Biomedical subjects

I Shibuya

Publications and source records attributed to I Shibuya.

At least 73 records · Page 4Linked to original sources

PubMed1997

Enhancement by baclofen of the Gs-coupled receptor-mediated cAMP production in Xenopus oocytes expressing rat brain cortex poly (A)+ RNA: a role of G-protein beta gamma subunits.

We investigated the mechanism by which GABA-B receptors enhance the Gs-coupled receptor-mediated cAMP production in Xenopus oocytes expressing poly (A)+ RNA derived from rat brain cortex. We expressed the cystic fibrosis transmembrane conductance regulator gene (CFTR) as a reporter for cAMP changes in oocytes. The GABA-B agonist (-)baclofen enhanced the adrenergic beta 2 agonist isoproterenol- or vasoactive intestinal peptide (VIP)-induced CFTR currents, whereas (-)baclofen alone did not cause any currents. The (-)baclofen-enhanced currents were inhibited by the GABA-B antagonist 2-OH saclofen. The enhancement by (-)baclofen was further augmented by coexpressing adenylyl cyclase (AC) type II, an isotype activated by G beta gamma and G alpha s, but not by coexpressing AC type III, an isotype insensitive to G beta gamma. Moreover, pretreatment of the oocytes with pertussis toxin (PTX) abolished the enhanced effect of (-)baclofen. These results indicate that upon GABA-B activation, the G beta gamma released from PTX-sensitive G-proteins activates the AC type II (or IV), and this process requires the G alpha s activation by Gs-coupled receptors.

Adrenergic beta-Agonists↗

PubMed1997

PTH-related peptide-like immunoreactivity in the median emminence, paraventricular and supraoptic nuclei in colchicine-treated rats.

The existence of PTH-related peptide (PTHrP) in the hypothalamus was examined by immunohistochemistry in colchicine-treated rats. Two days after intracerebroventricular administration of colchicine dense PTHrP-like immunoreactivity (LI) was observed in the external zone of the median emminence (ME). PTHrP-LI cells were found in the paraventricular nucleus, the supraoptic nucleus and the periventricular region of the third ventricule. The effects of PTHrP on intracellular Ca2+ concentrations ([Ca2+]i) were examined by a Ca2+ imaging method using fura-2 in perifused preparations of isolated rat anterior pituitary cells. The rise in [Ca2+]i induced by PTHrP was found in approximately 17% of the cells examined. These results suggest that PTHrP-LI cells in the hypothalamus may project to the ME and contribute to the anterior pituitary function.

Animals↗

PubMed1997

Upregulation of the expression of vasopressin gene in the paraventricular and supraoptic nuclei of the lithium-induced diabetes insipidus rat.

The expression of arginine vasopressin (AVP) gene in the paraventricular (PVN) and supraoptic nuclei (SON) was investigated in rats with lithium (Li)-induced polyuria, using in situ hybridization histochemistry and radioimmunoassay. The male Wistar rats consuming a diet that contained LiCl (60 mmol/kg) for 4 weeks developed marked polyuria. The Li-treated rats produced a large volume of hypotonic urine with low ionic concentrations. Plasma sodium concentrations were found to be slightly increased in the Li-treated rats compared with those in controls. Plasma concentration of AVP and transcripts of AVP gene in the PVN and SON were significantly increased in the Li-treated rats compared with controls. These results suggest that dehydration and/or the activation of visceral afferent inputs may contribute to the elevation of plasma AVP and the upregulation of AVP gene expression in the PVN and the SON of the Li-induced diabetes insipidus rat.

Animals↗

PubMed1997

Inhibition of spontaneous EPSCs and IPSCs by presynaptic GABAB receptors on rat supraoptic magnocellular neurons.

1. The function of presynaptic GABA receptors in the regulation of transmitter release in supraoptic nucleus (SON) magnocellular neurons was investigated by recording spontaneous postsynaptic currents from rat magnocellular SON neurons in a slice preparation (150 microns thick, 1.8 mm in diameter) using the whole-cell patch-clamp technique. 2. Both the spontaneous EPSCs and IPSCs were TTX resistant. The EPSCs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the IPSCs were abolished by picrotoxin, suggesting that the EPSCs and IPSCs are synaptic inputs from glutamatergic and GABAergic neurons, respectively. 3. The selective GABAB agonist, baclofen, reduced the frequency of both the EPSCs and IPSCs without affecting the amplitude. The time constant of the decay phase of both the EPSCs and IPSCs remained unchanged after baclofen application. 4. The reduction of the frequency of the synaptic currents by baclofen was dose dependent (10 nM to 100 microM) and the EC50 values were 5.8 and 8.5 microM for the EPSCs and IPSCs, respectively. 5. The effect of baclofen (10 microM) was antagonized by the selective GABAB antagonist, 2-hydroxy-saclofen (2OH-saclofen), at 300 microM. 6. When given alone, 2OH-saclofen (100 microM) increased the frequency of both the EPSCs and IPSCs without affecting their amplitude, suggesting that endogenously released GABA in the slice acts on presynaptic GABAB receptors. 7. The GABAA agonist, muscimol, reduced the frequency of EPSCs, and picrotoxin increased the frequency of the EPSCs, suggesting that GABAA receptors also participate in the presynaptic inhibition of glutamate release. 8. Taken together, these data suggest that GABAB receptors are present on the presynaptic terminals of both GABA and glutamate neurons in the SON, and that these presynaptic GABAB receptors play an important role in the regulation of the neuronal activity in SON magnocellular neurons.

Animals↗

PubMed1997

Increase of urocortin-like immunoreactivity in the rat supraoptic nucleus after dehydration but not food deprivation.

The effects of dehydration and food deprivation on urocortin-like immunoreactivity (Ucn-IR) in the rat supraoptic nucleus (SON) and the paraventricular nucleus (PVN) were examined by immunohistochemistry. Water deprivation for 48 h caused a significant increase in the number of Ucn-IR neurons in the SON, compared with control. Ucn-IR fibers and varicosities in the SON and the internal zone of the median eminence (ME) were increased, but a few and faint Ucn-IR neurons and fibers were observed in the PVN. On the other hand, food deprivation for 48 h caused a significant decrease in the number of Ucn-IR neurons in the SON, compared with control. Ucn-IR fibers and varicosities in the SON and the ME were fewer than those in controls. Ucn-IR neurons and fibers in the PVN were not detected after food deprivation. These results suggest that Ucn in the SON may be involved in the central regulation of water balance and nutrient homeostasis.

Animals↗

PubMed1997

Increase of urocortin-like immunoreactivity in the rat hypothalamo-neurohypophysial system after salt loading and hypophysectomy.

The effect of chronic salt loading on urocortin-like immunoreactivity (Ucn-IR) was investigated in the rat hypothalamo-neurohypophysial system. In control rats a few Ucn-IR neurons were observed scattered throughout the supraoptic nucleus (SON) and few in the paraventricular nucleus (PVN). A small number of Ucn-IR fibers were observed scattered in the median eminence (ME) and the posterior pituitary. However, after 5 days of chronic administration of 2% saline, a marked increase in the number of Ucn-IR perikarya and fibers was observed in the PVN and the SON. Additionally, Ucn-IR varicosities and fibers were found in the internal zone of the ME and in the posterior pituitary. To confirm the findings and examine the possible involvement of anterior pituitary function in synthesis of Ucn, surgical hypophysectomized rats were used. Five days after hypophysectomy, a marked increase in Ucn-IR was observed in the PVN, the SON, and both the internal and the external zone of the ME. These results suggest that Ucn in the hypothalamo-neurohypophysial system may be involved in the regulation of salt balance, and possibly in the stimulation of ACTH release from the anterior pituitary.

Animals↗

PubMed1997

Both GABAA and GABAB receptors participate in suppression of [CA2+]i pulsing in toad melanotrophs.

The receptor mechanisms involved in the inhibitory effect of gamma-aminobutyric acid (GABA) in suppressing spontaneous [Ca2+]i pulsing in melanotrophs of Xenopus laevis were investigated. The selective GABAB receptor agonist, baclofen reversibly arrested [Ca2+]i pulsing. This inhibition was unaffected by the selective GABAA receptor antagonist, bicuculline methiodide, but was blocked by the selective GABAB receptor antagonist, CGP 35348 (3-aminopropyl diethyoxymethyl phosphinic acid). The selective GABAA receptor agonist, muscimol, also arrested [Ca2+]i pulsing after causing a transient rise in [Ca2+]i. This biphasic response to muscimol was unaffected by CGP 35348, but was blocked by bicuculline. The inhibitory effect of GABA was unaffected by either CGP 35348 or bicuculline when given alone, but was blocked by both antagonists given together. In cells pretreated with pertussis toxin, the response to baclofen was completely lost, whereas responses to GABA and muscimol persisted; the response to GABA was blocked by bicuculline alone. Thus, both GABAA and GABAB receptors are involved in the inhibitory effect of GABA in suppressing spontaneous [Ca2+]i pulsing in Xenopus melanotrophs.

Animals↗

PubMed1997

Unbalanced membrane phospholipid compositions affect transcriptional expression of certain regulatory genes in Escherichia coli.

The amount of porin protein OmpF in the outer membrane of Escherichia coli was reduced to one-third by the pgsA3 mutation that diminishes the amount of phosphatidylglycerol and cardiolipin in the membrane, whereas a cls (cardiolipin synthase) null mutation had no effect. Osmoregulation of OmpF was functional in the pgsA3 mutant. As assessed by the beta-galactosidase activities of lacZ fusions, the ompF expression was not reduced at the transcriptional level but was reduced about threefold at the posttranscriptional level by pgsA3. This reduction was mostly restored by a micF null mutation, and the micF RNA that inhibits the ompF mRNA translation was present 1.3 to 1.4 times more in the pgsA3 mutant, as assayed by RNase protection and Northern blot analyses. Elevation of the level of micF RNA was not restricted to acidic-phospholipid deficiency: OmpF was hardly detected and micF RNA was present 2.7 to 2.8 times more in a pssA null mutant that lacked phosphatidylethanolamine. Other common phenotypes of pgsA3 and pssA null mutants, reduced rates of cell growth and phospholipid synthesis, were not the cause of micF activation. Salicylate, which activates micF expression and inhibits cell motility, did not repress the flagellar master operon. These results imply that an unbalanced phospholipid composition, rather than a decrease or increase in the amount of specific phospholipid species, induces a phospholipid-specific stress signal to which certain regulatory genes respond positively or negatively according to their intrinsic mechanisms.

Bacterial Outer Membrane Proteins↗

PubMed1997

Water deprivation increases the expression of pituitary adenylate cyclase-activating polypeptide gene in the rat subfornical organ.

The effect of water deprivation on the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) was examined in the rat subfornical organ (SFO), using a combination of immunohistochemistry and in situ hybridization histochemistry. In the euhydrated condition, PACAP-immunoreactivity (PACAP-IR) and the expression of PACAP gene was observed in the SFO. Water deprivation for 24 h and 48 h caused a significant increase in PACAP gene transcripts in the SFO, compared with euhydrated animals. Additionally, water deprivation for 48 h caused an increase in PACAP-IR. This increase of PACAP-IR was demonstrated in both nerve fibers and cell bodies. High correlation was found between the localization of PACAP-IR cell bodies and PACAP messenger RNA synthesizing cell bodies in the peripheral part of the SFO. These results suggest that PACAP in the SFO may play a role in the humoral and neural changes associated with the regulation of body fluid balance after water deprivation.

Analysis of Variance↗

PubMed1997

Pituitary adenylate cyclase-activating polypeptide potentiation of Ca2+ entry via protein kinase C and A pathways in melanotrophs of the pituitary pars intermedia of rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to stimulate melanotroph secretion, and PACAP-like immunoreactivity and expression of PACAP type I receptor messenger RNA have been identified in the pituitary pars intermedia (PI). The present study showed that PACAP messenger RNA is also expressed in the PI. To examine the mechanism of PACAP action in the PI, cytosolic Ca2+ concentrations ([Ca2+]i) and ionic currents were measured in acutely dissociated rat melanotrophs. In about 40% of the melanotrophs studied, PACAP induced an increase in [Ca2+]i, which was suppressed by extracellular Ca2+ removal; extracellular Na+ replacement; the blocker of L-type Ca2+ channels, nicardipine; or the secreto-inhibitory neurotransmitter, dopamine. The PACAP-induced [Ca2+]i increase was mimicked by activators of protein kinase A (PKA) and protein kinase C (PKC), Sp-diastereomer of cAMP and 1-oleoyl-2-acetyl-sn-glycerol, and was reduced by inhibitors of PKA and PKC, Rp-diastereomer of cAMP and staurosporine. Patch-clamp analysis revealed that PACAP caused inward currents with a reversal potential of -0.8 mV and facilitated voltage-dependent Ba2+ currents. It further revealed that PACAP-induced inward currents were mimicked by 1-oleoyl-2-acetyl-sn-glycerol and inhibited by staurosporine, and that Sp-diastereomer of cAMP facilitated Ba2+ currents. These results suggest that PACAP potentiates Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA.

Animals↗

PubMed1997

Production of recombinant Der fI with the native IgE-binding activity using a baculovirus expression system.

Der fI is a cysteine protease contained in feces of mites and is one of major mite allergens. Recombinant Der fI (reDer fI) that is produced using a baculovirus expression system contains pro-sequences of different lengths. Most of these can be removed by acid treatment. However, IgE-binding activity of acid-treated reDer fI is lower than that of native Der fI at high protein concentrations, and N-terminal amino acids of acid-treated reDer fI are not uniform. Now, a method for processing of the pro-sequence has been developed by producing reDer fI E(-1)K with baculovirus expression system in which the carboxy terminal amino acid of the pro-sequence (glutamate) was replaced by lysine using site directed mutagenesis. No difference in the amount of production was observed upon introducing the mutation into the pro-sequence. Addition of lysylendopeptidase into the culture medium led to processing of the pro-sequence of reDer fI E(-1)K and proceeded the degradation of the other proteins in the medium. Lysylendopeptidase-treated reDer fI E(-1)K was easily purified with an anion exchange column, resulting in 20% increase of the yield. Lysylendopeptidase-treated reDer fI E(-1)K obtained through these processes was compared with the native Der fI. Although some differences were found in protease activity and reactivity with lectins, their N-terminal amino acid and the IgE-binding activity were the same as those of the native one, indicating its usefulness for diagnostic purpose.

Animals↗

PubMed1996

PACAP type I receptor gene expression in the paraventricular and supraoptic nuclei of rats.

We have examined the distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor gene expression in the rat paraventricular (PVN) and supraoptic nuclei (SON) using in situ hybridization histochemistry. PACAP type I receptor gene was expressed moderately in the whole area of the PVN and the SON. In particular, PACAP type I gene transcripts were found in both the magnocellular and the parvocellular parts of the PVN. The results are consistent with the hypothesis that neurosecretory cells in the PVN and the SON might be modulated by PACAP through PACAP type I receptor.

Animals↗

PubMed1996

Effects of GABA on spontaneous [Ca2+]c dynamics and electrical properties of rat adrenal chromaffin cells.

A large fraction of rat adrenal chromaffin cells (about 60%) shows spontaneous [Ca2+]c oscillations and spontaneous action potentials. In the present study the effects of gamma-aminobutyric acid (GABA) on the spontaneous [Ca2+]c oscillations and electrical properties of rat adrenal chromaffin cells were investigated using Fura-2 [Ca2+]c imaging and patch clamp techniques. GABA inhibited the spontaneous [Ca2+]c oscillations in a reversible manner. The effect of GABA was mimicked by the GABAA and GABAC receptor agonist, muscimol, but not by the GABAB receptor agonist, baclofen. Moreover, the effect was antagonized by the selective GABAA receptor antagonist, bicuculline. The mode of the inhibition was all-or-none, and the threshold concentration at which the inhibition occurred varied widely (50 microM to over 1 microM) from cell to cell. GABA (100 microM) elicited a transient burst of action potentials of diminished amplitude, which was followed by arrest of action potentials. Further analysis showed that GABA (100 microM) induced inward whole-cell currents in voltage-clamp experiments and produced depolarization and membrane conductance increase in current-clamp experiments. The effects appear to be due to an increase in chloride ion conductance since the degree of GABA-induced depolarization depended on the pipette [Cl-]. These results suggest that GABA, acting through GABAA receptor, may play a role in the physiological regulation of rat adrenal chromaffin cells by directly modifying the discharge of spontaneous action potentials and spontaneous [Ca2+]c oscillations.

Action Potentials↗

PubMed1996

Proadrenomedullin N-terminal 20 peptide (PAMP) reduces inward currents and Ca2+ rises induced by nicotine in bovine adrenal medullary cells.

It has been recently reported that proadrenomedullin N-terminal 20 peptide (PAMP), which is secreted with adrenomedullin and catecholamines from the adrenal medulla, inhibits catecholamine release stimulated with nicotine. In the present study, to elucidate anticholinergic mechanisms of PAMP we employed the whole-cell patch-clamp and the intracellular Ca2+ imaging techniques in cultured bovine adrenal medullary cells. PAMP inhibited nicotinic currents and [Ca2+]i rises induced by nicotine in a dose-dependent manner (10(-9)-10(6) M). These inhibitions were selective, since PAMP alone did not induce any ionic currents, moreover it did not affect voltage-dependent Ba2+ currents or high K+ (50 mM)-induced [Ca2+]i rises. The onset of the inhibitory effect of PAMP (10(-6) M) was very rapid and reached a steady-state level within 10 sec. The effect of PAMP (10(-6) M) lasted for about 10-15 min. Desensitization process of the nicotinic current fitted to a single exponential function with a time constant of 6.4 +/- 0.3 sec. When PAMP (10(-6) M) simultaneously added with nicotine (10(-5) M), the desensitization process was facilitated and fitted to two exponentials with time constants of 0.46 +/- 0.08 and 2.5 +/- 0.8 sec. From the present results, the inhibition by PAMP of nicotinic currents which was well associated with that of nicotine induced [Ca2+]i rises leads to the attenuation of catecholamine release probably, at least in part, due to the facilitation of the desensitization process of the nicotinic currents.

Adrenal Medulla↗

PubMed1996

Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2+ release from intracellular stores and long lasting Ca2+ influx mediated by Na+ influx-dependent membrane depolarization in bovine adrenal chromaffin cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.

Adrenal Glands↗

PubMed1996

A regulatory mechanism for the balanced synthesis of membrane phospholipid species in Escherichia coli.

The mechanism that assures the balanced synthesis of zwitterionic (phosphatidylethanolamine) and acidic phospholipids (phosphatidylglycerol and cardiolipin) in Escherichia coli has been examined by genetically manipulating the two enzymes at the biosynthetic branch point, i.e., phosphatidylglycerophosphate synthase, encoded by pgsA, and phosphatidylserine synthase, encoded by pssA. A mutant in which the most part of the pssA gene was replaced with a drug resistance gene lacked phosphatidylserine synthase and phosphatidylethanolamine and required divalent metal ions for growth, as did a previously reported insertion-inactivated pssA mutant. When this mutant harbored a plasmid containing a Bacillus subtilis gene that encodes membrane-bound phosphatidylserine synthase, the phosphatidylethanolamine content was dependent on its activity, in contrast to that with the soluble E. coli counterpart. A defective mutation, pgsA3, caused reductions not only in acidic-phospholipid synthesis but also in phosphatidylethanolamine synthesis, despite the normal level of phosphatidylserine synthase activity. These results, together with previous observations, indicate that phosphatidylserine synthesis requires the membrane-associated form of phosphatidylserine synthase, which is related to the membrane-levels of acidic phospholipids, thus yielding balanced compositions of zwitterionic and acidic phospholipids.

Bacillus subtilis↗

PubMed1996

Production of recombinant mite allergen Der fI in insect cells and characterization of products--removal of pro-sequence is essential to IgE-binding activity.

Der fI is a major mite proteinaceous allergen found in house dust. We produced recombinant Der fI (reDer fI) in insect cells using a baculovirus expression system. Based on the molecular mass and N-terminal amino acid (aa) sequence analysis, reDer fI was found to be compromised of a mixture of two proteins, each of which includes a pro-sequence of different length. The reDer fI had IgE-binding activity at only 20% of that of native Der fI. The removal of the pro-sequence in an acidic solution drastically increased IgE-binding activity to almost the same as that of native Der fI, showing that the presence of the pro-sequence is inhibitory to the IgE-binding activity of Der fI.

Allergens↗

PubMed1996

Directed mutagenesis, Ser-56 to Pro, of Bacillus subtilis phosphatidylserine synthase drastically lowers enzymatic activity and relieves amplification toxicity in Escherichia coli.

Amino acid residue 56 of the phosphatidylserine synthase of Bacillus subtilis was changed from Ser to Pro by using modified primers in PCR amplification of its structural gene, pssBS. When an Escherichia coli mutant lacking its own phosphatidylserine synthase harbored a plasmid carrying this allele, the Mn(2+)-requiring Bacillus-type synthase activity, as assayed in vitro, was at least six-fold lower than that with the wild-type pssBS gene and the cellular phosphatidylethanolamine content was similarly lowered, indicating that the altered region of the enzyme is critically important for its activity. In contrast to the E. coli counterpart, amplification of the wild-type Bacillus enzyme increased both the relative and absolute contents of phosphatidylethanolamine and impaired cell growth. However, amplification of the mutant enzyme of the same level was much less toxic, implying that E. coli cells are more sensitive to the unbalanced accumulation of phosphatidylethanolamine than that of the hydrophobic enzyme molecules. Possible roles of the conserved region of the enzyme in its activity and the wild-type phospholipid composition in the proper membrane function are discussed.

Alleles↗