Cell surface trafficking of the antidepressant-sensitive norepinephrine transporter revealed with an ectodomain antibody

Savchenko, Valentina; Sung, Uhna; Blakely, Randy D.

doi:10.1016/s1044-7431(03)00235-5

Cited by 38 publications

(64 citation statements)

References 55 publications

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“…These axons may function in a high activity state, because they can synthesize NE and recycle both released NE and extracellular DA. These observations imply that NET and TH participate in regulated trafficking events directly linked to NE cell activity, an idea consistent with in vitro studies indicating that cellular activity modulates the surface distribution of NET (Savchenko et al, 2003).…”

Section: Introductionsupporting

confidence: 86%

“…Specificity has been extensively demonstrated by Western blot analysis, antigen preadsorption, absence of staining after selective NE lesions, light and electron microscopic distribution, dual labeling with dopamine-␤-hydroxylase-immunoreactive (D␤H-ir) profiles Miner et al, 2003), and absence of labeling in NET knock-out mice (Savchenko et al, 2003). NET is a selective marker for NE cells and processes, because NET mRNA is localized exclusively to D␤H-positive neurons (Lorang et al, 1994;Hoffman et al, 1998).…”

Section: Methodsmentioning

confidence: 99%

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Chronic Stress Increases the Plasmalemmal Distribution of the Norepinephrine Transporter and the Coexpression of Tyrosine Hydroxylase in Norepinephrine Axons in the Prefrontal Cortex

et al. 2006

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Norepinephrine (NE) potently modulates the cognitive and affective functions of the prefrontal cortex (PFC). Deficits in NE transmissionare implicated in psychiatric disorders, and antidepressant drugs that block the NE transporter (NET) effectively treat these conditions. Our initial ultrastructural studies of the rat PFC revealed that most NE axons (85-90%) express NET primarily within the cytoplasm and lack detectable levels of the synthetic enzyme tyrosine hydroxylase (TH). In contrast, the remaining 10 -15% of PFC NE axons exhibit predominantly plasmalemmal NET and evident TH immunoreactivity. These unusual characteristics suggest that most PFC NE axons have an unrecognized, latent capacity to enhance the synthesis and recovery of transmitter. In the present study, we used dual-labeling immunocytochemistry and electron microscopy to examine whether chronic cold stress, a paradigm that persistently increases NE activity, would trigger cellular changes consistent with this hypothesis. After chronic stress, neither the number of profiles exhibiting NET labeling nor their size was changed. However, the proportion of plasmalemmal NET nearly doubled from 29% in control animals to 51% in stressed rats. Moreover, the expression of detectable TH in NET-labeled axons increased from only 13% of profiles in control rats to 32% of profiles in stressed animals. Despite the consistency of these findings, the magnitude of the changes varied across individual rats. These data represent the first demonstration of activity-dependent trafficking of NET and expression of TH under physiological conditions and have important implications for understanding the pathophysiology and treatment of stress-related affective disorders.

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Section: Introductionsupporting

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Chronic Stress Increases the Plasmalemmal Distribution of the Norepinephrine Transporter and the Coexpression of Tyrosine Hydroxylase in Norepinephrine Axons in the Prefrontal Cortex

et al. 2006

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“…Less well-understood processes presumably also support insertion of transporters and link insertion to demand. Hints of a localized, activity-dependent process were recently covered by Savchenko and colleagues (48), who utilized a new NET-specific ectodomain antibody to visualize NET protein expression on the cell surface of transfected cells and cultured noradrenergic neurons. Labeling of live cells under detergent-free conditions was sensitive to prior treatments with PKC-activating phorbol esters, which, as expected, diminish surface expression and antibody labeling.…”

Section: A Transporter "Turn-on" Accompanies Focal Membrane Insertionmentioning

confidence: 99%

Biogenic Amine Neurotransmitter Transporters: Just When You Thought You Knew Them

2005

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Beginning in the early 1990s, a new era dawned in studies of neurotransmitter transporter structure, function, and regulation, illuminated by the cloning of transporter cDNAs and genes, the development of transporter-specific gene and protein probes, and the characterization of heterologous expression systems suitable for advanced biophysical analyses. Among the advances in this field over the next decade were the elucidation of critical domains and residues supporting substrate and antagonist recognition, the discovery that transporters exist as multimeric protein complexes, the definition of N-glycosylation and phosphorylation as important posttranslational modifications, the recognition that transporters exhibited ion-channel states, and the elucidation of the first human disorders associated with transporter mutations. Many reviews are available that highlight these and other areas (4-6, 8, 15, 20, 31, 51). Together, they illustrate a field reaching maturity, yet poised for renaissance, in which long-held ideas are challenged by new techniques and data. In this short review, we describe several new approaches and findings from our labs and from colleagues in the field that have challenged us to reconsider aspects of biogenic amine transporter structure, function, and regulation. Substrate Binding and Transport: Watching Transporters at WorkThe measurements of substrate flux through neurotransmitter transporters has relied extensively on radiolabeled substrates. Although such molecules are sensitive probes for transporter activity from populations of transporter proteins, radiolabeled substrates have limited utility for the real-time monitoring of either binding or transport; they cannot illuminate a substrate's local environment or how it changes during substrate flux, and they cannot be used to identify cellular subdomains or relate population properties to individual molecules. Amperometric approaches offer some solutions to these limitations, as we will note below, but they are limited to paradigms such as in vitro (26, 45) or in vivo clearance measurements (10,14), in which the presence or absence of oxidizeable amine changes significantly as a function of time. Binding and transfer events that occur before changes in extracellular levels of diffusible neurotransmitter are achieved cannot be resolved. To move beyond these approaches, Schwartz et al. 1548-9213/05 8.00

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“…NE transporter (NET) protein shifts NE from the synapse back into its vesicles for storage and later use (26). A chronic increase in CSNA post-MI may increase TH and NET expression in the heart and SG, since membrane depolarization upregulates TH and NET gene expression in sympathetic neurons in vitro and in vivo (39,42,53). A two-to threefold increase in NET and TH mRNA in the SG and in NET and TH protein in the noninfarcted base of the heart were observed in rats 1 wk after ischemia-reperfusion (33).…”

mentioning

confidence: 99%

Cardiac sympathetic innervation and PGP9.5 expression by cardiomyocytes after myocardial infarction: effects of central MR blockade

Drobysheva

Ahmad

White

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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Drobysheva A, Ahmad M, White R, Wang H, Leenen FH. Cardiac sympathetic innervation and PGP9.5 expression by cardiomyocytes after myocardial infarction: effects of central MR blockade. Am J Physiol Heart Circ Physiol 305: H1817-H1829, 2013. First published October 11, 2013; doi:10.1152/ajpheart.00445.2013.-Central mechanisms involving mineralocorticoid receptor (MR) activation contribute to an increase in sympathetic tone after myocardial infarction (MI). We hypothesized that this central mechanism also contributes to cardiac sympathetic axonal sprouting and that central MR blockade reduces cardiac sympathetic hyperinnervation post-MI. Post-MI, tyrosine hydroxylase (TH) and norepinephrine transporter protein content in the noninfarcted base of the heart remained unaltered. In contrast, protein gene product (PGP)9.5 protein was increased twofold in the base of the heart and sixfold in the peri-infarct area at 1 wk post-MI and was associated with increased ubiquitin expression. These changes persisted to a lesser extent at 4 wk post-MI and were no longer present at 12 wk. Cardiac myocytes rather than sympathetic axons were the main source of this elevated PGP9.5 expression. At 7-10 days post-MI, in the peri-infarct area, sympathetic hyperinnervation was observed with a fourfold increase in growth-associated protein 43, a twofold increase in TH, and a 50% increase in PGP9.5-positive fibers compared with the epicardial side of the left ventricle in sham rats. Central infusion of the MR blocker eplerenone markedly attenuated these increases in nerve densities but did not affect overall cardiac PGP9.5 and ubiquitin protein overexpression. We conclude that central MR activation contributes to sympathetic hyperinnervation, possibly by decreasing cardiac sympathetic activity post-MI, or by affecting other mechanisms, such as the expression of nerve growth factor. Marked PGP9.5 expression occurs in cardiomyocytes early post-MI, which may contribute to the increase in ubiquitin. myocardial infarction; cardiac sympathetic hyperinnervation; brain mineralocorticoid receptors; eplerenone; ubiquitin; protein gene product 9.5 CARDIAC REMODELING after myocardial infarction (MI) affects both infarcted and noninfarcted areas of the heart and contributes to the impairment of ventricular performance (19,43,44). Cardiac sympathetic hyperactivity enhances maladaptive cardiac remodeling post-MI and plays a major role in the development of chronic heart failure (CHF) (5, 31). In humans with mild CHF, there is a selective increase in cardiac sympathetic activity, which is followed by augmented sympathetic outflow to skeletal muscle and the kidneys as CHF progresses (16,38). In rats, direct recordings from cardiac sympathetic nerves demonstrate that cardiac sympathetic nerve activity (CSNA) increased twofold within hours after MI (40). In conscious sheep, CSNA was significantly increased by the second hour post-MI and remained elevated for at least 1 wk (14).Increased CSNA may also contribute to the development of cardiac sympathetic hyperi...

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Cell surface trafficking of the antidepressant-sensitive norepinephrine transporter revealed with an ectodomain antibody

Cited by 38 publications

References 55 publications

Chronic Stress Increases the Plasmalemmal Distribution of the Norepinephrine Transporter and the Coexpression of Tyrosine Hydroxylase in Norepinephrine Axons in the Prefrontal Cortex

Chronic Stress Increases the Plasmalemmal Distribution of the Norepinephrine Transporter and the Coexpression of Tyrosine Hydroxylase in Norepinephrine Axons in the Prefrontal Cortex

Biogenic Amine Neurotransmitter Transporters: Just When You Thought You Knew Them

Cardiac sympathetic innervation and PGP9.5 expression by cardiomyocytes after myocardial infarction: effects of central MR blockade

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