Biochemical Pharmacology. Vol. 31. No. 4. pp. 583-587, 1982. Printed in Great Britam. 00CS2952!82/040583-05 $03.0010 Pergamon Press Ltd. zyxwvut LOCALISATION OF [3H]CLONIDINE BINDING TO MEMBRANES FROM GUINEA PIG RENAL TUBULES GRANT A. MCPHERSON and ROGER J. SUMMERS* University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, 3084, Victoria, Australia zyxwvutsrqponmlkjihgfedcbaZYXWVUTS (Receiued 1 May 1981; accepted 17 August 1981) zyxwvutsrqponmlkjihgfedcbaZYXWVU Abstract-The selective radioligand [“Hlclonidine has been used to localise cuzadrenoceptors in guinea pig kidney. Chemical sympathectomy with 6-hydroxydopamine produced no significant change in the number of sites labeled by [‘Hlclonidine indicating that the majority of binding sites were not located on sympathetic nerve terminals. Binding was enhanced in membranes prepared from renal tubules and considerably reduced in preparations from glomeruli. Subcellular fractions of renal cortex revealed that binding was to plasma membranes and that the greatest binding capacity was present in the fraction rich in basal lateral membranes. It is concluded that the major concentration of renal 9 adrenoceptors are present on renal tubules and that they may be localised to a particular pole of the renal tubule cell. [3H] Clonidine has been used as a radioligand to study ai adrenoceptors in membranes prepared from the kidneys of guinea pig [I] and rat [2]. Within the guinea pig kidney the a~2 adrenoceptors are highly localised to membranes prepared from the renal cortex with membranes from medulla and papilla having much lower concentrations [3,4]. Recent autoradiographic studies have confirmed these find- ings and extended them to show that the CQ adren- oceptors are located on the proximal convoluted tubules [5]. The results are consistent with the pres- ence of (Y adrenoceptors on proximal convoluted tubules which mediate sodium reabsorption [6]. In the experiments described here [3H]clonidine binding within the guinea pig renal cortex has been localised using the techniques of chemical sympathectomy with 6-hydroxydopamine, preparation of membranes from glomeruli and tub- ules and subcellular fractionation. A preliminary account of some of this work has been previously described [7]. MATERIALS AND METHODS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF Chemical sympathectomy. Male guinea pigs (500-800 g) were given 6-hydroxydopamine (150mg/kg i.p.). Animals were killed and the kid- neys removed 5, 7 or 11 days after injection. The extent of sympathectomy was estimated by measure- ment of the catecholamine content of a sample of tissue by radioenzymatic assay [9]. Only kidneys in which the noradrenaline content was less than 5 per cent of the controls were used in this study. Preparation of glomeruli and tubules. Kidneys were removed from male guinea pigs and placed on ice. Renal cortex was dissected from medulla and papilla, minced with a razor blade, forced through a 212 micron nylon mesh and suspended in isotonic Tris 50 mM/sucrose 8%: pH 7.6 at 4”. This procedure disrupted the tubular structures and freed the glom- eruli which were then separated from the debris by * To whom correspondence should be sent. a combination of differential centrifugation and fil- tration. The glomerular suspension obtained after the initial sieving was centrifuged (1OOOg for 1 min), the supernatant removed and the pellet was resus- pended in fresh buffer. This procedure was repeated a further five times before passing the suspension through an 85 micron nylon mesh. The majority of the remaining tubule fragments passed through the mesh leaving the glomeruli (dia - 120 microns) on the surface. Tubules were prepared from renal cortex incu- bation in Krebs-Henseleit solution containing 0.05% collagenase (Sigma type II) for 30min at 37” with vigorous shaking (120 shakesimin) [lo]. After diges- tion the slices were disrupted by repeated passage into a Pasteur pipette. The suspension was then passed gently through a 212 micron nylon mesh to complete dispersion. After three washes in colla- genase free Krebs the suspension was layered onto a discontinuous sucrose gradient (75, 58 and 50%) and spun at 5000 g for 35 min. Tubules were collected from the interface (50:58) and purity checked by microscopic examination. Membranes from both tubules and glomeruli were prepared by homogen- ising the structures in Tris buffer (50mM, pH 7.6 at 4” and 25”) as previously described (41. Subcellularfractionation. Subcellular fractionation was carried out, using a homogenate of guinea pig renal cortex [ll]. Briefly, renal cortex was minced and homogenised in 10 vol. of 8% sucrose (pH 7.0) using 10 strokes of a Dounce homogeniser with a tight fitting pestle. A sample of this homogenate was retained (Hl). The remainder was centrifuged at 1OOOg for 10 min, the supernatant saved and the pellet resuspended in 2.5 vol. of 8% sucrose. After rehomogenisation and centrifugation the superna- tant was combined with that from the first spin and the pellet (Pl) retained. The supernatant was spun at 9000g for 10 min. The lower pellet (P2) was retained whereas the soft upper portion was com- bined with the supernatant and centrifuged at 47,000 g for 20 min. The clear supernatant from this spin was discarded. The upper lighter portion of the 583