Release of digestive enzymes from the crustacean hepatopancreas: effect of vertebrate gastrointestinal hormones.

Vertebrate gastrointestinal hormones were tested on their ability to liberate digestive enzymes from the crustacean midgut gland. CCK-8 (desulfated form), gastrin, bombesin, secretin, and substance P were detected to release enzymes. Maximal concentrations observed were 5 nM CCK for protease release, 1 nM gastrin for protease and 100 nM for amylase release, 100 nM bombesin for protease release, 10 nM secretin for amylase and protease release, and 100 nM substance P for protease release. Unlike in vertebrates, glucagon was unable to stimulate enzyme release in crustaceans, this also applies to the counterpart insulin. These results may support the assumption that Crustacea possess endogenous factors resembling the above mentioned vertebrate hormones, at least in such a way that the appropriate receptors have the capacity to accept these hormones.

Ecdysteroid biosynthesis in crayfish Y-organs: feedback regulation by circulating ecdysteroids

In crustaceans, ecdysteroid synthesis in the Y-organs is negatively regulated by the molt-inhibiting hormone (MIH). Reduction or cessation of MIH release from the sinus gland in the eyestalk, probably due to environmental cues, is one of possibly several signals for an increase of edysteroid production and subsequently enhancement of 20-hydroxyecdysone (20E) levels in the hemolymph. The present study asks the question whether the 20E peak in premoult stages D2/D3 is explained solely bythe cessation of MIH release or whether positive feedback mechanisms are also involved. Ecdysteroid production by the Y-organ of the crayfish Orconectes limosus was found to be under negative feedback control by circulating ecdysteroids. Exogenous 20-hydroxyecdysone (20E) as well as RH-5849, a non-steroidal ecdysteroid agonist, reduced ecdysteroid synthesis significantly when injected into intermoult animals. A direct, short loop inhibitory feedback effect was demonstrated by in vitro incubations of Y-organs with RH-5849. Thus, the results presented here do not point to a stimulatory effect of 20E on Y-organ activity but suggest that during intermolt a negative feedback by ecdysteroids plays a role in addition to MIH. Arch. Copyright 1999 Wiley-Liss, Inc.

Regulation of steroidogenesis in crayfish molting glands: involvement of protein synthesis.

The involvement of continuous protein synthesis in the mechanisms of crustacean steroidogenesis was investigated using crayfish molting glands (Y-organs). During intermolt, Y-organ steroidogenic activity is low. Eyestalk ablation initiates premolt which is characterized by a rapid increase in the production of ecdysteroids. In vitro incorporation of [14C]leucine into TCA-precipitable proteins was measured in Y-organs. A significant increase of de novo protein synthesis within 2 h and simultaneously led to a strong inhibition of the ecdysteroid synthesis. Sinus gland extracts (containing molt inhibiting hormone) also induced both a limited but reproducible inhibition of Y-organ protein synthesis and a pronounced inhibition of ecdysteroid production within 2 h. The results suggest a functional link between protein synthesis in the Y-organ and sustained ecdysteroid production. The analysis of autoradiographs from one-dimensional gel electrophoreses revealed an overall increase in de novo synthesis of glandular proteins in early premolt but also a more specific effect on distinct proteins (increase of 150, 140, 50-60, 22 and 15-18 kDa proteins) which may be more directly involved in the regulation of ecdysteroidogenesis.

Involvement of cAMP and cGMP in the mode of action of molt-inhibiting hormone (MIH) a neuropeptide which inhibits steroidogenesis in a crab.

In crustaceans, production of molting hormones (or ecdysteroids) by the molting glands (Y-organs; YO), is under negative control exerted by a neuropeptide, the molt-inhibiting hormone (MIH). MIH of the crab Carcinus maenas inhibits in vitro steroidogenesis of basal (intermolt crab) or activated (premolt crab) YO. MIH inhibits secretion of the two ecdysteroids synthesized by crab YO, ecdysone (E) secreted throughout the molting cycle, and 25-deoxyecdysone (25dE), secreted during the premolt period. At a MIH concentration of 10(-8) M, E is reduced about 50% and 25dE 94%. Regardless of the molting stage, this inhibition of steroidogenesis is reversible, dose dependent and measurable after 5 min. On intermolt YO, MIH induced cGMP increase and 8BrcGMP mimics the effect of MIH: at this stage cGMP seems to be involved with MIH inhibition of steroidogenesis. On premolt YO MIH induced a transient increase of cAMP (2-fold) and a long-lasting enhancement of cGMP (60-fold). On active YO, we demonstrated that a low concentration (10(-5) M) of dbcAMP, 8BrcAMP, 8BrcGMP, or agents increasing intracellular cAMP, mimic MIH effects and inhibit steroidogenesis. From these observations it is concluded that both cyclic nucleotides are involved in the mode of action of MIH on activated YO. At this premolt period, MIH/cAMP may act cooperatively with MIH/cGMP in the inhibitory control of steroidogenesis by crab YO.

Structure and biological activity of crustacean gastrointestinal peptides identified with antibodies to gastrin/cholecystokinin.

Four gastrin/cholecystokinin-like peptides (G/CCK) which cross-react with a specific C-terminal gastrin/CCK antiserum have been isolated from the stomach of the marine crustacean Nephrops norvegicus. The molecular weight of the four peptides was estimated between 1000 and 2000 Da by molecular sieving. By radioimmunoassay, the cross-reactivity of these peptides with human gastrin 17-I was found to be around 0.03%. Pure peptidic fractions were recovered after four successive steps of HPLC. Amino-acid analysis suggested a similarity between the four peptides identified which may belong to a new family. A limited homology between the C-terminus of one Nephrops peptide and vertebrate G/CCK was found after sequencing. Two of the peptides exhibited secretagogue effects on crustacean isolated midgut glands. The Nephrops peptides, although structurally distinct from the vertebrate G/CCKs, appear to serve similar biological functions in crustaceans.

Calmodulin-stimulated particulate guanylate cyclase in crayfish hepatopancreas.

1. A particulate guanylate cyclase from crayfish hepatopancreas membranes was investigated with respect to its dependence on Ca2+ and calmodulin. Addition of Ca2+ to EGTA-treated membranes increased cyclase activity by 100%. 2. Calmodulin stimulated the activity about 5-fold. 3. This effect could be abolished by the calmodulin antagonist compound 48/80. 4. These results present evidence that the particulate guanylate cyclase of crayfish hepatopancreas is a Ca2+/calmodulin-dependent enzyme. 5. The implications of this observation upon glycogen metabolism of crustaceans are discussed.

The crustacean hyperglycemic hormone (CHH) releases amylase from the crayfish midgut gland.

The present study aimed to investigate the role of eyestalk factors in the neuroendocrine control of the crustacean midgut gland concerning the release of amylase. The crustacean hyperglycemic hormone (CHH) is considered to be a candidate for this role. An optimum concentration (1.05 nM) CHH increased the in vitro release of amylase about 13-fold. CHH from Carcinus only slightly increased amylase release from Orconectes midgut glands, suggesting a species- or group-specificity. Studies on the possible mechanism of action concentrated on the role of Ca2+, cAMP and cGMP. Extracellular Ca2+ seems to be necessary to produce the amylase-releasing effect of CHH. Addition of dibutyryl derivatives of the cyclic nucleotides evoked the same effect as CHH. Additionally, the presence of forskolin in the incubation medium had an amylase-releasing effect, which points to a role of cAMP in the mode of action.

Ca2+-binding proteins in crayfish abdominal muscle. Evidence for a calmodulin lacking trimethyllysine.

A partially combined procedure for the isolation of some Ca2+-binding proteins from crayfish abdominal muscle is described, and some biochemical and biophysical data are reported. Crayfish calmodulin is similar to other calmodulins isolated from animal tissues, with the exception that it does not contain trimethyllysine. Besides calmodulin, an unknown protein is described which also binds to phenyl-Sepharose in a Ca2+-dependent manner. Despite its similarities with respect to subunit molecular weight and isoelectric point with 'sarcoplasmic calcium-binding proteins', its amino acid composition shows no similarities either with these proteins or with calmodulin. Furthermore, it is shown that sarcoplasmic Ca2+-binding proteins do not bind to phenyl-Sepharose under the same conditions.

Crayfish abdominal muscle adenylate cyclase. Studies on the stimulation by a Ca2+-binding protein.

A plasma-membrane preparation of crayfish muscle showed an adenylate cyclase activity which is inhibited to about 80% of its original activity by 100 microM-EGTA. Measurements of the enzyme activity in the presence of 100 microM-EGTA and various concentrations of Ca2+ revealed an increase in enzyme activity of about 400%, indicating an adenylate cyclase which is dependent on Ca2+ for activity. Fluphenazine (1 mM), a blocker of the Ca2+-binding protein calmodulin, decreased enzyme activity to zero. The enzyme can be re-activated by the addition of certain concentrations of calmodulin to the assay medium. This suggests that crayfish muscle adenylate cyclase is dependent on Ca2+ and calmodulin for activity.