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Biomedical subjects

M Fukase

Publications and source records attributed to M Fukase.

At least 19 recordsLinked to original sources
PubMed1992

Endothelin 1 hydrolysis by rat kidney membranes.

Hydrolysis of endothelin 1 by rat kidney membranes was investigated using a reverse-phase HPLC and an automated gas-phase protein sequencer. Endothelin 1 was hydrolyzed into four major fragments which were detected by HPLC. Phosphoramidon, an inhibitor of neutral endopeptidase 24,11, almost completely suppressed the production of three fragments, but one fragment was not affected by the inhibitor. Analysis of N-terminal sequences of the degradation products revealed that the phosphoramidon-sensitive fragments were generated by cleavage at the Ser5-Leu6 bond of endothelin 1 that was identical with its cleavage site by purified rat endopeptidase 24,11, reported previously. The phosphoramidon-insensitive fragment was produced by cleavage at Leu17-Asp18, which was distinct from the sites by endopeptidase 24,11, but corresponded to that by a phosphoramidon-insensitive metallo-endopeptidase recently isolated from rat kidney membranes by us [(1992) Eur. J. Biochem. 204, 547-552]. Kinetic determination of endothelin 1 hydrolysis by the isolated enzyme yielded values of Km = 71.5 microM and kcat = 1.49 s-1, giving a ratio of kcat/Km = 2.08 x 10(4) s-1.M-1. The Km value was much higher and the kcat/Km value was much lower than those for rat endopeptidase 24,11 reported previously. Thus, endopeptidase 24,11 appears to hydrolyze endothelin 1 more efficiently than the isolated enzyme does. Both enzymes may play physiological roles in the metabolism of endothelin 1 by rat kidney membranes in vivo.

Amino Acid Sequence↗
PubMed1992

The direct involvement of cAMP-dependent protein kinase in the regulation of collagen synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblast-like osteosarcoma cells (UMR-106).

The present study was performed to characterize the direct involvement of cAMP-dependent protein kinase (PKA) in the regulation of collagen synthesis by parathyroid hormone (PTH) and PTH-related peptide (PTHrP) in osteoblastic osteosarcoma cells, UMR-106. Sp-cAMPS (10(-4)M), a direct activator of PKA, as well as dibutyryl cAMP (dbcAMP, 10(-4)M) significantly inhibited collagen synthesis. Human (h) PTH-(1-34) (10(-7)M) and hPTHrP (10(-7) M) inhibited collagen synthesis to the same degree. Although Rp-cAMPS, which acted directly as an antagonist in the activation of PKA, did not affect collagen synthesis by itself, it significantly antagonized dbcAMP- and Sp-cAMPS-induced inhibition of collagen synthesis. Moreover, Rp-cAMPS antagonized PTH- and PTHrP-induced inhibition of collagen synthesis to the same degree. The present study first indicated that the activation of PKA was directly linked to the regulation of collagen synthesis by PTH in osteoblast and that PTHrP had the same effect on collagen synthesis presumably through the same mechanism as PTH.

Animals↗
PubMed1992

Immunohistochemical evaluation of parathyroid hormone-related protein (PTHrP) in the uterine cervix.

We investigated the immunohistochemical localization of parathyroid hormone-related protein (PTHrP), a major factor responsible for the humoral hypercalcemia of malignancy, in uterine cervical lesions. Formalin-fixed paraffin-embedded specimens from 16 cases of normal and reactive conditions, 45 cases of cervical intra-epithelial neoplasm (CIN) and 63 cases of invasive cancer were studied immunohistochemically by the avidine-biotin-peroxidase method, using an anti-PTHrP monoclonal antibody (MAb), 4B3. In normal and reactive conditions, PTHrP was positive in parabasal cells, squamous metaplasia, and hyperplastic reserve cells. In neoplastic conditions, 96% (43/45) of invasive squamous-cell carcinomas were positive for PTHrP, regardless of the patients' serum calcium levels. Two cases with hypercalcemia were strongly positive for PTHrP and showed prominent stromal interaction of the scirrhous type. In CIN, including koilocytic atypia, 76% (32/42) of cases were positive for PTHrP. In contrast, 91% (10/11) of adenocarcinomas were negative for PTHrP. In conclusion, we found, first, that in non-neoplastic conditions, the presence of PTHrP was correlated with the transformation of progenitor cells into squamous epithelia and with the maturation of keratinocytes and, second, that in squamous-cell carcinoma, the degree of keratinization and stromal interaction was higher in direct proportion to the apparent incidence of detectable PTHrP.

Animals↗
PubMed1992

The activation of cAMP-dependent protein kinase is directly linked to the stimulation of bone resorption by parathyroid hormone.

The present study was performed to characterize the direct involvement of cAMP in the stimulation of bone resorption by parathyroid hormone (PTH), using Sp-cAMPS and Rp-cAMPS, which were the direct agonist and antagonist in the activation of cAMP-dependent protein kinase (PKA), respectively. Bone resorbing activity was estimated as the number of pits formed on the dentine slice and total area of pits per slice in bone marrow cells derived from 2 week-old mice. Dibutyryl cAMP (dbcAMP)(10(-4)M) and Sp-cAMPS (10(-4)M) caused the remarkable stimulation of bone resorption. Although Rp-cAMPS (10(-4)M) did not affect bone resorption by itself, it significantly inhibited dbcAMP- and Sp-cAMPS-induced stimulation of bone resorption. Moreover, Rp-cAMPS (10(-4)M) antagonized 10(-7)M human PTH-(1-34)-induced stimulation of bone resorption, although it did not affect 10(-8)M 1,25(OH)2D3-induced stimulation of bone resorption. Present study indicates the direct involvement of PKA in the stimulation of bone resorption by PTH.

Animals↗
PubMed1992

Comparison of bone mineral content among Japanese, Koreans, and Taiwanese assessed by dual-photon absorptiometry.

Throughout the world the diagnosis and management of osteoporosis currently involves the measurement of bone mineral content. There are, however, no studies comparing bone mineral content among Asian people. This cross-sectional study was designed to quantify spine and femur bone mineral density (BMD) in Japanese and compare BMD among Asian people (Japanese, Koreans, and Taiwanese) using the same model dual-photon system (Norland Model 2600). Following a peak BMD in the third and fourth decades, the Japanese BMD values of the lumbar spine and femoral neck showed a clear decrease (annual loss of 0.99 and 0.74%, respectively) with age in females. On the other hand, Japanese BMD values were stable in males until the fifth decade. There was some decrease in BMD with age after the fifth decade, which was much less obvious than that in females. An age-dependent loss of BMD was clearly observed in Japanese and Korean but not in Taiwanese females. Korean males seemed to have a clearer age-dependent loss of BMD compared to Japanese males. Our findings indicate that differences may exist in the BMD of Asian people and that in addition to the quantitative determination of individual BMD, dual-photon absorptiometry may be useful for the comparison of BMD among different ethnic and cultural groups.

Absorptiometry, Photon↗
PubMed1992

Second messenger signaling in the regulation of cytosolic pH and DNA synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblastic osteosarcoma cells: role of Na+/H+ exchange.

The present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH-related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR-106 which possessed PTH-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amiloride-inhibitable Na+/H+ exchange system. Both human (h)PTH-(1-34) and hPTHrP-(1-34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose-dependent manner with a minimal effective dose of 10(-10) M. Dibutyryl cAMP (10(-4) M and Sp-cAMPS (10(-4) M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10(-6) M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP- and Sp-cAMPS-induced decrease in pHi but did not affect calcium ionophore-induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore-induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA-inhibited Na+/H+ exchange and increased cytosolic calcium-induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/H+ exchange system.

Amiloride↗
PubMed1992

The expression of parathyroid hormone-related protein (PTHrP) in normal parathyroid: histochemistry and in situ hybridization.

The expression and localization of parathyroid hormone-related protein (PTHrP), a major factor responsible for humoral hypercalcemia of malignancy (HHM), was investigated in 14 cases of surgically resected normal parathyroid glands. For light microscopic immunohistochemistry, formalin-fixed and paraffin-embedded specimens were stained with avidin-biotin-peroxidase complex (ABC) using the anti-PTHrP monoclonal antibody (MoAb), 4B3. Four percent paraformaldehyde (PFA)-fixed and OCT compound-embedded specimens were used for pre-embedded immunoelectron microscopy. For in situ hybridization, 4% PFA-fixed, frozen sections were studied using a bromodeoxyuridine (BrdU)-labeled PTHrP cDNA probe. Immunohistochemically, 12 of the 14 cases were positive for PTHrP, which was observed mainly in the oxyphil and transitional oxyphil cells. The chief and clear cells, on the other hand, were faintly positive. Electron microscopically, secretory granules positive for PTHrP were observed in cells containing abundant mitochondria. Consistent with the PTHrP immunoreactivity, transcripts of PTHrP were observed also in the oxyphilic cells by in situ hybridization. Thus the production and secretion of PTHrP was shown by the oxyphil cell lineage in the normal parathyroid glands.

Histocytochemistry↗
PubMed1992

Parathyroid hormone degradation by opossum kidney cells via receptor-mediated endocytosis and lysosomal hydrolysis.

The mechanisms involved in parathyroid hormone (PTH) degradation by proximal renal tubule cells were studied using an opossum kidney cell line possessing PTH receptors as an in vitro model system. One hour incubation of 5 nmol/l human (h) PTH-(1-84) with intact opossum kidney cells (4.0 x 10(6) cells) resulted in about 70% degradation and disappearance of hPTH-(1-84) from the medium, as determined by a two-site immunoradiometric assay. Preincubation with 100 nmol/l h[Nle8, Nle18, Tyr34]PTH-(1-34)amide for 6, 24, 48 and 72 h caused a 26, 47, 62 and 73% decrease, respectively, in PTH degradation by opossum kidney cells. Binding studies with 125I-labeled h[Nle8,Nle18,Tyr34]PTH-(1-34)amide as a radioligand showed that PTH receptor binding decreased with the time of pretreatment with the agonist. Pretreatments of the cells with monensin, an inhibitor of endocytosis, and the lysosomotropic agents such as chloroquine, ammonium chloride and leupeptin, inhibited degradation of hPTH-(1-84) by 87, 71, 76 and 72%, respectively. Concentrations of 5 nmol/l hPTH-(39-84) and hPTH-(39-68), which are known not to bind to PTH receptors appreciably, were not degraded by opossum kidney cells during 1 h incubations. Thus intact, biologically active PTH, but not its inactive fragments, is degraded by opossum kidney cells, by receptor-mediated endocytosis and lysosomal hydrolysis. A mechanism resembling the peritubular uptake of intact PTH by perfused kidneys reported previously appears to play a main role in PTH metabolism by cultured renal cells.

Animals↗
PubMed1992

Comparison of osteoporosis and calcium intake between Japan and the United States.

The number of osteoporotic females in Japan with vertebral bone mineral density measured by dual energy x-ray absorptiometry, defined as less than -3 SD of the peak bone mass, is approximately 10,000,000, corresponding to 8% of the whole population of Japan. While this value approximately corresponds to the prevalence of low bone mineral density in the United States, the incidence of hip fracture appears to be much less in Japan than in the United States, 50,000 per 125,000,000 per year compared with 250,000 for a population twice as large. This seems to be paradoxical because of the lower bone mineral density and lower calcium intake in Japan, with 400-500 mg/day mainly as soybean products, small fish with bones, and vegetables. The difference in hip fracture incidence, however, may not actually be as wide as it seems when the larger number of bedridden elderly subjects in Japan is taken into consideration. In these bedridden subjects with severe immobilization osteoporosis, hip fracture is only prevented by the fact that they are not ambulatory. Life-style difference may also offer an explanation. Sitting on a tatami mattress on completely flexed knees with frequent standing up, along with other household work, in a narrow home space may ensure a marked development of hip musculature and also provide skill in balancing oneself to prevent fails. The difference in fracture incidence should be analyzed from various angles to obtain a firm ground for the future prevention of hip fracture due to osteoporosis. Although osteoporosis universally affects all races and nationalities, conspicuous differences may be encountered in the severity of its manifestations and complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1992

New actions of parathyroid hormone through its degradation.

Parathyroid hormone degradation is intimately connected with its action. By the action of the unique renal neutral cytosolic PTH ase, PTH is split into 1-34 and 35-84 fragments, and further into 35-70 and 71-84 fragments. Amino-terminal 1-34 peptide was found to participate in the autoregulation of PTH secretion, suppressing the intact PTH secretion both in vivo in humans and in vitro in the dispersed bovine parathyroid cells. C-terminal fragment 35-84 and N-terminal fragment 1-34 both suppress the alkaline phosphatase production by ROS 17/2.8 cells to a lesser extent than the intact PTH 1-84, and the sum of the effects of the two fragments approximately equaled that of the intact hormone. Fragments 35-70 and 71-84 were devoid of such activity. Intracellular free calcium of human vascular endothelial cells was raised by intact 1-84, lowered on the contrary by C-terminal 35-84 fragment, but fragments 1-34, 35-70 and 71-84 had no effect. Fragments generated by the actions, supporting the physiological significance of PTH degradation by its target cells.

Animals↗
PubMed1991

Effect of elevated extracellular calcium on the proliferation of osteoblastic MC3T3-E1 cells:its direct and indirect effects via monocytes.

There has been evidence that elevated calcium concentration at the resorptive site of the bone directly regulates osteoclast function. In the present study, in order to clarify the role of elevated calcium concentration at the resorptive site in the regulation of osteoblast function, not only direct but also indirect effect via human monocytes of the increase in extracellular calcium (Cae) on the proliferation of osteoblastic MC3T3-E1 cells have been investigated in serum-free condition. The increase in Cae enormously stimulated osteoblast proliferation at the concentration of 3 to 20 mM. When human monocytes were cultured at the elevated Cae concentration, monocyte-conditioned medium-induced stimulation of osteoblast proliferation was significantly amplified. Present data demonstrate that elevated Cae has pronounced stimulatory effect on osteoblast proliferation not only directly but also indirectly via monocytes. Calcium released from bone matrix at the resorptive sites might be linked to the coupling of osteoclast and osteoblast functions.

Animals↗
PubMed1991

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization.

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.

Amino Acid Sequence↗
PubMed1991

The activation of cAMP-dependent protein kinase is directly linked to the inhibition of osteoblast proliferation (UMR-106) by parathyroid hormone-related protein.

The present study was performed to compare the effect of parathyroid hormone-related protein (PTHrP) on the proliferation of osteoblastic osteosarcoma cells (UMR-106) with that of PTH and characterize the direct involvement of cAMP in the change of osteoblast proliferation by PTHrP. Human(h)PTHrP-(1-34) (10(-11)-10(-7)M) dose-dependently inhibited [3H]thymidine incorporation (TdR) in the same manner as hPTH-(1-34). The simultaneous addition of PTHrP and PTH at a maximal effective dose of 10(-7) M did not cause additive suppressive effect on cell proliferation. Rp-cAMPs, which has been recently shown to act directly as antagonist in the activation of cAMP-dependent protein kinase (PKA), dose-dependently (10(-6)-10(-4)M) antagonized PTHrP-induced suppression of TdR in the same manner as PTH. Present study indicated that PTHrP has the same effect on osteoblast proliferation as PTH and that the activation of PKA is directly linked to the change of osteoblast proliferation by PTHrP.

Animals↗
PubMed1991

Effect of 17 beta-estradiol on the proliferation of osteoblastic MC3T3-E1 cells via human monocytes.

Effects of human monocyte-conditioned medium on the proliferation of osteoblastic MC3T3-E1 cells were investigated in serum-free cultured condition. Monocyte-conditioned medium significantly stimulated osteoblast proliferation at the concentration between 10 and 30%, compared to that in the absence of monocytes. 17 beta-estradiol directly stimulated osteoblast proliferation at the concentrations of 10(-8) and 10(-10)M. On the contrary, the conditioned medium prepared by monocytes cultured in the presence of 17 beta-estradiol at the concentrations of 10(-8) and 10(-10)M significantly inhibited osteoblast proliferation. Present data indicate that in addition to direct effect on osteoblasts, 17 beta-estradiol affected osteoblast proliferation presumably through modulating the release of several local regulators of bone turnover from monocytes. The effect on osteoblastic activity via monocytes might be linked to the coupling of osteoclast and osteoblast actions.

Animals↗
PubMed1991

Direct inhibitory effect of amino-terminal parathyroid hormone fragment [PTH(1-34)] on PTH secretion from bovine parathyroid primary cultured cells in vitro.

We have examined the possibility of direct inhibitory effect of PTH(1-34) on PTH secretion in bovine parathyroid cells. As low as 10(-12) M PTH(1-34) completely inhibited low calcium (0.5 mM Ca2+)-stimulated PTH secretion by these cells. In the presence of 1.25 mM Ca2+, 10(-12) M PTH(1-34) inhibited PTH secretion by about 14.3% of the basal value, while 10(-11) M or higher concentration of PTH(1-34) showed potent inhibitory effects equivalent to the inhibitory action of high calcium concentration (2.5 mM Ca2+) on PTH secretion. At 2.5 mM Ca2+, as much as 10(-9) M PTH(1-34) failed to inhibit PTH secretion further. These results suggest that PTH(1-34) might directly, not via calcium concentration, inhibit PTH secretion by parathyroid cells and that a cooperative mechanism could exist between calcium and PTH(1-34) to inhibit PTH secretion.

Animals↗
PubMed1991

Interleukin-1 enhances the response of osteoblasts to platelet-derived growth factor through the alpha receptor-specific up-regulation.

Both platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) are produced by activated macrophages and are thought to contribute to bone remodeling, but their precise roles remain to be clarified. The interaction between PDGF and IL-1 was, therefore, studied in normal osteoblast-like cells (MC3T3-E1). The expression of alpha- and beta-PDGF receptors on MC3T3-E1 cells was detected by RNA blot analysis and confirmed by immunoblot analysis. PDGF-induced chemotactic as well as mitogenic activities were synergistically enhanced by either IL-1 alpha or IL-1 beta (40 units/ml) pretreatment in serum-free medium, although IL-1 alone did not show any detectable chemotactic activities. This biological enhancement by IL-1 was accompanied by a selective increase of alpha-PDGF receptor expression, following the augmentation of alpha receptor autophosphorylation and inositol phosphate hydrolysis induced by PDGF-AA. These findings suggest that PDGF and IL-1 are jointly involved in the bone-remodeling microenvironment as local coupling factors.

Blotting, Northern↗