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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