European Journal of Pharmacology - Molecular Pharmacology Section, 266 (1994) 165-174 165 © 1994 Elsevier Science B.V. All rights reserved 0922-4106/94/$07.00

EJPMOL 90562

Coupling of human a2-adrenoceptor subtypes to regulation of cAMP production in transfected Sl15 cells

Chris t ian C. Jansson ,,a, A n n e Marjam~iki b, Kirsti L u o m a l a b, Ju h a -Ma t t i Savola c, Mika Schein in b o

and Kar l E .O. A k e r m a n a

a Department of Biochemistry and Pharmacy, BioCity, Abo Akademi University, P.O. Box 66, FIN-20521 Turku, Finland, b Department of Pharmacology, BioCity, University of Turku, FIN-20521 Turku, Finland, and c Orion Pharmaceuticals, FIN-20101 Turku, Finland

Received 30 June 1993, revised MS received 13 September 1993, accepted 28 September 1993

Stable Sl15 mouse mammary tumour cell lines, expressing separately a2A-C10, a2B-C2 and a2C-C4 adrenoceptors were used to compare the receptor binding properties of a2-adrenoceptor agonists with their potency in inhibiting cAMP production. All tested agonists detected high and low affinity binding sites in all three receptor subtypes. In the presence of the GTP analogue Gpp(NH)p (10 /zM), all displacement curves were shifted to the right and were best modelled by one-site fits, suggesting that the receptor subtypes are coupled to G-proteins. The extent of the Gpp(NH)p-induced shift was greatest in the a2A-C10 subtype, smaller in a2C-C4, and minimal in a2B-C2. All three receptor subtypes were also coupled to inhibition of forskolin-stimulated cAMP production through pertussis toxin-sensitive G-proteins. For the the full agonists noradrenaline, UK 14,304, and dexmedetomidine, the maximal inhibitory effect on cAMP production was smaller in the a2B-C2 subtype (35%) than in the aeA-C10 and a2C-C4 subtypes (50-70%). After treatment of cells expressing a2B-C2 receptors with pertussis toxin, cAMP production was increased by up to 58% by a2-adrenoceptor agonists. Similar stimulation of adenylyl cyclase activity could not be demonstrated at the other two receptor subtypes. In conclusion, these results demonstrate that (1) a2-adrenoceptor agonists may be characterized by an agonist-type binding pattern in homogenates of transfected Sl15 cells, (2) all three a2-adrenoceptor subtypes are coupled to inhibition of adenylyl cyclase in Sl15 cells through pertussis toxin-sensitive G-proteins, (3) the receptor-effector coupling in Sl15 cells is different among the subtypes so that the a2A-C10 subtype is coupled with high efficacy but with low sensitivity, the a2B-C2 subtype with low efficacy but high sensitivity, and the a2C-C4 subtype with both high efficacy and high sensitivity, and (4) at least aeB-C2 receptors may also be coupled to stimulation of adenylyl cyclase activity, presumably through G s.

a2-Adrenoceptor subtype; [3H]Rauwolscine; Ligand binding; Signal transduction; cAMP

I. Introduction

Activation of a2-adrenoceptors results in inhibition of adenylyl cyclase activity in most tissues and cell types (Regan and Cotecchia, 1992). This response has been shown to be mediated through pertussis toxin- sensitive inhibitory Gi-proteins. Three distinct Gi-pro- tein isotypes have been identified, based on differences in the a-subunit, termed Gia-1 , Gia-2 and Gia-3 (Yatani et al., 1988; Simonds et al., 1989; Codina et al., 1989). The a2A-C10 and a2C-C4 adrenoceptor sub- types have been demonstrated to activate all three Gi-proteins in reconstituted systems (Kurose et al., 1991), and there is strong evidence that both Gia-2 and Gia-3 may mediate inhibition of adenylyl cyclase activ-

* Corresponding author.

ity in cell membranes and intact cells expressing a2A- C10 and a2B-C2 (Simonds et al., 1989; McClue and Milligan, 1990, 1991; Gerhardt and Neubig, 1991). The inhibition of adenylyl cyclase is thought to be mediated either directly by Gia or indirectly by the fly-subunits through interaction with and deactivation of the a-sub- unit of G~ (Levitzki, 1987; Gilman, 1987; Federman et al., 1992).

In the present study we have investigated the cou- pling of all three subtypes of human aE-adrenoceptors to regulation of cAMP production, by using stable Shionogi 115 mouse mammary tumour cell lines ($115) transfected to separately express the receptor subtypes (Marjam~iki et al., 1993). Ten a2-adrenoceptor agonists were used to characterize the agonist binding proper- ties of the aE-adrenoceptor subtypes using [3H]rau- wolscine in competition studies. The purpose of this study was to perform a comparison between binding of different agonists to the three receptor subtypes and

SSDI 0 9 2 2 - 4 1 0 6 ( 9 3 ) E 0 1 5 6 - E

166

their coupling to regulation of cAMP production in the recombinant Sl15 cells.

2. Materials and methods

2.1. Materials

[3H]Rauwolscine (82.3 Ci/mmol)was from DuPont NEN. [3H]Adenine and [laG]cAMP were from Amer- sham. Clonidine, dexamethasone, forskolin, Geneticin (G418), 5'-guanylylimidodiphosphate (Gpp(NH)p), isobuthylmethylxanthine (IBMX), imidazole, (-)-nor- adrenaline, oxymetazoline, phentolamine, propranolol, quinacrine and testosterone were from Sigma. UK 14,304 (5-bromo-6-[2-imidazoline-2-ylamino]quinoxa- line), dexmedetomidine and l-medetomidine were from Orion Corporation Farmos, Turku, Finland.

The expression vector pMAMneo was from Clon- tech Laboratories, and Sl15 cells were originally from Drs. P.D. Darbre and R.B.J. King, The Imperal Cancer Research Fund, London, UK (Darbre and King, 1988).

2.2. Transfection, selection and cell culture

The construction of the expression vectors for a2A- C10, a2B-C2 and a2C-C4 has been reported previously (Marjam/iki et al., 1992, 1993). The constructs were transfected into Sl15 cells with the Lipofectin reagent kit (Gibco). Geniticin-resistant clones were selected from each transfection and examined for their ability to bind the a2-adrenergic antagonist radioligand [3H]rauwolscine. The clones with highest receptor den- sities were maintained in 200/zg/ml G418. Cells used for receptor analysis were grown to confluence without G418.

Recombinant Sl15 cells were cultured for ligand binding experiments in roller bottles (1400 cm 2, Nun- clon) in Dulbecco's modified Eagle medium (DMEM) (250 ml) supplemented with 20 mM Hepes, 20 mM NaHCO 3, 5% heat-inactivated fetal calf serum, 100 U / m l penicillin, 50 # g / m l streptomycin, 1 mM sodium pyruvate and 10 nM testosterone. Cells used for cAMP experiments were cultured in the same medium in tissue culture flasks (175 cm 2, Nunclon). All cells were grown in 5% CO2 at 37°C.

2.3. Ligand binding

Cells were harvested into chilled phosphate-buffered saline (PBS) and centrifuged at 1500 × g for 5 min. The cell pellet was suspended in ice-cold TME buffer (50 mM Tris-HCl, 10 mM MgCI 2, 1 mM Na2-EDTA, pH 7.4) and homogenized with an Ultra-Turrax ho- mogenizer (Model T25, Janke and Kunkel, 2 times 10

s, 9500 rpm). This homogenate was used as such for binding assays and protein determination.

Ligand binding assays were performed in TME buffer as described (Marjam~iki et al., 1992, 1993). In saturation studies, 30-100/zg of protein was incubated with [3H]rauwolscine (0.06-8 nM) in a final volume of 0.5 ml TME at 25°C for 30 min. Non-specific binding was determined by including 10 /xM phentolamine in parallel assays. Competition studies were performed using [3H]rauwolscine concentrations close to its K D at each receptor subtype and 16 concentrations of the competitors. The GTP dependence of agonist binding was investigated with the stable GTP analogue Gpp(NH)p; a Gpp(NH)p concentration of 10 tzM was selected for this study (Marjam~iki et al., 1992). Bound radioactivity was separated by filtration on Whatman G F / B filter strips, using a Brandel cell harvester (Model M-48R) and two 5 ml washes with ice-cold TME buffer. Radioactivity on the filters was deter- mined by liquid scintillation counting (Wallac 1410, Wallac-Pharmacia) in Optifluor O (Packard) with 48% efficiency. Protein concentrations were determined ac- cording to the method of Bradford (1976), using bovine serum albumin as reference. The saturation and com- petition data were analysed using the nonlinear least- squares computer programs LIGAND (McPherson, 1985) and GraphPAD (GraphPAD Software, San Diego, CA).

2.4. Measurement of cAMP production

The growth medium of confluent cultures was re- placed with one without fetal calf serum and supple- mented with 5 ixCi/ml of [3H]adenine. After incuba- tion for 2 h the cells were detached with EDTA (0.02%) and washed once in physiological assay buffer (137 mM NaCI, 5 mM KCI, 1.2 mM MgCI2, 0.44 mM KH2PO4, 4.2 mM NaHCO 3, 20 mM TES (2-{(3-hy- droxyl- 1,1-bis(hydroxymethyl)ethyl}ethane sulphonate), 10 mM glucose and 0.5 mM EGTA adjusted to pH 7.4). Thereafter the cells were suspended in the same medium and divided into aliquots of about 1 x 106 cells in 0.8 ml buffer. The ceils were preincubated with 0.5 mM IBMX (a phosphodiesterase inhibitor), 100 /zM propranolol (a /3-adrenoceptor antagonist) and 150/xM quinacrine (a phospholipase A 2 inhibitor) for 10 min at 37°C. Forskolin (60 IzM) and agonists in different concentrations (1 nM-100 /~M) were added. After 10 rain the cells were centrifuged for 1 min at 10000 × g, the medium was immediately removed and the reaction was terminated by resuspension in 1 ml 0.33 M perchloric acid, containing about 1600 cpm [14C]cA/VIP. The extent of conversion of [3H]ATP to [3H]cAMP was determined by sequential Dowex/ alumina ion exchange to isolate cAMP (Salomon et al., 1974). Conversion to [3H]cAMP was expressed as per-

centage of total cell-associated tritium and was normal- ized to the recovery of [14C]cAMP tracer (generally 70%). Radioactivity was determined by liquid scintilla- tion counting as described above. ECs0 values for a 2- adrenoceptor agonists were derived by computer-aided curve fitting.

3. Results

3.1. Ligand binding experiments

Stable Sl15 mouse mammary tumour cell lines ex- pressing separately the a2A-C10, a2B-C2 and azC-C4 adrenoceptor subtypes (Marjam~iki et al., 1992, 1993) were used to investigate the relationship between the binding properties of different az-receptor agonists

167

and inhibition of cAMP production. The binding affin- ity (K D) and capacity (Bma x) of [3H]rauwolscine in cell homogenates were as follows: a2A-C10, K o = 5.5 + 0.2 nM, Bma x = 1.2 _+ 0.2 p m o l / m g protein; a2B-C2 K D = 3.4 _+ 0.2 nM, Bma x = 7.3 _+ 0.3 p m o l / m g protein; and a2C-C4 K D = 0.9 _+ 0.2 nM, Bma x = 0.2 +_ 0.05 p m o l / mg protein. The displacement curves of the ten tested a2-adrenoceptor agonists at the three receptor sub- types were usually best modelled by two-site fits, and showed shallow slopes in one-site models (Hill num- bers < < 1) (Table 1). The addition of 10 /~M Gpp(NH)p induced a rightward shift in the high affin- ity component of the displacement curve (Fig. 1), and the curves were best modelled by one-site fits (Hill numbers closer to 1). The Gpp(NH)p-induced shifts of the curves to the right were greatest in the azA-C10 subtype. This is presented numerically in Table 2,

TABLE 1

Affinities of 10 different aa-adrenergic agents to three aa-adrenoceptor subtypes, separately expressed in Sl15 cells.

The competition studies were done in cell homogenates using [3H]rauwolscine as radioligand. Kill and KiL are inhibition constants for the high and low affinity sites in a two-site model. % H is the percentage of binding sites in the high affinity component. Apparent (App.) K i is the inhibition constant for a one-site-model. Slope indicates the Hill coefficient. The K i values are means_+S.E, from three independent experiments performed in triplicate.

Ligand Kill (nM) giL (nM) % H App. g i (nM) a Slope g i + Gpp (nM) Slope

A: Sl15, a2A-CIO Noradrenaline UK 14,304 Dexmedetomidine l-Medetomidine Oxymetazoline Clonidine Detomidine Para -iodoclonidine Guanfacine Guanabenz

B: $115, a2B-C2 Noradrenaline UK 14,304 Dexmedetomidine l-Medetomidine Oxymetazoline Clonidine Detomidine Para -iodoclonidine Guanfacine Guanabenz

C: $115, a2C-C4 Noradrenaline UK 14,304 Dexmedetomidine /-Medetomidine Oxymetazoline Clonidine Detomidine Para-iodoclonidine Guanfacine Guanabenz

3.20 _+2.10 920 _+270 49_+ 3 88.2 _+ 1.1 0.25_+0.10 1180 _ + 4 7 0 0.71_+0.07 0.09 _+0.08 29.5 _+ 1.3 28_+10 3.71_+ 0.79 0.48_+0.03 53.6 _+ 4.9 0.91_+0.20

0.41 pM+0.08 pM 2.54-+ 1.29 33-+ 8 0.78-+ 0.13 0.12+0.02 4.76_+ 1.17 0.87_+0.16 0.31 _+0.20 67.9 _+ 20.0 39_+ 1 21.7 _+ 10.85 0.41_+0.06 31.7 _+ 3.8 0.79_+0.03 0.13 _+0.12 13.9 _+ 0.1 31_+ 4 2.83_+ 0.85 0.76_+0.04 12.9 _+ 2.5 0.84_+0.06 1.10 _+0.60 43.6 _+ 7.3 42_+ 8 10.7 -+ 3.0 0.60_+0.06 33.1 _+ 3.8 1.00_+0.07 0.075_+0.04 15.9 _+ 3.1 39_+ 5 3.63_+ 1 . 0 1 0.43_+0.08 11.6 _+ 1.5 0.66_+0.09 0.12 _+0.04 10.7 _+ 3.2 39+ 6 1.54-+ 0.26 0.51-+0.09 10.6 -+ 0.7 1.06_+0.14 4.37 _+0.77 243 _+ 50 41+ 5 47.1 _+ 6.29 0.60_+0.04 214 _+ 8 0.97_+0.03 1.00 _+0.45 232 _+ 38 45_+ 5 5.45_+ 0.74 0.66_+0.03 20.1 _+ 1.1 0.94_+0.03

1.91 _+0.94 777 _+190 19_+ 0 475 + 72 0.64-+0.10 575 _ + 2 0 1 1.11+0.42 0.57 -+0.26 589 -+ 86 11-+ 3 512 -+ 2.7 0.91_+0.04 653 _+ 65 0.98_+0.12 0.66 _+0.29 8.21_+ 1.70 29_+10 3.77_+ 1.10 0.76+0.11 4.22-+ 0.34 0.70-+0.04 0.86 _+0.41 112 _+ 10 13_+ 4 84.2 _+ 8.7 0.77_+0.05 95.0 _+ 10.4 0.80_+0.08 2.12 _+0.14 4243 _+768 26_+ 5 1798 _ + 2 5 0 0.70_+0.07 2013 +170 0.76-+0.04 2.40 -+1.12 73.0 _+ 4.2 29_+ 8 39.7 _+ 3.9 0.83_+0.04 44.0 _+ 1.7 0.89_+0.06 1.29 _+0.42 36.5 _+ 5.0 27_+ 2 17.2 -+ 2.7 0.71_+0.03 10.8 _+ 1.3 0.85_+0.04 0.28 _+0.11 54.5 _+ 9.9 19_+ 6 31.5 _+ 6.2 0.63_+0.07 30.4 _+ 1.2 0.73_+0.05 5.34 _+4.68 628 _+111 30_+ 7 199 _+ 40 0.59-+0.01 375 _+ 73 1.00_+0.19

11.8 _+0.8 323 _+ 66 55+ 4 47.2 -+ 2.06 0.62-+0.06 168 + 44 0.69_+0.12

10.1 _+0.5 572 _+103 35_+ 5 191 _+ 6 0.56_+0.05 537 _+ 58 0.85 0.08 5.19 _+1.73 275 _+ 36 22_+ 0 120 _+ 13 0.71+0.02 361 -+ 47 0.90+0.01 0.53 -+0.10 21.6 -+ 4.2 31_+ 3 8.81-+ 3.20 0.63_+0.08 16.1 _+ 0.5 0.82_+0.02

10.5 +2.2 76.0 + 4.0 29+ 2 46.9 -+ 1.85 0.85-+0.03 51.1 -+ 1.0 1.00-+0.02 4.63 _+3.38 302 _+ 95 11_+ 1 220 + 38 0.90-+0.15 665 +300 1.08_+0.27 0.29 _+0.28 140 _+ 4 9_+ 1 134 _+ 4 0.82_+0.04 208 _+ 21 0.97_+0.10 1.16 _+0.47 25.4 _+ 1.4 18_+ 2 17.1 _+ 0.15 0.83+0.05 28.1 -+ 2.8 0.88-+0.08 0.66 _+0.12 113 + 62 28-+ 1 54.5 -+ 37.6 0.63-+0.01 50.3 _+ 5.3 0.91_+0.06 5.67 + 1.03 1453 + 180 14-+ 3 1025 _+ 69 0.79_+ 0.06 1597 + 160 0.91 -+ 0.08 1.50 +0.25 219 +101 26_+ 4 78.9 + 0.3 0.56-+0.02 120 + 9 0.85-+0.16

a One-site slope model.

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TABLE 2

GTP shift, subtype selectivity and potency of a2-adrenoceptor agonists in recombinant a2-adrenoceptor subtypes.

The extent of the GTP shift was calculated by dividing the K i value in the presence of Gpp(NH)p with the apparent one-site K i value without Gpp(NH)p. Apparent K i values of agonists at the three receptor subtypes were compared to assess subtype selectivity by K~ ratios. Agonist potency and affinity are compared by dividing the ECs0 values for inhibition of forskolin-stimulated cAMP production by the low-affinity binding coefficient (KiL). All K i values are from Table 1.

Ligand GTP-shift K i ratio ECs0/KiL K i + Gpp/App.K i App. K i /App. K i (a2A-C10)

azA-C10 o t 2 B - C 2 a2C-C4 a2A-CI0 a2B-C2 a2C-C4 a2A-C10 aeB-C2 a2C-C4

Noradrenaline 13.4 1.2 2.8 1.0 5.4 2.2 1.4 UK 14,304 14.4 1.3 3.0 1.0 138 32 6.4 Dexmedetomidine 11.6 1.1 1.8 1.0 4.8 11 1425 l-Medetomidine 1.5 1.1 1.1 1.0 3.9 2.2 348 Oxymetazoline 4.6 1.1 3.0 1.0 635 78 29 Clonidine 3.1 1.1 1.6 1.0 3.7 12 19 Detomidine 3.2 0.6 1.6 1.0 4.7 4.7 - Para-iodoclonidine 6.9 1.0 0.9 1.0 20 35 - Guanfacine 4.5 1.9 1.6 1.0 4.2 22 Guanabenz 3.7 3.6 1.5 1.0 8.7 14 -

1.6 0.7 0.8 0.6 2.4 19

16 297 4.6 57 1.9 3.0

where the ratios of the apparent one-site K i values in the presence and absence of Gpp(NH)p are shown for each ligand at each receptor subtype. The Gpp(NH)p- induced shifts were smaller in the a2C-C4 subtype than in the a2A-C10 subtype, and only minimal shifts were seen in the a2B-C2 subtype.

When comparing the apparent affinity constants de- rived from the one-site models, all ligands showed highest affinity for the a2A-C10 subtype. This was especially the case using dexmedetomidine which showed a very high apparent affinity (K m) for this subtype and a strong negative cooperativity as judged

from the Hill coefficient (0.12). Table 2 depicts this by comparing the apparent K i values for the a2B-C2 and a2C-C4 subtypes to the apparent K i values for the a2A-C10 subtype. Oxymetazoline was the best discrim- inator by having 635 times (comparing to a2B-C2) and 78 times (compared to a2C-C4) higher affinity for the a2A-C10 subtype. UK 14,304 also showed significant subtype selectivity, by having 138 times higher affinity for the a2A-C10 subtype compared with the a2B-C2 subtype and 32 times higher affinity for a2A-C10 than a2C-C4. Dexmedetomidine, clonidine, para-iodoc- Ion±dine, guanfacine and guanabenz were weaker dis-

TABLE 3

Conversion of [3H]ATP to [3H]cAMP (%) in Sl15 cells.

Some cells were Hincubated for 6 h with 500 ng/ml of pertussis toxin to ADP-ribolysate Gi-proteins. The values from the control experiment on a a-transfected cells are means + S.E. from 11 separate experiments performed in triplicate. The values from non-transfected cells are from two separate experiments performed in triplicate, and the values from the experiments using rauwolscine and pertussis toxin are from three separate experiments performed in triplicate. Buffer, assay buffer; FK, 60/xM forskolin; NA, 100 p~M noradrenaline.

Subtype Control + Rauwolscine (10/~M) Pertussis toxin

Buffer FK FK+NA % Inhibition FK+NA % Inhibition Buffer FK FK+NA % Change

Non-transfected 0.25 + 0.050 1.65 + 0.15 1.70 ± 0.34 . . . . . a2A-C10 0 .27_+0.14 2.46_+1.63 0.89_+0.12 71.7_+ 3.4 a 2.41_+0.19 2.3_+0.2 0.43_+0.02 1.86_+0.22 1.77_+0.15 - 6 . 3 + 0.5 a2B-C2 0.34_+0.13 2.58_+0.78 1.90_+0.28 30.4_+ 9.0 2.43_+0.26 6.7_+0.7 0.98_+0.20 1.96_+0.28 2.53_+0.58 +58 +16.2 a2C-C4 0.20-+0.10 1.86-+0.72 0.84±0.13 61.5+10.2 b 1.82_+0.18 2.4_+0.2 0.25_+0.09 0.88_+0.02 0.93_+0.03 +8.0_+ 3.4

a t-test: P < 0.001 compared to ct2B-C2; b t-test: P < 0.001 compared to ot2B-C2.

Fig. 1. A comparison of agonist binding curves in the presence (e) and absence (o) of 10 ~M Gpp(NH)p and the inhibition of forskolin-stimu- lated cAMP production ([]) for (a) noradrenaline, (b) dexmedetomidine, (c) UK 14,304, and (d) clonidine in the a2A-C10, a2B-C2 and a2C-C4 receptor subtypes. The binding curves indicate the percent inhibition of [3H]rauwolscine binding. The inhibition curves indicate percent inhibition of forskolin-stimulated cAMP production (maximal inhibitory effect taken as 100%). Data points from binding experiments are means _+ S.E. of three independent experiments performed in triplicate. Data points from cAMP experiments are from four independent

experiments performed in triplicate.

170

criminators, but had K i ratios between ot2A-C10 and OtEC-C4 in excess of 10, with intermediate affinity for a2B-C2. Noradrenaline, /-medetomidine, and detomi- dine were non-selective and had K~ ratios in the range of 2-5 between the subtypes. All receptor subtypes showed marked stereoselectivity in binding of the two isomers of medetomidine (Table 1).

3.2. cAMP experiments

To assess the coupling of the a2A-C10, a2B-C2 and a2C-C4 receptors to regulation of cAMP production, the percent of conversion of [3H]ATP to [3H]cAMP was determined in the absence and presence of forskolin (60 /xM) and a2-adrenoceptor agonists. The non-stimulated conversion of [3H]ATP to [3H]cAMP was similar in non-transfected cells and in transfected cells expressing the a2-adrenoceptor subtypes (0.20- 0.34% in 10 min) (Table 3). Treatment with 60 /xM forskolin for 10 min resulted in 6- to 9-fold increases in the rate of conversion. In forskolin-stimulated non- transfected cells 100 IzM noradrenaline had no signifi- cant effect on cAMP-formation. In aEA-C10 cells the inhibitory effect of 100/zM noradrenaline was 72%, in a2B-(~2 cells 31% and in a2C-C4 cells 62%. The inhibitory effect of 100 /zM noradrenaline was com- pletely antagonized by 10 /zM rauwolscine (Table 3). Pretreatment with 500 ng/ml pertussis toxin for 6 h abolished the inhibitory effect of 100 /xM nora- drenaline in aEA-C10 and a2C-C4-expressing cells, and resulted in a 58% stimulation by noradrenaline of forskolin-induced cAMP production in ol2B-C2 ceils (Table 3). The inhibitory effect of 100 /xM nora- drenaline increased with time to reach a maximum in all receptor subtypes at 10 min. A small decrease in the inhibitory effect was seen in all subtypes after 15 min (data not shown).

In addition to noradrenaline, also UK 14,304, dexmedetomidine, /-medetomidine, oxymetazoline and clonidine were also able to inhibit forskolin-stimulated cAMP production in a concentration-dependent man- ner in all three subtypes (Table 4, Fig. 2). The effect of

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Fig. 2. Concentration-dependent inhibition of forskolin-stimulated cAMP production by noradrenaline (/x ), UK 14,304 (0), dexmedeto- midine ([2), /-medetomidine ( • ) , oxymetazoline (.) and clonidine (11) in the ot2A-C10, a2B-C2 and ot2C-C4 receptor subtypes. Data points are means__S.E, from four independent experiments per-

formed in triplicate.

detomidine, para-iodoclonidine, guanfacine and guan- abenz were not tested. The maximal inhibitory effect obtained in cells expressing a2B-C2 receptors was around 35% with dexmedetomidine, noradrenaline and UK 14,304, while the corresponding inhibitory effects of these agonists were between 50 and 70% in the c~2A-C10 and a2C-C4 receptor subtypes (Table 4). The maximal inhibitory effect of clonidine was about 40%

TABLE 4

Inhibition of forskolin-stimulated cAMP production by different ligands in a2-adrenoceptor transfected Sl15 cells.

The ECso value is the concentration of each drug causing 50% of the maximal inhibitory effect (Max(%)) of that drug. The values are means 5: S.E. from four independent experiments performed in triplicate.

Ligand a 2 A-C 10 a 2 B-C2 a 2C-C4

ECs0 (/zM) Max (%) EC50 (/zM) Max (%) ECs0 (p,M) Max (%)

Noradrenaline 1.32 5:0.49 69.5 + 4.9 1.28 + 0.60 34.3 ± 3.5 0.40 + 0.18 62.9 + 5.5 UK 14,304 0.19 + 0.04 50.6 + 5.5 0.45 + 0.11 36.9 5:13.3 0.18 + 0.07 56.9 + 5.1 Dexmedetomidine 3.62 _+ 1.01 56.6 + 6.0 0.02 + 0.01 35.6 5:11.4 0.42 5:0.16 64.6 + 7.9 /-Medetomidine 23.6 + 3.40 11.1 + 2.0 1.76 5:0.45 17.4 + 2.1 22.6 5:3.30 14.2 + 2.3 Oxymetazoline 0.40 5:0.14 19.8 5:2.5 1.52 5:0.52 19.7 5:7.4 17.2 5:5.60 25.4 + 3.0 Clonidine 0.85 5:0.31 39.8 _+ 2.1 0.14 5:0.04 20.0 + 8.5 0.42 _+ 0.18 42.4 + 3.6

171

in a2A-C10 and a2C-C4 and 20% in ot2B-C2. The remaining two agonists, l-medetomidine and oxymet- azoline, were weak inhibitors of forskolin-stimulated cAMP production in all three subtypes, and no marked differences in the maximal inhibitory effect could be seen between the subtypes. Non-transfected cells and a2C-C4 transfected cells were also tested for inhibition of cAMP production by dexmedetomidine and l-med- etomidine to clarify whether non-adrenergic imidaz- oline receptors took part in the responses seen with these compounds. No inhibitory effect could be seen with these ligands in non-transfected cells, and the inhibitory effects seen in a2C-C4 transfected cells could be abolished by rauwolscine and pertussis-toxin treat- ment (data not shown).

The inhibition of cAMP production showed clear differences between the subtypes in its sensitivity to dexmedetomidine. Dexmedetomidine inhibited cAMP production with the highest sensitivity in the a2B-C2 subtype (ECs0 = 0.02 p-M), with an intermediate sensi- tivity in the a2C-C4 subtype (0.42 p-M) and with the lowest sensitivity in the a2A-C10 subtype (3.6 p-M) (Table 4). These data are in contrast with the very high apparent binding affinity for this subtype. The aEB-C2 subtype also showed the highest sensitivity for inhibi- tion by the other isomer of medetomidine, l-mede- tomidine (1.8 p-M) compared to the a2A-C10 (24 p-M) and the a2C-C4 (23 p-M) subtypes. Clonidine showed the same rank order of sensitivity as dexmedetomidine, ~2B-C2 (0.14 p-M) > t~2C-C4 (0.42 p-M) > a2A-C10 (0.85 p-M), but the differences were smaller. Oxymeta- zoline was most potent in the aEA-C10 subtype (0.40 p-M), intermediate in aEB-C2 (1.5 p-M), and clearly less potent in the a2C-C4 subtype (17 p-M). UK 14,304 seemed to inhibit forskolin-stimulated cAMP produc- tion with a relatively high potency in all three subtypes (aEA-C10 0.19 p-M; OtEB-C2 0.45 p-M; ot2C-C4 0.18 p-M). Noradrenaline inhibited cAMP production with slightly higher potency in ot2C-C4 (0.40 p-M) than in aEA-C10 (1.3 p.M) and c%B-C2 (1.3 p.M).

4. Discussion

The search for specific and subtype-selective a 2-

adrenoceptor ligands benefits greatly from the cloning of the a2-adrenoceptor genes, since recombinant tech- niques may now be employed to generate stable cell lines expressing single receptor subtypes. In addition to screening of new pharmacological agents, such heterol- ogous expression systems have proved to be very useful in the elucidation of receptor-coupled signalling mech- anisms (Buckley et al., 1990; Milligan et al., 1991; Eason et al., 1992; Federman et al., 1992; MacNulty et al., 1992; McClue et al., 1992). While antagonists may be succesfully characterized by performing binding as-

says (Marjam~iki et al., 1993), studies on agonists need to evaluate both binding properties and functional activity, since binding alone does not give sufficient information about agonist-receptor interactions. Therefore we have attempted to compare binding and agonist activity.

Stable recombinant Shionogi 115 mouse mammary tumour cell lines (Sl15) transfected to separately ex- press the three a2-adrenoceptor subtypes a2A-C10, a2B-C2 and a2C-C4 (Marjam~iki et al., 1992, 1993) were used to study agonist binding and the coupling of a2-adrenoceptor activator to regulation of cAMP pro- duction. Our results show that all three receptor sub- types, when expressed in Sl15 cells by recombinant techniques, are coupled to pertussis toxin-sensitive G- proteins and mediate inhibition of adenylyl cyclase. This inhibitory effect on forskolin-stimulated cAMP production was absent in non-transfected Sl15 cells and was blocked by an a2-adrenoceptor antagonist, confirming that the response was mediated by a 2- adrenoceptors.

Previous studies have also documented inhibition of cAMP formation by functional expression of recombi- nant a2-adrenoceptor subtypes in different mammalian cell lines, e.g. in NIH-3T3 fibroblasts (Lanier et al., 1991), LLC-PKI porcine kidney cells (Guyer et al., 1990), PS-120 Chinese hamster lung fibroblasts (Cotec- chia et al., 1990), and Chinese hamster ovary cells (CHO) (Eason et al., 1992). All three subtypes have previously been compared only in CHO cells. In these cells the three a2-adrenoceptor subtypes mediated in- hibition of forskolin-stimulated adenylyl cyclase activity with approximately equal efficiency. Pertussis toxin pretreatment revealed a stimulatory influence of the receptor subtypes on adenylyl cyclase activity, which increased with increasing receptor densities in the cell clones, and which was clearly more pronounced in the azA-C10 subtype than in a2C-C4 or a2B-C2 (Eason et al., 1992). Our results are at variance with this, since only azB-C2 was found to mediate stimulation of cAMP production, and the efficiency of a2B-C2 in inhibition of adenylyl cyclase was clearly weaker than that of a2A-C10 or a2C-C4. Since the expression levels were greatest in our azB-C2 cells, no definite conclusion can be drawn with respect to differences between subtypes in efficiency of coupling and the effects of receptor density. However, a new expression construct for the ot2C-C4 subtype was recently made, and Sl15 cells transfected with this construct showed a receptor den- sity of 5 pmol /mg protein, which is comparable with a 2 B-C2 (7.3 pmol/rag) (Marjam~iki et al., unpublished). This new cell line shows efficient az-adrenergic inhibi- tion of forskolin-stimulated cAMP production. The maximal inhibitory effect (76%) and the potency (ECs0 = 0.72 p-M) of noradrenaline were close to those ob- tained for the low-density a2C-C4 cell clone reported

172

in this investigation. This indicates that the weak in- hibitory effect of a2B-C2 on cAMP production in the current study would not be a consequence of the high expression level. Only a2B-C2 stimulated adenylyl cy- clase activity, which may have been due to the high receptor density in this cell line. Coupling of a2-adren- oceptors to stimulation of cAMP production has previ- ously been observed in several different cell lines (Jones et al., 1991; Fraser et al., 1989; Eason et al., 1992).

The ECs0 values for agonist-induced inhibition of cAMP production showed only small differences be- tween receptor subtypes with the exception of oxymetazoline, which had higher potency at the a2A- C10 and a2B-C2 subtypes as compared to the a2C-C4 subtype, and dexmedetomidine which showed higher potency at a2B-C2 than at the other two subtypes. On the other hand, the binding studies revealed some interesting differences in agonist interactions with the receptor subtypes. With few exceptions the a2A-C10 subtype had the highest affinity for all tested agonists. This was the case concerning apparent one-site K i values as well as when the affinity constants for high and low affinity binding sites were analyzed. The Hill coefficients of the a2A-C10 subtype also deviated most from hnity and the GTP shift was largest in this sub- type. Some agonists showed clear differences in their binding affinity to the three receptor subtypes. Oxymetazoline and UK 14,304 were able to discrimi- nate all three subtypes (a2A-C10 > a2C-C4 > a2B- C2), and dexmedetomidine, clonidine, para-iodocloni- dine, guanfacine and guanabenz were able to separate the a2A-C10 subtype from the other two. Previous reports have indicated that UK 14,304 has higher affin- ity for the a2A-C10 subtype than for the a2C-C4 subtype and that oxymetazoline has higher affinity for the a2A-C10 subtype than for the other two (Regan et al., 1988; Lomasney et al., 1990). The two isomers of medetomidine, dexmedetomidine and l-medetomidine, are known to bind to a2-adrenoceptors, but only dexmedetomidine is pharmacologically active in vari- ous in vivo and in vitro models (Savola and Virtanen, 1991). In our study dexmedetomidine bound to all three a2-adrenoceptor subtypes with higher affinity and inhibited cAMP production with higher efficacy and potency than l-medetomidine. Dexmedetomidine also seems to have isoreceptor selectivity by binding to the a2A-C10 subtype with higher affinity than to the other two subtypes, l-Medetomidine bound to all re- ceptor subtypes with similar affinity.

The comparison of ligand binding and inhibition of cAMP production indicated some interesting differ- ences between the subtypes. It has been suggested that the low affinity conformation of receptors is function- ally more relevant (Ross et al., 1977; Nathanson, 1983). The ECs0 value for inhibition of forskolin-stimulated cAMP production in intact cells was for all three

receptor subtypes and all ligands tested near or higher than the KiL values from two-site binding analysis of [3H]rauwolscine displacement in cell homogenates, which is in agreement with this suggestion. Therefore a comparison seems to be most relevant when it is done between the ECs0 value and the low-affinity binding component (KiL) of an agonist. When the binding curves and the dose-response curves for inhibition of forskolin-stimulated cAMP production are plotted to- gether for the full agonists noradrenaline, UK 14,304 and dexmedetomidine, differences are seen in the rela- tive efficacy of the receptor subtypes. With the excep- tion of noradrenaline the curves for inhibition of forskolin-stimulated cAMP production are clearly to the right of the low-affinity component of the binding curves of the a2A-C10. Dexmedetomidine showed the greatest discrepancy between binding and inhibition of cAMP production in this subtype. Thus the apparent KiL value was 2.5 nM while ECs0 for inhibition of cAMP production was 3.6/xM. These data are difficult to explain using a simple model for a G-protein cou- pled receptor. It appears that the real functional bind- ing affinity for this ligand is misjudged due to the very strong apparent negative cooperativity (Hill coefficient 0.12), suggesting that the binding characteristics of this ligand are complex. The inhibition curves for the a2B- C2 and a2C-C4 subtypes are near the low-affinity component of the corresponding binding curves. If an ECs0/KiL value is calculated a clearly lower value is also obtained for the a2B-C2 and a2C-C4 subtypes, suggesting a higher coupling efficacy (Table 2).

In the a2A-C10 subtype large GTP shifts were seen in the binding of the full agonists noradrenaline, UK 14,304 and dexmedetomidine, while the binding of partial agonists was less affected by Gpp(NH)p. This indicates that binding assays performed in the presence and absence of Gpp(NH)p would be predictive of agonist efficacy, which would be very useful for the screening of new agonist ligands. However, this predic- tive power was limited to the a2A-C10 subtype, as similar relationships could not be demonstrated in a2B-C2 and ot2C-C4. Thus, this simple method does not appear to be universally applicable.

In conclusion, these results demonstrate that (1) a2-adrenoceptor agonists may be characterized by an agonist-type binding pattern in homogenates of trans- fected Sl15 cells, (2) all three a2-adrenoceptor sub- types are coupled to inhibition of adenylyl cyclase in Sl15 cells through pertussis toxin-sensitive G-proteins, (3) the receptor-effector coupling in Sl15 cells may be different among the subtypes so that the a2A-C10 subtype tends to couple with high efficacy but with low sensitivity, the a2B-C2 subtype with low efficacy but high sensitivity, and the a2C-C4 subtype with both high efficacy and high sensitivity, and (4) at least a2B-C2 receptors may also be coupled to stimulation of aden-

173

ylyl cyclase activity, presumably through G s. Stimula- tion of type II adenylyl cyclase by the /3y-subunits released from the activated G i is an unlikely alterna- tive mechanism, since release of /3y-subunits should also be inhibited by pertussis toxin treatment.

Acknowledgements

This study was funded by the Technology Development Centre of Finland (TEKES) and also partly by grants from the Magnus Ehrn- rooth Foundation and the Borg Foundation.

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