Filtration disrupts synaptosomes during radiochemical analysis of serotonin uptake: comparison with chronoamperometry in SERT knockout mice.
Radiochemical methods have failed to reveal decreases in synaptosomal serotonin uptake in mice lacking one functional copy of the serotonin transporter (SERT) gene. By contrast, uptake rates determined by chronoamperometry in synaptosomes from SERT+/- mice show gene-related reductions. We revisited [(3)H]5-HT uptake in SERT knockout mice to determine the effects of inclusion of O(2) in the incubation buffer on the kinetic parameters obtained by this method. In oxygenated synaptosomes prepared from frontal cortex and striatum, modest 25 and 35% reductions in radiolabeled 5-HT uptake were detected in SERT+/- versus SERT+/+ mice. However, even in the presence of O(2), no differences in [(3)H]5-HT uptake were detected between SERT+/- and SERT+/+ mice in brain stem in contrast to 60% reductions determined by chronoamperometry. Moreover, while inclusion of O(2) modestly increased the rates of [(3)H]5-HT uptake, rates determined by chronoamperometry in the presence of O(2) were 40-fold greater than those determined radiochemically. We present evidence that the filtration process used in the radiochemical method leads to substantial loss of transported 5-HT resulting in lower apparent uptake rates. These findings explain the relative insensitivity of radiochemical methods for determining biologically important alterations in uptake such as those occurring between SERT+/- and SERT+/+ mice and in response to O(2).
Perez
Xiomara A
XA
Department of Chemistry, The Pennsylvania State University, University Park, 16802, USA.
Bianco
Laura E
LE
Andrews
Anne M
AM
D000465:Algorithms; D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D004566:Electrodes; D005374:Filtration; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D011854:Radiochemistry / Q000379:methods*; D012701:Serotonin / Q000032:analysis / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D013574:Synaptosomes / Q000737:chemistry / Q000502:physiology*
D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin