Hideshi Kobayashi

Isolation and amino acid sequence of urotensin I, a vasoactive and ACTH-releasing neuropeptide, from the carp (Cyprinus carpio) urophysis

Urotensin I (UI), a 41-residue mammalian hypotensive and fish or mammalian corticotropin-releasing peptide, isolated from 0.1 N HCI extracts of urophyses of the carp (Cyprinus carpio) was purified and the amino acid sequence was determined to be: H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu- Arg-Asn-Met-Ile-Glu-Met-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Leu-Asp-Glu-Val-NH2. When the extraction procedure included heating at 100 degrees C for 15 min, UI was cleaved at a highly acid labile Asp-Pro bond to give the fully active UI (4-41). Urotensin I shows close structural and biological homology with the recently isolated ovine hypothalamic corticotropin-releasing factor (CRF) and the frog skin peptide sauvagine and thus may be considered an evolutionary prototype of unique mammalian-hypotensive and vertebrate corticotropin-releasing factors.

Ecological adaptation of angiotensin-induced thirst mechanism in tetrapods

Abstract Dipsogenic action of angiotensin II (AII) was tested by intraperitoneal injection in 2 species of amphibians, 9 species of reptiles, 18 species of birds, and 8 species of mammals. The amphibians did not respond to AII by drinking. Most of the tetrapods other than the amphibians responded to AII (5–30 μg/100 g) by drinking. Among reptiles, birds, and mammals, however, the following species were relatively insensitive to AII: (1) the hibernating reptiles, Calotes versicolor and Natrix piscator , (2) the animals originated in arid areas, such as Testudo graeca, Melopsittacus undulatus, Lonchura malabarica, Meriones unguiculatus and Mus musculus , and (3) the carnivorous birds, Accipiter badius, Athene brama and Falco tinnunculus . Thus, it is likely that animals, which drink little water in nature, are relatively insensitive to AII in drinking. These observations suggest that AII has become adaptively involved in the physiological mechanisms inducing thirst during the evolutionary process. Evolution of receptive sites for AII in the brain is discussed in vertebrates.

Primary structures of multiple forms of urotensin II in the urophysis of the carp, Cyprinus carpio

Multiple forms of urotensin II (UII), one of the hormonal peptides of the caudal neurosecretory system of fishes, were purified from the urophyses of the carp, Cyprinus carpio. Three distinct peaks with UII activity (classified as UII-alpha, -beta and -gamma) were separated by reverse-phase high-pressure liquid chromatography (HPLC). Edman degradation as well as digestion with carboxypeptidase A revealed the primary structures of these peptides as UII-alpha: Gly-Gly-Gly-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val UII-beta: Gly-Gly-Ser-Asn-Thr-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val UII-gamma: Gly-Gly-Gly-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Ile The results of thin-layer chromatography, HPLC, amino acid analysis, and sequencing indicate that UII-alpha and -gamma are homogeneous. UII-beta appears, however, to be a mixture of two components, differing only at position 2. Thus, in the carp urophysis, four forms of UII appear to be present, although the separation of two components in UII-beta has not been obtained. Sequence of positions 6-11 is common to all forms of UII isolated from the carp, sucker (Catostomus commersoni) and goby (Gillichthys mirabilis).

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.

Urotensin II-immunoreactive neurons in the caudal neurosecretory system of freshwater and seawater fish

SummaryAntiserum generated against synthetic urotensin II of the goby, Gillichthys mirabilis, was used to localize urotensin II in the caudal neurosecretory system in six species of freshwater teleosts; Cyprinus carpio, Carassius auratus, Oreochromis mossambicus, Oreochromis niloticus, Salmo gairdneri and Plecoglossus altivelis, and six species of seawater teleosts: Acanthogobius flavimanus, Pagrus major, Paapristipoma trilineatum, Trachurus japonicus, Seriola dumerili and Seriola quinqueradiata. In the carp, urotensin II-immunoreactive perikarya were classified into three groups according to their size and shape. Small cells were located in the spinal cord dorsal to the urophysis, medium-sized cells immediately anterior to the urophysis, and large cells anterior to the medium-sized cells. In each group, a small number of nonreactive cells was found. Urotensin II-immunoreactive nerve fibers extended toward the urophysis and terminated around the blood vessels. Other species of teleosts showed a similar immunoreaction to that observed in the carp. The immunoreaction of the urophysis was stronger in seawater fish than freshwater fish. Urotensin II-immunoreactive elements could not be detected in the brains of the carp, goldfish and goby.

Immunohistochemical investigation of urotensins in the caudal spinal cord of four species of elasmobranchs and the lamprey, Lampetra japonica.

SummaryIn the four species of elasmobranchs examined (Triakis scyllia, Heterodontus japonicus, Scyliorhinus torazame, Dasyatis akajei), all identifiable caudal neurosecretory cells and their corresponding neurohemal areas showed urotensin II (UII)-immunoreactivity with varied intensity. To localize urotensin I (UI) in the caudal neurosecretory system of the dogfish, Triakis scyllia, h-CRF (1–20) antiserum that cross-reacts with UI was used in place of UI antiserum. CRF/UI-immunoreactivity was demonstrated in the neurosecretory cells and neurohemal areas. A considerable number of neurons showed both UII- and CRF/UI-immunoreactivities, suggesting that UII and UI are produced in the same neurosecretory cells. However, some neurons exhibited UII-immunoreactivity, but no CRF/UI-immunoreactivity. Cells immunoreactive only to CRF antiserum were not detected. At least two populations of neurons exist in the dogfish caudal neurosecretory system: (i) cells immunoreactive for both CRF/UI and UII, and (ii) cells immunoreactive for UII. The dorsal cells of the lamprey, Lampetra japonica, did not react with either UII or CRF antiserum.

Histochemical distribution of peroxidase in amphioxus and cyclostomes with special reference to the endostyle

The histochemical distribution of peroxidase was studied in amphioxus, ammocoetes larvae, adult lampreys, and hagfish. The endostyle of amphioxus displayed peroxidase activity in zone 5 and, in some individuals, in zone 1 as well. The endostyle of ammocoetes exhibited strong peroxidase activity in type 2c and type 3 cells. These peroxidase-positive regions coincide well with radioiodine-binding regions previously described. Thyroid follicles of adult lampreys stained strongly for peroxidase, but those of the hagfish did not. The branchial sac of amphioxus showed peroxidase activity, but the gill sac of cyclostomes did not. The intestine did not show peroxidase activity in amphioxus or in cyclostomes.

Water intake induced by water deprivation in the quail, Coturnix coturnix japonica.

SummaryMechanisms inducing drinking after water deprivation, and mechanisms terminating drinking after rehydration, were investigated in the quail,Coturnix coturnix japonica.1.Water intake was induced after 4 h of water deprivation, and the amount of water drunk increased in proportion to the period of water deprivation. Drinking occurred immediately after deprived birds were given access to water, and continued for periods proportional to the period of water deprivation.2.Plasma angiotensin II concentration increased, as did plasma osmolality and Na+ concentration, and blood volume decreased after water deprivation. The increase in plasma angiotensin II concentration and decrease in blood volume occurred soon after the start of water deprivation, whereas plasma osmolality and Na+ concentration did not increase until at least 4 h after the start of water deprivation.3.These results indicate that extracellular dehydration and angiotensin II are responsible for the significant drinking that follows 4 h of water deprivation, and that cellular dehydration is also involved in the stimulation of drinking that occurs after longer periods of water deprivation.4.Plasma osmolality and Na+ concentration in birds deprived of water for 48 h quickly returned to normal levels after the birds were allowed access to water. Plasma angiotensin II levels and blood volume also approached the values measured prior to water deprivation. However, the rate and degree of restoration of normal values were reduced, and normal values were not restored even after 1.5 h of rehydration when drinking terminated.5.The amount of water drunk over the course of 1.5 h by birds deprived of water for 48 h was much greater than the amount required to restore the changes in plasma osmolality and blood volume to normal, but neither excessive dilution of plasma nor abnormally high blood volumes were observed during drinking or 0.5 h after drinking terminated.

Histochemical distribution of peroxidase in ascidians with special reference to the endostyle and the branchial sac

The histochemical distribution of peroxidase was studied in 10 species of ascidians. In the endostyle, strong peroxidase activity was found in zone 7 in Ciona intestinalis, Ascidia zara, Ascidia sydneiensis samea, Cnemidocarpa areolata, Styela clava, and Pyrura vittata. The activity in zone 7 was weak in Styela plicata and Halocynthia hilgendorfi. Pyura michaelseni and Halocynthia roretzi showed only faint activity in zone 7, but showed strong activity in zone 9 and in the transitional zone, respectively. Pyura vittata exhibited peroxidase activity in zone 5 as well as in zone 7. Zone 8 was negative for peroxidase, but the cilia of zone 8 cells were distinctly stained for peroxidase in Ciona intestinalis and Cnemidocarpa areolata. These results show that wide species differences exist in the distribution of peroxidase in the ascidian endostyle. Peroxidase activity was also detected in the branchial sac, although here again wide species differences were noted in terms of peroxidase-positive sites. Peroxidase activity was also found in the postpharyngeal alimentary canal, but not in the tunic.

Immunohistochemical localization of urotensin I and other neuropeptides in the caudal neurosecretory system of three species of teleosts and two species of elasmobranchs.

SummaryIn three species of teleosts — carp Cyprinus carpio; grass carp Ctenopharyngodon idella; and crucian carp Carassius auratus — the caudal neurosecretory system displays small, medium-sized and large neurons. Urotensin I (UI)-immunoreactive and UI-nonreactive neurons were found in all three groups; in general, the number of the latter neurons exceeded that of the former. Noteworthy are: (i) UI-immunoreactive fibers in the caudal spinal cord and (ii) dense accumulations of UI-immunoreactive product around the capillaries of the urophysis. In two species of elasmobranchs — cat shark Heterodontus japonicas and swell shark Cephaloscyllium umbratile — neurosecretory neurons decreased in size in rostro-caudal direction. Most of the neurosecretory perikarya, their axons and the corresponding neurohemal areas were UI-immunoreactive, but a small number of secretory neurons was devoid of immunoreaction. Oxytocin, arginine vasopressin, substance P, somatostatin, neurotensin, vasoactive intestinal polypeptide and gastrin-releasing peptide were not detected in the caudal neurosecretory system of the carp.