QUANTITATIVE AUTORADIOGRAPHY OF THE DOPAMINE UPTAKE COMPLEX IN RAT-BRAIN USING [H-3] GBR-12935 - BINDING CHARACTERISTICS

The dopamine uptake complex was examined in the rat central nervous system using [H-3]GBR 12935 and in vitro quantitative autoradiography to determine all binding data. [H-3]GBR 12935 labels two unique binding sites, the dopamine uptake complex and a piperazine acceptor site. These two sites differ in their pharmacologic properties, anatomical distributions, densities, and response to lesions. Using appropriate binding conditions, [H-3]GBR 12935 can be used to specifically label the dopamine uptake complex. [H-3]GBR 12935 labeled a single binding site with characteristics of the dopamine uptake complex when mazindol (25-mu-M) was used as a blank. The specific binding and autoradiographic appearance of [H-3]GBR 12935 to the dopamine uptake complex was improved by including trans-flupentixol (0.75-mu-M) to displace binding to a previously described piperazine acceptor site, recently determined to be a site on cytochrome P450IID1. Binding was saturable and reversible to the dopamine uptake complex. The equilibrium dissociation constant (1.4 +/- 0.7 nM), maximal number of binding sites (6.0 +/- 1.3 pmol/mg protein), and Hill coefficient (1.1 +/- 0.1) of [H-3]GBR 12935 in rat striatum using mazindol to define non-specific binding was not significantly altered by the inclusion of trans-flupentixol (0.75-mu-M). Using GBR 12909 as a blank produced a greater maximal number of binding sites (8.4 +/- 2.3 pmol/mg protein), but no significant difference in the equilibrium dissociation constant (1.6 +/- 0.3 nM) or Hill coefficient (1.1 +/- 0.1). A series of drugs that bind to the dopamine uptake complex displaced [H-3]GBR 12935 in a rank order consistent with other binding and behavioral studies of this complex. The rank order of these drugs was GBR 12909 > mazindol > nomifensine > benztropine > desipramine > amphetamine > dopamine; all these drugs displayed a Hill coefficient near one and were best modeled as a single site. Cocaine and WIN 35,428 (a cocaine congener) were unique in their competition for [H-3]GBR 12935 binding, displaying biphasic curves, low Hill coefficients, and were best modeled as two site fits. Lesioning of the dopaminergic median forebrain bundle resulted in a dramatic loss of the dopamine uptake complex in the striatum, nucleus accumbens, olfactory tubercle, and substantia nigra. Other dopaminergic projection areas were decreased to a lesser extent. Striatal ibotenate lesions did not decrease the density of the dopamine uptake complex, despite a large decrease in the dopamine D1 receptor. [H-3]GBR 12935 can be used as an effective ligand to label the dopamine uptake complex for quantitative autoradiographic studies. It offers a number of advantages over previous autoradiographic assays for this complex including high specificity (> 95% specific binding in rat striatum), high sensitivity (detection of mazindol displaceable sites in the cerebral cortex), low background (comparable to film background), and low cost. This assay also supports the existence of two binding sites for cocaine on the dopamine uptake complex. The exact nature and differences between these two cocaine sites remains to be determined.