PharmacologyBiochemistry & Behavior. Vol. 32, pp. 831-834. ~ PergamonPress plc. 1989. Printedin the U.S.A. 0091-3057/89 53.00 + .00 BRIEF COMMUNICATION Buffer Effects on High Affinity [ H]-Prazosin Binding in Brain and Spinal Cord D. W. HERR* AND R. B. MAILMAN*+ ~ *Toxicology Curriculum, Universi O, of North Carolina, Chapel Hill, NC 27514 ~Departments of Psychiatry and Pharmacology, Biological Sciences Research Center University of North Carolina School of Medicine, Chapel Hill, NC 27514 Received 27 June 1988 HERR, D. W. AND R. B. MAILMAN. Buffereffects on high aff~niO' [~Hl-prazosin binding in brain and spinal cord. PHARMACOL BIOCHEM BEHAV 32(3) 831-834, 1989.--[3H]-Prazosin binding was characterized in cortical and spinal membranes from Fischer 344N and Sprague-Dawley rats. Estimates of Bma x and K,~ values were comparable with earlier studies of these regions in the central nervous system (CNS). However, the Ka obtained using Tris buffer system was greater than when HEPES or phosphate buffer was used. These data indicate that high affinity [3H]-prazosin binding in the homogenates of tissue from the CNS is affected critically by buffer selection. Buffer Prazosin Alpha-adrenoreceptor Radioligand binding Cerebral cortex Spinal cord RECEPTOR binding techniques allow the quantification and pharmacological characterization of specific receptor subtypes. Prazosin is an adrenergic antagonist which has been reported to have selectivity for % receptors both in the periphery (6,13) and central nervous system (CNS) (9,15). Prior to undertaking a study of toxicant-induced effects on [3H]-prazosin binding sites using a high specific activity (82 Ci/mmol) ligand, we examined the characteristics of binding of [3H]-prazosin to recognition sites in the cerebral cortex and spinal cord of two strains of rats, in several common buffers. We now report the unexpected finding that the buffer used has significant influence on the apparent characteris- tics of this ligand recognition site, and might influence results or introduce experimental artifacts. EXPERIMENTAL PROCEDURES Animals Male Fischer 344N rats (Charles River Laboratories, Raleigh, NC), approximately 9-13 weeks old, were housed in plastic home cages with corncob bedding in groups of four at the National Institute of Environmental Health Sciences. Food (NIH diet No. 31) and water were available at all times. The animal colony was maintained at a temperature of 21 _+2°C and 50 _+ 10% humidity, with a 12 hr light-dark cycle (lights on 0700-1900). Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), about 9-12 weeks of age, were housed in hanging wire cages in groups of four at the Biological Sciences Research Center at the University of North Carolina. Food (Rodent Laboratory Chow No. 5001, Purina Mills, Inc., St. Louis, MO) and water were continuously available. The animal facilities were main- tained at 22-+2°C and 45_+5% humidity, with a 12 hr light-dark cycle (lights on 0700-1900). Membrane Preparation Animals were decapitated, the brains removed, placed on ice, and the frontal cerebral cortex (anterior to the optic chiasm) removed. The spinal cord was removed hydraulically, dissected free from spinal roots, and a 2.5-3 cm portion of the lumbar region taken for assay. Tissue was frozen on dry ice and stored at - 70°C until binding was performed. The mean weights (_+SEM) of cortex and spinal cord were 386_ + 12 and 263_+7 mg, respec- tively. These regions of the CNS were selected because [3H]- prazosin binding had previously been characterized in these tissues (1,2, 18). Tissue from three rats was pooled in each of the assays. Tissue was homogenized on ice using a PCU-2 Polytron (setting 4) for 10 sec in 30 ml of a 50 mM buffer of either HEPES (N-2- 1Requests for reprints should be addressed to R. B. Mailman. 831