Meeting Abstract
P1.61 Saturday, Jan. 4 15:30 Analysis of nitric oxide production in the sinus gland of the green crab, Carcinus maneas, using a copper-based fluorescent ligand PITTS, N.L.*; CHANG, E.S.; MYKLES, D.L.; Colorado State University. Fort Collins, CO; Bodega Marine Laboratory, University of California Davis, Bodega Bay, CA; Colorado State University. Fort Collins, CO pittsn@rams.colostate.edu
Nitric oxide (NO) is a unique gaseous signaling molecule involved in a wide variety of biological processes. NO is synthesized from L-arginine, oxygen, and NADPH by nitric oxide synthase (NOS). In decapod crustaceans, we hypothesize that NO modulates the secretion of neuropeptides such as molt-inhibiting hormone and crustacean hyperglycemic hormone, from the sinus gland (SG) in the eyestalk ganglia. A copper fluorescent ligand (CuFL) was used to measure NO production in the SG of the green crab, Carcinus maneas. Using confocal microscopy, NO-CuFL fluorescence was distributed throughout the SG. The effects of an NO scavenger (cPTIO), NO donor (SNAP), and an NOS inhibitor (L-NAME) on total fluorescence were quantified using Metamorph analysis of whole SGs in vitro. CuFL had a higher affinity for NO than cPTIO, preferentially binding NO when the compounds were present in the same solution. Fluorescence was maximally reduced in a solution containing both cPTIO and L-NAME; fluorescence was recovered one hour after loading SGs with CuFL in the absence of cPTIO. L-NAME partially inhibited post-cPTIO CuFL fluorescence recovery, indicating that NO is generated from NOS. However, the lack of complete inhibition of CuFL fluorescence recovery by L-NAME suggests that NO is released from endogenous storage molecules. SNAP had no effect on the recovery of fluorescence after cPTIO treatment, suggesting that endogenous NO production and release was sufficient to saturate CuFL. These experiments showed that CuFL can be used to localize and quantify NO in the SG. NO metabolism is complex and we hypothesize that NO produced by NOS can bind and activate NO-dependent proteins or bind to NO storage proteins. Supported by NSF (IOS-1257732).
