Characterization of the Binding and Comparison of the Distribution of Benzodiazepine Receptors Labeled with Ph1diazepam and [3H]Alprazolam L&S K

fIivIoPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.4 305 Characterization of the Binding and Comparison of the Distribution of Benzodiazepine Receptors Labeled with pH1Diazepam and [3H]Alprazolam l&s K. Wamsley, Ph.D., Lisa L. Longlet, B.S., MaryAnne E. Hunt, Ph.D., Donald R. Mahan, IfiMilrio E. Alburges, Ph.D. Ttrbinding characteristics of [3H]diazepam and benzodiazepine drugs and apparently do not overlap onto �prazolam were obtained by in vitro analysis of other subtypes of receptors. These experiments were IIfiorrsof rat brain. Dissociation, association, and performed by both binding assay in tissue sections and by sDlItionaTUllyses were performed to optimize the light microscopic autoradiography. The major difference GdilWnsfor obtaining selective labeling of between the labeling of the two compounds is· represented llaldiDzepinerecept ors with the two tritiated by the peripheral benzodiazepine sites, which are GIIpOUnds. Both drugs approached equilibrium rapidly recognized by [3H]diazepam, but not occupied by nntro. Rosenthal analysis (Scatchard plot) of the [3H]alprazolam (at nanomolar concentrations). This amiondata indicated a similar finite number of difference was readily apparent in the auto radiograms. IIII;otSwas being occupied by both ligands. Other pharmacokinetic or pharmacodynamic properties Clapetitionstudies, using various ligands to inhibit both must distinguish these two benzodiazepines. fH}bu.tpam and [3H]alprazolam indicated that these [Neuropsychopharmacology 8:305-314, 1993J tIIIcmnpoundsbind to the tissue sections as typical lIT WORDS: Benzodiazepine receptors; Anxiolytics; in the treatment of panic disorder (Feighner et a1. 1983; 0upmI;Alprazolam; Autoradiography; Localization; Pitts et a1. 1983; Shehi and Patterson 1984; Rickels et Drtsity;Binding characteristics a1. 1985; Alexander and Alexander 1986; Leibowitz et 1986; 1986), A¥azolamis a triazolobenzodiazepine that is distinct al, Mendels and SchIess whereas many DIIother cornpounds in its class due to its unique clin other typical benzodiazepines are not as efficaciousin g profIle (Fawcett and Kravitz 1982; Dawson et a1. these latter two areas, but are remarkably effective 114;Cir aulo et a1. 1986; Dunner et a1. 1986). Alprazo anxiolytics (others are used as sedative-hypnotics or an .. hasbeen used as an anxiolytic, antidepressant, and ticonvulsants). Results from several studies have sug gested alprazolam may be recognizing other receptor sites in addition to benzodiazepine receptors (Cer fan the Neuropsychiatric Research Institute, Fargo; and the nansky et al, 1982; Sethy and Hodges 1982; Charney tsof Pharmacology and Neuroscience, University of North IIIfIrtmen and Heninger 1985; Eriksson et al, 1986; Kostowski et 1Iika.School of Medicine, Grand Forks and Fargo, North Dakota; alDIeUp john Company, Kalamazoo, Michigan. al, 1986; Soderpalm and Engel 1989), in the central ner Addressre print requests to: James K. Wamsley, Ph.D., Director vous system (CNS). The in vitro analysis of [3H]al d_ScienceResearch, Neuropsychiatric Research Institute, 700 prazolam binding in synaptosomal preparations of rat "Avenue South, Fargo, North Dakota 58103. IDivedSept ember 3, 1991; revised March 17, 1992; accepted April brain has not been able to verify this as a direct effect -\J9I12. (McCabe et a1. 1990). To analyze the potential for over- • n93American College of Neuropsychopharmacology Mished by Elsevier Science Publishing Co., Inc. 5Avenue of the Americas, New York, NY 10010 0893-133X/93/$6.00 NEUROPSYCHOPHARMACOLOGY VOL. 306 J.K. Wamsley et ai. 1993- 8, NO.� lap of [3Hlalprazolam onto other sites, a receptor au incubation of groups of sections in various concenm toradiographic approach (Kuhar et al. 1985) was used tions of the radioactive ligands to establish saturatioa in the present study. Previous investigations, aimed at Competition studies were performed by labeling sec· analyzing the distribution of benzodiazepine receptors tions with a 2-nmollL concentration of the radiolabel� in the CNS, have principally involved the use of ligand using the parameters established in the previ pHlflunitrazepam as the ligand of choice (Young and ously outlined experiments, in the additional presena Kuhar 1979, 1980). Relatively little information is avail of 10-4 to 10-11 mollL concentrations of the follo� able on the binding of [3Hldiazepam after its initial compounds: clonidine, CL218,872, sulpiride, desipra characterization in membrane preparations (Braestrup mine, imipramine, 4-0H alprazolam, or flurazepant and Squires 1977; Gallager et al. 1981). We sought to The ICso values were obtained by plotting the percent use a typical anxiolytic compound for comparison with of radioligand bound versus the percent of radioligand alprazolam and to provide the binding conditions and bound multiplied by the molar concentration of the characteristics for obtaining selective labeling of both competing ligand. In all cases, adjacent sections wert [3Hldiazepam and [3H]alprazolam to slide-mounted incubated in the presence of 10-6 mollL clonazepam tissue sections for autoradiographic analysis. to establish nonspecifIc binding. For the autoradiographic studies, slide-mounted sections (20 J.1 in thickness) of whole brain were labeled MATERIALS AND METHODS using the optimum binding conditions and, insteadd wiping the sections from the slide, the sections wert Male Long-Evan's rats (weighing between 180 and 200 dried by blowing cool dry air over the tissue surface, gm) were deeply anesthetized with C02 and perfused These sections were subsequently exposed to tritium intracardially with ice-cold isotonic saline. The brain tis sensitive film (Amersham Hyperfilm, Arlington sues of the animals were rapidly dissected free from Heights, IL). After an appropriate exposure period, the surrounding structures and frozen onto microtome fIlms were developed and analyzed by computerized chucks by slow immersion in isopentane at -70°C. Sec microdensitometry (Palacios et al. 1981) using anMOD tions, 10 J.1 in thickness, either frontal or sagittal plane, system (Imaging Systems Corporation, St. Catherines, were cut on a cryostat microtome and thaw-mounted Ontario). Tritium standards (Amersham, Arlington onto subbed microscope slides. The brains of some Heights, IL) were included in the exposure of each DIm animals were removed from the skull and gently to quantitate femtomoles of ligand bound per milligram homogenized in a glass tube with a Teflon-coated pes tissue. Another group of sections was incubated ina tle. Frozen sections of this homogenate were cut in the Kd concentration of the radioactive ligand (inan effort cryostat as before. This procedure assured a uniform to occupy a major portion of the sites recognized bylbe distribution of different cell types in each section so radioactive compound) in the additional presence ofjn. more consistent data could be obtained. Alltissues were creasing concentrations of the other benzodiazepine, stored overnight in the presence of desiccant prior to Thus, incubations were performed with [3Hldiazepam being utilized in the incubation procedures. in the presence of increasing concentrations of unla For the biochemical assays, slide-mounted tissue beled alprazolam and [3Hlalprazolam was used to la sections of homogenized brain were initially given a bel sections inthe presence of nonradioactive diazepam. 3-minute preincubation period in distilled water to os These sections were analyzed by receptor autoradiog· motically disrupt the cells and release any gamma raphy to determine if the radioactive compound oc· aminobutyric acid (GABA) or other constituents that cupied sites where the binding was not inhibited by the may interfere with the subsequent binding of the ra other benzodiazepine. dioactive ligand (McCabe et al. 1988). This was followed by two 5-minute rinses, after which individual groups of sections were incubated for 60 minutes in the pres RESULTS ence of the radioactive ligand in Tris HCl buffer (0.17 mollL, pH 7.6) at between 0 and 4°C. Initially, a 2 Comparison of the binding characteristics of [3H]al· nmollL concentration of pHlalprazolam (15 to 45 prazolam and [3Hldiazepam to slide-mounted tissue Ci/mmol, supplied by the Upjohn Company, Kalama sections of rat brain indicated many similarities. The zoo, MI) or [3Hldiazepam (85.2 Ci/mmol, Dupont initial osmotic shock of the slide-mounted tissue sec· NEN, Boston, MA) was used to label the sections. The tions dramatically reduced the binding of pH]alprazo. rinse time was varied and the tissue sections were wiped lam. The magnitude of this reduction in binding was from the microscope slides with fIlter paper and the as high as 50% and affected only specifIc binding bound radioactiVity determined by liquid scintillation (nonspecifIcbinding remained unchanged). Bindingd counting. Next, the incubation time was varied, with [3Hldiazepam was also reduced by the osmotic shock, the rinse time held constant. This was followed by the but not to the extent of the alprazolam binding. The Nn!aoPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.4 Comparison of [3H]Diazepam and [3H]Alprazolam 307 II!beIingwithboth ligands was temperature dependent Fig. 1). A BMAX of 189.8 and 195.7 fmol of receptor mdaJI subsequent experiments were performed at ice bound per milligram tissue was obtained for [3H]al WIte mperature. Analysis of the dissociation data (Fig. prazolam and [3H]diazepam, respectively. Inhibition ,wasperformed using a nonlinear regression program curves (Fig. 2) generated by incubating various com

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