Kuniaki Takagi

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions.

Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2′,5′-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.

P2X7 Receptor-Dependent Cell Death Is Modulated during Murine T Cell Maturation and Mediated by Dual Signaling Pathways

Extracellular ATP causes apoptosis and/or necrosis of the hemopoietic lineage through the activation of P2X7 receptors. In this study, we investigated P2X7 receptor-mediated cell death during murine T cell maturation. The expression level and activity of P2X7 receptors, as measured by induction of cell death and pore formation, were higher in splenocytes than thymocytes. Flow cytometric analysis revealed that cell shrinkage was induced by activation of the P2X7 receptor in murine lymphocytes and the responding cells were T cells. Splenic T cells were more responsive than their thymic counterpart. These observations indicate that the system of P2X7 receptor-mediated cell death in T cells could be modulated during T cell maturation. Furthermore, decreased extracellular Cl− suppressed ATP-induced cell shrinkage in splenocytes without inhibiting ERK1/2 phosphorylation, which is reported to mediate necrotic cell death. Treatment with U0126 (a MEK inhibitor) suppressed ATP-induced ERK1/2 phosphorylation without inhibiting cell shrinkage. Moreover, decreased extracellular Cl− and treatment with U0126 suppressed ATP-induced cell death. These observations indicate that the activation of P2X7 receptor leads to T cell death by two independent pathways, one of which is cell shrinkage dependent and the other of which involves the phosphorylation of ERK1/2. In conclusion, we demonstrate increasing P2X7 receptor activity during T cell maturation and the existence of two essential pathways in P2X7 receptor-mediated T cell death. Our findings suggest that ATP-induced cell death of peripheral T lymphocytes is important in P2X7 receptor-regulated immune responses.

Nitric oxide blocks the cell cycle of mouse macrophage-like cells in the early G2+M phase

The effects of nitric oxide produced by macrophage‐like cells (Mml) on the cell cycle were investigated. Mml cells lost proliferative activity in the presence of interleukin‐6 (IL‐6) and a subpopulation accumulated in the G2+ M phase. This level increased in proportion to the incubation time. The DNA content of the cells was slightly lower than that of Mml cells treated with vinbrastine or demecolcine, drugs which block the cell cycle in the M phase. The peak of the early G2+M phase was reduced by treatment with N G‐mono‐methyl‐l‐arginine. However, after treatment with exogenous nitric oxide or sodium nitroprusside, the G0/G1 phase increased, but the early‐G2+M and the S phase decreased. The flow cytometry pattern in IL‐6‐treated Mml was the same as that of cytochalasin B‐treated Mml. These data suggest that endogenous nitric oxide affects the microfilament system of IL‐6‐treated Mml cells and blocks the cell cycle in the early G2+M phase.

Reorganization of ZO‐1 by sodium‐dependent glucose transporter activation after heat stress in LLC‐PK1 cells

Heat stress (HS) induces activation of high‐affinity sodium‐dependent glucose transporter (SGLT1) in porcine renal LLC‐PK1 cells. In this study, we investigated the roles of SGLT1 activation in reorganization of zonula occludens‐1 (ZO‐1), a cytosolic tight junction (TJ) protein, after HS. HS (42°C, 3 h) caused decrease in transepithelial electrical resistance (TER). Subsequent incubation at 37°C for 12 h increased TER above pre‐HS level. The treatment of phloridzin, a potent SGLT1 inhibitor, or the replacement of glucose with a nonmetabolizable glucose analog blocked the recovery of TER and increased the transepithelial flux of FITC‐dextran (4,000 Da). Immunofluorescent staining of ZO‐1 showed that HS diffused ZO‐1 from cell contact to cytosolic sites. Furthermore, the fraction of ZO‐1 was distributed from the Triton X‐100 insoluble to the Triton X‐100 soluble pool. After incubation at 37°C for 12 h, cell contact and ZO‐1 extractability with Triton X‐100 returned to pre‐HS conditions, but the recovery was completely prevented by phloridzin. Tyrosine kinases activity was increased by HS that was inhibited by phloridzin. Genistein and CGP77675, tyrosine kinases inhibitors, blocked the recovery of TER and increased the transepithelial flux of FITC‐dextran. Furthermore, these inhibitors prevented the recovery of cell contact and ZO‐1 extractability with Triton X‐100 as same as phloridzin. These findings suggested that the activation of SGLT1 reorganized ZO‐1 mediated by elevation of tyrosine kinases activity after heat injury.

Inhibitory mechanisms of antibody production by nitrogen oxides released from activated macrophages during the immune response: relationship to energy consumption.

We investigated the relationship between the sensitivity of mouse splenocytes in immune response to nitrogen oxides and energy consumption rate of the cells. Macrophage‐like cells (Mm 1) pretreated with lL‐6 served as the source of the nitrogen oxides. The antibody production of both 2,4,6‐trinitrophenyl‐keyhole limpet haemocyanin‐primed splenocytes and B cell hybridomas was markedly reduced; about 20–40% of splenocytes and B cell hybridomas were killed by co‐culture with IL‐6‐treated Mm 1. Cell viability and antibody production were completely restored by the addition of NG‐monomethyl L‐arginine to the culture medium. The cytotoxicity of the nitrogen oxides was correlated with the distance between effector and target cells. Under conditions of low cytotoxicity, antibody production by B cell hybridomas was suppressed by the nitrogen oxides, this suppression not being correlated with the reduction in cell growth. The sensitivity of the target cells differed in co‐cultures of antigen‐primed splenocytes and B cell hybridomas with IL‐6‐treated Mm 1. The nitric oxide‐sensitivity of the cells corresponded to their 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide reducing activity and ATP consumption rate. These findings suggest that nitrogen oxides act as regulatory molecules in immune response in three ways: cytostasis, reduction of cell growth and suppression of antibody synthesis.

Suppression of Cytokine Production in T Helper Type 2 Cells by Nitric Oxide in Comparison with T Helper Type 1 Cells

We examined the effect of nitric oxide (NO) on cytokine production in T helper (Th) cell subsets, using murine splenic CD4+ T cells and two types of Th clones. Interferon‐gamma‐treated murine peritoneal exudate cells (IFN‐PEC) suppressed DNA synthesis to 60% of the control level in CD4+ T cells stimulated with the anti‐CD3 monoclonal antibody. The production of IL‐2 and IL‐4 in the CD4+ T cells decreased to 63.2% and 9.2%, respectively, of the control value by co‐culture with IFN‐PEC. The addition of NG‐monomethyl‐L‐arginine (L‐NMMA) partially recovered the suppression of DNA synthesis. In the presence of indomethacin, the suppression of DNA synthesis was partially inhibited and the reduction in the cytokine production caused by IFN‐PEC was partially recovered. The simultaneous addition of NG‐monomethyl‐L‐arginine (L‐NMMA) and indomethacin completely inhibited not only the suppression of DNA synthesis but also the reduction in the cytokine production caused by IFN‐PEC. Moreover, DNA synthesis in the Th2 clone was suppressed to a greater extent than that in the Th1 clone by co‐culture with IFN‐PEC. This suppression in the Th1 clone was inhibited by the addition of L‐NMMA, whereas the DNA synthesis in the Th2 clone was not recovered by L‐NMMA. In addition, sodium nitroprusside (SNP) suppressed IL‐4 production in the Th2 clone but had no effect on IL‐2 production in the Th1 clone. In the experiment of the co‐culture with IFN‐PEC, the inhibitory‐effect of NO on T cell activation was not clarified by the influence of prostaglandins. However, in conclusion, cytokine production in Th2 cells may be more susceptible to NO than that in Th1 cells.

Angiotensin II Receptor Blockade Ameliorates Mesangioproliferative Glomerulonephritis in Rats through Suppression of CTGF and PAI-1, Independently of the Coagulation System

Background: Previously we observed that the coagulation system promotes matrix protein accumulation through transforming growth factor (TGF)-β and connective tissue growth factor (CTGF) expression in rat mesangioproliferative glomerulonephritis (MsPGN). Angiotensin II receptor blockers (ARBs) are known to suppress matrix accumulation in experimental MsPGN. In the present study, we investigated whether ARB suppresses MsPGN through inhibition of these profibrotic cytokines, and in relation to coagulation and fibrinolytic systems. Methods: MsPGN was induced in Wistar rats by intravenous injection of anti-Thy-1.1 monoclonal antibody, OX-7. As an ARB, olmesartan was orally administered in rat feed from the day of OX-7 injection (day 0) to day 8, when rats were sacrificed and kidney specimens were collected. The degrees of cellular proliferation, matrix production, coagulation factors, and inhibitory factor of fibrinolysis were evaluated. Results: Although blood pressure did not change in the normal, disease control, or treatment groups, the amount of urinary protein was significantly decreased in the ARB-treated groups, compared with the disease control group (p < 0.05). α-Smooth muscle actin expression was suppressed significantly in the treatment groups (p < 0.001). Blue-staining areas of trichrome, the number of proliferating cell nuclear antigen (PCNA)- or ED-1-positive cells, fibronectin and plasminogen activator inhibitor type 1 in glomeruli significantly decreased in the treatment groups (p < 0.05, respectively); however, fibrin-related antigen and factor V depositions were not suppressed in the treatment groups. Conclusions: These results suggest that the ARB drug would ameliorate MsPGN in vivo, at least partly through CTGF and plasminogen activator inhibitor type 1 suppression, and independently of the local coagulation system in glomeruli.

Induction of nitrite production in mouse spleen cells by immunization

Changes of nitrite production in mouse spleen cells of in vitro secondary antibody response were investigated. Mouse spleen cells immunized with gamma globulin fraction of rat serum produced nitrite 3 days after in vitro challenging with the same antigen. Nitrite production of rabbit IgG-challenged spleen cells was found to be about 2.9-times higher than that of spleen cells primed with the gamma globulin fraction of rat serum. Nitrite production in this system was completely suppressed by T cell depletion (99.7% inhibition). Furthermore, nitrite production in these cells significantly decreased by addition of anti-interferon gamma antibody (62.9% inhibition). These data indicate that nitrite production in antigen-immunized spleen cells is affected with the immunogenicity of an antigen and regulated by T cells, especially interferon (IFN) gamma.

Development of a saliva collection device for use in the field

BACKGROUND For monitoring biomarkers, saliva has several distinct advantages over other biological fluids. Saliva sampling is relatively non-invasive and enables the collection from either adults or infants under many different circumstances. However, there is no collection device that can be speedily used for analysis in the field. The aim of the present study was to evaluate the suitability of a new device, termed Muddler, compared with commercially available collection devices. METHODS Saliva was collected from healthy volunteers. The collection devices such as Muddler, eye sponge, Salivette® Cotton (SC) and Salivette® Synthetic (SS) were evaluated in terms of the volume and/or composition of the collected saliva. The amounts of immunoglobulin A (IgA) and lactofferin in saliva were assessed by the enzyme-linked immunosorbent assays with the corresponding antibodies. Amylase activity was measured using a commercially available kit, and high molecular weight complexes including mucin were assessed by SDS-PAGE staining. RESULTS A newly developed Muddler, which was made of plastic plate, was the best device for collecting a constant volume of saliva among all the devices examined in the present study. Furthermore, Muddler can collect without change in composition of salivary proteins such as IgA, lactoferrin, amylase, and mucin complex, whereas the levels of the salivary proteins obtained with all the commercial devices used were clearly different from those in original saliva. CONCLUSIONS The newly developed Muddler was the best collection device in terms of the accuracy of collection and the reliability of measurements among all the devices examined in the present study.

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.