Br. J. Pharmacol. (1989), 98, 890-897 Differences between the cx2-adrenoceptor in rat submaxillary gland and the a2A- and a2B-adrenoceptor subtypes 1Anton D. Michel, Dana N. Lodry & Roger L. Whiting Institute of Pharmacology, Syntex Research, 3401 Hillview Avenue, Palo Alto, CA, 94303, U.S.A. 1 The a2-adrenoceptors of rat submaxillary gland, labelled with [3H]-rauwolscine, were character- ized by use of a range of subtype selective ligands and were compared to rabbit spleen a2A-adrenoceptors and rat kidney a2B-adrenoceptors. 2 In rat submaxillary gland, [3H]-rauwolscine labelled an apparently homogeneous population of binding sites with relatively low affinity (Kd= 11.65 nM) compared to the affinity in rat kidney (Kd = 2.18 nM) and rabbit spleen (Kd = 4.64 nM). 3 In competition studies using 16 ligands the a2-adrenoceptors in rat submaxillary gland appeared to differ from both the x2A-adrenoceptor of rabbit spleen (r = 0.62) and also the a2B-adrenoceptor of rat kidney (r = 0.28). 4 The affinity data obtained with benoxathian, imiloxan and WB 4101 indicated the presence of an a2B-adrenoceptor in rat submaxillary gland. However, data for chlorpromazine, oxymetazoline, spiroxatrine and xylometazoline indicated that submaxillary gland a2-adrenoceptors were of the a2A subtype. The affinity estimate for prazosin in rat submaxillary gland was intermediate between its affinity at the ae2A- and a2B-adrenoceptors while affinity estimates for idazoxan and phentolamine in rat submaxillary gland were greater than those obtained at either the c2A- or x2B-adrenoceptor. 5 These data indicate that rat submaxillary gland a2-adrenoceptors differ from the CX2A- and a2B-adrenoceptors found in rabbit spleen and rat kidney, respectively. Introduction Recent studies on the ligand binding properties and and chlorpromazine, while a2B-adrenoceptors molecular biology of the X2-adrenoceptor have indi- display the converse selectivity for these agents cated that a2-adrenoceptor subtypes exist. Regan et (Bylund, 1988). al. (1988) have cloned two genes that encode We have previously demonstrated that rabbit a2-adrenoceptors and have shown that these two spleen and rat kidney possess a2-adrenoceptors that receptors can be differentiated on the basis of their display the pharmacology of the a2A- and affinity for a range of ligands. These molecular bio- a2B-adrenoceptor, respectively (Michel et al., 1989). logical studies complement the data obtained from In the present study we extend our characterization ligand binding studies conducted in several labor- of the rabbit spleen and rat kidney X2-adrenoceptors atories (Nahorski et al., 1985; Bylund, 1985; 1988) and report on the characterization of an which have demonstrated that a2-adrenoceptors can a2-adrenoceptor subtype present in rat submaxillary be separated into a2A and o2B adrenoceptor subtypes gland that displays significant differences from the depending upon their affinity for a range of ligands. a2A- and O2B-adrenoceptors described to date. According to the present classification scheme, 22A-adrenoceptors display high affinity for oxymeta- zoline and WB 4101 and low affinity for prazosin Methods Membrane preparation 1 Author for correspondence at present address: Depart- ment of Neuropharmacology, Glaxo Group Research All membrane preparation procedures were con- Limited, Park Road, Ware, Hertfordshire, SG12 ODP. ducted at 40C and used ice cold buffers. Kidneys and © The Macmillan Press Ltd 1989 a2-ADRENOCEPTOR SUBTYPE OF RAT SUBMAXILLARY GLAND 891 submaxillary glands from adult Sprague-Dawley rats Materials and spleens from adult New Zealand White rabbits were obtained from Pel-Freeze (Arkansas, U.S.A.). [3H]-rauwolscine (Specific activity 82 Ci mmol') Tissues were homogenized for 30 s in 3 volumes (w/v) was obtained from New England Nuclear. Chlor- of 50 mM Tris, 5 mM EDTA homogenizing buffer promazine, noradrenaline and oxymetazoline were (pH 7.4 at 40C) in a Waring Blender at maximum obtained from Sigma chemical company as were all setting. The homogenates obtained were further chemicals and reagents used. Phentolamine was homogenized in a Polytron PlO tissue disrupter obtained from Ciba-Geigy while prazosin was (setting 10; two 10s bursts) and filtered through a obtained from Pfizer. Benoxathian, spiroxatrine and double layer of cheesecloth. The homogenates were WB 4101 (2-(2,6-dimethoxyphenoxyethyl) amino- centrifuged at 5OOg for 10min and the supernatants methyl - 1,4 - benzodioxane) were obtained from reserved. The pellets were resuspended in 2 volumes Research Biochemical Inc. Idazoxan, imiloxan, UK of homogenizing buffer and rehomogenized in the 14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quin- polytron P10 tissue disrupter and centrifuged at oxaline bitartrate) and SKF 104078 (6-chloro-9-[(3- 500g for 10min. The supernatants from this step methyl- 2- butenyl)oxyl - 3 - methyl - 1H - 2,3,4,5 - tetra- were combined with the original supernatants and hydro-3-benzazepine) were synthesized by Dr R. centrifuged for 12min at 43,000g. The crude mem- Clark, IOC, Syntex. brane pellets obtained were washed by resuspending in homogenizing buffer and centrifuging at 43,000g foz 12min. The pellets from this step were washed Results twice, in a similar manner, but with ice cold 50mM Tris, 0.5 mm EDTA assay buffer (pH 7.4 at 40C). The Saturation studies final pellets were resuspended in assay buffer (pH 7.4 at 25°C) and stored under liquid nitrogen until Results of saturation binding studies conducted in required. rat kidney, rat submaxillary and. rabbit spleen mem- branes are presented graphically in Figure 1. In rat submaxillary gland, [3H]-rauwolscine labelled a single population of binding sites (B,,, = 137.5 Ligand binding assays + 12.2 fmol mg 1protein; n = 4) with low affinity (Kd = 11.65 + 1.0nM). The Kd value obtained in this Membranes, [3H]-rauwolscine and competing drugs preparation was significantly higher than the Kd were incubated in a final volume of 0.25 ml of assay values obtained in either rat kidney (Kd = 2.18 buffer (pH 7.4 at 25°C) for 45 min at room tem- + 0.46 nM; n = 4) or rabbit spleen (Kd = 4.64 perature (220C). In competition studies the final + 0.78 nM; n = 4). assay concentration of radioligand was 1-2 nm while in saturation studies the concentration of [3H]-rau- Competition studies wolscine was varied between 0.1 and 105 nm. In all experiments non-specific binding was defined by use Affinity estimates for a range of compounds at of 1O gM phentolamine. Incubations were terminated M2-adrenoceptors of rat submaxillary gland were by vacuum filtration over 0.1% polyethyleneimine determined in competition studies. These results are pretreated glass fibre filters using a Brandel cell har- presented in Table 1 along with affinity estimates vester. The filters were washed for 10s with ice cold obtained in both rat kidney and rabbit spleen. O.1M NaCl and radioactivity retained on the filters Results in rat kidney and rabbit spleen for several of was determined by liquid scintillation spectrometry. the ligands have been described previously (Michel Protein was determined by the dye binding et al., 1989). Representative displacement isotherms method (Bio-Rad) with bovine gamma globulin as for idazoxan, spiroxatrine and WB 4101 in the three standard. tissues are shown in Figure 2. Displacement isotherms for most compounds dis- played Hill coefficients that were close to unity and Data analysis the data could only be described assuming the presence of a single population of binding sites. For Competition and saturation binding data were WB 4101 and oxymetazoline in rabbit spleen, and analysed as described previously by use of iterative for oxymetazoline in rat kidney, Hill coefficients curve fitting techniques (Michel & Whiting, 1988). were less than unity and we have previously shown IC50 values were converted to Ki values using the that these data are better described by assuming the Cheng-Prusoff approximation (Cheng & Prusoff, presence of two populations of binding sites (Michel 1973). et al., 1989). WB 4101 and oxymetazoline displayed 892 A.D. MICHEL et al. .'300a pKi values of 8.71 and 8.60, respectively, for 70-75% 7o /of the a2-adrenoceptors labelled using [3H]-rauwol- 0 scine in rabbit spleen. 250/ The x2-adrenoceptor of rat submaxillary gland displayed relatively high affinity for imiloxan with E the value being similar to that obtained at the E 200 /a2B-adrenoceptorpKi of rat kidney. For benoxathian and WB 4101, affinity estimates in rat submaxillary n150 / / gland were also similar to those obtained at the o / / a2B-adrenoceptor of the rat kidney. .0 * /For oxymetazoline, spiroxatrine and xylometazol- / X 5C 100 ine, affinity estimates in rat submaxillary gland were la 502 fox 7 Q ° similar to those obtained at the x2A-adrenoceptor in o 0 the rabbit spleen. Finally, for idazoxan and phentolamine, affinity Do estimates at the rat submaxillary gland a2-adren- 0Io.r oceptor were significantly higher than those obtained 0 20 40 60 80 120 140 at either the M2A- or CX2B-adrenoceptors. For prazosin, Free ligand concentration (nM) the affinity estimate in rat submaxillary gland was z500b intermediate between affinity estimates obtained 5 50 -thea2 and M2B-adrenoceptor. :at 0~ T 400 Correlation between affinity estimates obtained in rat D 400/ kidney, rat submaxillary gland and rabbit spleen o 0 In Figures 3, 4 and 5 correlations between affinity 300/ estimates obtained in the three preparations are pre- *at / sented. As can be seen from Figure 3, there was a 200 / /poor correlation (r = 0.066) between affinity esti- 200) mates obtained in rat kidney and rabbit spleen. Fur- thermore, 7 of the compounds examined displayed c0,//ogreater than 10 fold selectivity for one or the other of 0 100 _/. of the a2-adrenoceptors compared (Table 1, column A). LEO0 /° Rat submaxillary gland x2-adrenoceptors differed co substantially (r = 0.276) from the M2B-adrenoceptor 20 on , , , , ,of rat kidney (Figure 4) with 4 compounds dis- 0 20 40 60 80 100 120 playing greater than 10 fold selectivity for either the Free ligand concentration (nM) rat kidney a2B-adrenoceptor or rat submaxillary c c gland (Table column 250 The x2-adrenoceptorrat submaxillary gland1 x2-adrenoceptorC).