Haruko Uemura

Immunocytochemical localization of endothelin in cultured bovine endothelial cells

SummaryTo investigate the intracellular localization of endothelin in cultured endothelial cells, an immunocytochemical study was carried out by the post-embedding protein A-gold technique with endothelin-specific antiserum. Gold particles were seen on the rough endoplasmic reticulum, the Golgi cisternae, the Golgi vesicles, small vesicles beneath the cell membrane, and the lysosomes. By contrast, no secretory granules were observed. These results suggest that endothelin is secreted by a constitutive pathway and that the lysosome may play an important role in regulating the biological activity of endothelin.

Connexin43 in rat pituitary: localization at pituicyte and stellate cell gap junctions and within gonadotrophs

Immunohistochemical methods were employed to investigate the cellular and ultrastructural localization of the gap junction protein connexin43 (Cx43) in rat pituitary. Western blots of pituitary homogenates probed with anti-Cx43 antibodies showed the presence of Cx43 in both anterior and posterior pituitary lobes. By light microscopy (LM), Cx43-immunoreactive (Cx43-IR) puncta were found in all areas of the posterior lobe, but at greater concentrations in peripheral regions of this structure. By electron microscopy (EM), immunogold labelling for Cx43 was seen at gap junctions between thin cytoplasmic processes of pituicytes. No immunoreactivity was detected in the intermediate lobe. The anterior lobe contained puncta similar to but more sparsely scattered than those in the posterior lobe, and by EM analysis these were demonstrated to correspond to labelled gap junctions between stellate cells. In addition, anti-Cx43 antibodies produced intracellular labelling in a small percentage of endocrine cells, which were distributed throughout the anterior lobe and determined by double immunostaining methods to be cells containing luteinizing hormone. By EM, labelling within these cells was associated with predominantly large secretory granules and other loosely organized organelles. The results indicate that gap junctions in the pituitary are composed of Cx43 and that this or a related protein may have a novel intracellular function within gonadotrophs.

Drinking induced by angiotensin II in fishes.

Among 20 species of freshwater fishes examined, Pseudorasbora parva, Rhodeus ocellatus, Cobitis anguillicaudatus, Carassius auratus, Oryzias latipes, Gambusia affinis, and Gyrinocheilus anymonieri were found to drink water like seawater fishes, while 13 remaining species did not drink. For fish species found exclusively in fresh water, angiotensin II (AII) treatment did not induce drinking. In contrast, those freshwater fishes which survive in estuarine brackish water (Leuciscus hakonensis, C. carassius, Parasilurus asotus, G. affinis, Chaenogobius annularis, Tridentiger obscurus, and G. anymonieri responded to AII by drinking. Furthermore, some freshwater fishes which survive either in hypertonic water (C. auratus) or in sea water (Anguilla japonica and O. latipes) also responded to AII by drinking. Of 17 seawater fishes examined, Eptatretus burgeri, Triakis scyllia, and Heterodontus japonicus failed to drink water, and for Trachurus japonicus, Platichthys bicoloratus, and Glossogobius giuris fasciatopunctatus, water intake was minor (similar to freshwater fishes). The 11 remaining seawater fishes drank water. AII did not induce drinking in fishes living exclusively in sea water. However, seawater fishes which survive either in tide pools (Chasmichthys dolichognathus gulosus) or in brackish water (Sillago japonica, Mugil cephalus, G. giuris fasciatopunctatus) responded to AII by drinking. P. bicoloratus, Acanthopagrus schlegeli, and Fugu niphobles were exceptional, in that they survive in brackish water, but did not respond to AII. Although some exceptions exist, it is generally concluded that a drinking response to AII is characteristic of fishes which encounter water more hypertonic than that in which they typically reside. Accordingly, a drinking mechanism induced by AII may be a compensatory emergency reaction to dehydration stress.

Immunoreactive atrial natriuretic peptide in the fish heart and blood plasma examined by electron microscopy, immunohistochemistry and radioimmunoassay

SummaryThe immunoreactivity of atrial natriuretic peptide and ultrastructure of cardiocytes were examined in 5 species each of freshwater and seawater teleosts, as well as in 2 species each of elasmobranchs and cyclostomes. Immunoreactivity was strong in the atria of Cyprinus carpio, Anguilla japonica and Conger myriaster, rather weak in atria of Channa maculata, Lepomis macrochirus, Salmo gairdneri, Oplegnathus fasciatus and Eptatretus burgeri, very weak in atria of Pagrus major, Trachurus japonicus and Triakis scyllia, and not detectable in atria of Hexagrammos otakii, Narke japonica and Lampetra japonica. The immunoreactivity of the atrial cardiocytes was generally stronger in freshwater than seawater fish. Ventricular immunoreactivity was detected only in 7 species, always being weaker than that observed in the atrium. Ultrastructurally, however, secretory granules were found in atria and ventricles of all species examined, being more frequent in the former than the latter. By radioimmunoassay, immunoreactive ANP was detected in the extracts of blood plasma and both atrial and ventricular tissues of all species examined. There were no statistically significant differences in the values between freshwater and seawater species.

Endothelin B receptor-like immunoreactivity is associated with LHRH-immunoreactive fibers in the rat hypothalamus

Antisera were raised against amino-acid residues 425-439 of the rat endothelin B (ETB) receptor. Massive ETB receptor immunoreactive fibers were observed in the median eminence and organum vasculosum lamina terminalis of rats. Double labeling studies with anti-luteinizing hormone-releasing hormone (LHRH) antiserum revealed that ETB receptor-like immunoreactivity associated with LHRH-immunoreactive fibers in these areas. However, ETB receptor-like immunoreactivity was not detected in LHRH-immunoreactive somata. This result suggests that endothelin affects the LHRH neuronal systems via ETB receptors on the axon at the terminal field.

Neuropeptide Y-immunoreactive neuronal system and colocalization with FMRFamide in the optic lobe and peduncle complex of the octopus ( Octopus vulgaris )

Abstract. The distribution of neuropeptide Y (NPY)-like immunoreactivity and its colocalization with FMRFamide were investigated in the optic lobe and peduncle complex of the octopus (Octopus vulgaris) by using immunohistochemical techniques. In the optic lobe cortex, NPY-immunoreactive (NPY-IR) fibers were observed in the plexiform layer, although no NPY-IR somata were observed in the outer or inner granular cell layers. In the optic lobe medulla, NPY-IR somata were seen in the cell islands, and abundant NPY-IR varicose fibers were observed in the neuropil. Most of the NPY-IR structures in the medulla showed FMRFamide-like immunoreactivity. In the peduncle lobe, abundant NPY-IR and FMRFamide-IR (NPY/FMRF-IR) varicose fibers were seen in the basal zone neuropil of the peduncle lobe. In the olfactory lobe, NPY/FMRF-IR varicose fibers were also abundant in the neuropil of the three lobules. NPY/FMRF-IR somata, with processes running to various neuropils, were scattered in the median and posterior lobules. In the optic gland, many NPY/FMRF-IR varicose fibers formed a honeycomb pattern. These observations suggest that NPY/FMRF-IR neurons in the optic lobes participate in the modulation of visual information and that those in the optic gland are involved in the regulation of endocrine function.

Immunohistochemical investigation of neuropeptides in the central nervous system of the amphioxus, Branchiostoma belcheri

The immunohistochemical localization of nine different neuropeptides was studied in the central nervous system of the amphioxus, Branchiostoma belcheri. In the brain, perikarya immunoreactive for urotensin I and FMRFamide were localized in the vicinity of the central canal. One of the processes of each of these perikarya was found to cross the dorso ventral slit-like lumen of the central canal. Oxytocin-immunoreactive short fibers, but not perikarya, were detected in the ventral part of the brain. Perikarya immunoreactive for arginine vasopressin/vasotocin, oxytocin and FMRFamide were widely distributed in the spinal cord. Arginine vasopressin/vasotocin-immunoreactive fibers often made contacts with Rohde cell axons. Angiotensin II-immunoreactive perikarya were observed in the posterior half of the spinal cord, and urotensin I-immunoreactive perikarya were found in the caudal region of the spinal cord. Cholecystokinin/gastrin-immunoreactive fibers, but not perikarya, were detected in the spinal cord; some extended as far as the ependymal layer of the cerebral ventricle. No colocalization of the peptides examined was observed. No immunoreactivity for atrial and brain natriuretic peptides nor for urotensin II was detected. The present study indicates that there are at least six separate neuronal systems that contain different peptides, respectively, in the central nervous system of the amphioxus. Their functions remain to be determined.

Galanin-immunoreactive neuronal system and colocalization with serotonin in the optic lobe and peduncle complex of the octopus (Octopus vulgaris)

Immunohistochemical techniques were used to investigate the distribution of galanin-like immunoreactivity and colocalization with serotonin (5-HT) in the optic lobe and peduncle complex of the octopus, Octopus vulgaris. Galanin immunoreactive (Gal-IR) fibers, but not cells, were seen in the plexiform layer of the optic lobe cortex. Gal-IR cells were scattered in the cell-islands of the optic lobe medulla and Gal-IR varicose fibers were observed to be abundant in the neuropil surrounding the islands. All Gal-IR cells were immunoreactive for 5-HT, and a few cells showed only 5-HT-like immunoreactivity. In the peduncle lobe, no Gal-IR cells were seen in the basal zone or spine, but in the basal zone, many Gal-IR fibers were seen. In the anterior olfactory lobule, only a few pyramidal Gal-IR cells were observed in the cell layer, and their apical processes were traced to the central neuropil. In the median olfactory lobule, ovoid Gal-IR cells were scattered in the peripheral cell layer. All Gal-IR cells in the anterior and median olfactory lobules showed 5-HT-like immunoreactivity. In the posterior olfactory lobule, ovoid and triangular Gal-IR cells were scattered in the cell layer. Some of them showed 5-HT-like immunoreactivity. Western blot analysis indicated an Gal-IR band at approximately 15.4 kDa. These results suggest the association of galanin-like substance and 5-HT with the visual system of octopus and that the main form of the octopus galanin might have a different molecular weight from vertebrate galanins.

Adenosine deaminase in rodent median eminence: detection by antibody to the mouse enzyme and co-localization with adenosine deaminase-complexing protein (CD26)

Adenosine deaminase in the hypothalamic tuberomammillary nucleus and median eminence of rat and mouse brains was investigated with two different antibodies generated against the enzyme derived from either calf or mouse. Both antibodies labelled neurons in the tuberomammillary nucleus and, as determined in rat, they immunolabelled the same neurons. In the median eminence, immunopositive fibres and terminals were detected with anti-mouse adenosine deaminase in both rat and mouse, while no such staining was seen in either species with antibody against the calf enzyme. These fibres were most concentrated in the external median eminence, had a more restricted distribution than those containing either galanin or tyrosine hydroxylase and only partially overlapped with oxytocin-positive fibres. By electron microscopy, adenosine deaminase was found in terminals containing both small, clear vesicles with diameters of 35 to 45 nm and large dense-core vesicles with diameters of 100 to 140 nm. Preadsorption of antibodies with purified enzyme derived from the species against which they were directed eliminated all staining in rat, while antibody adsorptions across species were less effective. Preadsorption of anti-mouse adenosine deaminase antibody with the mouse deaminase led to increased labelling in mouse median eminence, suggesting an interaction between tissue components and antibody-linked enzyme. Tests for the presence of adenosine deaminase-complexing protein (CD26) with an antibody against this protein gave positive labelling in the median eminence of both species and this labelling was co-distributed with that seen for adenosine deaminase. These results confirm the expression of adenosine deaminase in restricted populations of neurons in rodent brain as revealed with a novel antibody, suggest the presence of a distinct form or localization of the enzyme in the median eminence, and raise the possibility that it contributes, perhaps along with CD26, to purinergic regulation of hormone secretion in this structure.

The distribution of an immunoreactive endothelin-like substance in the nervous system of the nereid, Neanthes diversicolor (Annelida)

SummaryThe distribution of an immunoreactive endothelin-1-like peptide was investigated in the nereid, Neanthes diversicolor, using an antiserum raised against synthetic endothelin-1. Immunoreactive perikarya were localized in the brain, and nerve fibers containing endothelin-1-like material were found in the neuropil occupying the central portion of the brain. No immunostained fiber elements were traced in the circumesophageal connectives. Immunoreactive perikarya occurred in the subesophageal ganglion. From this ganglion, specifically stained fibers run posteriorly toward the ventral nerve cord. In each segmental ganglion, immunoreactive neurons were observed in medio-ventral and latero-ventral regions, and one or two marked fibers extended to the parapodium. In the parapodium, small immunoreactive perikarya and fiber elements were visible. Immunolabeled fibers occurred in the stomatogastric nerves, in the wall of the buccal cavity, and in the pharynx, esophagus, intestine and its anal region. Immunoreactive perikarya and nerve fibers were visualized between the circular muscle layer and epithelial cell layer in the esophagus and intestine. The endothelin-1-like substance shown to occur in N. diversicolor appears to function as a neurotransmitter or neuromodulator.