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178 Neurokinin 3 Receptor antagonist reduces senktide-induced internalization of NEUROKININ 3 RECEPTOR in supraoptic neurons.
  1. A Foianini,
  2. C. D. Sladek
  1. Department of Physiology and Biophysics, University of Colorado Health Science Center, Aurora, CO

Abstract

Magnocellular neurons in the supraoptic nucleus (SON) of the hypothalamus are known to express neurokinin 3 receptors (NK-3R). Senktide (an NK-3R agonist) stimulated vasopressin (VP) release from explants of the hypopthalamoneurohypophyseal system (HNS) in vitro and induced internalization of NK-3R when injected immediately above SON in vivo. A1 neurons projecting from medulla to SON express substance P (SP), and SP stimulates VP and oxytocin (OT) release from HNS explants. However, pretreatment with an NK-3R antagonist (SB23575) did not prevent SP-stimulated VP release. In order to confirm the antagonistic actions of SB23575 in SON neurons, senktide-induced internalization of NK-3R in SON neurons was evaluated in the presence and absence of SB23575. Rats were surgically prepared with a unilateral cannula located immediately above SON. Four days later, animals were anesthetized with avertin (2,2,2-tribromoethanol), and 60 min later, 2 μL of antagonist (100 μM) or vehicle (PBS) was microinjected above the SON, followed 15 minutes later by injection of senktide (2 μL, 100 nM). Animals were perfused 5 minutes later with 4% paraformaldehye and 3% acrolein; their brains were frozen, sectioned at 30 microns, and stained for NK-3R immunoreactivity. Images were captured using confocal microscopy and the number of NK3-R-IR endosomes was counted in 5.62 μm regions of cytoplasm in 10 SON neurons on each side of the brain. PBS/senktide-treated animals showed an increase in cytoplasmic endosome localization (10.3 P138} 0.42 vs 4.58 ± 0.35 on the nontreated side, p < .005, paired t-test). Animals pretreated with SB23575 showed no significant increase (5.18 ± 0.78 vs 3.38 ± 0.28, p = .115). Additionally, vehicle-pretreated animals showed significantly more senktide-induced internalization than animals pretreated with SB23575 (10.3 ± 0.42 vs 5.18 ± 0.78, p < .005). The effectiveness of SB23575 in blocking senktide-induced internalization suggests that SP may be exerting its effects on OT and VP release through a mechanism other than NK-3R.

Supported by NIH RO1 NS44835 to CDS and a NIDDK short-term traineeship.

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