l t s ( O k a j i m a et al., I 9 8 9 a ) . In l A P - t r e a t e d cells (results not shown) no stimulato W effect by adenosine analogues for either aract'ddonate release or phosphoinositol production could be obser.,ed (Okajima
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7 6 S Agontst~; (-lo~ M} Fig, 2. Do~=re~po~se cu~'e~ of adenosine agoinsts concerning inhibitlon {A) and stim~alation {B) of cAMP accumulation in control and tAP-treated cells, respectively. ©. PlAt e, aden©sine: ~x. NECA: A. CAD©: ~. CHA In [A) TSH (2 nMt and the phosphodiesterase inlubitor R© 20-i721 (t00 #M) were added together ~ith the agents. In (B) TSH was omitted. Aden©sine deamina.~e (0.5 units/rrd) was added to al! but the adeeosine preparations, hicubafion time was 10 mJn. gDs0 v a l ~ (in nM (A)) were for PfA 1.86±0.09. for adenosine 25.1 ± 2.4. far NECA 4~25 ±0.38. for CAD© 25.6 ± 2.5, and for CHA 3.5 zO.3. In (A} the TSH value (1395 pmo]/mg/proiein) and in (B) the IAP bec.aI ~alue (9.9 pro©l/rag/protein) was taken to be 10O~. Values are the means of al least sLx experiments earned out in duplicate. S.E. values were ~I0% arid therefore no error bra's were dravrn.
not go belt~w 30~ even at higher doses. Whether different experimental conditions (adenosine deaminase being left out) or still other factors account for this different behavior could not be determined. In any event, this rank order of potency is in good agreement with that of agonist potencies for A~ receptors (Van Calke~ et al.. 1979: Londos et al., 1980). In lAP-treated cells (fig. 2B). the same agonists induced an increase in c A M P uniess T S H was present. The order of potency for agonists was N E C A > C A D O = adenosine > PIA > C H A , which is
~
3.3. Influence of adenosine antagonists on arachidonate release and cA MP accumulation
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(B) Control
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Fig. 3. Dose-response curves of adenosine receptor antagonists. )n A, antagonists DPX, (o): CPX. (11); CPT. t~; IBMX, (AI were added at the indicated concentrations m medium cont,'fi~g 300/aM CTP and 0.1 .aM P1A. In B, antagonists were added together with P1A (0.l .aM), TSH (2 nM). adenosine deamina~ t0.5 units/mlj and RO 20-1724 (100 .aM). In C, lAP-treated ceils were tested w;~ha~ta~or,]Ms in the same way as in B except that the PIA concentration was 10 ;aM and that TSH was left out. Incubation time was 10 rain. Data are expressed as PIA effect ~qthout antagonists equaling 100%. lf~)% values were 12.1 × 103 dpm (A). t096 pmo!/mg protein (Bi and 36.9 pmol/mg protein (C). Experiments are repr~entative of at lea.st two more duplicate determinations. Apparem K, values (nM~ were for: DPX, 102 (A) and 137 (B); CPX, 0.30 CA) and 0.24 (B): CPT, 29 (A) and 19 (B). and we-'~c~,tculated accc~rdingto the lot)owing equation: K i [C50~antago~tlX ED~c~eL~I/(ED~ptAi-[C] (PIA)). PIA ED~3was estimated to be 18.5 nM (from fig. 1B) and L86 rum (from fig. 2A). =
characteristic of adenosine A 2 receptors (fig. 2) (Van Calker et al., 1979: Londos et al., 1980). Thus. there are two types of P~ receptors in FRTL-5 cells: A~ and A 2 receptors, which are coupled to adenylate cyclase in an inhibitor3" and stimulator 3' fashion, respectively. When comparing the potentiation of GTP-activated phospholipase C (fig. I A ) and arachidonate release (fig. 1B) responses with c A M P inhibition (fig. 2A) and c A M P stimulation (fig. 2B). it is obvious that the order of potency is roughly identit.a! between the potentiafion of G T P effects and c A M P inhibition, although there is a one order potency shift to the left for c A M P inhibition (fig. 2A). These findings suggest that adenosine-induced potentiation of G T P actions and adenylate cyclase inhibition are mediated by the same receptor, the At-type receptor,
T o further confirm that the A 1 and not the A 2 receptor is involved in the permissive action of adenosine on GTP-induced stimulation of phospholipase C and its related responses, four adenosine antagonists were tested for their ability to antagonize PIA actions with respect to arachidonate release (fig. 3A) and the inhibition (fig. 3B) and stimulation (fig. 3C) of c A M P accumulation. All these PIA-induced responses were antagonized by adenosine antagonists. The order of antagonist potency was C P X > C P T > D P X >> I B M X for PlA-induced arachidonate release (fig. 3A) a n d inhibition of c A M P (fig. 3B). In lAP-treated cells (fig. 3C), the rank order of antagonists to inhibit the stimulation of c A M P accumulation was D P X = C P X > C P T > I B M X and thus clearly different from the one for arachidonate release and c A M P inhibition. Since I B M X also acts as a phosphodiesterase inhibitor, its behavior is difficult to interpret. In any event, the high potency of C P X in fig. 3A and 3B argues for an At-type receptor. A shift of potency to the right can be observed for c A M P inhibition (fig. 3B) when compared to the araehidonate release (fig. 3A), which might be caused by ditferent app.zrent potencies by which P I A induced the respective respenses. In fact, the K i values (see legend to fig. 3) were the same. These findings support the idea that PI.'_-induced inhibition of c A M P accumulation and pote,~tiation of the arachidonate release response are m e O a t e d by the same receptor, the A~ receptor.
4. Discussion T h e present study shows that the potentiation of phospholipase C and arachidonate release induced by adenosine (P~)-reeeptor stimulation is of the adenosine
51 A~-subtype r e c e p t o r . T h r e e lines of e v i d e n c e s u p p o r t ttfis finding. Firstly. P I A a n d CI-tA w e r e m o r e p o t e n t t h a n N E C A , C A D O a n d a d e n o s i n e in i n d u c i n g this effect. Secondly, the r a n k o r d e r o f a g o n i s t - i n d u c e d c A M P i n h i b i t i o n , w h i c h is by d e f i n i t i o n an A~ receptor-mediated r e s p o n s e , m a t c h e d t h a t of t h e agonist-induced arachidonate release and phosp h o i n o s i t i d e t u r n o v e r . T h i r d l y , the p e r m i s s i v e effect was competitively antagonized by xanthine derivatives with the s a m e r a n k o r d e r for c A M P inhibition and a r a c h i d o n a t e release, b u t a d i f f e r e n t o n e for the l a t t e r and c A M P s t i m u l a t i o n . A s i m i l a r p o s i t i v e c r o s s - t a l k w a s r e p o r t e d for a d e n o sine in g u i n e a - p i g c e r e b r a l cortex, w h e r e it e n h a n c e d histamine-stimulated phosphatidylinositol hydrolysis ( H o l l i n g s w o r t h et al.. 19865. C o n v e r s e l y . the s a m e effect was d e s c r i b e d to b e n e g a t i v e l y m o d u l a t e d by a d e n o s i n e in m o u s e c e r e b r a l cortex ( K e n d a l l a n d H i l l . 1988). W h e t h e r t h e c r o s s - t a l k is c a . n e d o u t in a p o s i t i v e ( s t i m u l a t o r y ) o r n e g a t i v e ( i n h i b i t o r y ) m a n n e r s e e m s to be tissue- a n d species-specific. M o r e e x a m p l e s of n e g a tive c r o s s - t a l k are d o p a m i n e , w h i c h m o d u l a t e s a n g i o tensin ( J o u r n o t e t a l . , 1987) a n d T R H r e s p o n s e s in rat p i t u i t a r y ( V a l l a r e t a l . , 19885, a n d e x c i t a t o r y arcfino a c i d s to i n h i b i t n o r e p i n e p h r i n e ( N i c o l e t t i e t a l . , 19861 a n d h i s t a m i n e ( B a n d r y e t a l . , 19865 a c t i o n s in rat hipp o c a m p a s . In b o t h p o s i t i v e a n d n e g a t i v e c r o s s - t a l k . I A P - s e n s i t i v e G - p r o t e i n s s e e m to be i n v o l v e d in the transduction (Okajima etal., 1989a.b; Delahunty etal.. 1988). The mechanism by which IAP-snbstrate G-proteins potentiate the phospholipase C activation and a r a c h i d o n a t e release r e m a i n s u n c l e a r . T h e a d e n o s i n e r e e e p t o r - l A P - s u b s t r a t e G - p r o t e i n t r a n s d u c e r s y s t e m has r e c e n t l y b e e n s h o w n to b e the l i n k in c a u s i n g the i n h i b i t i o n o f C a -~+ c h a n n e l s a n d t h e s t i m u l a t i o n o f K c h a n n e l s a s well a s the i n h i b i t i o n o f a d e n y l a t e cyc!ase ( F r e d h o l m a n d D u n w i d d i e . 19885. T h e q u e s t i o n w h e t h e r this novel p e r m i s s i v e effect o f a d e n o s i n e is j u s t c a u s e d s e c o n d a r i l y by a c h a n g e in c A M P o r C a : - levels h a s b e e n a d d r e s s e d in p r e v i o u s w o r k s ( O k a j i m a e t a l . , 1989a. b) a n d c o u l d be r u l e d out. H o w e v e r , an a c t i v i t y c h a n g e in K ÷ c h a n n e l s may" be a c c o m p a n i e d b y a s ~ i t c h o f the p l a s m a m e m b r a n e p o t e n t i a l , a n d it c o u l d t h e r e b y m o d u l a t e the a c t i v i t y o f the p h o s p h o l i p a s e C - C a 2 system ( D i V i r g i l i o et ok, 1987t. T h i s possibility is now u n d e r investigation.
AeknoMedgements This ~ork wos supported by a Gram-in-Aid for Scientific Research and a Grant-in-Aid for Scientific Research on Pnorit) Are~-, from the Minist~' of Education. Science and Culture of Japan. M.N. is a Japan Society for the Promotion of Science Fellow.
References All. H., J.R. Cunha-Melo, W.F. Saul and M.A. Beaven. 1990, Activation of phospholipase C via adenosine receptors provides syner~stic signals for secretion in amigen-stimulated RBL-gH3 cells, J. Biok Chem. 265, 745. Ambesi-lmpiombalo. F S.. 1. A.M. Parks and H.G. Coon. 1980. Culture of hormone-dependem functional epithelial cells from rat thyroids. 8roc. Natl. Acad. Set. U.S.A. 77. 3455. Baudry. M., J, Evans and G. Lynch- 1986. Excitatou amino acids inhibit ~timulation of phosphatidylinositol metabolism by aminergic agonists in hippocampus, Nature 319, 329 Burnstock, G., t987. A basis for distinguishing two types of punner~c receptors, in: Cell membrane receptors for drugs and hormones. eds. E. Bolts and R.N, gtraub IRaxen Press. New York! p. 107. Bushfield. M.. A. McNico] and D.E. Mactmyre. 1987. Possible mechanisms of the potentiation of blcod-platelet activation by adrenNine. Biochem. J. 241. 671. Crouch, M,F. and E.C. Lapetina. 1988. A role for G! in control of thrombin receptor-phospholipase C coupling in human platelets. J. Biol. Chem 263. 3363. De Chaffo5 de Courcetles. D.. P. Ko=~ens. H. Van Belle and F. De Clerck. 1987. The synerostic effect of serotonin and epinephrine on the human platelet at the level of ~ignal ~ransduction. FEBS Lett 219. 283. DeVoahunty. T.M, M.J Cro~in and J. Linden. t988. Regulation of G H ~-cetl function ~ia adenosine A I receptors. Biechem. J. 255. 69. Di Virgflio. F.. P,D. Low-. T. Anderson and T. Pozzan, 1987, Plasma membrane potential modulates chemotactic peptide-stimulated cs"to.,~olic free Ca 2" changes in human neutrophils. 262, 4574. Fredhotm. B.B. and T.V Dunwiddie, 1988. Ho'.~ does adenosine inhibit transmitter release?, Trends Pharmacok Sci 9. 130. Hill. SA. and D.A. Kendall, 1987. Studieb on the adenosine receptor mediating the augmentation of histamine-induced inositol phospholipid bydrolysis in guinea plg cerebral cortex. Br. J. Pharmacoi. 91. 661. Hollings:~er~h. E.I3. R.A De La Cn~ and J W DalV. !986. Accumulation of ino~itol phosphate, and cyclic AMP in brain slices; ~nergisnc interactions of h~stamine and 2-chloradenosine. European J Pharmacoh i22. 45. Hon no. M.. T. Satoh. J Takeza~a and 5,1. Ui. 1977. An uhra.~ensiti~e rletbod for the si,~uhaneou~ determination of cychc AMP and c-,~hc (.iMP in ~mait-~olurne sample~ from blood and tissue. B~crchem. Med. 18. 257. Jot~aot. L. V Hombtirger, C PantaIoni. M. Pnam. J. Bockaert and : . Enjalbert, 1987, An t~et activating proton-sensitive G protein i. mvol~ed m dopamine mhibit~on of emNotensin and lhryrotr(~ I m-r¢lea~mg ho.,~,ii~ne->imlu,ated illoMlo{ phosphate production i . ante,;o: pituitar} cet!~. J. Biol. Chern 262, !51Ck6. Kendall. D.A and S.J. Hill. 1988. Adenosine inhibition of histm'nine~timuiated inositol phosphclipid hydrolysis in mouse cerebral codex. J. Nearc,chern. 50. 497. kondos. C.. D.M,F Cooper and J. Wolff, t9g0. Subcla.s~e~ of e,~lernal adenosine rc-cept,~r~, Pn~c. Nod Acad Sck US.A. 77. 2551, N~olem, F, MA. !adaro]a. JT. Wrobte~sk~ ~nd E Costa, 1986. k×citator~ Amino acid recx~',ltlon ~ite+coupled with ino+i{ol phosphohp+d moraN+loom: De~etopmen'at changes and imeracnon ~ith a+-adrenoreceptor+: P+c¢. Nalk Acad. Sci. U.S.A. 83+ 1931+ Okajima. F.+ K. Sho and Y. Kondo. 1988. Inhibition by i>let-acti~"ating protem, pertu~qs toxin, of Pz-punnergic receptor-mediated iodide efflux and phosphoinos~tide turnover ia FRTL-5 cent. Endocnnologb 123, 1035. Okmima. F.. K, Sa~< M. N~.zarea. K. Sho rand Y. Kt~ndo. 1989a. A perrai~si~e rifle of per~uss~s to~in qubstrate G-I~rotein in Pzpunnerg~c stimulation of phosphoinc)~itide turnover and arachidonate r¢]ea,e in FRTL-5 thyroid ceils, J. Biol. Chem. 264, ] 3029.
Okajima. F,, K. Sato. K. Sho and Y. Kondo. lC~89b. S[imulalion of adenosine receptor enhances al-adrener~c receptor-mediated activation of phospholipa~e C and Ca z ÷ mobilization in a pert,assis toxin-sensitive manner irt FRTL-5 thyroid cells, FEBS Lett. 248, 145. Paris. S., £-C. Chambard and J. Pou~,ssegur. 1988. Tbrosine kina~e activating growth factors poten0a[e tbrombin- and AIF~ -induced phosphoinosidde breakdown in hamster fibroblasts. J BioL Chem. 263, 12893. Ribeiro. J.A. alad A.M. Sebastiao. 1986, Adenosme receplor~ and calcium: basis for proposing a third (A~) ademJ~m~ receplor, Progr. NeurobioL 26, 179. Sho. K.. F. Ok;~jima. H Aklvama. Y Shoda. [. Kobasa~hi and Y Kondo. 1989. Requirement of in~uhn-like gro~th factor I plu, hydrocorfsone for the regeneration of th?,rotropin ~TSHt depen-
dent mechani,m of 1 -efflu~, and Ca z• mobilization in FRTL-5 cells during TSH depletion. Endc~--rinology 124. 598. Sleen. V M . O-B. Tysne~ and H Hoimsen, igg8, Synergism between thrombin and adrenahne (epinephrine) in human platelets. Biochem. J. 253. 581 Vallar. L. L.M. Vicenfini and J. Meldolesi. 1988, Inhibition of ino~itol phosphate production ix a late, Ca 2*-dependent effect of D2 dopaminergic receptor acP~a0on in rat lactotroph cells, J. Biol. Chem 263. 10127 '~,'an Calker. D.. M. Muller and B. Hamprecht. 1979, Adenosinc t,*gulate, xla t ~ o dfffc~'en[ types of receptors, the accumulation of Q.dlc AMP in cultured brain cells. J. Neurosci. 33, 999. Yora. ]. and N L:t. 1978. Punfica0on and subfractionation of bovine th~rotropin m d ]utropin using radioimmuno-assa~, for evaluation of tile punficaaon processe~..! Bi0chem. 83. 1173.
European Journal of Pharmacolo,~"
47
ADONIS 09224!069100056Q
EJPMOL °:9131
P,-purinergic activation of phosphoinositide turnover is potentiated by A :receptor stimulation in thyroid cells Martina Nazarea. Fumikazu Okajima and Yoichi Kondo Departmem of P]~swal Bl~cheml~lrv, Institute of End:~ rl nol~)~. Gunma t "ni~'eMm. ~1acha~h~ : 71. J~l~an
Recei'~ed 4 July 1990. revised MS received 4 Seplernbe~ 1990. accepIed 25 September t990
We recently discovered that in FRTL-5 cells the Pi-purinergic agonist PIA tphenylisopropyladenosinel markedly enhanced P2-purinergic agonist-induced responses in an lAP (islet-activating protein or pertu~sis tovJnt-sensitive manner. ~n thi> study we tested PIA and other P~ agonists for their permissive effects on GTP (a P: agonistl-m,'luced inositol phosphate production and arachidonate release and found that the order of potency was PIA = CHA (cyclnhex',ladenosine} > NECA (N-ethylearboxa~ndoadenosine) = CADO (chtoradenosine). The P~ agonists also caused an inhibition of thyrotropin-induced cAMP increase in FRTL-5 cells as well as a stimulation of cAMP accumulation in lAP-treated cells. The order of potency was very :.imflar for phosphoinositide turnover, arachidonate release and cAMP inhibition, and therefore suggestive of an adenosine A1 receptor thee. As for cAMP stimulation, CADO. PIA and CHA were weaker than NECA and thus in agreement v.ith the A : receptor t;~e. The order of potency of four adenosine antagonists also revealed a similarity between arachidonate release and cAMP inhibition and a difference for arachidonate release and cAMP stimulation. These results indicate that ,Both A l- and A:-recepmr subtypes are present in FRTL-5 cells and that extracethilar adenosine enhances the P:-purinergic agonist-induced response> by >timulating an A 1 receptor which is coupled to an IAP-sensiti;e G-protein,%). Adenosine; Adenosine A 1 receptor: FRTL-5 thyroid cells: Phosphoinositide turnover: ArachiJonate release: Purine ~ receptor
1. Introduction
Purine nucleoside and nucleotide receptors are currently classified into two subclasses (Burnstock, 1987). namely the purine Pt receptor which has a high affinity for extracellular adenosine, and the purine P= receptor at which A T P binds more strongly. For the former, two further subclasses can be distinguished by a negative (A t) or positive (A z) coupling to adenytate cs~tase (Van Calker et at.. 1979; Londos et at.. 1980). T N s classification is also based on the relative potency of adenosine derivatives including phenyfisopropyladenosine (P]A) and N-ethylcarboxamidoadenosine ( N E C A ) to influence c A M P levels. The former is the strongest agonist at A~. receptors, whereas the latter is most potent at A : receptors. Both receptor types can be competifivdy antagonized by xanthine derivatives. A t~ird ( A ) ) subtype. which is characterized by its linkage to calcium metabolism rather than adenylate cydase, has been postulated by Ribeiro and Sebastiae (!986). In FRTL-5 coils purine ~ receptor stimulation caused
Correspondence to: Manina Nazm-ea. Department of Physical Biochemis,.D', institute of Endocnnology, (.~unma Univerxity~Maeb~sba 371. Japan.
phospholipase C activation which ~.~as accompanied by Ca r* mobdization, aracbJdonate release and I efftux (Okajima et al.. t988: Sho et al.. !989). A simultaneous stimulation of the adenosine receptor by' PIA. which by i~_self causes no such stimutatoQ effects, led to an enhancement of phosphotipase C and its re!ated responses (Okajima et ai.. t989a}. For cq-adrenoceptors the same potentiation was inducible by PIA ( O k ~ i m a . 1989b}. A similar potentiation induced by adenosine has been described for RBL-2H3 mast cells (All et ai., 1990) and guinea-pig cerebra! cortex (Holiingsworth et ai.. 1986: Hill and K e n d a l l !987}. Besides adenosine, other neuromoduiator> have been found to induce a similar positive cross-talk. For instance, epinephrine potentiates reactions induced by serotonin (De Chaffoy de Cource!tes el aI.. 1987L ~asopressin {Busi~field e~ aL, 1987l and thrombin (green et al.. !988: Crouch and kapetina, t988) in human platelets. In CCL39 fibroblast~ {Paris et aL. 1988) thrombin reactions have been described to become enhanced by a number of growth factors. In some cases (All et M.. t990: Okajima et ak, 1989a.b) G : p r o t e i n involvement for the perwdssive effect has been demonstrated. In FRTL-5 ceils, for instance, I A P (islet-activating protein or pertussis toxin) treatment, which inactivates IAP-st~bstrate G-proteins
by ADP ribosytation, had almost no influence on the Pz-purinergic receptor-induced phospholipase C activation and the subsequent responses, but completely abolished the permissive effect induced by PIA. The aim of the present study was to characterize the adenosine (P1)-receptor subtype which potentiates the P,-receptor-mediated phosphoinositide turnover and arachidonate release in FRTL-5 cells in an IAP-sensirive manner. Judging from agonist and antagonJ ~, orders of potency, we conclude that the Aj receptor md,Jced the above-described potentiation.
2.3. A rachidonic acid release measurement Cells labeled for 6 h with [*H]arachidonate were washed three times at t0-min intercals at 37°C with HEPES-buffered medium which consisted of (mM): HEPES 10 (pH 7.4). NaCI 134, KCI 4.7, KH2PO4 1,2, MgSO4 1.2, CaCI: 2.0. NaHCO3 2.5 and glucose 5, and bovine serum albumin (fraction V) 0,1%. Cells were incubated for I0 rain with the indicated agents in 750 #1. Radioactivity of the medium (0.5 ml) was measured in a liquid scintilhtion spectrometer (Beckman LS 7500). Data were expre,,sed as percentages of total radioactivity incorporated into the cells.
2. Materials and methods 2.1. Drugs and abbrel,iations From Sigma. adenosine. N~-(L-2-phen31isopropylladenosine (PIA), 5"-N-ethylcarboxamidoadenosine (NECA), N6-cyclohexytadenosine (CHA). 2-chloradenosine (CADO). 3-isobutyl-l-methylxanthine (IBMX) and adenosine deaminase were bought. From Research Biochemicals Inc. (Wayland, MA) we purchased 8-cyclopentyl-l,3-dipropylxanthine (CPX). 8-cyctopentyl-l.3-dimethylxanthine (CPT). and L3-diethyl-8-phenyl×anthine (DPX), GTP was obtained from Yamasa Shoyu Co. (Choshi, Japan). Bovine TSH was purified by the method of Yora and Ui (1978) and had an activity of 2'3 U / m g based on its follicle-reconstructing activity in culture. Myo-[2-3H]inositol (15.6 Ci/mmol) and [5.6,8.9Al,12,14.15-3H]arachidonic acid (100 Ci/mmol) were obtained from Du Pont-New England Nuclear. lAP and RO 20-1724 (4-(3-butoxy-4methoxybenzyl)-2-imidazolidinone) were gifts from Dr. Michio Ui (Tokyo Univer-~ity, Tokyo, Japan) and Nippon Roche Research Center (Kamakura, Japan), respectively. The reagents for the cAMP radioimmunoassay were generously provided by Yamasa Shoyu Co. (Choshi, Japan). Sources of reagents for cell culture and other purposes were those described by Okafima etal. (i98~).
2.4. cA M P measurement Cel!s were washed once with HEPES-buffered medium and incubated for 10 rain at 37°C in the same buffer in a final volume of 750 #1, A 10-min incubation was terminated by adding 10/tl of 1 N HC1 and cAMP content was measur~.~dby radioimmunoassay (Honma et al.. 1977). 2.5. Measurement of mositol phosphate production [~H]inositol-labele6 cells were washed once and proincubated for 10 miE with HEPES-buffered medium. The indicated agents were added in a final volume of 750 #1 together with i0 mM LiC1, GTP (300/zM) and adenosine deaminase (0.5 units/ml; it was omitted, however, in adenosine preparations) and incubated for 30 min. The reaction was terminated by aspirating the medium, adding 700 ul of 0.i N HC1 and overnight fieezing. The separation of [3H]inositol phosphate was performed as described in Okajima et al. (1988).
3. Results 3.1, Adenosine derit,ati~,es enhance phosphoinositide and arachidonate release J'esponses to GTP
2,2. Cell ctdture FRTL-5 ceils, a cell fine deri:ed from normal rat thyroid (Ambesi-lmpiombato et ak. 1980). were obtained from Interthyr Research Foundation (Baltimore, MD) and grown as described by Okajima et al, (1989a). When cells were 60-70% confluent in 12-welI plates, the cahure medium (Coon's F-12 containing 5% calf serum and six hormones including TSH) wa:; changed for a further 2 days to Ham's F-10 containing only 5% calf serum (TSH depletion). Where indicated, this medium was supplemented with ehher 30 ng/mf lAP or [-~Htinositol (2 #Ci/ml), or both. For arachidonate release measurement, [-~H]arachidonate (0.1 #Ci/ml) was added 6 h prior to agent addition,
GTP stimulates phosphoiipase C in FRTL-5 cells via a P2-purinergic receptor (Okajima, 1989a). This stimulation was hardly affected by IAP treatment of the cells. Here, GTP was added together with adenosine or adenosine analogues. Although these adenosine analogues alone had no effect on either inositol phosphate production or arachidonate release, they enhanced both of these GTP-induced responses in a dose-dependent manner, As for the inositol phosphate production (fig. IA). the order of potency was CHA = PIA > adenosine = CADO = NECA and was roughly the same as for arzehidonate release (fig. 1B) with P1A and CHA being the most potent agonists (see EDs0s in legend to fig. 1B) and NECA, CADO and adenosine approximately one
49 eta!., 1 9 8 9 a ) a n d this s u g g e s t s t h e i n v o l v e m e n t o f I A P - s u b s t r a t e G - p r o ' , e i n s in a d e n © s i n e - r e c e p t o r - m e d i a ted p o t e n t i a t i o n o f G T P r e c p o n s e s .
lOO
//
_g o so
3.2. c A M P receptors
/~e t
i
t
i
reveal both A,;- and A 2 - o T e
By r a i s i n g c A M P levels w i t h T S H in p r e v i o u s t } r T S H - d e p l e t e d c e l l s (fig. 2 A ) w e c o u l d o b s e r v e a n i n h i b i t i o n b y a d e n o s i n e a n d its a n a l o g u e s . T h e o r d e r o f potency for the four adenosine derivatives was PIA > C H A > N E C A > C A D © . A d e n o s i n e itself w a s r a t h e r e f f e c t i v e at l o w e r c o n c e n l r a t i o n s a n d t h i s i n h i b i t i o n d i d
0
100
measurements
t
'
{A) Controt o / "
100 I
1 9
8 7 6 S Agonlsts (-log M) Fig. 1. The effect of adenosine agonists on inositol phosphate production (A) and arachidonate release (B). ©. PIA; e. adenosine; a. NECA; A. CAD©; [2. CHA. GTP (300 vM) had been added to all and adenosine deaminase (0.5 u.,fits/ml, to all but the adenosine preparations. Incubation times were 30 min (A) and t0 .win (BL In the absence of adenosine derivatives. GTP increased the inositol phosphate production from 3.7 x 103 dpm to 20.2 x 10 ~ dpm IA) and the arachidonate release from 73 dpm to 832 dpm (B). Adenosine derivatives alone had no effect on basal values (they were in the range of 3.6-3.9 x I03 dpm for inositol phosphate production and bet~een 70 and 90 dpm for arachidonate release), but enhanced GTP-induced actions roughly twofold in the case of inositol phosphate production and fivefold for arachidonate release. The increments of responses to GTP induced by adenosine derivatives were plotted as at pet~.~ntage of the increment induced at 10 t~M PIA (for inositol phosphate production 27.0x103 dpm and for arachidonate release 3368 dpm). Data represent the means of two (A) and three (B) experiments carried out in duplicate or triplicate. In {B) S.E. values were smaller than tot{ and EDso values+S.E. (nM) were 18,5 ±0.7 for P1A, 89.1 ±6,4 for adenosine, 50.3±4.5 for NECA. 70.85:7.0 for CAD© and 24.2+_20 for CHA.
o r d e r o f p o t e n c y w e a k e r in i n d u c i n g a p o t e n t i a t i o n o f the GTP-induce.d stimulation of phospholipase C and a r a c h i d o n a t e r elease. T h i s f i n d i n g w a s c o n s i s t e n t w i t h p r e v i o u s r e s u l t s ( O k a j i m a et al., I 9 8 9 a ) . In l A P - t r e a t e d cells (results not shown) no stimulato W effect by adenosine analogues for either aract'ddonate release or phosphoinositol production could be obser.,ed (Okajima
"o
o
f 79°°~
~ //
i
r
i
i
imlAp
°
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100 ~- e-..q m
'
7 6 S Agontst~; (-lo~ M} Fig, 2. Do~=re~po~se cu~'e~ of adenosine agoinsts concerning inhibitlon {A) and stim~alation {B) of cAMP accumulation in control and tAP-treated cells, respectively. ©. PlAt e, aden©sine: ~x. NECA: A. CAD©: ~. CHA In [A) TSH (2 nMt and the phosphodiesterase inlubitor R© 20-i721 (t00 #M) were added together ~ith the agents. In (B) TSH was omitted. Aden©sine deamina.~e (0.5 units/rrd) was added to al! but the adeeosine preparations, hicubafion time was 10 mJn. gDs0 v a l ~ (in nM (A)) were for PfA 1.86±0.09. for adenosine 25.1 ± 2.4. far NECA 4~25 ±0.38. for CAD© 25.6 ± 2.5, and for CHA 3.5 zO.3. In (A} the TSH value (1395 pmo]/mg/proiein) and in (B) the IAP bec.aI ~alue (9.9 pro©l/rag/protein) was taken to be 10O~. Values are the means of al least sLx experiments earned out in duplicate. S.E. values were ~I0% arid therefore no error bra's were dravrn.
not go belt~w 30~ even at higher doses. Whether different experimental conditions (adenosine deaminase being left out) or still other factors account for this different behavior could not be determined. In any event, this rank order of potency is in good agreement with that of agonist potencies for A~ receptors (Van Calke~ et al.. 1979: Londos et al., 1980). In lAP-treated cells (fig. 2B). the same agonists induced an increase in c A M P uniess T S H was present. The order of potency for agonists was N E C A > C A D O = adenosine > PIA > C H A , which is
~
3.3. Influence of adenosine antagonists on arachidonate release and cA MP accumulation
"~. 0
(B) Control
~_~ 0! I
ff
p
F __
r
(c~ tAP I¢ 1 O0 .
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9
8 ? 6 A n , ag o n l st s (-log M t
5
Fig. 3. Dose-response curves of adenosine receptor antagonists. )n A, antagonists DPX, (o): CPX. (11); CPT. t~; IBMX, (AI were added at the indicated concentrations m medium cont,'fi~g 300/aM CTP and 0.1 .aM P1A. In B, antagonists were added together with P1A (0.l .aM), TSH (2 nM). adenosine deamina~ t0.5 units/mlj and RO 20-1724 (100 .aM). In C, lAP-treated ceils were tested w;~ha~ta~or,]Ms in the same way as in B except that the PIA concentration was 10 ;aM and that TSH was left out. Incubation time was 10 rain. Data are expressed as PIA effect ~qthout antagonists equaling 100%. lf~)% values were 12.1 × 103 dpm (A). t096 pmo!/mg protein (Bi and 36.9 pmol/mg protein (C). Experiments are repr~entative of at lea.st two more duplicate determinations. Apparem K, values (nM~ were for: DPX, 102 (A) and 137 (B); CPX, 0.30 CA) and 0.24 (B): CPT, 29 (A) and 19 (B). and we-'~c~,tculated accc~rdingto the lot)owing equation: K i [C50~antago~tlX ED~c~eL~I/(ED~ptAi-[C] (PIA)). PIA ED~3was estimated to be 18.5 nM (from fig. 1B) and L86 rum (from fig. 2A). =
characteristic of adenosine A 2 receptors (fig. 2) (Van Calker et al., 1979: Londos et al., 1980). Thus. there are two types of P~ receptors in FRTL-5 cells: A~ and A 2 receptors, which are coupled to adenylate cyclase in an inhibitor3" and stimulator 3' fashion, respectively. When comparing the potentiation of GTP-activated phospholipase C (fig. I A ) and arachidonate release (fig. 1B) responses with c A M P inhibition (fig. 2A) and c A M P stimulation (fig. 2B). it is obvious that the order of potency is roughly identit.a! between the potentiafion of G T P effects and c A M P inhibition, although there is a one order potency shift to the left for c A M P inhibition (fig. 2A). These findings suggest that adenosine-induced potentiation of G T P actions and adenylate cyclase inhibition are mediated by the same receptor, the At-type receptor,
T o further confirm that the A 1 and not the A 2 receptor is involved in the permissive action of adenosine on GTP-induced stimulation of phospholipase C and its related responses, four adenosine antagonists were tested for their ability to antagonize PIA actions with respect to arachidonate release (fig. 3A) and the inhibition (fig. 3B) and stimulation (fig. 3C) of c A M P accumulation. All these PIA-induced responses were antagonized by adenosine antagonists. The order of antagonist potency was C P X > C P T > D P X >> I B M X for PlA-induced arachidonate release (fig. 3A) a n d inhibition of c A M P (fig. 3B). In lAP-treated cells (fig. 3C), the rank order of antagonists to inhibit the stimulation of c A M P accumulation was D P X = C P X > C P T > I B M X and thus clearly different from the one for arachidonate release and c A M P inhibition. Since I B M X also acts as a phosphodiesterase inhibitor, its behavior is difficult to interpret. In any event, the high potency of C P X in fig. 3A and 3B argues for an At-type receptor. A shift of potency to the right can be observed for c A M P inhibition (fig. 3B) when compared to the araehidonate release (fig. 3A), which might be caused by ditferent app.zrent potencies by which P I A induced the respective respenses. In fact, the K i values (see legend to fig. 3) were the same. These findings support the idea that PI.'_-induced inhibition of c A M P accumulation and pote,~tiation of the arachidonate release response are m e O a t e d by the same receptor, the A~ receptor.
4. Discussion T h e present study shows that the potentiation of phospholipase C and arachidonate release induced by adenosine (P~)-reeeptor stimulation is of the adenosine
51 A~-subtype r e c e p t o r . T h r e e lines of e v i d e n c e s u p p o r t ttfis finding. Firstly. P I A a n d CI-tA w e r e m o r e p o t e n t t h a n N E C A , C A D O a n d a d e n o s i n e in i n d u c i n g this effect. Secondly, the r a n k o r d e r o f a g o n i s t - i n d u c e d c A M P i n h i b i t i o n , w h i c h is by d e f i n i t i o n an A~ receptor-mediated r e s p o n s e , m a t c h e d t h a t of t h e agonist-induced arachidonate release and phosp h o i n o s i t i d e t u r n o v e r . T h i r d l y , the p e r m i s s i v e effect was competitively antagonized by xanthine derivatives with the s a m e r a n k o r d e r for c A M P inhibition and a r a c h i d o n a t e release, b u t a d i f f e r e n t o n e for the l a t t e r and c A M P s t i m u l a t i o n . A s i m i l a r p o s i t i v e c r o s s - t a l k w a s r e p o r t e d for a d e n o sine in g u i n e a - p i g c e r e b r a l cortex, w h e r e it e n h a n c e d histamine-stimulated phosphatidylinositol hydrolysis ( H o l l i n g s w o r t h et al.. 19865. C o n v e r s e l y . the s a m e effect was d e s c r i b e d to b e n e g a t i v e l y m o d u l a t e d by a d e n o s i n e in m o u s e c e r e b r a l cortex ( K e n d a l l a n d H i l l . 1988). W h e t h e r t h e c r o s s - t a l k is c a . n e d o u t in a p o s i t i v e ( s t i m u l a t o r y ) o r n e g a t i v e ( i n h i b i t o r y ) m a n n e r s e e m s to be tissue- a n d species-specific. M o r e e x a m p l e s of n e g a tive c r o s s - t a l k are d o p a m i n e , w h i c h m o d u l a t e s a n g i o tensin ( J o u r n o t e t a l . , 1987) a n d T R H r e s p o n s e s in rat p i t u i t a r y ( V a l l a r e t a l . , 19885, a n d e x c i t a t o r y arcfino a c i d s to i n h i b i t n o r e p i n e p h r i n e ( N i c o l e t t i e t a l . , 19861 a n d h i s t a m i n e ( B a n d r y e t a l . , 19865 a c t i o n s in rat hipp o c a m p a s . In b o t h p o s i t i v e a n d n e g a t i v e c r o s s - t a l k . I A P - s e n s i t i v e G - p r o t e i n s s e e m to be i n v o l v e d in the transduction (Okajima etal., 1989a.b; Delahunty etal.. 1988). The mechanism by which IAP-snbstrate G-proteins potentiate the phospholipase C activation and a r a c h i d o n a t e release r e m a i n s u n c l e a r . T h e a d e n o s i n e r e e e p t o r - l A P - s u b s t r a t e G - p r o t e i n t r a n s d u c e r s y s t e m has r e c e n t l y b e e n s h o w n to b e the l i n k in c a u s i n g the i n h i b i t i o n o f C a -~+ c h a n n e l s a n d t h e s t i m u l a t i o n o f K c h a n n e l s a s well a s the i n h i b i t i o n o f a d e n y l a t e cyc!ase ( F r e d h o l m a n d D u n w i d d i e . 19885. T h e q u e s t i o n w h e t h e r this novel p e r m i s s i v e effect o f a d e n o s i n e is j u s t c a u s e d s e c o n d a r i l y by a c h a n g e in c A M P o r C a : - levels h a s b e e n a d d r e s s e d in p r e v i o u s w o r k s ( O k a j i m a e t a l . , 1989a. b) a n d c o u l d be r u l e d out. H o w e v e r , an a c t i v i t y c h a n g e in K ÷ c h a n n e l s may" be a c c o m p a n i e d b y a s ~ i t c h o f the p l a s m a m e m b r a n e p o t e n t i a l , a n d it c o u l d t h e r e b y m o d u l a t e the a c t i v i t y o f the p h o s p h o l i p a s e C - C a 2 system ( D i V i r g i l i o et ok, 1987t. T h i s possibility is now u n d e r investigation.
AeknoMedgements This ~ork wos supported by a Gram-in-Aid for Scientific Research and a Grant-in-Aid for Scientific Research on Pnorit) Are~-, from the Minist~' of Education. Science and Culture of Japan. M.N. is a Japan Society for the Promotion of Science Fellow.
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