Axel Willig

The role of biogenic amines in the control of blood glucose level in the decapod crustacean, Carcinus maenas L.

Abstract 1. Administration of biogenic amines into intact Carcinus maenas induces dose- and timedependent elevation of hemolymph glucose level. 2. Removal of the neurosecretory centre containing the crustacean hyperglycemic hormone (CHH) by ablation of the eyestalks did not induce hypoglycemia. 3. Injection of dopamine (DA) into eyestalkless crabs showed no hyperglycemic effect, while serotonin (5-HT), epinephrine (E), norepinephrine (NE), and octopamine (OA) elevated glucose levels. 4. The dopaminergic effect was significantly reduced by administration of trifluoperazine (TFP). 5. 5-HT and OA were found to be strong elevators of glucose levels, while the other biogenic amines had moderate effects only. 6. The results indicate, that DA exerts its hyperglycemic effect by stimulating the release of CHH from the eyestalk neurosecretory centre. Elevation of hemolymph glucose level by 5-HT, OA, E, and NE, occurs independently of CHH.

Purified crustacean enkephalin inhibits release of hyperglycemic hormone in the crab Carcinus maenas L.

Abstract 1. The release of the crustacean hyperglycemic hormone (CHH) is inhibited by synthetic Leu-Enkephalin (L-Enk). 2. Endogenous enkephalinergic material was isolated by reversed phase liquid chromatography. 3. The endogenous substance inhibits CHH release as demonstrated by bioassay in vivo and in vitro . 4. The decrease in CHH release is antagonized by naloxone. 5. L-Enk immunoreactivity is demonstrated in the eyestalk of Carcinus maenas . 6. The results suggest a modulatory role of L-Enk in the control of neuropeptide release.

Cytological effects of platelet-derived growth factor on mitochondrial ultrastructure in fibroblasts.

The goal of this study was to evaluate morphofunctional changes in mitochondrial ultrastructure after platelet-derived growth factor application in fibroblasts as an indicator of mitochondrial activation in processes like wound healing. NRK-49F fibroblasts were synchronized, incubated with PDGF (platelet-derived growth factor) and studied by electron microscopy. Volume density (Vv), numerical density (Nv) and surface density (Sv) were measured by stereological analysis. Application of PDGF on NRK-49F caused an increase in mitochondrial volume density by 57% and surface area of cristae per mitochondrion by 65%. The numerical density of the mitochondria was decreased in the PDGF-treated cells by 23%, but at the same time their mean volume was increased. Furthermore, the mitochondria had a complex and highly variable shape both in control and PDGF-treated cells, possibly indicating the existence of a mitochondrial reticulum. The results demonstrated that biochemically active membrane systems in fibroblast mitochondria are enlarged as a direct effect of small doses of platelet-derived growth factor and support the concept that this factor and related peptides serve as mitogens for connective tissue forming cells. Thus, in mitogenic processes like wound healing, the high energy demand of fibroblasts is provided by the increase of the inner surface of mitochondria.

Sex pheromones in marine polychaetes: Steroids from ripe Nereis succinea ☆

A number of neutral marine steroids such as desmosterol, campesterol, brassicasterol, gorgosterol, and other trace steroids were isolated from the coelomic fluid of ripe Nereis succinea and checked for biological activity as sex pheromones on swarming specimens of Platynereis dumerilii and Nereis succinea. No significant influence of synthetic gorgosterol or a natural extract of gorgosterol or the other identified steroids on the swarming behavior was observed.

Opioid receptor types for endogenous enkephalin in the thoracic ganglion of the crab, Carcinus maenas

In crustaceans, the endogenous opioid peptides, enkephalins, are known to be concentrated in the thoracic ganglion, although they have been demonstrated in all parts of the nervous system. Bmax and Kd measurements have been obtained for the binding of ligands used to characterize delta- and kappa-type opioid receptors in vertebrates. High- and low affinity binding of [3H] [2-D-Pen5-D Pen] enkephalin ([3H]DPDPE) has been measured with a Kd = 9.2 +/- 2.4 nM, Bmax = 153 fmol/mg, and Kd = 243 +/- 27 nM, Bmax = 1.785 pmol/mg, respectively. In addition a kappa-type receptor with Kd 85.5 +/- 12.6 nM and Bmax = 21.138 pmol/mg protein has been recorded. Binding characteristics of several ligands were monitored. Electrophoretic studies of affinity chromatographically purified receptor fractions revealed a molecular mass of 60 kDa. Isoelectric focusing showed a specific binding of [3H]DPDPE to thoracic ganglion membranes at a pl of 5.5.

Differential localization of leu-enkephalin and hyperglycemic hormone in axon terminals of the sinus gland of the crabsCarcinus maenas, eriocheir sinensis, andUca pugilator

SummaryLeu-enkephalin containing secretory granules were demonstrated in axon terminals of immunogoldlabeled electron-microscopic sections of the sinus gland of three brachyuran crustaceans. These granules have a diameter of 120+-15 nm and differ in electron density from those located in adjacent terminals containing hyperglycemic or molt-inhibiting hormone. These neurohormones do not show co-localization with leu-enkephalin. The cross-reactivity of leu-enkephalin antiserum with met-enkephalin is less than 1%. The sinus glands of the three species examined show no immunoreactivity for FMRF-amide. A modulatory activity of endogenous enkephalin by paracrine mechanisms is suggested.

Autoradiographic localization of opioid binding sites combined with immunogold detection of Leu-enkephalin, crustacean hyperglycaemic hormone and moult inhibiting hormone at the electron microscopic level in the sinus gland of the shore crab,Carcinus maenas

Double labelling experiments were performed on the same tissue section at the electron microscopic level, in order to show the involvement of the opioid leucine-enkephalin (Leu-enk) in the modulation of crustacean hyperglycaemic hormone (CHH) mobilization. Both neuropeptides were stored in distinct axon terminals of the sinus gland ofCarcinus maenas. A post-embedding immunogold cytochemical technique for Leuenk, CHH and the CHH neurohormone related moult inhibiting hormone (MIH) was combined with a scintillator intensified autoradiographic method to demonstrate binding of the opioid antagonist [3H] naloxone. Ultrathin sections were successively incubated with antisera against Leu-enk, CHH or MIH, and the corresponding colloidal gold labelled antisera, followed by autoradiographic processing. At the ultrastructural level [3H] naloxone binding sites were easily recognized by their silver tracks after development. Opioid binding sites for [3H] naloxone were visualized only at membranes of CHH-containing axon terminals. These results provide morphological evidence for direct enkephalinergic control of CHH containing neurons in the sinus gland ofC. maenas and are furthermore the first autoradiographic demonstration of opioid binding sites in the nervous system of invertebrates.

Delta and kappa opioid receptors in eyestalk ganglia of a crustacean

Crustacean eyestalk ganglia are part of the protocerebrum and have been demonstrated to produce numerous neurohormones. 3H(2-D-Pen, 5-D-Pen)-enkephalin, 3H-(-)-ethylketocyclazocine and 3H(D-Ala2-NMePhe-Glyol5)-enkephalin were used as ligands for opioid receptors on neuronal membrane preparations of eyestalk ganglia under consideration of their stereospecific binding properties. In context with saturation binding isotherms, association and dissociation plots, we demonstrate here two opioid receptors; a delta-type receptor with high affinity (Bmax 68.5 fmol/mg protein, Kd = 4.0 nM) and low affinity (Bmax 493 fmol/mg, Kd = 83.6 nM) and a second receptor of kappa-type with Bmax of 3.1 pmol/mg protein and Kd = 68.6 nM.

Isolation and purification of calmodulin from the shrimp, Crangon crangon

Calmodulin was isolated and purified from shrimp abdominal muscle by heat precipitation, ion exchange and hydrophobic interaction chromatography. The purified calmodulin was homogeneous when evaluated by polyacrylamide gel electrophoresis. A still remaining contaminant was eliminated by high performance liquid chromatography on a phenyl column. The biological and physicochemical properties of shrimp calmodulin such as amino acid composition, molecular weight and the ability to activate calmodulin-deficient bovine heart phosphodiesterase were compared to those of other invertebrate calmodulins.

Biological properties and partial purification of a growth factor from porcine spleen.

3T3-Fibroblast growth is enhanced in a dose-dependent way by a factor isolated from porcine spleen after limited proteolysis. The factor is not dialyzable and is stable for 5 min at 100 degrees C and at pH 2 at room temperature. Trypsin or collagenase treatment does not affect its biological activity. The stimulation of cell growth is independent of serum concentration in the culture medium but is accelerated by insulin supplement. The biochemical properties of this factor indicate a novel growth promotor different from other growth factors.