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Biomedical subjects

E Ogata

Publications and source records attributed to E Ogata.

At least 145 records · Page 8Linked to original sources
PubMed1991

Comparison of effects of HGF and EGF on cellular calcium in rat hepatocytes.

We compared the effects of HGF and EGF on cytoplasmic free calcium concentration, [Ca2+]c, and inositol trisphosphate production in rat hepatocytes. HGF induced a prompt and transient elevation of [Ca2+]c. EGF also induced an immediate increase in [Ca2+]c, the magnitude of which was greater than that by HGF. In contrast, in the presence of 1 microM extracellular calcium EGF increased [Ca2+]c to a lesser extent than HGF. When cells were pretreated with EGF, the effect of HGF on [Ca2+]c was greatly enhanced. However, such enhancement was not observed in medium containing 1 microM extracellular calcium. In hepatocytes prelabeled with [3H]-inositol, both HGF and EGF increased [3H]inositol trisphosphate. HGF and EGF acted synergistically to stimulate production of inositol trisphosphate. These results indicate that both HGF and EGF increase [Ca2+]c by a mechanism involving phosphoinositide turnover and that the actions of HGF and EGF on hepatocyte calcium metabolism are not totally identical.

Animals↗
PubMed1991

Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation.

We have localized a G protein activator region of the human beta 2-adrenergic receptor to region beta III-2 (from Arg259 to Lys273). The synthetic beta III-2, corresponding to the C-terminal end of the third cytoplasmic loop, activates Gs at nanomolar concentrations and weakly activates Gi. beta III-2 activates adenylyl cyclase at nanomolar concentrations in wild-type S49 lymphoma membranes, but not in membranes of unc mutant S49 cells, in which Gs is uncoupled from beta-adrenergic stimulation. Phosphorylation of beta III-2 by cAMP-dependent protein kinase A, which is involved in the desensitization of the beta-adrenergic receptor from Gs, drastically reduces the effect of beta III-2 on Gs while potentiating its action on Gi, resulting in a total loss of adenylyl cyclase-stimulating activity. These findings indicate that this receptor sequence is a multipotential G protein activator whose G protein specificity is regulated by protein kinase A.

Adenylyl Cyclases↗
PubMed1991

Negative regulatory elements in the human parathyroid hormone gene.

We have identified and characterized a pair of negative regulatory elements far upstream of the transcription start site of the human parathyroid hormone (hPTH) gene. Transfection of various types of cultured cells with a fusion plasmid containing 4.7 kilobase pairs of the 5'-flanking portion of the hPTH gene linked to the chloramphenicol acetyltransferase (CAT)-coding sequence generated only 10% of the CAT activity of a plasmid containing 684 base pairs of the 5'-flanking region of the hPTH gene. Deletion analyses reveal that there are at least two separate upstream DNA elements in the hPTH gene responsible for the negative regulation. We find that these cultured cells possess nuclear factors which specifically bind to several short DNA sequences within these elements and that these sequences can suppress transcription of the hPTH gene.

3T3 Cells↗
PubMed1991

Prostaglandin F2 alpha stimulates proliferation of clonal osteoblastic MC3T3-E1 cells by up-regulation of insulin-like growth factor I receptors.

Prostaglandin F2 alpha (PGF2 alpha) stimulates proliferation of clonal osteoblastic MC3T3-E1 cells mainly via the stimulation of phospholipase C. These cells constitutively produced and secreted insulin-like growth factor I (IGF-I). In addition, a neutralizing anti-IGF-I antibody completely abolished DNA synthesis stimulated by PGF2 alpha in MC3T3-E1 cells, suggesting that IGF-I indeed mediates the PGF2 alpha effect. However, PGF2 alpha decreased the expression of IGF-I mRNA and the secretion of immunoreactive IGF-I into the medium, whereas progression activity in the conditioned medium was not affected by PGF2 alpha. Although IGF-I alone did not stimulate DNA synthesis in MC3T3-E1 cells, when PGF2 alpha was added to the cultures, IGF-I stimulated their proliferation. Thus, PGF2 alpha may potentiate the action of IGF-I. At the same time, PGF2 alpha increased the number of high affinity binding sites (molecular mass of 130 kDa) for IGF-I in a dose-dependent manner. The increase in IGF-I-binding site number preceded the elevation of DNA synthesis by approximately 3 h. Furthermore, MC3T3-E1 cells secreted at least three species of IGF-binding proteins (IGFBPs) with molecular masses of 24, 30, and 34 kDa. In the early period of PGF2 alpha exposure, PGF2 alpha attenuated the secretion of all of these IGFBPs, whereas thereafter, it markedly increased their secretion, especially that of the 34-kDa IGFBP, suggesting a modulation of metabolism and action of IGF-I. These effects of PGF2 alpha on IGF-I receptor number and IGFBP secretion may play a role in the synergism between PGF2 alpha and IGF-I that results in the stimulation of DNA synthesis in MC3T3-E1 cells.

Amanitins↗
PubMed1991

Distinct mode of G protein activation due to single residue substitution of active IGF-II receptor peptide Arg2410-Lys2423: evidence for stimulation acceptor region other than C-terminus of Gi alpha.

Arg2410-Lys2423 (RVGLVRGEKARKGK, peptide 14) of the human insulin-like growth factor II receptor directly activates Gi and deletion of C-terminal 4 residues from peptide 14 nullifies this activity. A study was thus made of the effects of peptides modified in the C-terminal structure. RVGLVRGEKAAKGK and RVGLVRGEKARKGA scarcely activated Gi, whereas RVGLVRGEKARAGK (peptide A5) activated Gi as much as peptide 14 did. However, peptide A5 action did not depend on Mg2+ concentration and was little affected by pertussis toxin modification of Gi alpha. Peptide A5 may thus recognize the region on Gi alpha that is distinct from the extreme C-terminus. It is consequently considered that (i) the first and the last basic residues in the C-terminal motif of peptide 14 determine the capacity for recognition of Gi and (ii) there is a region different from the C-terminus of Gi alpha, through which the C-terminal second basic residue-altered peptide 14 activates Gi in a Mg(2+)-independent manner.

Amino Acid Sequence↗
PubMed1991

Guanine nucleotide-binding protein interacting but unstimulating sequence located in insulin-like growth factor II receptor. Its autoinhibitory characteristics and structural determinants.

RVGLVRGEKARKGK (peptide 14) from residues 2410-2423 of the human insulin-like growth factor II receptor (IGF-IIR) directly activates adenylylcyclase-inhibitory guanine nucleotide-binding proteins (Gi proteins) whereas RGEKARKGK (peptide 9) has no stimulatory action. However, peptide 9 inhibited the actions of peptide 14 on both GDP release from and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to Gi-2 in an aqueous system. Peptide 9 also inhibited peptide 14-induced Gi-1 activation with a similar profile. The peptide 9 action was competitive for peptide 14 action and determined by the first arginine residue and the C-terminal RKGK. In reconstituted IGF-IIR-Gi-2 vesicles, peptide 9 blocked the G protein stimulation by IGF-II with a potency similar to that observed in the action of peptide 9 on peptide 14; and peptide 9-induced inhibition was also observed in IGF-IIR-Gi-1 vesicles. In pertussis toxin-treated K562 cell membranes supplemented with Gi-2, peptide 9 inhibited IGF-II-induced reduction in pertussis toxin-catalyzed ADP-ribosylation of Gi-2 with an IC50 of 30 microM. This inhibitory effect of peptide 9 was competitive for the concentration of these IGF-IIR-positive/IGF-I receptor-negative cell membranes. Therefore, peptide 9 inhibits the Gi-activating function of IGF-IIR possibly by interacting with the putative peptide 14 recognition site of Gi proteins. The significance of this sequence is discussed.

Adenosine Diphosphate Ribose↗
PubMed1991

Possible role of sodium-hydrogen antiport in acetylcholine-induced relaxation of rat aorta.

The decreased extracellular Na concentration (25mM) attenuated vasodilator effect of acetylcholine (ACh) in norepinephrine-treated aortic ring. This attenuation was greater in the low Na medium substituted by Li, which can exchange intracellular H through Na-H antiport, as compared with that substituted by choline, which cannot. 10 microM amiloride canceled the difference between the two low Na mediums. Thus the inhibition of Na-H antiport may counteract the suppressive effect of decreased intracellular Na on ACh vasodilation, suggesting a possible role of Na-H antiport in a release of vasoactive substance(s) from endothelial cells.

Acetylcholine↗
PubMed1991

Activin A/erythroid differentiation factor induces thromboxane A2 synthetic activity in murine erythroleukemia cells.

Activin A, a protein homologous to transforming growth factor beta, was shown to induce hemoglobin synthesis in murine erythroleukemia (MEL) cells and was also termed erythroid differentiation factor (EDF) (Eto, Y., Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y., and Shibai, H. (1987) Biochem. Biophys. Res. Commun. 142, 1095-1103). We found that activin A/EDF also induced thromboxane (TX) A2 synthetic activity in these cells. Synthesis of TXA2 from arachidonic acid is catalyzed by cyclooxygenase and TX synthase. Activin A/EDF induced the latter TX synthase activity, whereas the cyclooxygenase activity was constitutively expressed. The induction of this enzyme activity was inhibited by cycloheximide, suggesting that activin A/EDF induced de novo protein synthesis of TX synthase. Furthermore, we studied the relationship between the induction of TXA2 synthetic activity and erythroid differentiation in MEL cells, since the former is not an erythroid phenotype. We found 1) that the two responses to activin A/EDF were distinctly affected by the initial cell density; 2) that the dose-response curves for activin A/EDF were similar (ED50 = approximately 100 pM), whereas the time course of induction of TXA2 synthetic activity was much faster; and 3) that other erythroid differentiation inducers of MEL cells, namely dimethyl sulfoxide and hexamethylene bisacetamide, had little or no effect on TXA2 synthesis. These results indicate that activin A/EDF induces TXA2 synthetic activity independently of erythroid differentiation.

Acetylcholinesterase↗
PubMed1991

Regulation of transmembrane signal transduction of insulin-like growth factor II by competence type growth factors or viral ras p21.

In BALB/c 3T3 cells pretreated with platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) (primed-competent cells), insulin-like growth factors I and II (IGF-I and IGF-II) bind to their own receptors (IGF-IR and IGF-IIR) and stimulate calcium influx and DNA synthesis by a mechanism involving a 40-kDa pertussis toxin substrate. In contrast, these IGFs do not act on unprimed quiescent cells. In this study, the 40-kDa pertussis toxin substrate was identified as Gi-2 alpha using anti-G protein antibodies. We analyzed the quality of signal transduction from IGF-II to Gi-2 alpha. There was no difference in the amount of Gi-2 alpha between quiescent and primed-competent cells, and both of these cells had similar Kd values and numbers of IGF-II-binding sites. Whereas IGF-II did not alter pertussis toxin-catalyzed ADP-ribosylation of Gi-2 alpha in quiescent cells, IGF-II reduced the pertussis toxin substrate activity by 35-50% via the IGF-IIR in primed-competent cells. The action of IGF-II lasted for up to 3 h when IGF-II was present in the medium, and it disappeared when IGF-II was removed. These results suggest that the signaling pathway triggered by IGF-II is uncoupled between the IGF-IIR and Gi-2 alpha in quiescent cells and that PDGF and EGF restore the IGF-IIR-Gi-2 coupling. This study also indicates that low concentrations of IGF-I reduce the pertussis toxin substrate activity of Gi-2 alpha in primed-competent cells in a time course slower than that of IGF-II, but not at all in quiescent cells. However, both of these cells had similar Kd values and numbers of IGF-I binding sites. Therefore, the IGF-I signaling pathway may also be uncoupled between the IGF-IR and Gi-2 alpha in quiescent cells and restored by PDGF and EGF. In BALB/c 3T3 cells transfected with temperature-sensitive Kirsten sarcoma virus bearing the v-Ki-ras gene (ts cells), a 40-kDa pertussis toxin substrate was also identified as Gi-2 alpha. In nonpermissive ts cells, IGF-II was without effect on the pertussis toxin substrate activity of Gi-2 alpha or on calcium influx.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Role of ascorbic acid in the regulation of proliferation in osteoblast-like MC3T3-E1 cells.

Proliferation of osteoblast-like MC3T3-E1 cells was minimal in serum-free Eagle's minimum essential medium (MEM) but was enhanced by about 3.5-fold in serum-free alpha-modification of MEM (alpha-MEM). By adding back each of the extra constituents present in alpha-MEM to MEM, it was found that ascorbic acid was responsible for the sustained proliferation of MC3T3-E1 cells without serum. Ascorbic acid also stimulated the synthesis of collagen and increased the hydroxyproline content of MC3T3-E1 cell cultures markedly. Inhibitors of collagen synthesis, L-azetidine-2-carboxylic acid, cis-4-hydroxyproline, and 3,4-dehydroproline, almost completely eliminated the stimulatory effect of ascorbic acid on DNA synthesis of MC3T3-E1 cells. The dose response of the effect of L-azetidine-2-carboxylic acid on the hydroxyproline content closely paralleled that on DNA synthesis of MC3T3-E1 cells. Furthermore, a 10 times higher concentration of proline, which competes with L-azetidine-2-carboxylic acid for the incorporation into procollagen molecules, reversed the inhibition of DNA synthesis by L-azetidine-2-carboxylic acid. These results are consistent with the assumption that the stimulatory effect of ascorbic acid on the proliferation of MC3T3-E1 cells is mediated through its effect on the synthesis of collagen or some related protein. Furthermore, a fibronectin attachment peptide, GRGDTP, that competes with matrix proteins for specific binding to cell surface adhesion receptors also inhibited the stimulation of proliferation by ascorbic acid almost completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1991

Histological changes of the gallbladder and liver after extracorporeal shock wave lithotripsy for gallbladder.

The histological changes of the gallbladder and liver following extracorporeal shock wave lithotripsy (ESWL) were investigated in a canine model. After performing ESWL on human cholesterol stones placed in the gallbladder of 8 dogs, the gallbladder and livers were removed for pathological study. The mucosa of the gallbladder showed erosion and ulceration in three of the eight dogs. Edema and bleeding were found in the stroma of the gallbladder beds. Hemorrhagic necrosis and vacuolar degeneration occurred in liver parenchyma adjacent to the gallbladder bed, but the portal area was fairly well preserved. It can be said that ESWL caused damage to both the liver and the gallbladder, but its effects on the liver may be only slight because of the preservation of the portal area.

Animals↗
PubMed1991

Stimulation by 1,25-dihydroxyvitamin D3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells.

Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH)2D3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH)2D3, and a significant stimulation of 45Ca accumulation was observed with 3 x 10(-10) M 1,25(OH)2D3. Although 1,25(OH)2D3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH)2D3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D3 nor human PTH(1-34) affected mineralization in the presence or absence of 1,25(OH)2D3. These results demonstrate that 1,25(OH)2D3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH)2D3 has a direct stimulatory effect on bone mineralization process.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1991

Regulation of parathyroid hormone synthesis in chronic renal failure in rats.

To clarify the mechanism of secondary hyperparathyroidism in chronic renal failure at the parathyroid hormone (PTH) synthesis level, we measured PTH messenger RNA (mRNA) levels in parathyroid glands in a rat model of chronic renal failure. Four weeks after 7/8 nephrectomy, hyperplasia of parathyroid glands was evident and serum PTH levels were elevated. Serum concentration of calcium, inorganic phosphate, and 1,25-dihydroxyvitamin D (1,25(OH)2D) of rats with chronic renal failure were not detectably different from those of sham-operated rats. In chronic renal failure rats, PTH mRNA levels were elevated both per RNA and per DNA of parathyroid cells, suggesting increased PTH mRNA levels per cell. The elevated levels of PTH mRNA were returned to normal levels by achieving supraphysiological concentrations of 1,25(OH)2D3 given i.p. twice at 24 and 48 hours before sacrifice, although this was attended by slight hypercalcemia. A synthetic analogue of vitamin D, 22-oxa-1,25(OH)2D3, also suppressed PTH mRNA to normal levels, but without hypercalcemia. These data suggest that secondary hyperparathyroidism in early chronic renal failure may be due in part to the resistance of parathyroid cells to the physiological concentration of 1,25(OH)2D in circulation on PTH synthesis and that 22-oxa-1,25(OH)2D3 may be useful in the management of secondary hyperparathyroidism of chronic renal failure.

Animals↗
PubMed1991

Importance of chloride in the development of salt-induced angiotensin II hypertension in rats.

This study attempted to evaluate the effect of anion associated with sodium loading on the development of angiotensin II (AII)-induced hypertension in rats. Hypertension was induced by intraperitoneal infusion of AII(125 ng/min) for 12 days via miniosmotic pump (systolic blood pressure on day 12, 143 +/- 3 mm Hg). High dietary intake of sodium chloride significantly augmented the AII-induced hypertension (systolic blood pressure on day 12, 166 +/- 4 mm Hg), but equimolar sodium loading provided as sodium citrate failed to enhance AII hypertension (systolic blood pressure on day 12, 136 +/- 8 mm Hg). Thus, the data suggest that the full expression of salt (NaCl) sensitivity in AII hypertension depends on high dietary intake of both sodium and chloride.

Angiotensin II↗
PubMed1991

Mechanisms of endothelin-1 mRNA and peptides induction by TGF-beta and TPA in MDCK cells.

Mechanisms of prepro-ET-1 mRNA expression and mature endothelin-1 (ET-1) peptide secretion in MDCK cells (dog collecting duct origin) were investigated. MDCK cells constitutively expressed prepro-ET-1 mRNA (approximately 2.3 kb). TGF-beta time-dependently increased prepro-ET-1 mRNA levels between 30 min and 6 h. Induction of the mRNA 6 h following TGF-beta addition was dose-dependent with a half-maximal concentration of 10 pM. The half-life of prepro-ET-1 mRNA was 15 min in controls when the cells were treated with 10 micrograms/ml of actinomycin D, whereas it was extended to 30-45 min by TGF-beta treatment. Prepro-ET-1 mRNA was rapidly (15-30 min) induced by 0.5 microM TPA, one of the phorbol esters, but downregulated below baseline after 1 h. Our data show that MDCK cells constitutively secrete ET-1 and increase its production in response to TGF-beta. An axis of TGF-beta-ET-1-collecting duct may play an important role in regulation of electrolyte transport and cell growth of the renal tubules.

Animals↗
PubMed1991

Polarized secretion of endothelin-1 and big ET-1 in MDCK cells is inhibited by cell Na+ flux and disrupted by NH4Cl.

We recently found that TGF-beta increases ET-1 secretion in MDCK, a renal tubular cell line. The secretion of ET-1, by a confluent monolayer of MDCK cells grown on a filter, to the basolateral side of the epithelium was two times greater than that to the apical side. However, TGF-beta-increased ET-1 and big ET-1 secretion were exclusively released to the basolateral side. We investigated the mechanism of polarized secretion of ET-1 and big ET-1 in MDCK cells. After 24 h of incubation with 80 pM TGF-beta, basolateral secretion of both ET-1 and big ET-1 increased two to threefold without a significant increase on the apical side. TGF-beta-stimulated ET-1 and big ET-1 secretion in the basolateral bath were inhibited when 10 microM amiloride (Na+ channel blocker) or 1 microM ouabain (Na+, K(+)-ATPase inhibitor) was added for 3 h. Polarized secretion of ET-1 and big ET-1 was not affected. In contrast, 10 mM NH4Cl or 0.2 mM chloroquine (both lysosomal function inhibitors) reduced TGF-beta-stimulated ET-1 secretion in the basolateral bath whereas big ET-1 secretion in the apical bath increased two times. Thus, the polarity of big ET-1 secretion was reversed. In addition, the conversion rate from big ET-1 to ET-1 was significantly decreased from 80 to 55% by inhibiting lysosomal function. Prepro-ET-1 mRNAs 3 h after these perturbations were virtually unaltered. Our data show that ET-1 and big ET-1 secretion may be regulated by cell Na+ flux and that lysosomal functions may play some roles in the conversion of big ET-1 to mature ET-1 and in polarized secretion of big ET-1. Translational or posttranslational regulation may play an additional role in ET-1 secretion as well as the mRNA level.

Ammonium Chloride↗