Evidence for parathyroid hormone-related peptide as a cause of hypercalcemia in myeloma.
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Biomedical subjects
I Kojima
Publications and source records attributed to I Kojima.
Stimulation of follistatin production by epidermal growth factor in cultured rat hepatocytes.
Production of follistatin in cultured rat hepatocytes was studied by measuring follistatin release with a protein-binding assay using [125I]activin A. Follistatin was detected in conditioned medium of cultured hepatocytes. Ligand blotting using [125I]activin revealed that follistatin released into the medium consisted of two different forms with molecular weight of approximately 40 K-Da. Epidermal growth factor (EGF) elicited dose-dependent increases in DNA synthesis and follistatin release. Dose-response relationship for EGF-induced follistatin release correlated well with that for EGF-induced DNA synthesis. In EGF-stimulated cells, a marked increase in DNA synthesis occurred after 48 hrs. Similarly, follistatin release was markedly augmented after 48 hrs. Amount of cell-bound follistatin was not changed by the treatment with EGF. These results indicate that cultured hepatocytes synthesize and release follistatin. The activin-follistatin system operates in cultured rat hepatocytes and may modulate DNA synthesis by altering the action of activin A.
Activins↗
Studies on the effect of parathyroid hormone (1-84) on glucose output in the liver: comparison of effects in isolated hepatocytes and in perfused liver.
This study was conducted to determine the action of parathyroid hormone (1-84) (PTH(1-84)) on glucose output both in perfused liver and in isolated hepatocytes. In isolated rat hepatocytes, PTH(1-84) stimulated glucose output in a concentration-dependent manner. The action was detected at 10(-11) M and, at 10(-9) M, PTH produced its maximal effect. The magnitude of the maximal effect of PTH(1-84) was about 65% of that of phenylephrine. In contrast, PTH(1-84) had no effect on glucose output in perfused rat liver. Concentration of PTH(1-84) in effluent of perfused liver was less than that in the inflow. However, when the effluent obtained from liver perfused with 10 nM PTH(1-84) was added to isolated hepatocytes, a considerable amount of glucose was released, which was reversed by PTH(7-34), a competitive inhibitor of PTH receptor. These results indicate that PTH(1-84) increases glucose output in isolated hepatocytes but not in intact liver. It is suggested that the action of PTH(1-84) is blocked in intact liver by a yet unknown mechanism.
Animals↗
Granulosa cell luteinizing hormone receptor expression is modulated by ganglioside-specific ligands.
The ganglioside GM1 (Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3] Gal beta 1-->4Glc beta 1-->1Cer) was synthesized during granulosa cell development in vitro, and the effect of the interaction between cell-surface GM1 and its ligands on the luteinizing hormone (LH) receptor expression was investigated. GM1 synthesis, demonstrated by metabolic labeling of glycosphingolipids with [3H]galactose and binding studies using the 125I-B-subunit of cholera toxin, a specific ligand for GM1, was increased in follicle-stimulating hormone (FSH)-treated granulosa cells. When granulosa cells were cultured for 72 h in a medium containing the B-subunit of cholera toxin, FSH-induced LH-receptor contents determined by measuring the binding of 125I-deglycosylated human chorionic gonadotropin to intact cells, was augmented. The stimulatory effect of the B-subunit was dependent on the FSH concentration and culture duration. The augmentation was observed after culture for 48 h, and marked increases were evident after 72 h, which coincided with an increase of the 125I-B-subunit binding capacity. Scatchard analysis of the LH-receptor binding indicated that treatment with the B-subunit increased the number of LH-binding sites (6580 sites/cell after treatment with 20 ng/ml FSH; 11,290 sites/cell after FSH plus 100 ng/ml B-subunit), but did not alter the binding affinity. A specific antibody against GM1 mimicked the stimulatory effect of the B-subunit. The augmentation was not accompanied by granulosa cell proliferation. These findings suggest that binding of exogenous or possible endogenous ligands to cell-surface GM1 produces signals and modulates the cellular behavior during granulosa cell development.
Animals↗
Rate of calcium entry determines the rapid changes in protein kinase C activity in angiotensin II-stimulated adrenal glomerulosa cells.
The present study was conducted to monitor precisely the activity of protein kinase C (PKC) in adrenal glomerulosa cells stimulated by angiotensin II (ANG II). PKC activity in cells was monitored by measuring phosphorylation of a synthetic KRTLRR peptide, a specific substrate for PKC, immediately after the permeabilization of the cells with digitonin [Heasley and Johnson J. Biol. Chem. (1989) 264, 8646-8652]. Addition of 1 nM ANG II induced a gradual increase in KRTLRR peptide phosphorylation, which reached a peak at 30 min, and phosphorylation was sustained thereafter. When the action of ANG II was terminated by adding [Sar1,Ala8]ANG II, a competitive antagonist, both Ca2+ entry and KRTLRR phosphorylation ceased rapidly, whereas diacylglyercol (DAG) content was not changed significantly within 10 min. Similarly, when blockade of Ca2+ entry was achieved by decreasing extracellular Ca2+ to 1 microM or by adding 1 microM nitrendipine, KRTLRR peptide phosphorylation was decreased within 5 min. In addition, restoration of Ca2+ entry was accompanied by an immediate increase in KRTLRR peptide phosphorylation. Under the same condition, DAG content did not change significantly. We then examined the role of the PKC pathway in ANG II-induced aldosterone production. Ro 31-8220 inhibited ANG II-induced KRTLRR phosphorylation without affecting the activity of calmodulin-dependent protein kinase II. In the presence of Ro 31-8220, ANG II-mediated aldosterone production was decreased to approx. 50%. Likewise, intracellular administration of PKC19-36, a sequence corresponding to residues 19-36 of the regulatory domain of PKC known to inhibit PKC activity, attenuated ANG II-mediated activation of PKC and aldosterone output. These results indicate a critical role of Ca2+ entry in the regulation of PKC activity by ANG II.
Aldosterone↗
Enhancement by tocoretinate of epidermal growth factor-induced DNA synthesis in human intestinal epithelial cells.
Tocoretinate (TR) is a hybrid compound composed of alpha-tocopherol esterified with retinoic acid. The present study was conducted to clarify whether or not TR affects DNA synthesis in a human intestinal cell line, FHs 74 Int. In these cells, addition of 10% serum resulted in an approximately 10-fold increase in DNA synthesis as assessed by [3H]thymidine incorporation. Epidermal growth factor (EGF) augmented DNA synthesis three- to fourfold. When EGF was added together with insulin-like growth factor-I (IGF-I), which had only a small effect by itself, a combination of these growth factors reproduced the effect of serum. In quiescent FHs 74 Int cells, TR had no effect on DNA synthesis by itself. However, when quiescent cells were pretreated with TR for 24 hr, DNA synthesis induced by EGF was markedly enhanced. Thus, in TR-pretreated cells, EGF stimulated DNA synthesis to the same extent as 10% serum. The effect of TR was dose-dependent and the maximal effect was obtained by 10(-7) M TR. Pretreatment with TR enhanced the effect of EGF on DNA synthesis but did not change the dose-response relationship for EGF-mediated DNA synthesis. All-trans-retinoic acid had similar stimulatory effect on EGF-induced DNA synthesis. When the medium was changed during the treatment with TR, the effect of TR on DNA synthesis was reduced. In addition, pretreatment with TR resulted in release of immunoreactive IGF-I into medium. Finally, the effect of TR was attenuated by an addition of antibody against IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)
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Bimodal effect of transforming growth factor-beta on insulin secretion in MIN6 cells.
The effects of transforming growth factor-beta (TGF-beta) on insulin secretion were investigated using a glucose-responsive clonal cell line, MIN6. One hundred pM TGF-beta stimulated insulin release during 0.5-24 h of incubation in the presence of 5.5 mM glucose, but not after 48 h; 1 nM TGF-beta also stimulated insulin release up to 2 h of exposure, but the effect was not seen after 6 h of exposure. When cells were incubated with 25 mM glucose for 24 h, 100 pM TGF-beta significantly inhibited glucose-stimulated insulin release, whereas insulin release was not altered at 0 or 2.8 mM glucose. On the contrary, forskolin- (10 microM) and tolbutamide- (40 microM) induced insulin release were not affected by TGF-beta. TGF-beta affected neither the cell growth nor the cellular insulin content. An addition of 1 microM nitrendipine abolished TGF-beta-induced insulin secretion at 5.5 mM glucose. The presence study shows that TGF-beta exerts a bimodal effect on glucose-induced insulin secretion from MIN6 cells, depending on dose, time of exposure and concentrations of coexisting glucose. These effects might be mediated by the Ca(2+)-dependent mechanism.
Animals↗
Basic fibroblast growth factor induces luteinizing hormone receptor expression in the presence of insulin-like growth factor-I in ovarian granulosa cells.
Effect of basic fibroblast growth factor (bFGF) on the expression of receptors for luteinizing hormone (LH), a marker of differentiation, was studied using estrogen-primed rat ovarian granulosa cells in primary culture. bFGF had no effect by itself but dose-dependently induced expression of functional LH receptors in the presence of insulin-like growth factor-I (IGF-I). The effect of a combination of bFGF and IGF-I was delayed in onset and the magnitude of the response was smaller when compared to the action of follicle-stimulating hormone (FSH). Scatchard analysis revealed that dissociation constant (Kd) and number of LH receptors induced by bFGF and IGF-I were 0.47 nM and 6.48 fmol/10(6) cells, respectively. Unlike FSH, bFGF plus IGF-I did not cause an immediate increase in cAMP release, however, considerable amount of cAMP release was observed in cells incubated for 72 h with bFGF plus IGF-I. Indomethacin, an inhibitor of cyclooxygenase, attenuated both LH receptor expression and cAMP release induced by bFGF plus IGF-I but had little effect on the action of FSH. Finally, a combination of bFGF and IGF-I increased production of prostaglandin E2 in granulosa cells. These results indicate that bFGF is capable of inducing LH receptor in the presence of IGF-I by a mechanism involving production of prostaglandin E2.
Animals↗
Activin A: negative regulator of amylase secretion and cell proliferation in rat pancreatic acinar AR42J cells.
Activin A, a member of the transforming growth factor-beta supergene family, exists in secretory granules of non-B-cells of rat pancreatic islet (H. Yasuda, K. Inoue, H. Shibata, T. Takeuchi, Y. Eto, Y. Hasegawa, N. Sekine, Y. Totsuka, T. Mine, E. Ogata, and I. Kojima. Endocrinology 133: 624-630, 1993). Because functions of exocrine pancreas are influenced by hormones in pancreatic islet, it is possible that activin A affects the function of pancreatic acinar cells. To examine this possibility, we studied the effects of activin A on amylase secretion and DNA synthesis in AR42J cells. In these cells, dexamethasone (Dx) induces increases in secretory organelles and secretion of amylase (C. D. Logsdon, J. Moessner, J. A. Williams, and I. D. Goldfine. J. Cell Biol. 100: 1200-1208 1985). Activin A did not change the rate of amylase release by itself nor affect the cholecystokinin-stimulated amylase release from Dx-treated differentiated AR42J cells. However, when activin A was added together with Dx, activin A inhibited Dx-induced increase in amylase content in a dose-dependent manner. In the presence of 1 nM activin A, the effect of Dx was abolished. In the absence of Dx, amylase content of the cells was also reduced by activin A in a dose-dependent manner. The maximum inhibitory effect was obtained by 10 nM activin A, and at this concentration amylase content became undetectable. In addition, activin A potently inhibited DNA synthesis as assessed by [3H]thymidine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)
Activins↗
Physical exercise increases bone mineral density in postmenopausal women.
To examine whether physical exercise is beneficial in preventing postmenopausal osteoporosis, we measured bone mineral density (BMD) in three distinct groups of healthy postmenopausal Japanese women aged 49-61 yrs: 11 volleyball players (V) and 5 joggers (J), and 9 controls (C) who had not been participating in regular physical activity. BMD was measured at the lumbar spine (L2-L4) and proximal femur using dual energy X-ray absorptiometry, and at the radius using single X-ray photon absorptiometry. Serum levels of estradiol (E2), parathyroid hormone (PTH) and calcitonin were also measured by radioimmunoassay. Osteocalcin was determined by enzyme immunoassay. BMD in the lumbar spine was greater in the V and J groups than in the C group (P < 0.01). The J group had a significantly lower PTH level than the C group. In contrast to weight-bearing bones, we found no significant differences in BMD at the radius among the three groups. BMD at the distal radius was negatively correlated with years after menopause in both the V group and the J group significantly. These results indicate that regular physical exercise has a positive effect on the maintenance of bone mineral in postmenopausal women and that the protective action is localized in skeletal sites used predominantly for the sport without opposing the negative regulation caused by estrogen deficiency in systemic bones.
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Co-ordinate actions of FSH and insulin-like growth factor-I on LH receptor expression in rat granulosa cells.
The actions of FSH and Insulin-like growth factor-I (IGF-I) were studied in cultured rat ovarian granulosa cells. Cells became differentiated and expressed LH receptors when they were incubated for 72 h with 200 micrograms FSH/l (high FSH) but not 20 micrograms FSH/l (low FSH). Treatment with high but not low FSH increased the release of both immunoreactive and bioactive IGF-I into the medium. A combination of low FSH and IGF-I reproduced the effect of high FSH on LH receptor expression. We then examined the critical time when low FSH and IGF-I exerted their effects. In the presence of continuous low FSH, IGF-I was capable of inducing LH receptor expression even when added 24 h after the addition of low FSH. However, when IGF-I was added at 36 h, LH receptor expression measured at 72 h was greatly reduced. In contrast to the action of IGF-I, continuous exposure to low FSH was required for LH receptor expression, and IGF-I had no effect when FSH was not included for the entire 72 h of culture. DNA synthesis as assessed by both [3H]thymidine incorporation and nuclear bromodeoxyuridine labelling was moderate at the beginning of culture and markedly reduced at 24 h both in the presence and absence of either high FSH or low FSH plus IGF-I. In the presence of either high FSH or a combination of low FSH plus IGF-I, DNA synthesis remained decreased for up to 72 h whereas it began to increase in the absence of either high FSH or a combination of low FSH plus IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)
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Human growth hormone augmentation of epidermal growth factor binding sites on rat granulosa cells.
The effect of human GH (hGH) on the regulation of epidermal growth factor (EGF) receptor was investigated during differentiation of FSH-treated rat granulosa cells, which has been reported to be mediated by a cAMP-dependent mechanism. By measuring the binding of [125I]iodo-EGF to the intact cells, FSH was shown to cause increases in the number of EGF binding sites after culture for 72 h. When granulosa cells were cultured with hGH, the number of FSH-induced EGF binding sites was augmented, with a half-maximal effect at about 10 micrograms hGH/l and a maximal stimulatory concentration of 100 micrograms/l. The stimulatory effect of hGH was absolutely dependent on insulin which by itself showed stimulatory effects on EGF binding sites. Scatchard analysis of EGF binding sites indicated that treatment with hGH increased the number of EGF binding sites (17,200 sites/cell after treatment with FSH; 31,700 sites/cell after FSH plus hGH), but did not alter the binding affinity. The augmentation was observed after culturing for 48 h and increased progressively with time, reaching 280% of the level after FSH treatment by 120 h. Although progesterone synthesis was increased by hGH, the markers of cell differentiation such as cAMP synthesis and LH binding sites were suppressed, indicating hGH inhibition of the cAMP-mediated signal. The action of hGH on the EGF binding sites was not accompanied by cell proliferation. These findings indicate that hGH has a novel action on the regulation of rat granulosa cell EGF binding sites and that the granulosa cell may possess both cAMP-dependent and -independent mechanisms for expression of EGF binding sites.
Animals↗
A rapid and simple screening method for HIV-1 protease inhibitors using recombinant Escherichia coli.
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Amino Acid Sequence↗
Mer-NF5003B, E and F, novel sesquiterpenoids as avian myeloblastosis virus protease inhibitors produced by Stachybotrys sp.
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Amino Acid Sequence↗
Signaling pathway other than phosphatidylinositol turnover is responsible for constant expression of c-myc gene in primary cultures of rat hepatocytes.
We investigated signal transduction pathways involved in c-myc activation, using rat hepatocytes in primary culture. c-Myc mRNA was constantly expressed in the cultured hepatocytes regardless of the conditions present. When the expression was examined in the presence of various agents modulating intracellular signals, isoflavonoids (genistein, psi-tectorigenin, and orobol) significantly decreased c-myc mRNA levels, in a dose dependent manner. However, genistein did not decrease Li+ induced inositol phosphate accumulation using [3H]inositol-labeled cultured hepatocytes. In addition, we have shown that these isoflavonoids increase cytoplasmic free Ca2+, when measured using aequorin-loaded hepatocytes. In light of these observations, the persistent basal level of c-myc expression seems to be maintained by mechanisms other than phosphatidylinositol turnover.
Animals↗
Stimulation of calcium entry is prerequisite for DNA synthesis induced by platelet-derived growth factor in vascular smooth muscle cells.
The present study was conducted to determine a role of calcium entry in the growth promoting action of platelet-derived growth factor (PDGF) in cultured rat vascular smooth muscle cells. Addition of PDGF to quiescent cells induced a rapid elevation of cytoplasmic free calcium concentration, [Ca2+]c, followed by a sustained plateau of [Ca2+]c. Removal of extracellular calcium abolished the sustained phase, confirming that PDGF augments calcium entry. When calcium influx rate was determined by measuring initial uptake of [45Ca], PDGF induced a gradual increase in the rate of calcium entry. PDGF-mediated calcium entry became maximal after 90 min of the addition of PDGF, which persisted for at least 8 hrs. Calcium entry induced by PDGF was attenuated by nickel ion but not by a calcium channel blocker, nifedipine. When PDGF-induced calcium entry was blocked for a few hrs by either reducing extracellular calcium or by adding nickel, rate of entrance of S phase was reduced. In particular, VSMC stop progression when calcium entry is blocked in the middle of G1 phase. These results indicate that stimulation of calcium entry is prerequisite for initiation of DNA synthesis induced by PDGF in vascular smooth muscle cells.
Animals↗
Activin A increases intracellular free calcium concentrations in rat pancreatic islets.
Activin A stimulated insulin secretion in rat pancreatic islets, an effect that was attenuated by reduction of extracellular Ca2+ and abolished by either nitrendipine or verapamil. Activin A increased intracellular the free Ca2+ concentration, [Ca2+]i in fura-2-loaded islets. Activin A-mediated elevation of [Ca2+]i was abolished by the reduction of extracellular Ca2+ or the addition of nifedipine. In addition, activin A did not increase [Ca2+]i in the presence of diazoxide, an opener of ATP-sensitive K+ channels. These results suggest that activin A increases insulin secretion by stimulating Ca2+ entry.
Activins↗
Role of calcium entry and protein kinase C in the progression activity of insulin-like growth factor-I in Balb/c 3T3 cells.
We previously reported that insulin-like growth factor-I (IGF-I) stimulated calcium entry (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1988) J. Biol. Chem. 263, 16561-16567) and production of 1,2-diacylglycerol in IGF-responsive "primed competent" Balb/c 3T3 cells (Kojima, I., Kitaoka, M., and Ogata, E. (1990) J. Biol. Chem. 265, 16846-16850). The present study was conducted to determine a role of protein kinase C (PKC) in the progression activity of IGF-I. To monitor the activity of PKC in intact cells, we measured phosphorylation of a synthetic KRTLRR peptide, a substrate of PKC, immediately after the permeabilization of the cells with digitonin. When 1 nM IGF-I was added to primed competent cells, KRTLRR peptide phosphorylation was augmented. IGF-I induced more than 2-fold increase in KRTLRR peptide phosphorylation that was blocked by PKC19-36, a pseudosubstrate of PKC, which blocks the activity of the kinase, and Ro31-8220, an inhibitor of PKC. The phosphorylation remained elevated for up to 6 h. To assess the role of PKC in cell cycle progression, IGF-I-induced nuclear labeling was measured in the presence of Ro31-8220. Ro31-8220 reduced the rate of entrance into S phase when added in the first quarter of the G1 phase, but did not affect cell cycle progression when added at the second quarter or later. In contrast, reduction of extracellular calcium completely blocked cell cycle progression when done in the first, second, and third quarter but had no effect in the last quarter. These results indicate that IGF-I persistently activates PKC in primed competent cells, but the activation is required only for the initiation of progression. We conclude that IGF-I promotes cell cycle progression by calcium-dependent mechanisms that are largely independent of PKC.
3T3 Cells↗