50 mM Tris –H Cl buf fer (pH 7.5 ) con tai nin g 10 mM MgCl

2

with [

3

H] CGS 21 680 (5 nM ) in the pr es enc e or ab sen ce of unl ab ele d 5

0

-

N

-ethylcarbo xamid oadenosi ne (NECA; 10

l

M) for dete rmi na- tio n of non spe ciﬁ c bin di ng. After 90 mi n at 25



C, the bin di ng reaction was stopped by ﬁltration through GF/C ﬁber glass ﬁlters (Wha tma n, Mai dsto ne, Kent , UK) , and then the mem bran e was was hed ﬁve tim es with 5 mL ice-co ld buffer. Radio acti vity of the punc hed -out wet ﬁlters was coun ted after adding 5 mL of Ulti ma Gold scintillation cocktail (Perkin–Elmer, Waltham, MA, USA).

2.3. Cell culture and transient expression of adenosine A

2A

receptors in CHO cells

Cells were grown in Dulbecco’s modiﬁed Eagle’s medium con- taini ng 10% heat-ina ctiv ated fetal bovi ne seru m and 1% pen icil- lin –st rep tom yci n at 37



C i n a 1 0 % C O

2

inc ub at or at 95 % hum id ity . Th e pl asm id co nta ini ng the hum an ad eno sin e A

2A

recep- tor (OriGene, Rockville, MD, USA) was transiently transfected into Chin ese ham ster ovar y CHO cells with Hilyma x tran sfec tion re- agent (Dojindo, Seoul, Korea) according to the manufacturer’s pro- toco l. Tran sfect ions wer e perf orm ed in 100- mm tissu e cultu re dishes and cells were treated with limonene for subsequent cAMP, PKA activity, and calcium assays.

2.4. Cyclic AMP measurement

Cells were washed with phosphate-buffered saline and treated with vari ous conc entr atio ns of lim one ne. Afte r incub atio n with lim onen e for 30 min , intra cell ular cAMP was quan tiﬁe d with a com mer ciall y avai labl e cAM P rad ioim mun oass ay syste m (Enz o Life Sciences, Plymouth Meeting, PA, USA) according to the manufac- turer’s recommendations.

2.5. PKA activity assay

PKA activit y was determ ined usin g a com mer ciall y avai labl e kit (Enzo Life Sciences) according to the manufacturer’s instructions.

2.6. Measurement of limonene-induc ed cytosolic calcium release

Cells were seeded at a density of 30,000 cells/well into black 96-well plates with clear bottoms to allow cell visualization and ﬂuor esce nce me asur eme nts fro m the bott om of each wel l. The dy e-l oad ing bu ffe r con sis ted of a ﬁn al con cen tra tio n of 4

l

M 4-(6-acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-4

0

-methyl- 2,2

0

-(ethylenedioxy)dianiline-

N

,

N

,

N

0

,

N

0

-te tra ace tic aci d tet ra kis (acetoxymethyl) ester (ﬂuo-3-acetomethyl ester; Sigma, St. Louis, MO , US A) in ser um -fr ee cel l cul tur e ma int en anc e me diu m co nta in- ing 20 mM HEPE S and 2.5 mM prob enec id. Assay conditi ons were as describe d previousl y [14] . Brieﬂy, each 96-well plate containing dy e-l oad ed cel ls wa s pla ced int o a mu lti lab el pl ate rea de r (VICT OR3 ; Per kin– Elm er) and lase r inten sity was adj usted to an opti mum level to obta in basa l valu es of app rox ima tely 10,000 ﬂuorescence units. After determining the baseline ﬂuorescence of the ﬂuo -3-C a

2+

com plex , lim onen e and NEC A wer e add ed into the 96-w ell plat e. Fluo resc ence read ings wer e obta ined ever y 10 s for 100 s. The maxim um ﬂuor esce nt signal was recor ded and normalized to a positive control of 10

l

M NECA. Each experiment was perfo rm ed in quad rupl icate on every plate. Each 96- wel l plat e contained four wells dedicated to a positive control (10

l

M NECA) and four wells to a negative control (assay buffer alone).

2.7. Immunoblotting analysis

Ce ll s we re ly se d in RI PA bu ff er (1 0 mMTri s– HC l, 1% NP -4 0, 0. 1% sod iumdeox ych ola te, 0.1 % SD S, 150 mM Na Cl, 1 mM ED TA , pH 7.5 ) containing protease inhibitor cocktail at 4



C. Samples were sepa- rat ed usi ng 7.5 % SD S– PA GE an d blo tte d on to a nit ro cel lul os e me m- bra ne. Me mb ra nes we re inc uba ted wi th pr im ary ant ibo di es over nigh t at 4



C. Afte r was hing severa l time s with 0.5% TBS -E was h buff er, the me mbr ane was incubate d with the seco nda ry ant ibo dy for 1 h at ro om tem pe ra tur e. Im mu no re act ive ba nds we re dete cted by enha nced chem ilum ines cenc e Wes tern blot ting dete c- tion reag ents (Am ersh am- Phar mac ia Kor ea, Seo ul, Kor ea) and quantiﬁed with ChemiDoc™ XRS system (Bio-Rad, USA) using the Quantity One software.

3. Results and discussion

We demonstrated for the ﬁrst time that a cyclic terpene, limo- nene, was a direct ligand for A

2A

recepto rs and activated recep- tor -m ed iat ed sig nal ing pa thw ays , as con ﬁr me d by cyt oso lic cAMP and calcium concentration s, and induced phosphorylation of CREB transcription factor. Taken together, our data demonstrate that limonene is an A

2A

receptor agonist. First , we perf orm ed a rad ioli gandbindingassay to quan titate the binding afﬁnity of limonene to A

2A

receptors with a rat brain cere- bral cort ex mem bran e frac tion. Spe ciﬁc ally , we emp loye d com peti - tiv e bin di ng of lim on ene wi th a kn ow n sel ect ive A

2A

receptor agonist, [

3

H]CGS21680. We found that [

3

H]CGS21680 displayed a sat ur abl e bin din g pr oﬁ le in the abse nce of lim on ene , and the bin d- ing of [

3

H]CGS21680 to A

2A

receptors decreased with increasing concentrations of limonene in a concentration-dependent manner ( Fi g. 1 ). At the hig hes t lim on ene con cen tra tio n, [

3

H]CGS21680 bind ing decr ease d by 91.2 % as compa red to the cont rol gro up. This ind ica ted tha t lim on ene co mp eti tiv el y bo und to the [

3

H]CGS 21680-binding site of the A

2A

receptor. Therefore, we found that limonene has a high afﬁnity toward A

2A

receptors and can act as a ligan d for them . In the next step, we inve stiga ted whether limon ene binding activated the A

2A

receptor -med iated signaling pathway . A series of exp erim ents was perfor med with CHO cells trans fecte d with the human A

2A

receptor gene. A

2A

receptors are coupled to a G

s

G protein [15] and activate adenylyl cyclase [16] . The activated G

s

sub uni t can co upl e to do wn str eam eff ect or s to re gul ate the amo unt of seco nd mes seng ers, inclu ding cAMP and PKA . cAM P- indu ced gene tran scri ptio n is gene rall y acce pted to be med iated thr oug h the act iva tio n of PK A, wh ose tw o reg ula tor y sub uni ts bin d to cAM P and indu ce a conf orm atio nal chan ge, whi ch pro duce s subu nit diss ocia tion resu lting in enzy mat ic activ atio n [17] and subsequent phosphorylation of the transcription factor CREB.

Fig. 1.

Competitive binding assay of limonene with [

3

H]CGS21680 in a rat brain membrane fraction. Binding of [

3

H]CGS21680, an A

2A

receptor ligand, was reduced by limonene in a conce ntrat ion-de pend ent manner. Values are means ± SEM. 346