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Am J Physiol Gastrointest Liver Physiol 295: G442-G451, 2008. First published July 17, 2008; doi:10.1152/ajpgi.90280.2008
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NEUROREGULATION AND MOTILITY

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

Center for Swallowing and Motility Disorders, Veterans Affairs Boston Healthcare System and Harvard Medical School, Boston, Massachusetts

Submitted 4 April 2008 ; accepted in final form 11 July 2008

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 nNOS dimers, both of which lacked LC8. These results suggest that LC8 may dissociate from the 320-kDa nNOS dimer upon binding to membrane, thus supporting the view that LC8 may transport nNOS dimer to the varicosity membrane for participation in nitrergic neurotransmission.

protein inhibitor of nNOS (PIN), dynein light chain (DLC8 or LC8), nNOS; membrane-bound nNOS; cytosolic nNOS; nitrergic neurotransmission