NDLI: PIN/LC8 is associated with cytosolic but not membrane-bound nNOS in the nitrergic varicosities of mice gut: implications for nitrergic neurotransmission.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Chaudhury, Arun Rao, Y. Manjula Goyal, Raj K.
Description	Country affiliation: United States Author Affiliation: Chaudhury A ( Center for Swallowing and Motility Disorders, Veterans Affairs Boston Healthcare System and Harvard Medical Center, Boston, MA, USA.)
Abstract	This investigation demonstrates the presence and binding of the protein LC8 (described as 'protein inhibitor of nNOS' or PIN in some reports) to different components of neuronal nitric oxide synthase (nNOS) in nitrergic varicosities of mice gut. Whole varicosity extracts showed three (320-, 250-, and 155-kDa) nNOS bands with anti-nNOS(1422-1433) antibody and a 10-kDa band with anti-LC8 antibody. The LC8 immunoprecipitate (IP) showed three nNOS bands, suggesting that LC8 was bound with all three forms of nNOS but dissociated from them during SDS-PAGE. Studies using LC8 IP and supernatant and probed with anti-CaM showed that LC8 was not associated with CaM-bound 320-kDa nNOS but was present in the CaM-lacking fraction. Probing these fractions with anti-serine847-P-nNOS showed that 320-kDa serine847-phosphorylated-nNOS consisted of LC8-bound and LC8-lacking components. Subsequent studies with varicosity membrane and cytosolic fractions separately showed that membrane contained CaM-bound and CaM-lacking, serine847-phosphorylated 320-kDa nNOS; both these fractions lacked LC8. On the other hand, the cytosolic fraction contained CaM-lacking, serine847-phosphorylated 320-kDa, 250-kDa, and 155-kDa nNOS bands that were all associated with LC8. These studies, along with in vitro nitric oxide assays, show that in gut nitrergic nerve varicosities 1) all cytosolic serine847-phosphorylated nNOS was catalytically inactive and bound with LC8, and 2) membrane-associated nNOS consisted of catalytically active, CaM-bound and catalytically inactive, CaM-lacking, serine847-phosphorylated nNOSalpha dimers, both of which lacked LC8. These results suggest that LC8 may dissociate from the 320-kDa nNOSalpha dimer upon binding to membrane, thus supporting the view that LC8 may transport nNOSalpha dimer to the varicosity membrane for participation in nitrergic neurotransmission.
File Format	HTM / HTML
ISSN	01931857
e-ISSN	15221547
DOI	10.1152/ajpgi.90280.2008
Journal	AJP: Gastrointestinal and Liver Physiology
Issue Number	3
Volume Number	295
Language	English
Publisher	American Physiological Society
Publisher Date	2008-09-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physiology Discipline Gastroenterology Cell Membrane Enzymology Cytosol Dyneins Metabolism Enteric Nervous System Gastrointestinal Tract Nitrergic Neurons Nitric Oxide Synthase Type I Nitric Oxide Synaptic Transmission Animals Calmodulin In Vitro Techniques Isoenzymes Mice Mice, Inbred C57bl Phosphorylation Presynaptic Terminals Protein Binding Serine Subcellular Fractions Research Support, N.i.h., Extramural Research Support, U.s. Gov't, Non-p.h.s.
Content Type	Text
Resource Type	Article
Subject	Hepatology Physiology Physiology (medical) Gastroenterology

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PIN/LC8 is associated with cytosolic but not membrane-bound nNOS in the nitrergic varicosities of mice gut: implications for nitrergic neurotransmission

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