Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]dihydrotetrabenazine.

Abstract

[2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-llb-H-benzo[a]-quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane ofpurified bovine chromaffin granules. The chromaffin granules of the adrenal medulla and the ghosts derived from their membranes accumulate catecholamines by a two-step ATP-dependent process (1-4): (i) inward translocation of protons by an electrogenic ATP-dependent H' pump (5-10), and (ii) a monoamine carrier driven by the H' electrochemical gradient (9,(11)(12)(13)(14)(15) and specifically blocked by reserpine or tetrabenazine (16). Whereas the H' pump has been functionally and structurally related to the mitochondrial ATPase complex (17,18), the catecholamine carrier has not yet been characterized. It is assumed to be a protein because the transport is temperature sensitive, shows some stereoselectivity (19), and is affected by the histidine-specific reagent diethylpyrocarbonate (20). The carrier has been solubilized and reconstituted in lipid vesicles (21,22), but this technique has not allowed purification. The carrier activity is difficult to assay because vesicles have to be energized to accumulate catecholamines, thus introducing possible artifacts.An alternate approach to the study of the carrier is the use ofbinding techniques. We have recently shown that, of several inhibitors (tetrabenazine, reserpine, haloperidol, and chlorpromazine), tetrabenazine has the most specific interaction with the carrier (23). In the present communication, we describe the binding of 2-[2-3H]hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3, 4,6,7-hexahydro-llb-H-benzo[a]quinolizine (dihydrotetrabenazine, [3H]TBZOH), a derivative of tetrabenazine to bovine chromaffin granule membrane. A preliminary account of some of these experiments has been published (24).[3H]Dihydrotetrabenazine MATERIALS AND METHODS TBZOH was obtained by reduction of tetrabenazine (Fluka) by NaBH4 in methanol (25).[3H]TBZOH (12 Ci/mmol; 1 Ci = 3.7 x 1010 Bq) was prepared as described (24). Its purity was periodically checked by TLC and, when necessary, the product was repurified (24). Stock solutions (80 ,AM) were made in 100 mM HCl and diluted in water.Bovine chromaffin granule membranes were prepared by osmotic lysis of granules isolated by centrifugation on a 1.6 M sucrose layer (26,27). Membranes were frozen in liquid nitrogen and were stored at -80°C. They were rapidly thawed at 370C, centrifuged at 100,000 x g for 15 min, and resuspended -in 20 mM Hepes KOH buffer containing 0.3 M sucrose.For [3H]TBZOH binding studies, membranes (5-15 ,ug of protein per ml) were incubated at 25°C with various concentrations of [3H]TBZOH in 0.3 M sucrose/20 mM KOH Hepes, pH 7.5. Bound ligand was measured by filtration on Millipore HAWP filters or by centrifugation at full speed in a Beckman Airfuge with cellulose propionate tubes. For filtration, 0.2-to 2.0-ml aliquots of the incubation mixture were diluted in 4 ml of ice-cold 0.3 M sucrose/10 mM KOH Hepes, pH 7.5, containing 125 ...

Expression of a bovine vesicular monoamine transporter in COS cells

“…2B show, for instance, that ketanserin at 20 nM fully occupies its site without any inhibition of ATP-dependent noradrenaline uptake. This is in contrast with the correlation, previously reported, between the occupancy of TBZ binding sites and the inhibition of monoamine uptake [16]. The most likely explanation is an heterogeneity in the transporter molecules.…”

“…TBZOH binding was measured as described [16], using ['HITBZOH (11.4 Ci/mmol) from C.E.A. (Saclay, France) and HAWP filters (Millipore).…”

Serotonin organelles of rabbit platelets contain synaptophysin

“…As shown in Fig. 4, the profile of synaptophysin enrichment was similar to that obtained for the granular amine transporter, measured by [3H]dihydrotetrabenazine binding [20,251. However, in the latter case a higher purification factor in respect to rabbit platelet homogenate was found.…”

“…The amount of amine granular transporter in rabbit platelet subcellular fractions was estimated by determining the maximal binding (Bmax) of the selective ligand [3H]dihydrotetrabenazine (CEA, Gyf-sur-Yvette, France; 15 Ci/mmol) [20]. B,,, values were calculated from saturation experiments using nine different radioactivity concentrations.…”

Visualizing pancreatic β-cell mass with [11C]DTBZ

“…Previous studies have shown that DTBZ specifically targets the VMAT2 [37][38][39][40]. Using in situ hybridization, immunohistochemistry and confocal microscopy, Weihe et al [20], Maffei et al [25] and Anlauf et al [27] have shown that VMAT2 immunoreactivity colocalizes with insulin or is expressed with other β-cell markers and is absent from human islet cells stained with antiglucagon, somatostatin and pancreatic polypeptides.…”