© 2001 by The Society for Integrative and Comparative Biology
Further Studies on Signaling Pathways for Ecdysteroidogenesis in Crustacean Y-Organs1
1 Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
2 Department of Biology, Kenyon College, Gambier, Ohio 43022
The Y-organs of crustaceans secrete ecdysteroids (molting hormones) and are regulated (negatively) by a neurosecretory peptide, molt-inhibiting hormone (MIH). Signaling path(s) in Y-organs were explored that connect MIH receptors ultimately with suppression of receptor number for the uptake of cholesterol (ecdysteroid precursor) and of gene expression of steroidogenic enzymes. Experiments were conducted in vitro with Y-organs of crabs (Cancer antennarius, Menippe mercenaria) and crayfishes (Orconectes sp.). It was confirmed in all species that steroidogenesis occurs in the absence of external calcium (Ca++), but increases to a maximum as Ca++ is increased to 1 to 10 mM and is substantially inhibited at higher Ca++ concentrations. MIH does not require external Ca++ for inhibitory action, but inhibition is eliminated by high Ca++concentrations. Several experimental approaches failed to find evidence of phospholipase C activation, turnover of inositol triphosphate or diacylglycerol generation connected with steroidogenesis. Unbinding or chelation of intracellular Ca++ with thapsigargin or TMB-8, respectively both caused dose-dependent inhibition of ecdysteroid output. Blockade of Ca++ channels with verapamil, nifedipine or nicardipine also inhibited steroidogenesis; highest doses inhibited profoundly to below Ca++-free basal levels. Inhibition also was obtained with all doses of the Ca++ channel agonist/antagonist (–) BAY K 8644 in crabs, but in crayfishes lower doses were stimulatory. However, if the crayfish cells were depolarized, allowing greater Ca++ influx, the previously stimulatory doses of BAY K 8644 became inhibitory. Y-organ protein kinase C (PKC) is Ca++-sensitive. Activation of PKC was uniformly stimulatory in crabs, but inhibitory in crayfishes. Cytochalasin D, which disrupts the actin cytoskeleton, and which causes moderate Ca++ influx, stimulated hormone formation. These results are interpreted to indicate a regulatory role for Ca++ in ecdysteroidogenesis, involving a local, submembrane circulation of Ca++ through ion channels and Ca++ pumps and interaction with PKC in phosphorylating key proteins. An optimal local Ca++ environment fostering hormone synthesis is evident since too little or too much Ca++ is inhibitory.
Methyl farnesoate (MF) had no effect on ecdysone production in crab or crayfish Y-organs in 24-hr incubations with MF at 100 pM to 10 µM.