Dopamine transporters (DATs) undergo intracellular sequestration and functional down-regulation upon exposure to psychostimulant substrates. To investigate the potential mechanism underlying these responses, we examined the acute in vitro and in vivo effects of amphetamine and methamphetamine (METH) on phosphorylation and down-regulation of rat DAT using wild type and N-terminal truncation mutants. Phosphorylation of DAT assessed by 32 PO 4 metabolic labeling was increased up to 2-fold by in vitro treatment of rDAT LLC-PK 1 cells with amphetamine or METH and was similarly increased in rat striatal tissue by in vitro application or in vivo injection of METH. The dopamine transport blocker (؊)-cocaine did not affect DAT phosphorylation but prevented the phosphorylation increase induced by METH. Phosphorylation of DAT induced by METH was also prevented by the protein kinase C blocker bisindoylmaleimide I and was absent in an N-terminally truncated protein that lacks the first 21 residues including 6 serines that also represent the site of phorbol ester induced phosphorylation. Down-regulation of transport induced by METH was also cocaine-and protein kinase C-dependent but was retained in the N-terminal truncation mutant. These results demonstrate that transport or binding of METH stimulates DAT phosphorylation and down-regulation by a mechanism that requires protein kinase C but that METH-induced down-regulation can occur independently of direct transporter phosphorylation. The finding that DAT phosphorylation is stimulated by amphetamines reveals a previously unknown effect of these drugs that is not produced by cocaine and may be related to reinforcement.
The dopamine transporter (DAT)2 is a phosphoprotein expressed in dopaminergic neurons that clears extracellular dopamine (DA) from the synapse following neurotransmitter release. This regulates transmitter availability for DA receptors and is crucial for the temporal and spatial shaping of dopaminergic neurotransmission (1). Neurological disorders such as schizophrenia, depression, and attention deficit disorder are associated with abnormal DA levels that may result from functional dysregulation of DAT (2). DAT is also a major site of action for numerous psychostimulant and neurotoxic drugs that modulate DA levels or lead to dopaminergic neurodegeneration (3). Amphetamine (AMPH) and methamphetamine (METH) are DAT substrates that raise extracellular transmitter levels by competing with endogenous DA for transport and inducing DA efflux through transport reversal (4, 5). Many DAT substrates, including AMPH, METH, and 6-hydroxydopamine, are toxins that induce production of free radicals and neurodegeneration after transport into dopaminergic cells (2). Other drugs such as cocaine are not transported by DAT but bind to the protein and elevate synaptic DA concentrations by inhibiting substrate translocation.DAT transport activity, plasma membrane expression, and phosphorylation level are acutely modulated by exogenous kinase and phosphatase treatments (6, 7), but the...
The SA algorithm-developed MA protocols are currently in use in our laboratory and they rapidly detect SE, reducing the number of samples requiring correction and improving patient safety.
BACKGROUND: During pregnancy, human chorionic gonadotropin (hCG) immunoreactivity in urine consists of intact hCG as well as a number of hCG variants including the core fragment of hCG (hCGcf). We identified 3 urine specimens with apparent false-negative results using the OSOM® hCG Combo Test (Genzyme Diagnostics) qualitative hCG device and sought to determine whether an excess of 1 of the fragments or variants might be the cause of the interference.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.