Biomedical subjects

S Kikuyama

Publications and source records attributed to S Kikuyama.

At least 19 recordsLinked to original sources

PubMed1992

The alpha-subunit of glycoprotein hormones exists in the prolactin secretory granules of the bullfrog (Rana catesbeiana) pituitary gland.

Our recent finding that the number of immunoreactive alpha-subunit cells was invariably greater than the total number of immunoreactive gonadotropin (GTH) and thyrotropin (TSH) cells in the bullfrog (Rana catesbeiana) pituitary gland raises the possibility that the alpha-subunit also exists in pituitary cells other than GTH and TSH cells. The present study demonstrates that there are a considerable number of immunoreactive prolactin (PRL) cells that are also stained with antibody against the alpha-subunit when adjacent sections are immunocytochemically examined. Neither immunoreactive growth hormone nor adrenocorticotropin cells are stained with the antibody against the alpha-subunit. The specificity of the antibody against the alpha-subunit and of that against PRL was demonstrated by preabsorption test, non-competitive binding test, and immunoblot analysis. Double-immunolabeling with gold particles of different sizes for the alpha-subunit and PRL revealed that most of the immunolabeled PRL-secretory granules are also labeled with the alpha-subunit antibody. The gold particles indicating the presence of the alpha-subunit were mostly found in the peripheral zone of the secretory granules.

Animals↗

PubMed1992

The origin of the luteinizing hormone-releasing hormone (LHRH) neurons in newts (Cynops pyrrhogaster): the effect of olfactory placode ablation.

Neurons containing luteinizing hormone-releasing hormone (LHRH) are first detected in newt embryos (Cynops pyrrhogaster) in the olfactory epithelium and ventromedial portion of the olfactory nerve, after which they sequentially appear in the intracerebral course of the terminal nerve at prometamorphosis, and in the septo-preoptic area at postmetamorphosis. In adults, however, LHRH-immunoreactive cells are rarely seen in the nasal region, and their distribution shifts into the brain, suggesting their migration. In order to ascertain the origin and possible migration route of these neurons in newt larvae, the effect of unilateral or bilateral olfactory placodectomy on the LHRH neuronal system has been studied. Removal of the olfactory placode results in the absence of LHRH-immunoreactive cells in the nasal and brain regions of the operated side, whereas the subsequent growth and the LHRH-immunoreactive cellular distribution in the contralateral side are identical to those of normal larvae. Following bilateral placodectomy, no LHRH immunoreactivity is detected on either side of the olfactory-brain axis. These results suggest that LHRH neurons of the newt, Cynops pyrrhogaster, originate in the olfactory placode and then migrate into the brain during embryonic development.

Animals↗

PubMed1992

Hormonal control of in vitro vitellogenin synthesis in Rana esculenta liver: effects of mammalian and amphibian growth hormone.

Estradiol 17-beta is known to induce hepatic synthesis and secretion of vitellogenin in all species studied and in Rana esculenta, previous experiments demonstrated the involvement of pituitary in these processes; indeed, in addition to estradiol 17-beta, homologous pituitary homogenate directly stimulated male and female liver to produce vitellogenin in tissue cultures. Therefore, the effect of ovine growth hormone (o-GH) and Rana catesbeiana growth hormone (f-GH) on hepatic vitellogenin synthesis was investigated. In the present in vitro experiments, both o-GH and f-GH positively stimulated vitellogenin synthesis, in female and male liver, in a dose-related fashion. No significant differences were found in VTG levels induced by o-GH and f-GH. The GH stimulatory effects, found during the different phases of the reproductive cycle, displayed different trends related to season and sex.

Animals↗

PubMed1992

Pharmacological characterization of vasotocin stimulation of phosphoinositide turnover in frog adrenal gland.

In a previous report we demonstrated the presence of a vasotocin (AVT)-like peptide in chromaffin cells of the amphibian adrenal gland and showed that synthetic AVT is a potent stimulator of corticosterone and aldosterone secretion by frog adrenocortical cells. In the present study we evaluated the relative potency of various AVT analogs and investigated the mechanism of action of AVT on frog interrenal (adrenal) tissue. Several AVT agonists, including hydrin 2, oxytocin (OXT), arginine vasopressin (AVP), Lys-conopressin G, and mesotocin (MT), were able to mimic the stimulatory effect of AVT on steroid secretion, but AVT was by far the most potent stimulator of steroidogenesis. In the series of analogs studied, the order of potency was: AVT greater than hydrin 2 greater than OXT greater than AVP greater than Lys-conopressin G greater than MT greater than [deamino-Cys1,D-Arg8]AVP greater than [d(CH2)5,Tyr(OMe)2] AVP. The effect of AVT (5 x 10(-10) M) was totally blocked by both the antidiuretic V2 antagonist [d(CH2)5,D-Phe2,Ile4,Ala9-NH2]AVP (10(-6) M) and the oxytocinergic antagonist [d(CH2)5,Tyr(OMe)2,Orn8]AVT (10(-6) M); the V2 antagonist was approximately twice as potent as the OXT antagonist. In contrast, the V1 antagonist 1-(1-mercapto-4-phenylcyclohexaneacetic acid)-AVP (10(-6) M) did not affect the response of the interrenal tissue to AVT. Indomethacin (5 microM), a cyclooxygenase inhibitor, induced a dramatic decrease in the spontaneous secretion of corticosteroids, but did not impair the stimulatory effect of AVT (5 x 10(-9) M) on corticosterone and aldosterone secretion. In addition, AVT did not stimulate the production of prostaglandin E2, suggesting that prostaglandins are not involved in the mechanism of action of AVT. Concurrently, AVT did not modify cAMP production by frog adrenal slices. In contrast, AVT induced both an increase in inositolphosphate production and a reduction of membrane phospholipid content. We conclude that in the frog adrenal gland, the stimulatory effect of AVT on steroid secretion is mediated through activation of receptors related to the mammalian V2 and/or OXT receptors, which are positively coupled to phosphoinositide-specific phospholipase C.

Adrenal Cortex Hormones↗

PubMed1992

Cloning of a bullfrog growth hormone cDNA: expression of growth hormone mRNA in larval and adult bullfrog pituitaries.

A GH cDNA was specifically amplified from cDNAs constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses employing the DNA polymerase chain reaction. Sequencing analysis revealed that the cDNA clone thus obtained was 654 bp in length, and included an open reading frame encoding the entire sequence of mature GH, with its signal peptide. Slight discrepancies were noted between the deduced amino acid sequence and that determined by direct protein sequencing of purified bullfrog GH or that deduced from the nucleotide sequence reported previously. The length of the bullfrog GH mRNA was estimated to be about 1.2 kb by Northern blot analysis. Homologies of nucleotide and amino acid sequences between GH and prolactin of bullfrog origin were 48% and 26% respectively. Using the cDNA as a probe, the content of GH mRNA in the pituitary of larval and adult bullfrogs was measured. GH mRNA levels were relatively low at the preclimax stage, and rose markedly during climax. In juvenile frogs, GH mRNA levels in the pituitary were extremely high and declined towards adulthood. This finding suggests that the increase in plasma and pituitary GH levels reported previously accompanies the increase in GH synthesis.

Amino Acid Sequence↗

PubMed1991

The complete amino acid sequence of growth hormone of the bullfrog (Rana catesbeiana).

The primary structure of growth hormone (GH) isolated from the adenohypophysis of the bullfrogs (Rana catesbeiana) was determined. The hormone was reduced, carboxymethylated and subsequently cleaved with cyanogen bromide. Intact bullfrog GH was also digested with lysyl endopeptidase and trypsin. The resulting fragments were separated by reverse-phase high-performance liquid chromatography and subjected to sequence analysis using an automated gas-liquid sequencer employing the Edman method. Bullfrog GH was found to consist of 190 amino acid residues. The amino acid sequence determined is in accord with that deduced from bullfrog GH cDNA by Pan and Chang (1988) except for nine residues at positions 43-48, 73, 80 and 87. Sequence comparisons revealed that bullfrog GH is more similar to tetrapod GHs (e.g., 69% homology with sea turtle GH, 66% with chicken GH and 61% with ovine GH) than to GHs of teleosts (e.g., 35% homology with chum salmon GH and 33% with bonito GH) except for eel (52% identity). Bullfrog GH and prolactin exhibit a sequence homology of 25%.

Amino Acid Sequence↗

PubMed1991

The complete amino acid sequence of prolactin from the bullfrog, Rana catesbeiana.

The complete amino acid sequence of prolactin (PRL) from an amphibian species, the bullfrog (Rana catesbeiana), has been determined and conserved residues and domains were analyzed by sequence comparison of PRLs from 15 species of five major vertebrate classes. The bullfrog PRL consists of 197 amino acid residues with three disulfide linkages formed between residues 4-11, 58-172, and 189-197. The bullfrog PRL shows the highest identity with sea turtle PRL (75%); lower identities with chicken PRL (72%), pig, horse, and fin whale PRLs (68%), human, cattle, sheep, and elephant PRLs (60-58%), and rat and mouse PRLs (50%); and significantly lower identity with teleost PRLs (about 30%). It is apparent that all tetrapod PRLs characterized so far contain three disulfide bonds in homologous positions and differ from teleost PRLs which lack the N-terminal disulfide loop. The tetrapod and teleost PRLs share 34 common residues and these conserved residues are clustered in six domains (PD1 to PD6), suggesting that these common residues, or at least part of them, are responsible for the activities common to all PRLs. On the other hand, PD5 is conserved significantly within tetrapod PRLs, but to a lesser extent in teleost PRLs, suggesting that the PD5 contributes to the activities specific to tetrapod PRLs.

Amino Acid Sequence↗

PubMed1991

Arginine vasotocin (AVT) and AVT-related peptide are major aldosterone-releasing factors in the bullfrog neurointermediate lobe.

Two major components which stimulate aldosterone release from Xenopus adrenocortical tissue were isolated from an acid-acetone extract of the neurointermediate lobes of the bullfrog (Rana catesbeiana) using C18 Sep-Pak cartridges, Sephadex G-50, and reverse-phase HPLC columns. One of the components was identified as arginine vasotocin (AVT) from its HPLC profile and amino acid sequence analysis. The other was an AVT-like decapeptide with an extra glycine residue at the C-terminus of nonamidated AVT, which was recently termed hydrin 2. The yields of these two peptides were almost the same. They also showed equipotent activity in stimulating water flux from the isolated urinary bladder of the toad (Bufo japonicus).

Aldosterone↗

PubMed1991

Distribution and characterization of immunoreactive growth hormone (GH) in the pituitary of the frog Rana ridibunda using an antiserum against purified bullfrog GH.

The presence of growth hormone (GH) in the pituitary of the frog Rana ridibunda was investigated using an antiserum raised against purified bullfrog GH. The immunofluorescence technique revealed that GH-containing cells are exclusively located in the dorsal area of the distal lobe of the pituitary. The relative abundance of these GH-positive cells, which correspond to acidophilic type 2 cells, was 18 +/- 1% of the total population of endocrine cells of the pars distalis. Frontal sections of the distal lobe indicated that GH-producing cells are distributed in an arc of a circle occupying all of the dorsal part of the lobe. At the electron microscopic level, GH-immunoreactive material was sequestered in large polymorphic granules (200-700 nm). GH was quantified in R. ridibunda pituitary extracts using a radioimmunoassay for bullfrog GH. The displacement curves obtained with serial dilutions of pars distalis extracts were not strictly parallel to the standard curve made with purified bullfrog GH. In contrast, Western blot analysis revealed that GH from R. ridibunda had a molecular weight (22 kDa) similar to that of bullfrog GH. In the pars distalis, the apparent amount of GH was 0.61 +/- 0.14 microgram per lobe, corresponding to 0.92 +/- 0.17% of total proteins in the extracts. In contrast, frog neurointermediate lobe or hypothalamus did not contain significant concentrations of immunoreactive GH (less than 0.006% of total proteins in the extracts). Taken together, these results validate the use of an antiserum to bullfrog GH to investigate the regulation of GH secretion in R. ridibunda.

Animals↗

PubMed1991

Involvement of prolactin in the regulation of plasma calcium levels in the newt, Cynops pyrrhogaster.

In the newt, Cynops pyrrhogaster, parathyroidectomy (PTX) brought about a marked decrease in the concentration of plasma calcium. The animals recovered from the hypocalcemia by 15 days after the operation if the pituitary gland was left intact. After PTX, no significant changes in the plasma sodium concentration were observed. Experiments were then conducted to obtain direct evidence that endogenous prolactin (PRL) is involved in this recovery process. Recovery of the calcium level after PTX was blocked by administration of an antiserum raised against newt PRL. In newts deprived of both the pituitary and parathyroid glands, no recovery from hypocalcemia was observed. Administration of newt or ovine PRL to parathyroidectomized-hypophysectomized newts significantly elevated the blood calcium level. After PTX, the concentration of immunoassayable PRL in the blood rose to 10 times the value in sham-operated animals. These results indicate the involvement of PRL in calcium homeostasis in newts with a shortage of parathormone.

Animals↗

PubMed1991

Amphibian prolactins: activity in the eft skin transepithelial potential bioassay.

The effects of purified prolactins isolated from frogs (fPRL; Rana catesbeiana) and newts (nPRL; Cynops pyrrhogaster) were compared with those of ovine prolactin (oPRL) and thyrotropin releasing hormone (TRH) in the eft (Notophthalmus viridescens) skin transepithelial potential (TEP) bioassay. At total doses as low as 0.4 micrograms/animal, both fPRL and nPRL were as effective as oPRL in reducing eft skin TEP. By contrast, TRH at total doses as high as 400 micrograms/animal was ineffective in altering eft skin TEP. These data provide the first direct evidence that amphibian PRLs can exert significant physiological control over salamandrid integumental sodium transport.

Animals↗

PubMed1991

Homologous radioimmunoassay for bullfrog growth hormone.

Antiserum against bullfrog growth hormone (fGH) was produced by immunizing rabbits with the highly purified fGH obtained from adenohypophyses of adult bullfrogs. Histological studies on bullfrog adenohypophyses revealed that the cells that immunologically reacted with the antiserum against fGH corresponded to the ones positively stained with the antiserum against rat GH. The antiserum together with fGH and 125I-fGH was employed to develop a radioimmunoassay (RIA) for fGH. Several dilutions of plasma and of pituitary homogenate of both adult and larval bullfrogs yielded dose-response curves which were parallel to the standard curve. Ovine prolactin (PRL), and growth hormone (GH); eel and salmon GHs; and bullfrog LH, FSH, TSH, PRL, and neurointermediate lobe homogenate did not react in this assay. Plasma from hypophysectomized bullfrogs had no detectable immunoreactive GH. Pituitary homogenates of Bufo japonicus, Xenopus laevis, and Cynops pyrrhogaster gave inhibition curves which did not parallel the standard. The homologous RIA for bullfrog GH thus developed was applied for the determination of plasma and pituitary GH levels in the larvae and adults. Plasma GH levels were relatively low during preclimax period and rose as metamorphosis progressed. Plasma GH concentrations were maximum in the juvenile frogs and decreased as the animals grew up. Pituitary GH concentrations also increased as metamorphosis progressed. After metamorphosis, pituitary GH concentrations declined as the frogs gained weight. There was no sex difference in plasma and pituitary GH levels in the adult.

Aging↗

PubMed1990

Development and application of homologous radioimmunoassay for newt prolactin.

A specific and sensitive homologous radioimmunoassay (RIA) for newt (Cynops pyrrhogaster) prolactin (PRL) was developed. PRL isolated from newt pituitary glands was used for generating antiserum in a rabbit, for radioiodination, and for the standard. Several dilutions of plasma and pituitary homogenate of newts yielded dose-response curves which were parallel to the standard curve. Plasma from hypophysectomized newts showed the least amount of cross-reaction. Pituitary homogenates of other species of urodeles such as Ambystoma mexicanum and Onychodactylus japonicus gave inhibition curves which were parallel to the standard curve. Purified PRLs of anurans such as Rana catesbeiana and Bufo japonicus gave inhibition curves which did not parallel the standard. Bovine PRL and ovine PRL showed no inhibition of binding even at relatively high doses in this RIA. The RIA was applied to the determination of plasma and pituitary PRL levels in the adult newts treated with dopamine agonist (bromocriptine) and/or antagonist (pimozide). Pimozide enhanced PRL levels and bromocriptine antagonized it, while pituitary PRL levels were not appreciably changed.

Animals↗

PubMed1990

Purification and properties of newt prolactin.

A highly purified prolactin (PRL) was obtained from pituitary glands of newts, Cynops pyrrhogaster, by extraction of acetone-dried powder with acid acetone and high-performance liquid chromatography (HPLC) on Mono-Q (anion exchange), Superose-12 (gel filtration), and TSK-gel ODS-120T (reverse-phase) columns with a yield of 4.5 mg per 325 mg of protein starting material. Purification was monitored by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting analysis employing antiserum against bullfrog PRL. Newt PRL thus obtained has a molecular weight of 23,000 as determined by SDS-PAGE. The isoelectric point is 4.7 as determined by isoelectric focusing. The amino acid composition closely resembles that of anuran PRLs. This hormone was as potent as bovine PRL in stimulating collagen synthesis in the bullfrog tadpole tail fin. Antiserum against newt PRL was produced by immunizing a rabbit. Histological studies on newt adenohypophyses revealed that the cells that immunologically reacted with the antiserum against newt PRL correspond to the ones positively stained with the antiserum against bullfrog PRL.

Amino Acids↗

PubMed1990

Molecular cloning and nucleotide sequence analysis of complementary DNA for bullfrog prolactin.

A prolactin cDNA was cloned from a cDNA expression library constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses by immunoscreening with antiserum against bullfrog prolactin. The cDNA clone thus obtained contained a 249 bp insert. Using this clone as a probe, plaque hybridizations were performed and two additional clones obtained. These clones had a polyadenylation site different from that of the first obtained clone, suggesting that the 3'-untranslated sequence was heterogeneous in length. The longest clone contained 830 bp, which encoded part of the signal peptide and the entire sequence of mature prolactin. The deduced amino acid sequence was in good accord with that determined by direct protein sequencing of purified bullfrog prolactin. The length of the bullfrog prolactin mRNA was estimated by Northern blot analysis to be about 1.0 kb. Homologies of prolactin nucleotide and amino acid sequences between bullfrog and other vertebrates were 64 and 65% for man, 66 and 68% for pig, 61 and 52% for rat, 69 and 74% for chicken, and 50 and 35% for salmon respectively. Highly conserved regions reported for mammalian prolactins also existed in bullfrog prolactin. Homologies of nucleotide and amino acid sequences between prolactin and GH of bullfrog origin were 49 and 25% respectively. Using the cDNA, the content of prolactin mRNA in the pituitary glands of metamorphosing tadpoles was measured. Prolactin mRNA levels rose at the mid-climax stage, suggesting that the increase in plasma and pituitary prolactin levels known to occur at the climax stage accompanies the increase in prolactin synthesis.

Amino Acid Sequence↗

PubMed1989

Development of Xenopus laevis skin glands producing 5-hydroxytryptamine and caerulein.

The granular glands in Xenopus laevis skin are known to contain large quantities of biogenic amines and bioactive peptides which closely resemble mammalian brain-gut peptides. We studied the development of glands producing 5-hydroxytryptamine (5-HT) and caerulein using immunohistochemistry, HPLC-fluorometric systems and RIA. The immunoreactivities of 5-HT and caerulein were first detected in the spherical gland rudiments in the stratum spongiosum at St. 58 (Nieuwkoop and Faber stage), or at the beginning of metamorphosis. Both immunoreactivities appeared in the same rudiment at the same time. Some of the gland rudiments have a small lumen filled with both immunoreactive materials at St. 58-59. During the rest of the metamorphic period, the glands grow in size, accumulating immunoreactive materials in the lumen. The concentrations of 5-HT and caerulein in the skin of tadpoles were below 1 ng per mg wet tissue at St. 58-59, increased as metamorphosis proceeded and reached 63 and 134 ng per mg wet tissue at St. 66, or at the end of metamorphosis, respectively. The amphibian granular glands where large quantities of biogenic amines and hormone-like peptides are rapidly synthesized may provide a useful model for the study of the development of amine- and peptide-producing cells including neurons and paraneurons.

Animals↗

PubMed1989

Effects of vertebrate prolactins and growth hormones on thyroxine 5'-monodeiodination in the eel (Anguilla anguilla): a potential bioassay for growth hormone.

Growth hormones (GHs) and prolactins (Prls) purified from representatives of each vertebrate class from bony fish onwards were tested for their ability to stimulate in vivo peripheral deiodination of labeled thyroxine (T4*) into triiodo-L-thyronine (T3*) in the eel. Plasma T3*/T4* ratio was used as parameter. All GHs significantly increased T3*/T4*, the magnitude of the response being unrelated to the phylogenic position of species. No significant stimulation was shown with the various Prl, with the exception of ovine Prl, suggesting a heterosomatotropic effect of this preparation in the eel. Furthermore, both tilapia and ovine GH produced a dose-related effect on plasma T3*, T4*, and T3*/T4*. The stimulation of the peripheral deiodination of T4* into T3* estimated in vivo in the eel could become a specific, sensitive, and rapid fish bioassay for GH.

Anguilla↗

PubMed1989

Localization and characterization of prolactin-like immunoreactivity in the pituitary of the frog Rana ridibunda.

Distribution and quantification of PRL in the pituitary gland of the frog Rana ridibunda were investigated using a high-affinity antiserum raised against bullfrog prolactin (PRL). The immunoreactive PRL-producing cells were distributed throughout the pars distalis, the highest density of cells being observed in the rostral region of the adenohypophysis facing the neurointermediate lobe. The dorsal region of the pars distalis contained only a few scattered PRL-immunoreactive cells. At the electron microscopic level, PRL-containing cells were visualized using the immunogold procedure. PRL-immunoreactive material was exclusively stored in secretory granules (size ranging from 200 to 700 nm in diameter). Neither the rough endoplasmic reticulum nor the Golgi apparatus were immunolabeled. Using a radioimmunoassay method we have compared the displacement curves obtained with bullfrog PRL and acetic extracts from Rana ridibunda pituitary. The two binding curves were not completely parallel, suggesting the existence of slight variations of the amino acid sequences of PRL in the two species. The concentration of PRL in the green frog adenohypophysis appeared somewhat higher (35.3 +/- 8.8 micrograms/mg protein) than that in the bullfrog pituitary. These results validate the use of an antiserum to bullfrog PRL to investigate the regulation of PRL secretion in Rana ridibunda.

Animals↗