Norepinephrine transporter immunoblotting and radioligand binding in cocaine abusers.
The norepinephrine transporter (NET) is a membrane protein responsible for transporting extracellular norepinephrine. The cocaine and tricyclic antidepressant-sensitive NET belongs to a family of sodium and chloride coupled transporters that include the monoamines dopamine and serotonin and the amino acids GABA and glycine. The regional distribution of the NET has been defined by synaptosomal uptake of norepinephrine and by autoradiographic approaches in rodent and primate brain. However, the NET has not been well characterized in the human brain due to the overall low abundance of protein expressed in axon terminals. Recently, immunolocalization studies have been used to identify the regional distribution of the cytoplasmic NET epitope in rodent brain. We report here on the characteristics of drug interactions with the native NET protein in human postmortem brain. Antisera raised against a 17-amino acid peptide from the N-terminus of the hNET recognized an 80 kDa species in human cerebral cortex. Chronic exposure to cocaine upregulated NET protein expression and [3H]nisoxetine binding sites in the insular cortex from brains of cocaine addicts. These results demonstrate that immunologic and radioligand binding approaches afford specific labeling of the native transport protein in postmortem human brain.
Mash
Deborah C
DC
Department of Neurology (D4-5), School of Medicine, University of Miami, 1501 N.W. 9th Avenue, Miami, FL 33136, USA. dmash@newssun.med.miami.edu
Ouyang
Qinjie
Q
Qin
Yujing
Y
Pablo
John
J
D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D001923:Brain Chemistry / Q000187:drug effects / Q000502:physiology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D003042:Cocaine / Q000494:pharmacology*; D019970:Cocaine-Related Disorders / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D005473:Fluoxetine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D009638:Norepinephrine / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011869:Radioligand Assay / Q000379:methods*; D027981:Symporters / Q000032:analysis* / Q000187:drug effects* / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D000906:Antibodies; D018765:Dopamine Uptake Inhibitors; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; D027981:Symporters; D005473:Fluoxetine; C011386:nisoxetine; D003042:Cocaine; D009638:Norepinephrine