The goal of this application is to develop background information to determine the physiological significance of the unexpected observation that a physiological stimulus (hypotension) and a selective neurokinin 3 receptor (NK3-R) agonist (senktide) induce the appearance of NK3-R immunoreactivity (ir) in the nucleus of neurons in the supraoptic nucleus (SON) of the hypothalamus. Since NK3-Rs are G-protein coupled receptors (GPCR), this suggests the intriguing possibility that extracellular NK3-R ligand induces translocation of NK3-R from the cell membrane to the nucleus for direct regulation of gene expression by a GPCR. This is exciting, because it has important implications for other GPCRs, and therefore could impact on numerous GPCR-regulated cellular functions. Also, the observation could be important to a wide range of CNS functions, because NK3-Rs are widely distributed in the CNS and have been implicated in diverse and important CNS functions and pathophysiology including motivated behaviors such as salt, cocaine and alcohol intake; emotional states such as anxiety and depression; locomotion and dystonia; fluid, electrolyte, and cardiovascular homeostasis; and analgesia. Therefore, this observation has the potential to impact the treatment of diverse and debilitating neurologic, psychiatric, and homeostatic disorders. In order to assess the potential importance of this observation, we must 1) fully characterize the NK3-R-ir observed in the nucleus of SON neurons, and 2) determine if the same phenomenon occurs in other CNS regions. Therefore, the Specific Aims for this proposal will test the following hypotheses: Hypothesis 1: The ligand-induced appearance of NK3-R-ir in the nuclei of SON neurons represents all or a portion of NK3-R that translocates to the nucleus following receptor internalization. To test this hypothesis, we have generated antibodies to different epitopes in the N-terminal and C-terminal regions of NK3-R. These antibodies will be used for immunohistochemical (IHC) and western blot analysis of SON. SON from normotensive, hypotensive, and senktide (a selective NK3-R agonist)-treated rats will be compared. Western blots will be used to analyze cytosolic and nuclear extracts of SON to determine the size and cellular location of NK3-R-ir. Hypothesis 2: NK3-R activation in other brain regions induces nuclear localization of NK3-R immunoreactivity. Senktide injection into the lateral ventricle has been shown to activate cfos in numerous regions of the CNS that also express H3-senktide binding and NK3-R mRNA and immunoreactivity. This approach will be used to screen for nuclear translocation of NK3-R-ir in areas such as lateral septum, amygdala, hypothalamus (median preoptic nucleus, paraventricular, supraoptic, and arcuate nuclei), substantia nigra, ventral tegmental area, periaqueductal gray, and nucleus tractus solitarius.
The proposed studies have potential significance to a wide range of human diseases as a result of 1) the possibility that a new method of regulation of gene expression by neurokinin 3 receptors (NK3-R) may be identified that is applicable to other G-protein coupled receptors and 2) the evidence that NK3-Rs are involved in a large number of neurological and psychiatric disorders including addiction, depression, anxiety, movement disorders, and hypertension. ? ? ?
| Sladek, C D; Song, Z (2012) Diverse roles of G-protein coupled receptors in the regulation of neurohypophyseal hormone secretion. J Neuroendocrinol 24:554-65 |
| Sladek, C D; Stevens, W; Levinson, S R et al. (2011) Characterization of nuclear neurokinin 3 receptor expression in rat brain. Neuroscience 196:35-48 |
