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Biomedical subjects

K Chihara

Publications and source records attributed to K Chihara.

At least 145 records · Page 8Linked to original sources
PubMed1996

Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers.

Specific receptors for brain-gut peptide hormones, cholecystokinin (CCK) and gastrin, are expressed in a variety of human tumor cells. CCK and gastrin promote the growth of NIH3T3 cells into which the CCK-B/gastrin receptor had been introduced via a eukaryotic expression vector. In this study, we have examined the effect of CCK-8 on the actin cytoskeleton by using two mouse fibroblast cell lines expressing human CCK-B/gastrin receptors. Treatment with very low concentration of CCK-8 (10(-10) M) induced the formation of actin stress fibers within one minute. Stress fiber formation increased for 30 min. In contrast, a potent mitogen for fibroblasts, platelet-derived growth factor (PDGF), initially induced membrane ruffling and, later, a weak formation of stress fibers. Microinjection of rho GDP dissociation inhibitor or Clostridium botulinum ADP-ribosyltransferase C3 which is known to impair the function of a small GTP-binding protein, rho p21, inhibited the stress fiber formation by CCK-8 as well as by PDGF. These results indicate that CCK-B/gastrin receptor could regulate stress fiber formation in a rho p21-dependent manner. The signals from CCK-B/gastrin receptor might affect cell growth as well as cell motility or adhesion by regulating the actin cytoskeleton.

3T3 Cells↗
PubMed1996

Identification of a putative target for Rho as the serine-threonine kinase protein kinase N.

Rho, a Ras-like small guanosine triphosphatase, has been implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid (LPA) to form stress fibers and focal contacts. The form of RhoA bound to guanosine triphosphate directly bound to and activated a serine-threonine kinase, protein kinase N (PKN). Activated RhoA formed a complex with PKN and activated it in COS-7 cells. PKN was phosphorylated in Swiss 3T3 cells stimulated with LPA, and this phosphorylation was blocked by treatment of cells with botulinum C3 exoenzyme. Activation of Rho may be linked directly to a serine-threonine kinase pathway.

3T3 Cells↗
PubMed1996

Prostaglandin E2 stimulates osteoclast-like cell formation and bone-resorbing activity via osteoblasts: role of cAMP-dependent protein kinase.

Prostaglandin E2 (PGE2) is an important local regulator in bone. The present study was performed to investigate the effect of PGE2 on osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts in the presence or absence of osteoblasts, PGE2 (10(-8) to 10(-6) M) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although it did not affect osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in osteoblast-free mouse spleen cell cultures. The conditioned medium from osteoblastic UMR-106 cells pretreated with PGE2 (10(-8) and 10(-6) M) significantly stimulated osteoclast-like cell formation from hemopoietic blast cells. PGE2 also significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures. In contrast, PGE2 significantly inhibited the bone-resorbing activity and osteopontin mRNA expression in isolated rabbit osteoclasts. Rp-cAMPS, a direct protein kinase (PKA) antagonist, significantly inhibited PGE2-stimulated osteoclast-like cell formation and the bone-resorbing activity of mature osteoclasts, although protein kinase C inhibitors, dantrolene (an inhibitor of calcium release from the intracellular calcium pool) and voltage-dependent calcium channel blockers did not affect PGE2-stimulated osteoclast-like cell formation. In conclusion, PGE2 stimulated osteoclast-like cell formation and bone-resorbing activity in mouse bone cell cultures presumably through osteoblasts. The activation of PKA is linked to PGE2-stimulated osteoclast-like cell formation and bone-resorbing activity.

Animals↗
PubMed1996

High extracellular calcium stimulates osteoclast-like cell formation and bone-resorbing activity in the presence of osteoblastic cells.

The present study was performed to examine the effect of the high concentration of extracellular calcium ([Ca2+]e) on osteoclast-like cell formation and bone-resorbing activity in the presence or absence of osteoblasts. High [Ca2+]e (3 and 5 mM) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although high [Ca2+]e did not affect the formation of osteoclast-like cells from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in mouse spleen cell cultures. The osteoclast-like cells, newly formed by high [Ca2+]e in the presence of osteoblasts, possessed the ability to form pits on the dentine slices. The conditioned medium from osteoblastic MC3T3-E1 cells treated with high [Ca2+]e (5 mM) significantly increased the formation of osteoclast-like cells from hemopoietic blast cells, compared with the control medium. Dantrolene, an inhibitor of calcium mobilization from the intracellular calcium pool, and indomethacin significantly blocked high [Ca2+]e-stimulated osteoclast-like cell formation in the presence of osteoblasts, although voltage-dependent calcium channel blockers and anti-insulin-like growth factor I antibody did not affect it. High [Ca2+]e, however, significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures, although high [Ca2+]e inhibited bone-resorbing activity in isolated rabbit osteoclasts. An increase in the extracellular magnesium concentration (5 mM) affected neither osteoclast-like cell formation nor bone-resorbing activity. In conclusion, high [Ca2+]e stimulated osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts, presumably via osteoblasts.

Animals↗
PubMed1996

Refractory thrombocytopenia with chromosome 11q23 abnormality.

Although cytopenia is a common manifestation of myelodysplastic syndrome (MDS), isolated thrombocytopenia is rare. The term "refractory thrombocytopenia" (RTC) has been proposed as a counterpart of refractory anemia. We describe here a case of RTC associated with chromosome abnormality on 11q23. A 59-year-old man was admitted because of severe thrombocytopenia. A bone marrow examination revealed an increased number of micromegakaryocytes and the absence of normal-sized megakaryocytes without obvious dysplasia in either the myeloid or the erythroid lineage. A remarkable increase of GPIIb/IIIa (CD41a)-positive precursor cells in the bone marrow was observed. Cytogenetic examination detected the chromosome abnormality, an addition on chromosome 11q23. Translocation of the HRX gene was not detectable by Southern blot analysis. The diagnostic problems of isolated thrombocytopenia and possible participation of gene(s) on chromosome 11q23 in megakaryocytopoiesis as well as early hematopoiesis are discussed.

Biopsy, Needle↗
PubMed1996

Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells.

We studied the transport mechanism of pirarubicin (THP) in HL60 and its THP-resistant (HL60/THP) cells, which showed no expression of mdr1 mRNA on Northern blot analysis. Under physiological conditions, the uptake of THP by both types of cell was time- and temperature-dependent. The amount of drug transport in the resistant cells was significantly less than that in the parent cells within 3 min of incubation. THP uptake was significantly higher in the presence than in the absence of 4 mM 2,4-dinitrophenol (DNP) in glucose-free Hanks' balanced salt solution in both HL60 and HL60/THP cells and the increases were approximately equal. In the presence of DNP, the uptake of THP by both types of cell was concentration-dependent, and there were no significant differences in the apparent kinetic constants (Michaelis constant (Km), maximum velocity (Vmax) and Vmax/Km) for THP uptake between HL60 and HL60/THP cells. Additionally, THP transport was competitively inhibited by its analogue doxorubicin. The efflux of THP from HL60/THP cells was significantly greater than that from HL60 cells, and the release from both types of cell was completely inhibited by decreasing the incubation temperature to 0 degrees C and by treatment with DNP in glucose-free medium. In contrast, the P-glycoprotein inhibitors verapamil and cyclosporin A did not inhibit THP efflux. However, genistein, which is a specific inhibitor of multidrug resistance-associated protein (MRP), increased the THP remaining in the resistant cells, and the value was approximately equal to that of the control group in the sensitive cells. These results suggest that THP is taken up into HL60 and HL60/THP cells via a common carrier by facilitated diffusion, and then pumped out in an energy-dependent manner. Furthermore, the accelerated efflux of THP by a specific mechanism, probably involving MRP, other than the expression of P-glycoprotein, resulted in decreased drug accumulation in the resistant cells, and was responsible, at least in part, for the development of resistance in HL60/THP cells.

Analysis of Variance↗
PubMed1996

Growth hormone (GH)-releasing peptide and GH releasing hormone stimulate GH release from subpopulations of somatotrophs in rats.

The synthetic hexapeptide GH-releasing peptide (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2; GHRP-6) and GH releasing hormone (GHRH) are both potent stimulators of GH release in rats. Using reverse hemolytic plaque assay (RHPA), we have compared the effects of human GHRH and GHRP-6 on GH release from the dispersed individual cells of rat anterior pituitary. In a single RHPA, we quantified the percentage of plaque forming cells (% PFC) and their mean plaque area (MPA) after 30 min-incubation, and calculated a total secretion index (TSI) by multiplying % PFC and MPA. 10 nM GHRH and 100 nM GHRP-6 each caused a significant increase in % PFC (%) (GHRH 39.15, GHRP-6 29.4, vs vehicle 24.3, P < 0.01), MPA (x 10(-2) microns2) (GHRH 124.04, GHRP-6 94.80, vs vehicle 44.57, P < 0.01) and TSI (x 10(-2)) (GHRP-6 32.87, vs vehicle 10.84, P < 0.01). Simultaneous addition of both secretagogues caused a further increase in GH release (%PFC 46.4, MPA 142.55, TSI 69.82, P < 0.01 vs vehicle), although the effect was additive but not synergistic. Somatostatin analog, SMS201-995 (SMS) partially suppressed all parameters in GH secretion after stimulation by GHRH and/or GHRP-6. A double RHPA was then performed to test whether all somatotrophs respond equally to GHRH and GHRP-6 or some cells formed plaques only be either GHRH or GHRP-6. There were somatotrophs responsive to only GHRH (23.3% vs control 6.2%, P < 0.01), those responsive to only GHRP-6 (11.9% vs control 6.1%, P < 0.01), and those responsive to both GHRH and GHRP-6 (7.8% vs control 0.2%, P < 0.01). These results confirmed the previous findings that GHRP-6 and GHRH directly but independently stimulate GH release from the pituitary cells, and further suggest that presence of at least three functionally distinct somatotroph subpopulations concerning the responsiveness to GHRP-6 and GHRH in rats.

Animals↗
PubMed1996

Antiproliferative effect of a novel cholecystokinin-B/gastrin receptor antagonist, YM022.

Cholecystokinin (CCK)-B and gastrin receptors are expressed on a variety of human tumor cells. Recently, we have demonstrated that the human brain CCK-B receptors are identical to the gastrin receptors derived from the stomach mucosa, and that the brain-gut peptides, CCK-8 and gastrin I are mitogenic for mouse NIH 3T3 fibroblasts expressing human CCK-B/gastrin receptors (N-hCCKBR). In this report, we evaluated the antiproliferative potency of CCK-B/gastrin receptor antagonists by using N-hCCKBR cells. Among several antagonists, a benzodiazepine derivative, YM022 had the most potent activities in competing with [125I]CCK-8 or [125I]gastrin I binding, inhibition of CCK-8- or gastrin I-induced phosphoinositide hydrolysis and increasing cytoplasmic free calcium. Interestingly, a potent antagonist for rat CCK-B/gastrin receptors did not have such activities in N-hCCKBR cells. YM022 inhibited the CCK-8- or gastrin I-induced [methyl-3H]thymidine incorporation of N-hCCKBR cells in a dose-dependent manner. In the absence of exogenous peptide ligands, YM022 also inhibited the proliferation of several human cancer cell lines expressing the genes for both gastrin and its receptor. These results suggest that YM022 could intervene in the autocrine stimulation of human tumor cell lines through CCK-B/gastrin receptors. N-hCCKBR cells are an excellent tool to screen for novel human CCK-B/gastrin receptor antagonists possessing antiproliferative activity for human cancer cells.

3T3 Cells↗
PubMed1996

Identification of a growth hormone mutation responsible for short stature.

A heterozygous single base missense substitution in exon 4, which converted codon 77 from arginine to cysteine, has been identified in a child with severe growth retardation. The mutant GH fails to stimulate GH signal transduction by itself, despite possessing a higher affinity than the wild-type molecule for GHBP, and appears to have a dominant antagonistic effect on the protein phosphorylation activity of wild-type GH.

Body Height↗
PubMed1996

Human rib cage distortability.

In five normal men, we divided the rib cage into lung-apposed [pulmonary rib cage (RCp)] and diaphragm-apposed [abdominal rib cage (RCab)] compartments and calculated their absolute cross-sectional areas (Arc,p and Arc,ab) by anteroposterior and lateral dimensions measured by magnetometry. Distortion was quantified as the displacement of RCp and RCab produced by diaphragmatic twitches away from the relaxed configuration. We measured transdiaphragmatic pressure as the difference between gastric and esophageal pressures. Distortability was expressed as percent distortion per transdiaphragmatic pressure and varied among individuals from 0.02 to 0.23%/cmH2O. The pressure acting to restore the distorted rib cage back to its relaxed shape (Plink) varied from 0.1 to 31.3 cmH2O/%distortion. Distortion correlated positively (r = 0.92) and Plink per percent distortion negatively (r = -0.90) with RCab compliance during the relaxation maneuver (delta Arc, ab/delta gastric pressure). We conclude that rib cage distortability varies widely among normal subjects and is closely linked to RCab compliance.

Abdomen↗
PubMed1996

Effect of intravenous or intracerebroventricular injections of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on GH release in conscious, freely moving male rats.

The present study was designed to examine the effects of intracerebroventricular injection of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP) on GH secretion in freely moving conscious male rats and to determine whether the central action of GHRP is mediated by increased GHRH and/or somatostatin (SRIF) release. A significant and dose-related suppression of natural fluctuations in plasma GH occurred immediately after intracerebroventricular injection of GHRP at the doses of 1, 3 and 10 mu g/rat and was sustained for more than 1 h. An intracerebroventricular injection of GHRP also suppressed plasma GH rises after an intravenous injection of [D-Ala2, Nle27] human GHRH (1-28)agmatine (hGHRH analog), but the GH suppression by intracerebroventricular GHRP was significantly blunted in rats treated with antirat SRIF gamma-globulin (SRIF-ab). In addition, the suppression by intracerebroventricular GHRP of hGHRH-analog-induced GH release was observed even in rats treated with anti-rat GHRH goat gamma-globulin (GHRH-ab) which does not cross-react with hGHRH analog, and again its suppression was significantly attenuated by simultaneous treatment with SRIF-ab and GHRH-ab. Furthermore, intracerebroventricularly injected GHRP was effective enough to inhibit the central-nervous-system (endogenous GHRH)-driven natural GH-secretory surges in SRIF-ab-treated rats. When 10 mu g/kg GHRP was injected intravenously every 1 h during a 7-hour observation period in control rats without antibody treatment, plasma GH levels were increased with the peak 10 min after each injection during the surge period of the GH-secretory rhythm, but the plasma GH response to GHRP was frequently absent during its trough period. In addition, in SRIF-ab-treated rats, basal GH levels as well as plasma GH peaks after GHRP injections were markedly elevated and the obvious plasma GH rises after GHRP could be observed even during the trough period. In GHRH-ab-treated rats, basal GH levels were lower, and the plasma GH response to GHRP was significantly attenuated as compared with those in control rats. Plasma GH peaks after GHRP injections in rats simultaneously treated with SRIF-ab and GHRH-ab were higher than those in rats given GHRH-ab alone, but its difference in GH response was obviously smaller than that observed between SRIF-ab-treated and control rats, suggesting a crucial role of endogenous GHRH in GHRP-induced GH release. To further clarify the interaction of GHRH and GHRP in GH release, hGHRH analog was injected intravenously alone or simultaneously with GHRP in rats treated with SRIF-ab and GHRH-ab. The peak GH values after simultaneous injection of hGHRH analog and GHRP were significantly higher than the sum of GH peaks after each injection of hGHRH analog and GHRP. In conclusion, an intracerebroventricular injection of GHRP suppresses GH secretion by a central mechanism which is likely to include increased SRIF release, reduced GHRH release or both. We have confirmed that intravenous injection of GHRP can stimulate GH release by itself in an immunologically nullified condition of circulating endogenous GHRH and SRIF in rats, and that endogenous SRIF modestly suppresses the GH-stimulating effect of intravenously injected GHRP while both endogenous and exogenous GHRH markedly enhance GH release by intravenously injected GHRP in a synergistic manner.

Amino Acid Sequence↗
PubMed1996

Paracrine stimulation of cell growth by cholecystokinin/gastrin through cholecystokinin-B receptor on GH3 cells in vitro.

Cholecystokinin (CCK) is detected in pituitary tumors but its role remains unknown. On the hypothesis that CCK may facilitate the cell growth in pituitary tumors, we have examined the effect of CCK on cell growth using a rat pituitary tumor cell line, GH3, cultured in a serum-free, chemically defined medium. Addition of sulfated CCK-(26-33) (CCK-8) in two different concentrations (0.5 approximately 1 nM) caused a significant increase in the number of GH3 cells. The antagonist (1 microM) for CCK-B receptor, but not CCK-A receptor, significantly inhibited the number of GH3 cells. Northern blot analysis revealed a significant expression of CCK-B receptor mRNA in GH3 cells, but not in normal rat pituitary glands. In addition, immunoreactive CCK/gastrin was detected by RIA in the GH3 cell extracts as well as the serum-free culture medium. In GH3 cell extracts, both CCK-8 and gastrin like peptides were identified by gel chromatography. These findings provided the first evidence for an autocrine/paracrine role of CCK and gastrin on stimulation of GH3 cell growth through the CCK-B receptor.

Animals↗
PubMed1996

Stimulatory effect of growth hormone on bone resorption and osteoclast differentiation.

Although the actions of GH on osteoblasts have been extensively investigated, its effects on osteoclasts remain unknown. In the present study, the effects of GH on bone resorption and osteoclast differentiation were examined in vitro. Bovine GH (bGH; 1-100 ng/ml) significantly stimulated bone resorption by preexistent osteoclasts in stromal cell-containing mouse bone cell cultures, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When bGH was added to unfractionated bone cells after degeneration of preexistent osteoclasts, it concentration dependently stimulated osteoclast-like cell formation. GH also enhanced 1,25-dihydroxyvitamin D3-induced osteoclast-like cell formation. Moreover, osteoclast-like cells newly formed from unfractionated bone cells in the presence of bGH possessed the ability to form pits on dentine slices. The conditioned medium from osteoblastic MC3T3-E1 cells or MC3T3-G2/PA-6 stromal cells pretreated with bGH stimulated osteoclast-like cell formation from mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. On the other hand, the PCR products corresponding in size to the mouse GH receptor were detected in mouse hemopoietic blast cells as well as liver. GH concentration dependently stimulated osteoclast-like cell formation from these hemopoietic blast cells in the absence of stromal cells, and these osteoclast-like cells formed pits on dentine slices in the presence of MC3T3-G2/PA-6 stromal cells. The present study indicated for the first time that GH stimulates osteoclastic bone resorption through both its direct and indirect actions on osteoclast differentiation and through its indirect activation of mature osteoclasts, possibly via stromal cells, including osteoblasts.

Animals↗