Abstract
Classical studies using bovine chromaffin granules have defined the physiologic and pharmacologic properties of the vesicular amine transporter that packages monoamine transmitters into intracellular vesicles for subsequent regulated release. The recent isolation of two distinct but closely related cDNA clones encoding vesicular amine transport suggests that the activity expressed in the brain (synaptic vesicle amine transporter or SVAT) may differ significantly from the previously described adrenal gland activity (chromaffin granule amine transporter or CGAT). A direct comparison of the two transporters now shows that SVAT has a higher affinity than CGAT for monoamine substrates, in particular for histamine. In addition, SVAT shows approximately 10-fold greater sensitivity to tetrabenazine than CGAT. [3H]Dihydrotetrabenazine shows no detectable binding to CGAT but does bind to SVAT, accounting for the differential sensitivity. Furthermore, methamphetamine preferentially inhibits transport by SVAT relative to CGAT, apparently by competing at the site of amine recognition rather than by disrupting the vesicular pH gradient. These previously unsuspected differences in the storage of monoamine transmitter in the central nervous system and the adrenal gland may help to account for several classic pharmacological observations.
Highlights
Classical studies using bovine chromaffin granules nantly centralmonoamine stores (Carlsson, 1963)
Two distinct but closely related cDNA clones encoding Bovine chromaffin granules express an easilydetectable vesicular amine transport suggests that the activity exm-onoamine transport activity and have provided a model syspressed in the brain (synaptic vesicle amine transpotretmerto studyvesicular transport (Johnson,1988)
Competition or SVAT)may differ significantly from the previously studies show that a single transport activity expressedin these describedadrenalglandactivity(chromaffingranule granules recognizes each of the amine transmitterswith high amine transporteror chromaffin granule amine transporter (CGAT))
Summary
Dansient Expression-The CGAT and SVATcDNAs subcloned into the expression vector CDM8 were transfected into COS cells by electroporation. Dunsport Assay-COS or CHO transfectants were harvested and homogenized at 0.01-mm clearance in cold0.32 M sucrose, m HEPES-KOH,(pH 7.4) (SHbuffer)containing 5 m Mg-EGTA, 2 p g h l leupeptin, and 0.2 m diisopropylfluorophosphate.After removalof the cell debris by centrifugation at 3500 x g for 5 min, 10 1.1 of the supernatant (- 100pg of protein) was added to 200 pl of SH buffer containing. Aliquots of 10 pl (-100 pg of protein) were added to 200 pl of SH buffer containing 4 m KC1,4 m MgSO,, 5 m ATP, and 2 m [3H]reserpine.After incubation at 30 "C for 5 min in the pressing CGAT and SVAT were homogenized at 0.01-mm clearance in cold 0.15 M NaC1, 0.01 M K-HEPES, pH 7.4 (HBS).After centrifugation at low speed to remove cell debris, the supernatant was collected by centrifugation at 35,000 rpm in a SW 50.1 rotor (Beckman)for 20 min. Case of competition studies, free reserpine was separated from reserpine bound to cell membranes by centrifugationfor 2 min using a prepacked
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