Binding sites of crustacean hyperglycemic hormone and its second messengers on gills and hindgut of the green shore crab, Carcinus maenas:: A possible osmoregulatory role

To determine the possible involvement of crustacean hyperglycemic hormone (CHH) in osmoregulation in crustaceans, ligand binding and second messenger assays were performed on gills and hindgut preparations of the green shore crab Carcinus maenus, whilst midgut gland, previously known as one of the target tissues of CHH served as a control tissue. Classical receptor binding analyses using [I-125]CHH by saturation and displacement experiments from membrane preparations from gills, hindgut, and midgut glands demonstrated that CHH binding characteristics involved one site, highly specific, saturable, and displaceable kinetics: (gills: K-D 5.87 +/- 2.05 x 10(-10) and B-MAX 6.50 +/- 1.15 x 10(-10), hindgut: KD 3.54 +/- 1.49 x 10(-10) and Bm,, 2.31 +/- 0.44 x 10(-10), and midgut gland: KD 7.28 +/- 0.9 x 10(-10) and B-MAX 3.28 +/- 0.25 x 10(-10)) all expressed as M/mg protein. No differences, in terms of displacement were observed between the two CHH isoforms (N-terminally blocked pGlu and unblocked Gln) variants. CHH binding sites appeared to be coupled to a second messenger system involving cGMP in all the tissues examined. Exposure of crabs to dilute seawater increased levels of cGMP, glucose in gills and circulating CHH levels. Other crustacean neuropeptides including crustacean cardioactive peptide, molt inhibiting hormone, L-enkephalin, FMRF-amide, proctolin, and crustacean hyperglycemic hormone precursor-related peptide were tested with regard to possible osmoregulatory roles with reference to changes in second messenger (cAMP and cGMP) concentrations in gill, hindgut, and midgut tissues in vitro, following application at 2 x 10(-8) M but all were found to be inactive. Thus, it seems likely that CHH is a pertinent neurohormone involved in osmoregulation, thus expanding its many functions as a pleiotropic hormone in crustaceans. (c) 2006 Elsevier Inc. All rights reserved.