Dietrich Sedlmeier

The Mode of Action of the Crustacean Neurosecretory Hyperglycemic Hormone I. Involvement of Cyclic Nucleotides

Abstract The influence of the crustacean hyperglycemic neurohormone (CHH) on cyclic nucleotide levels has been studied in heart, hepatopancreas, integumentary tissue, antennal gland, and abdominal muscle of the crayfish, Orconectes limosus . In all tissues studied, both cAMP and cGMP were elevated after hormone administration in vivo . Relative to resting levels, the increase of cGMP was more pronounced than that of cAMP. In antennal gland and integumentary tissue, peak levels were observed 2 min after hormone injection, in heart and hepatopancreas after 5 min. A longer duration of the response was observed in abdominal muscle. Hepatopancreas tissue was also studied in vitro . Cyclic AMP is raised unspecifically by preincubation in vitro and could not be altered by hormone addition; however, cGMP, which did not change during the preincubation period, was elevated markedly by the hormone. This response was followed by glucose release into the medium. It is proposed that cGMP may be, at least in some tissues, like the hepatopancreas, the second messenger of the hyperglycemic hormone.

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.

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.

Ecdysteroid biosynthesis in moulting glands of the crayfish Orconectes limosus: evidence for the synthesis of 3-dehydroecdysone by in vitro synthesis and conversion studies

Abstract Ecdysteroids secreted in vitro by the Y-organs of Orconectes limosus were measured by radioimmunoassay after HPLC-separation of culture media. Ecdysone and 3-dehydroecdysone were found as secretory products. Metabolic studies with [ 3 H]ecdysone and [ 3 H]3-dehydroecdysone showed that Y-organs are not able to convert 3DE into E or vice versa. The putative precursors 5β-ketodiol and 5-β-diketol (“3-oxoketodiol”) were both converted by Y-organs. [ 3 H]5-β-ketodiol was converted into E. [ 3 H]5β-diketol was converted into 3DE and E. Ketodiol was also observed as a conversion product of 5β-diketol. Our data suggest, that in Orconectes 5β-diketol is a good candidate for an intermediate in the ecdysteroid biosynthetic pathway and that there is a branching of the pathway leading to 3DE as well as to E, due to the activity of a 3-oxoecdysteroid-3β-reductase specific for less hydroxylated compounds.

The mode of action of the crustacean neurosecretory hyperglycemic hormone (CHH): II. Involvement of glycogen synthase

Abstract A study on the influence of the neurosecretory hyperglycemic eyestalk hormone of crustaceans (CHH) on glycogen synthase activity was performed. Extirpation of the eyestalks, and therefore elimination of the hormone, resulted in an increase of the synthase I activity in the abdominal muscle from 30 to 70% of the total activity. Total activity remains unchanged during this procedure. Injection of CHH into eyestalkless animals lowers I activity to normal values within 15 min. Concomitantly, the cyclic nucleotide levels in the abdominal muscle are increased with a maximal response observed about 10 min after hormone injection. Dose-response curves suggest a close correlation between the degree of glycogen synthase inactivation and cAMP increase. Injection of cyclic nucleotides into destalked animals has an effect corresponding to that of CHH, suggesting a second messenger function of the cyclic nucleotides.

THE ROLE OF HEPATOPANCREATIC GLYCOGEN IN THE ACTION OF THE CRUSTACEAN HYPERGLYCEMIC HORMONE (CHH)

Abstract 1. 1. The results presented here demonstrate that in the crayfish Orconectes limosus the hepatopancreas is a target tissue of the crustacean hyperglycemic hormone (CHH). 2. 2. Hepatopancreatic glycogen stores can be prelabeled by incubation with ( 14 C)-glucose. 3. 3. The incorporation rate is linear with respect to time and glucose dose. 4. 4. The incorporation rate is reduced about 30% when CHH is present in the incubation medium. 5. 5. Application of CHH (0.1 sinus gland equivalents) to prelabeled hepatopancreatic glycogen decreases the label to about 50%. 6. 6. This reaction can be mimicked by cyclic nucleotides, such as dBcAMP or dBcGMP. 7. 7. This supports the idea that cyclic nucleotides are a necessary link in the mode of action of CHH.

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.

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.

Variations in epidermal cyclic Nucleotide-dependent protein kinase activity during moult cycle of the crayfish Orconectes limosus and hormonal control of kinase activity by 20-hydroxyecdysone

1. 1. In correlation to changes in ecdysone hemolymph titers during moulting of the crayfish high kinase activity is observed in the moult stages D0−D23 and in postmoult stage B. 2. 2. Both basal (measured in the absence of cyclic nucleotides) and total (in the presence of cyclic nucleotides) protein kinase activity were enhanced, probably caused by an increase in tissue Levels of cAMP and cGMP and furthermore by the disapperance of a kinase inhibirory protein. 3. 3. Injection of 20-hydroxyecdysone into intermoult animals increased integumental kinase activity in a time- and dose-dependent manner, maximal activity occurring after 5 hr at a dose of 1 μg/g fresh weight. 4. 4. In vitro incubation of an integumental extract with 20-hydroxyecdysone showed no enhancement of cAMP and cGMP levels in the tissues.

Protein phosphorylation in the moulting gland of the crayfish, Orconectes limosus: Role of cyclic nucleotides, calcium, and moult inhibiting hormone (MIH)

Summary Y-organs of the crayfish Orconectes limosus revealed a phosphorylation pattern which varied in different moulting stages. A 95-kDa-phosphoprotein increased in abundance as moulting proceeded, becoming the predominant phosphoprotein in stage D1. Phosphorylation of proteins with apparent molecular masses of 68 (pp68) and 17 kDa decreased during the moulting cycle. Phosphorylation of pp68 was evoked by incubating Y-organs of premoult animals with sinus gland extract. The presence of Ca2+ induced a decrease in overall kinase activity in Y-organ homogenates and also caused reduced phosphorylation of endogenous Y-organ proteins, most of them (200, 40, 31, and 17 kDa) exhibiting cAMP-dependent phosphorylation. cAMP-dependent phosphorylation of a 34- and 31-kDa-phosphoprotein (pp34, pp31) was detectable only in Y-organs of premoult animals. A 40-kDa-protein may be a substrate for an endogenous cGMP-dependent protein kinase since its phosphorylation can be stimulated by cGMP as well as cAMP.