Toyoji Kaneko

Localization of calcitonin gene-related peptide in the small intestine of various vertebrate species

SummaryCalcitonin gene-related peptide (CGRP) was found extensively in the small intestine of both non-mammalian and mammalian vertebrates using radioimmunoassay and immunocytochemistry. By radioimmunoassay, the levels of CGRP in rats, mice, chickens, bullfrogs and rainbow trout were found to range from 91.5 to 419.1 ng/g tissue. To localize CGRP in the small intestine, we used three different tissue preparations for immunocytochemistry: whole-mount preparations, and frozen and Paraplast sections. The combination of three tissue preparations made it easier to visualize the three-dimensional structure and reduced the possibility of missing the immunoreaction. Immunoreactive cell bodies were found in the plexi in the mammalian species. Dense and regular networks of CGRP fibers were observed in the smooth muscle layers, when examined in whole-mount preparations. In non-mammalian species, however, immunoreactive cell bodies could not be detected, although immunoreactive fibers were present, forming less dense and regular networks. Our results indicate that CGRP-immunoreactive fibers are present in the smooth muscle layers of the intestine from fish to mammals, suggesting that CGRP may be involved in regulating gastrointestinal smooth muscles in vertebrates.

Characterization of antisera raised against hypocalcin (teleocalcin) purified from corpuscles of stannius of rainbow trout, Salmo gairdneri

Highly specific antisera were raised against hypocalcin, a 54-kDa glycoprotein purified from the corpuscles of Stannius (CS) of rainbow trout, Salmo gairdneri. The specificity of the antisera was determined by using Ouchterlonys double immunodiffusion test, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunocytochemistry. In the double immunodiffusion test, a single precipitin line was formed between the antisera and hypocalcin. Both RIA and ELISA studies showed that serial dilutions of hypocalcin, teleocalcin, and CS extracts produced dose-response curves, whereas rat FSH, chicken LH, bovine TSH, salmon calcitonin, bovine parathyroid hormone, and human angiotensins I and II failed to cross-react with the antiserum. Immunoreactive hypocalcin was demonstrated in the plasma of a teleost (flounder), but not of an elasmobranch (dogfish). In the immunocytochemistry, most of the gland cells showed strong immunoreaction with the antisera, whereas some cells displayed no immunoreactivity.

Calcitonin gene-related peptide in the bullfrog, Rana catesbeiana: localization and vascular actions

Calcitonin gene-related peptide (CGRP) was found using radioimmunoassay in the brain and spinal cord of the bullfrog, Rana catesbeiana. The brain extracts were found to contain 241.7 +/- 68.1 pg/mg tissue, and the spinal cord contained 1753.0 +/- 96.8 pg/mg tissue. An intense immunocytochemical reaction was observed in the dorsal spinal cord. Vascular studies using helical strips of the dorsal aorta, iliac artery, and femoral artery showed CGRP to exert a vasorelaxant effect which was most pronounced in the femoral artery and minimal in the aorta. As in the rat, CGRP was shown to exert its vasorelaxant effect by inhibiting the mobilization of intracellular calcium.

Immunocytochemical detection of parathyroid hormone-like substance in the goldfish brain and pituitary gland

Parathyroid hormone (PTH)-like substance was immunocytochemically detected in the goldfish brain and pituitary gland using the peroxidase-antiperoxidase method with an antiserum raised against bovine PTH1(-84). Immunoreactive cell bodies were found in the nucleus preopticus in the preoptic area, and immunoreactive fibers could be traced from these cell bodies to the pituitary, in which immunoreactivity was detected in the neurohypophysis. These findings suggest that PTH-like substance is biosynthesized in the neuroendocrine cells in the brain and released in the neurohypophysis in the pituitary gland. Our results are discussed in relation to the hypercalcemic regulation of the pituitary gland in teleosts.

Immunocytochemical localization of hypocalcin in the endocrine cells of the corpuscles of Stannius in three teleost species (trout, flounder and goldfish)

SummaryIn order to identify the cell-type responsible for the production of hypocalcin (the recently isolated hypocalcemic hormone of teleost fish), the corpuscles of Stannius (CS) of trout, flounder and goldfish, were immunocytochemically stained with antisera raised against trout hypocalcin. The secretory granules of the type-1 cells of the CS, considered to be the hypocalcin-producing cells, showed intense immunoreactivity in all species examined. However, in trout and flounder, the secretory granules produced by the type-2 cells, which have been suggested to represent a functionally different cell-type, also showed an intense immunoreactivity. In goldfish, no type-2 cells were observed. We tentatively conclude that type-1 and type-2 cells represent structurally different forms of the same functional cell-type.

Isolation and characterization of carp prolactin

Prolactin (PRL) was extracted with acid-acetone from common carp (Cyprinus carpio) pituitary glands and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography (HPLC) on TSK-gel TMS 250 with a yield of 0.7 mg/g wet tissue. At each stage of purification, fractions were monitored by HPLC on TSK-gel ODS 120T and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Carp PRL was almost equipotent with ovine PRL in retaining plasma Na concentrations in the hypophysectomized killifish, Fundulus heteroclitus. Immunocytochemistry at both the light and electron microscopic levels revealed that carp PRL antiserum specifically stained cells in the goldfish rostral pars distalis. No cross reaction with putative growth hormone (GH) cells in the proximal pars distalis was observed. The specificity of the carp PRL antiserum was confirmed by immunoblot studies. Although immunostaining of both carp and salmon PRL was observed, there was no cross reaction to GHs from these species. Carp PRL had a sole N-terminal residue of valine, a molecular weight of 23 kDa in SDS-PAGE, and an isoelectric point of 7.3 by gel electrofocusing. Based on these results, together with the knowledge of physicochemical properties of salmon and tilapia PRLs, we propose a standard procedure for isolation of fish PRLs.

Immunocytochemical demonstration of a novel system of neuroendocrine peptidergic neurons in the pond snail Lymnaea stagnalis, with antisera to the teleostean hormone hypocalcin and mammalian parathyroid hormone

Immunocytochemical staining with antisera raised against trout hypocalcin, the hypocalcemic hormone of the Stannius corpuscles and against bovine parathyroid hormone (bPTH1-84), revealed a new system of neuroendocrine neurons in the pond snail Lymnaea stagnalis. The neurons are located in small groups or single cells in the visceral, parietal, and pedal ganglia of the central nervous system. The axons of these cells are running to the periphery of the pleuroparietal, visceroparietal, and pleuropedal connections, the dorsopedal commissure, and to several nerves originating in the visceral, parietal, and pedal ganglia. The axons are ending with characteristic axonal distensions in the periphery of these connectives, commissure, and nerves. These regions probably act as neurohaemal areas. The affinity of this neuroendocrine system for both the anti-hypocalcin and anti-PTH sera is another indication for a special relationship between hypocalcin and PTH, which possess some immunological resemblance and similar biological activities, although no similarity in primary structure.

Calcitonin gene-related peptide: an inhibitor of bullfrog (Rana catesbeiana) gallbladder contraction in vitro.

The presence of a calcitonin gene-related peptide (CGRP)-like material was demonstrated in the gallbladder of the bullfrog, Rana catesbeiana, using immunocytochemistry and confirmed by radioimmunoassay. An intense immunocytochemical reaction was observed in nerves located in the smooth muscle layers and associated with blood vessels. No immunoreactive nerve fibers were associated with ganglia, nor were immunoreactive cell bodies observed. Radioimmunoassay showed that 25.03 +/- 2.5 pmol/g tissue of CGRP-like material was present. In vitro tension studies using gallbladder strips showed that CGRP exerted an inhibitory effect on both acetylcholine- and cholecystokinin octapeptide-induced tension but had no effect on KCl-, norepinephrine-, or cerulein-induced tension. CGRP may act directly on the gallbladder smooth muscle to inhibit contraction.