title: Nitric oxide production and sequestration in the sinus gland of the green shore crab Carcinus maenas.
abstract: Molting in decapod crustaceans is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced in the X-organ (XO)/sinus gland (SG) complex of the eyestalk ganglia (ESG). Pulsatile release of MIH from the SG suppresses ecdysteroidogenesis by the molting gland or Y-organ (YO). The hypothesis is that nitric oxide (NO), a neuromodulator that controls neurotransmitter release at presynaptic membranes, depresses the frequency and/or amount of MIH pulses to induce molting. NO synthase (NOS) mRNA was present in Carcinus maenas ESG and other tissues and NOS protein was present in the SG. A copper based ligand (CuFL), which reacts with NO to form a highly fluorescent product (NO-FL), was used to image NO in the ESG and SG and quantify the effects of NO scavenger (cPTIO), NOS inhibitor (l-NAME), and sodium azide (NaN3) on NO production in the SG. Pre-incubation with cPTIO prior to CuFL loading decreased NO-FL fluorescence ~30%; including l-NAME had no additional effect. Incubating SG with l-NAME during pre-incubation and loading decreased NO-FL fluorescence ~40%, indicating that over half of the NO release was not directly dependent on NOS activity. Azide, which reacts with NO-binding metal groups in proteins, reduced NO-FL fluorescence to near background levels without extensive cell death. Spectral shift analysis showed that azide displaced NO from a soluble protein in SG extract. These data suggest that the SG contains NO-binding protein(s) that sequester NO and releases it over a prolonged period. This NO release may modulate neuropeptide secretion from the axon termini in the SG.
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Natalie L
authors.initials: NL
authors.lastname: Pitts
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA Donald.Mykles@ColoState.edu.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Mykles
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000378:metabolism; D003386:Brachyura / Q000254:growth & development / Q000502:physiology*; D005088:Exocrine Glands / Q000378:metabolism; D005786:Gene Expression Regulation; D008297:Male; D018978:Molting / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D019810:Sodium Azide / Q000494:pharmacology
chemical_list: D060829:Arthropod Proteins; D012333:RNA, Messenger; D009569:Nitric Oxide; D019810:Sodium Azide; D019001:Nitric Oxide Synthase
keywords: Arthropoda; Brachyura; Confocal microscopy; Copper fluorescent ligand; Crustacea; Eyestalk ganglia; Nitric oxide; Nitric oxide synthase; Protein expression; Sinus gland; Tissue distribution