Osteoporosis is highly prevalent in Japanese males with chronic obstructive pulmonary disease and is associated with deteriorated pulmonary function.

Osteoporosis has recently been recognized as a major comorbidity in chronic obstructive pulmonary disease (COPD). We conducted a cross-sectional study in a cohort of 136 Japanese males with COPD to evaluate the prevalence of vertebral fracture (VF) and to explore its relationship with pulmonary function parameters. VFs were present in 108 (79.4%); multiple and severe (SQ grade 2 or 3) VFs were found in 77 (56.6%) and 25 (18.4%), respectively. Multivariate logistic regression analyses revealed that decrease in forced expiratory volume in one second (FEV1.0)/forced vital capacity (FVC) [odds ratio (OR) 0.963, 95% confidence interval (CI) 0.929-998, p = 0.036] was associated with the presence of VF after adjustment for age and that FVC (OR 0.462, 95% CI 0.220-0.968, p = 0.041) and current smoking (OR 2.992, 95% CI 1.128-7.940, p = 0.028) were associated with VF severity (grade 2-3 vs. 1). We also found that FEV1.0 was the sole independent determinant of the number of VFs by stepwise multivariate linear regression (p < 0.001). Bone mineral density (BMD) values were available in 49 subjects. Mean T scores were -2.0 ± 1.2 in femoral neck, -1.4 ± 1.2 in total hip and -1.1 ± 1.4 in lumbar spine. Nineteen patients (38.8%) had a BMD T score less than -2.5. BMD Z scores of all the sites showed a progressive decrease as GOLD stage of COPD advanced (p < 0.05). Our results indicate a high prevalence of osteoporosis in Japanese male COPD patients and a strong inter-relationship between the two diseases, re-emphasizing the urgent need for appropriate intervention to maintain both bone and lung health.

Down-regulation of molecular chaperone 78-kd glucose-regulated protein/immunoglobulin-binding protein expression involved in enhancement of human RS cell mutability.

OBJECTIVES: Enhancement of cell mutability via extracellular materials of cancer cells is a crucial event leading to the development of cancers; however, the activation process of mutability is still not well understood. In this study, to identify the regulatory mechanism of cell mutability, we investigated mutability modulated in response to human pancreatic cancer cell-conditioned medium and identified the candidates for cellular molecules involved in the mutability modulation. METHODS: To test the mutation-modulating effects of the conditioned medium, human RS cells were cultured with medium derived by culturing human pancreatic cancer KP-4 cells, followed by irradiation with UV (mainly 254 nm in wavelength). Mutations were detected by phenotypic ouabain resistance and genetic base substitution of K-ras codon 12. Messenger RNA differential display was used to identify genes that were differentially expressed between conditioned medium-treated and mock-treated RSa cells. The influence of 78-kd glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) expression on mutability was assessed by the down-regulation of GRP78/BiP using antisense oligonucleotides or antisense complementary DNA. RESULTS: The UV-induced mutagenicity in RS cells was strengthened by preculture with KP-4 cell-conditioned medium. Messenger RNA differential display revealed that GRP78/BiP expression was suppressed in RS cells after treatment of the conditioned medium. Furthermore, the level of UV-induced mutations was elevated significantly in GRP78/BiP down-regulated cells. CONCLUSIONS: Culture of human RS cells with pancreatic cancer KP-4 cell-conditioned medium resulted in increased UV mutagenicity, possibly via the down-regulation of GRP78/BiP.

Upregulation of sodium-dependent vitamin C transporter 2 expression in adrenals increases norepinephrine production and aggravates hyperlipidemia in mice with streptozotocin-induced diabetes.

The hyperglycemia and hyperoxidation that characterize diabetes lead to reduced vitamin C (L-ascorbic acid, AA) levels in diabetic humans and animals. We examined the possibility that diabetes-induced low plasma AA levels impair AA distribution to various tissues and that these changes are closely related to the development of diabetic complications. AA levels were markedly decreased in the plasma and increased in the adrenals of mice with streptozotocin (STZ)-induced diabetes. Consistently with these results, in [1-(14)C]AA accumulation assays, the efficiency of [1-(14)C]AA accumulation was significantly higher in the adrenals (which had the greatest ability to accumulate [1-(14)C]AA) of diabetic mice than in those of controls. Expression of sodium-dependent vitamin C transporter (SVCT)-2, a transporter of AA, was upregulated in diabetic adrenals. Furthermore, increased AA incorporation into the diabetic adrenals by SVCT-2 led to increased plasma norepinephrine, triglyceride and free fatty acid levels in mice with STZ-induced diabetes. Therefore, oversupplementation with AA could be deleterious in diabetic patients, because overexpression of adrenal SVCT-2 in diabetes could lead to excessive AA uptake, thus enhancing norepinephrine production and exacerbating some diabetic complications. Interestingly, however, treatment with AA dose-dependently abolished the increased expression of adrenal SVCT-2 and normalized the abovementioned plasma parameters in diabetic mice. These results suggest SVCT-2-mediated increases in AA uptake by the adrenals followed by excessive production of plasma norepinephrine may play a pivotal role in the development of diabetic complications.

Degradation of soluble proteins including some allergens in brown rice grains by endogenous proteolytic activity during germination and heat-processing.

The effect of germination and subsequent heat-processing on the degradation of soluble proteins, including some allergenic proteins, in brown rice grains was investigated. The content of soluble proteins, including 14-16-kDa and 26-kDa allergens, in the germinated and processed brown rice grains (GPR) was much lower than that of non-germinated brown rice. These proteins in brown rice grains were also much lower after subsequent heat-processing during the manufacturing process. The protease activity of germinated brown rice (GR) was detected and increased 1.5 times after germination. The optimum pH values for degradation of the 26-kDa and 14-16-kDa allergens in the GR grains were 4 and between 5 and 7, respectively. These results suggest that the decrease in the soluble proteins and allergens was induced in part by proteolytic degradation. The presence of a detergent enhanced the proteolytic degradation of the soluble proteins, especially of the 26-kDa allergen, in the brown rice grains. The degradation of the 26-kDa allergen was weakly inhibited by NEM, suggesting cysteine protease(s) may have been involved in its degradation. These results suggest that the two abundant allergens were degraded in a different manner and probably by different proteases in the grains during germination.

X-ray absorption fine structure analysis of the local environment of zinc in dentine treated with zinc compounds.

It has been reported that zinc oxide (ZnO) inhibits dentine demineralization. By using the X-ray absorption fine structure (XAFS) technique, our aims in this study were to provide information about the local environment of zinc atoms in dentine that had been treated with zinc compounds. We measured the Zn K-edge X-ray absorption near-edge structure (XANES) and the extended X-ray absorption fine structure (EXAFS) of dentine specimens treated with zinc chloride or ZnO. In XAFS analyses, the spectra of dentine specimens treated with ZnO (D-ZO) or with zinc chloride (D-ZC) were similar and obviously different from the reference ZnO spectrum. This suggests that most of the zinc atoms detected in D-ZO are not derived from particles of ZnO. The spectra of D-ZO and D-ZC were similar to the spectrum of the synthetic, zinc-containing hydroxyapatite, but were not similar to that of zinc in ZnCl2-treated collagen. The results of this study suggest that most of the zinc atoms detected were attached to hydroxyapatite and not to collagen.

Regulation of an inwardly rectifying K+ channel by nitric oxide in cultured human proximal tubule cells.

We investigated the effects of nitric oxide (NO) on activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells, using the cell-attached mode of the patch-clamp technique. An inhibitor of NO synthases, N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM), reduced channel activity, which was restored by an NO donor, sodium nitroprusside (SNP; 10 microM) or 8-bromo-cGMP (8-BrcGMP; 100 microM). However, SNP failed to activate the channel in the presence of an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM). Similarly, the SNP effect was abolished by a protein kinase G (PKG)-specific inhibitor, KT-5823 (1 microM), but not by a protein kinase A-specific inhibitor, KT-5720 (500 nM). Another NO donor, S-nitroso-N-acetyl-D,L-penicillamine (10 microM), mimicked the SNP-induced channel activation. In contrast to the stimulatory effect of SNP at a low dose (10 microM), a higher dose of SNP (1 mM) reduced channel activity, which was not restored by 8-BrcGMP. Recordings of membrane potential with the slow whole cell configuration demonstrated that l-NAME (100 microM) and the high dose of SNP (1 mM) depolarized the cell by 10.1 +/- 2.6 and 9.2 +/- 1.0 mV, respectively, whereas the low dose of SNP (10 microM) hyperpolarized it by 7.1 +/- 0.7 mV. These results suggested that the endogenous NO would contribute to the maintenance of basal activity of this K(+) channel and hence the potential formation via a cGMP/PKG-dependent mechanism, whereas a high dose of NO impaired channel activity independent of cGMP/PKG-mediated processes.

Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3-E1 osteoblasts.

Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca2+-, PO4(3-)-stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in Vmax with no change in Km. Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM beta-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization.

Modulation of the Ca2+-activated large conductance K+ channel by intracellular pH in human renal proximal tubule cells.

The Ca2+-activated and voltage-sensitive large conductance K+ channel (BK channel) with a slope conductance of about 300 pS is present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs). In this study we examined the effects of cytoplasmic pH (pH(i)) on activity and gating kinetics of the BK channel by using the inside-out configuration of the patch-clamp technique. At a constant cytoplasmic Ca(2+) concentration ([Ca2+]i), membrane depolarization raised channel open probability (P(o)), and lowering pH(i) shifted the P(o)-membrane potential (V(m)) relationship to the positive voltage direction. However, the value of the gating charge was not affected by changes in pH(i), suggesting that the effects of pH(i) on P(o) were not due to an alternation of the voltage sensitivity. At constant V(m), lowering pH(i) suppressed the [Ca2+]i-dependent channel activation and shifted the P(o)-[Ca2+]i relationship in the direction of higher [Ca2+]i with a reduction of maximal P(o). Furthermore, both the mean open and mean closed times of the BK channels at pH(i) 6.3 in the presence of 10(-4) M [Ca2+](i) were shorter than those at pH(i) 7.3 in the presence of 10(-5) M [Ca2+]i, even though these two different conditions gave a similar P(o). The data indicate that cytoplasmic H+ suppresses P(o) of the BK channel in RPTECs, which involves the mechanism independent of Ca2+ activation. Our preliminary kinetic analysis also supported this notion.

Protein kinase G activates inwardly rectifying K(+) channel in cultured human proximal tubule cells.

An ATP-regulated inwardly rectifying K(+) channel, whose activity is enhanced by PKA, is present in the plasma membrane of cultured human proximal tubule cells. In this study, we investigated the effects of PKG on this K(+) channel, using the patch-clamp technique. In cell-attached patches, bath application of a membrane-permeant cGMP analog, 8-bromoguanosine 3',5'-monophosphate (8-BrcGMP; 100 microM), stimulated channel activity, whereas application of a PKG-specific inhibitor, KT-5823 (1 microM), reduced the activity. Channel activation induced by 8-BrcGMP was observed even in the presence of a PKA-specific inhibitor, KT-5720 (500 nM), which was abolished by KT-5823. Direct effects of cGMP and PKG were examined with inside-out patches in the presence of 1 mM MgATP. Although cytoplasmic cGMP (100 microM) alone had little effect on channel activity, subsequent addition of PKG (500 U/ml) enhanced it. Furthermore, bath application of atrial natriuretic peptide (ANP; 20 nM) in cell-attached patches stimulated channel activity, which was blocked by KT-5823. In conclusion, cGMP/PKG-dependent processes participate in activating the ATP-regulated K(+) channel and producing the stimulatory effect of ANP on channel activity.