Peter P. Jaros

Species or group specificity in biological and immunological studies of crustacean hyperglycemic hormone.

Abstract Molecular differences of the hyperglycemic hormone have been reported previously for several decapod crustaceans. The relationships of these neuropeptides have been tested now, comparing their biological response by cross-injection experiments and their immunological properties by immunodiffusion. The data revealed cross-reactivity between several species within the appropriate group.

Immunocytochemical identification of hyperglycemic hormone-producing cells in the eyestalk of Carcinus maenas.

SummaryAntiserum raised in rabbits against extracts of sinus glands from Carcinus and shown by several criteria to contain antibodies directed against the neurosecretory hyperglycemic hormone was used to locate the hormone-producing perikarya in the optic ganglia. By means of the double antibody fluorescence technique, selective staining of the large neurosecretory perikarya of the medulla terminalis ganglionic X-organ (MTGXO) and their axons is obtained. The axon endings of the sinus gland are also stained. None of the other groups of neurosecretory cells in the eyestalk shows fluorescence. Preabsorption of the antiserum with pure hyperglycemic hormone abolishes the fluorescence.

The hyperglycemic neuropeptide of the terrestrial isopod, Porcellio dilatatus. I. Isolation and characterization

From isolated sinus glands of Porcellio dilatatus, a hyperglycemic neuropeptide (CHH) was purified by means of a single, two-step purification procedure which consisted of gel chromatography on Sephadex G-50, followed by high-performance liquid chromatography. The 5800- to 6100-Da peptide contains 50-52 amino acids residues. The amino acid composition is (Pro, Ile)1; His1-2; (Thr, Ser, Gly, Met, Tyr)2; (Val, Phe, Lys)3; (Ala, 1/2Cys, Leu, Arg)4; Glx5; Asx7; Trp, n.d. The amino acid composition differs from that of two decapod CHHs analyzed thus far. The N-terminus is blocked. The neuropeptide exhibits little or no interspecific hyperglycemic effect in the brachyuran, Uca pugilator, and its cross-reactivity and potency in the RIA for Carcinus-CHH is very low.

Tracing of neurosecretory neurons in crayfish optic ganglia by cobalt iontophoresis

SummaryThe axonal connections between the medulla terminalis ganglionic X-organ (MTGXO) and the sinus gland are traced by iontophoretic application of cobalt dye to the neurosecretory system in the eyestalks of the crayfish, Orconectes limosus. The MTGXO consists of about 15 large perikarya, forming a distinct subgroup of neurosecretory cells in the medulla terminalis and giving rise to a prominent fibre bundle. Additional axons reaching the sinus gland from the medulla interna, the medulla externa and the optic nerve are less conspicuous.

Occurrence of immunoreactive enkephalins in a neurohemal organ and other nervous structures in the eyestalk of the shore crab, Carcinus maenas L. (Crustacea, Decapoda)

SummaryLight-microscopical observations with immunofluorescence and peroxidase staining procedures revealed leu-enkephalin-like immunoreactivity in axon profiles of the sinus gland (SG) and in single small neurons in the optic ganglia of the eyestalk of Carcinus maenas. Electron microscopy of the SG showed reactivity to be associated with neurosecretory granules 82±23 nm in diameter. High performance liquid chromatography of SG-extracts revealed radioimmunoreactive substances with the retention times of synthetic met- and leu-enkephalin and met-enkephalin-Arg6-Phe7, respectively.

Immunocytochemical demonstration of the neurosecretory X-organ complex in the eyestalk of the crab Carcinus maenas

SummaryAn antiserum was obtained by immunizing rabbits with sinus gland extracts from Carcinus maenas. The antiserum is almost exclusively directed against neurosecretory material in the medulla terminalis X-organ (MTGXO), as demonstrated by the peroxidase—antiperoxidase (PAP) staining method in light and electron microscopic studies. Radioimmunological binding studies indicate the presence of antibodies against the crustacean hyperglycemic hormone (CHH) or the black pigment dispersing hormone (BPDH) in the antiserum. The results suggest that the neurosecretory perikarya of the MTGXO are the sites of production of CHH and/or BPDH.

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.

Evidence for a crustacean hyperglycemic hormone-like molecule in the nervous system of the stick insect, Carausius morosus

SummaryHistological sections of the brain, suboesophageal ganglion, and the corpora cardiaca/corpora allata complex were examined for the presence of crustacean hyperglycemic hormone-like immunoreactive material. With the use of an antiserum directed against the hyperglycemic hormone of Carcinus maenas, immunofluorescence was found in the median portion of the pars intercerebralis, and the corpora cardiaca. Extracts of corpora cardiaca were examined by radioimmunoassay for competitive binding to the antiserum; one pair of corpora cardiaca contains at least 7 pg crustacean hyperglycemic hormone-like material.