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Nesfatin-1: Distribution and Interaction with a G Protein-Coupled Receptor in the Rat Brain

Department of Pharmacology (G.C.B., S.L.D., E.B., S.I., N.J.D.), Temple University School of Medicine, Philadelphia, Pennsylvania 19140; and Phoenix Pharmaceuticals, Inc. (J.Y., J.K.C.), Burlingame, California 94010

Address all correspondence and requests for reprints to: G. Cristina Brailoiu, M.D., Department of Pharmacology, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania 19140. E-mail: gbrailou{at}temple.edu.

Nesfatin-1 is a recently identified satiety molecule detectable in neurons of the hypothalamus and nucleus of solitary tract (NTS). Immunohistochemical studies revealed nesfatin-1-immunoreactive (irNEF) cells in the Edinger-Westphal nucleus, dorsal motor nucleus of vagus, and caudal raphe nuclei of the rats, in addition to the hypothalamus and NTS reported in the initial study. Double-labeling immunohistochemistry showed that irNEF cells were vasopressin or oxytocin positive in the paraventricular and supraoptic nucleus; cocaine-amphetamine-regulated transcript or tyrosine hydroxylase positive in arcuate nucleus; cocaine-amphetamine-regulated transcript or melanin concentrating hormone positive in the lateral hypothalamus. In the brainstem, irNEF neurons were choline acetyltransferase positive in the Edinger-Westphal nucleus and dorsal motor nucleus of vagus; tyrosine hydroxylase positive in the NTS; and 5-hydroxytryptamine positive in the caudal raphe nucleus. The biological activity of rat nesfatin-1 (1–82) (100 nM) was assessed by the Ca²⁺ microfluorometric method. Nesfatin-1 elevated intracellular Ca²⁺ concentrations [Ca²⁺]_i in dissociated and cultured hypothalamic neurons. The response was prevented by pretreating the cells with pertussis toxin (100 nM) or Ca²⁺-free solution and by a combination of the L-type and P/Q-type calcium channel blocker verapamil (1 µM) and omega-conotoxin MVIIC (100 nM). The protein kinase A inhibitor KT 5720 (1 µM) suppressed nesfatin-1-induced rise in [Ca²⁺]_i. The result shows that irNEF is distributed to several discrete nuclei in the brainstem, in addition to the hypothalamus and NTS reported earlier, and that the peptide interacts with a G protein-coupled receptor, leading to an increase of [Ca²⁺]_i, which is linked to protein kinase A activation in cultured rat hypothalamic neurons.

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