European Journal of Pharmacology - Molecular Pharmacology Section, 226 ( 19921169-173 169 Elsevier Science Publishers B.V. EJPMOL 8(11177 Short communication The AT z subtype of the angiotensin II receptors has differential sensitivity to dithiothreito| in specific brain nuclei of young rats Keisuke Tsutsumi, Stcfan Zorad and Juan M. Saavedra Section on Pharmacolo.~.3', Laborato~, of Clinical Science, National Institute of Mental I{ealth, Bethesda, MD 208q2, USA Received 8 JanuaD' 1992, revised MS received 23 March 1992. accepted 31 March 1992 We studied the effect of the sulfhydryl reducing agent dithiothreitol on the binding of the angiotensin II agonist [12Sl][Sar~]-angiotensin II to A'i-. receptors in sclectcd brain areas of young (2-week-old) rats. In the inferier olive and the hypoglossal nucleus, angiotensin !I binding to AT, receptors was insensitive 1o 5 mM dithiothrcitol. Conversciy, in the ventral and mediodersal thalamic, mcdial gcniculate, and oculomotor nuclei, the superior colliculus and the ccrebellar cortex, incubatkm with 5 mM dithiothreitol significantly decreased angiotcnsin 11 binding to ATe. receptors to about ae.e,.,j :c of control. These data ........... , ,u.., • AW receptors are holort~oono¢~! ~: I~.J;tlarospo¢~ m thoir ,;one llvitv 1o ,~ulfhvctrvl ~eduein~_ a~_ents. ,~uss,-,,~ -,-, bral~ "'2 ...... e, ....................... . . . ~ Angiotcnsin Ii receptor subtypes; AT 2 receptors; Sulfhydryl reducing agents; Dithiothreitol; Losartan (DuP 753): Brain d,cvclop_mcnt 1. lntroduclion (Tsutsumi and Saavedra, 199!c). The DTT sensitivity of other brain AT 2 receptors in the brain of young rats, Angiotensin II (Ang II) receptors have recently been however, has not yet been studied. We used quantita- classified into two s~btypes (AT~ and AT 2) based on tire autoradiography to further characterize the DTT their differential affinity for selective antagonists sensitivity in AT 2 receptor-rich areas of young, 2-week- (Whitebread et al., 1989; Bumpus et a!., 1991; Timmcr- old rats. We now report that brain AT, receptors in mans ct al., 1991}. In peripheral tissues, Ang II binding young rats are heterogeneous with respect to their to ATj receptors is decreased by st dfhyd~yl reducing sensitivity to DTY. agents such as dithiothreitol (DTT) while binding to AT, receptors has been reported to be enhanced (Whitebread et al., 19891. 2. Materials and methods The brain contains both AT m and AT, receptor subtynes (Chang et al., 1990; Tsutsumi and Saavedra, 2.1. Animals 1991a,b,c). DTT inhibits Ang I1 binding to brain AT t receptors (Spetb et a!., 1991; Tsutsumi and Saavedra, Groups of 10-day-old Sprague-Dawley male rats, 1991b). A controversy exists as to whether Ang II along with lactating females, were purchased from Zivic Miller (Alliston Park, PA, USA) and were provided binding to brain AT 2 receptors is enhanced (Speth et with standard rat food and water ad libitum, with a al. 1991) or not changed (Tsutsumi and Saavedra, 1991b) in the presence of DTT. This controversy sug- 12:12 h light-dark cycle with lio,hts on at 06.00 h. Rats were killed when 14 days old, by decapitation, between gested the possibility of heterogeneity of brain AT, receptors in their sensitivity to DTT. - 09.011 and 10.00 h, and the brains were removed imme- AT 2 receptors are very highly expressed in several diately. brain nuclei of young rats (Tsutsumi and Saavedra, 2.Z Quantitative autoradiography 1991a). In the inferior olive of immature rats~ AT 2 receptors have been found to be insensitive to DTI" For binding studies, the brains were frozen immedi- ately after removal in isopentane at -30°C, and stored at -70°C for less than 1 week. Coronal sections (16 ~orrespcmdence to: Juan M. Saavedra, MD, Section on Phar.,nacol- #m thick) were cut in a cryostat at - 15°C, thaw- ogy. NIMH, 9,~g) Rockviile Pike, Bldg 10, Room 2D-45, Bethesda, mounted on gelatin-coated glass slides and dried MD 21/892, USA. Tel. 301-496-0160; Fax 301-402-0337. overnight in a desiccator at 4°C.