Trafficking of tachykinin neurokinin 3 receptor to nuclei of neurons in the paraventricular nucleus of the hypothalamus following osmotic challenge

Abstract

Tachykinin NK3 receptor (NK3R) is a g-protein coupled receptor that is heavily expressed by magnocellular neurons of the paraventricular nucleus of the hypothalamus (PVN). Osmotic challenge is reported to activate NK3R expressed by magnocellular neurons and cause the NK3R to be internalized to the cytoplasm and perhaps the cell nucleus. In this study we show using immunoelectron microscopy that isolated nuclei from neurons in the PVN of osmotic challenged animals show a robust labeling for the NK3R. NK3R immunoreactivity was detected by Western blot in isolated nuclei of PVN neurons following the 2 M NaCl injection. No nuclear NK3R immunoreactivity was detected in control animals. NK3R antibody specificity was confirmed by small interfering (SI) RNA technology. This study establishes that the NK3R is trafficked to the nucleus of PVN neurons following a peripheral osmotic challenge. KeywordsG-protein coupled receptor; Cell signaling; Electron microscopy; siRNA Magnocellular neurons of the hypothalamus synthesize and secrete two key hormones: vasopressin (VP) and oxytocin (OT). The release of these hormones is influenced by a number of transmitters and magnocellular neurons express a number of receptors. Noteworthy is the tachykinin, NK3 receptor (NK3R) which is heavily expressed by magnocellular neurons of the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus (Ding et al., 1999). NK3R expressed by magnocellular neurons appears to have a functional role in the release of vasopressin and oxytocin. Stimulation of NK3R results in an increase in Ca ++ conductance (Buell et al., 1992), induction of c-Fos expression in magnocellular neurons, and causes the systemic release of both hormones (Ding et al., 1999;Smith and Flynn, 2000;Bealer and Flynn, 2003). Furthermore, the release of the endogenous ligand, presumably neurokinin B (NKB), and binding to the NK3R results in the internalization of the peptide-receptor complex (Chawala et al., 1997;Colin et al., 2002;Lessard et al., 2004;Johnson et al., 2004) that can be tracked immunohistochemically. NK3R expressed by magnocellular neurons are membrane bound in control (isotonic saline treated) animals. After hyperosmotic or Direct correspondence to: Francis W. Flynn, 1000 E. University Ave., Dept 3166, Laramie, WY 82071, tel: 307-766-6446, e-mail: flynn@uwyo.edu. Section Editor: Dr. Miles Herkenham Systems Neuroscience, Bethesda, MD, USA Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2009 July 31. Published in final edi...

Stress-induced dendritic internalization and nuclear translocation of the neurokinin-3 (NK3) receptor in vasopressinergic profiles of the rat paraventricular nucleus of the hypothalamus

“…This is consistent with previous studies showing no or weak nuclear NK 3 R labeling in PVN neurons of normal animals (Jensen et al, 2008;Misono and Lessard, 2012). In contrast, our results showed that an acute restraint stress session increased the nuclear NK 3 R density in AVP neurons of the PVN.…”

“…The internalized receptor is then recycled back to the membrane or degraded in the cytoplasm by endopeptidases (Regoli et al, 1994;Khawaja and Rogers, 1996). Moreover, our group and others have located NK 3 Rs in the neuronal nuclei in the rat PVN following physiological challenges and in the ventral tegmental area (VTA) of normal rats (Howe et al, 2004;Jensen et al, 2008;Lessard et al, 2009). NK 3 Rs now belong to a sub-group of nuclear GPCRs, along with the apelin, angiotensin AT1 and bradykinin B2 receptors but not the neurokinin-1 or neurokinin-2 receptor (Lee et al, 2004;Gobeil et al, 2006).…”

“…Conversely, hyperosmolarity evokes internalization of PVN neurokinin-3 receptors (NK 3 Rs), confirming their potential role in osmotic balances through the release of AVP (Haley and Flynn, 2006). Interestingly, an osmotic challenge also evoked nuclear translocation of PVN NK 3 Rs (Jensen et al, 2008). The association of nuclear NK 3 Rs with acetylated histones in hypothalamic neurons is highlighting a new signaling pathway by which NK 3 Rs might change gene transcription in AVP or other phenotypes of PVN neurons.…”

The Tachykinin Peptide Neurokinin B Binds Copper Forming an Unusual [CuII(NKB)2] Complex and Inhibits Copper Uptake into 1321N1 Astrocytoma Cells

“…NK3-R and NK1-R appear as dimers, which has been observed previously. 51,52 We also confirmed that copper up to 50 μM had a negligible effect on astrocyte viability ( Figure S4, Supporting Information).…”

Blockade of NK3R signaling in the PVN decreases vasopressin and oxytocin release and c-Fos expression in the magnocellular neurons in response to hypotension

“…Other neurotransmitter systems carry particular information, such as osmolarity or blood volume, and alter magnocellular neuron function under specific physiological conditions; however, NK3R appears to have a broad or general role in VP and OT release. The NK3R is a unique receptor on these neurons, as it translocates to the nucleus following activation by specific receptor agonists and physiological challenges (19,29,30). To date, no other G protein-coupled receptor has been shown to translocate to the nucleus of cells in vivo.…”