In vivo Ca2+ dynamics induced by Ca2+ injection in individual rat skeletal muscle fibers

Citation: Wakizaka, M., Eshima, H., Tanaka, Y., Shirakawa, H., Poole, D. C., & Kano, Y. (2017). In vivo Ca2+ dynamics induced by Ca2+ injection in individual rat skeletal muscle fibers. Physiological Reports, 5(5), 10. doi:10.14814/phy2.13180In contrast to cardiomyocytes, store overload-induced calcium ion (Ca2+) release (SOICR) is not considered to constitute a primary Ca2+ releasing system from the sarcoplasmic reticulum (SR) in skeletal muscle myocytes. In the latter, voltage-induced Ca2+ release (VICR) is regarded as the dominant mechanism facilitating contractions. Any role of the SOICR in the regulation of cytoplasmic Ca2+ concentration ([Ca2+](i)) and its dynamics in skeletal muscle in vivo remains poorly understood. By means of in vivo single fiber Ca2+ microinjections combined with bioimaging techniques, we tested the hypothesis that the [Ca2+](i) dynamics following Ca2+ injection would be amplified and fiber contraction facilitated by SOICR. The circulation-intact spinotrapezius muscle of adult male Wistar rats (n = 34) was exteriorized and loaded with Fura-2 AM to monitor [Ca2+](i) dynamics. Groups of rats underwent the following treatments: (1) 0.02, 0.2, and 2.0 mmol/L Ca2+ injections, (2) 2.0 mmol/L Ca2+ with inhibition of ryanodine receptors (RyR) by dantrolene sodium (DAN), and (3) 2.0 mmol/L Ca2+ with inhibition of SR Ca2+ ATPase (SERCA) by cyclopiazonic acid (CPA). A quantity of 0.02 mmol/L Ca2+ injection yielded no detectable response, whereas peak evoked [Ca2+](i) increased 9.9 +/- 1.8% above baseline for 0.2 mmol/L and 23.8 c 4.3% (P < 0.05) for 2.0 mmol/L Ca2+ injections. The peak [Ca2+](i) in response to 2.0 mmol/L Ca2+ injection was largely abolished by DAN and CPA (-85.8%, -71.0%, respectively, both P < 0.05 vs. unblocked) supporting dependence of the [Ca2+](i) dynamics on Ca2+ released by SOICR rather than injected Ca2+ itself. Thus, this investigation demonstrates the presence of a robust SR-evoked SOICR operant in skeletal muscle in vivo

In vitro characterization of rat bone marrow-derived dendritic cells and their precursors

Although the rat is commonly used for basic immunology and transplantation research, phenotypic and functional characterization of rat dendritic cells (DCs) lags behind similar studies in the human and mouse. Therefore, these features were examined using DCs propagated from cultures of rat bone marrow maintained in a medium supplemented with granulocyte-monocyte colony-stimulating factor. Analysis of cytospin preparations of cultured cells showed that DCs arise from OX7+ myelomonocytic precursors. Typical mature rat DCs were morphologically similar to their mouse and human counterparts and expressed major histocompatibility complex (MHC) class II (common part determinant of Ia), OX62 (integrin molecule), OX7 (CD90), ICAM-1 (CD54), and CTLA4 counterreceptor, but were negative for OX8 (CD8), OX19 (CD5), W3/25 (CD4), and ED2, a rat macrophage marker. Functional analysis of OX62+ sorted DCs showed that they could effectively present the soluble antigen ovalbumin to naive T cells in vitro. A combination of anti-MHC class II monoclonal antibody and CTLA4-immunoglobulin inhibited allostimulatory ability more effectively than either reagent alone. Implications for studying the role of DCs in immune responses in the rat are discussed

A new protocol for the propagation of dendritic cells from rat bone marrow using recombinant GM-CSF, and their quantification using the mAb OX-62

Bone marrow (BM)-derived dendritic cells (DC) are the most potent known antigen (Ag) presenting cell in vivo and in vitro. Detailed analysis of their properties and mechanisms of action requires an ability to produce large numbers of DC. Although DC have been isolated from several rat tissues, including BM, the yield is uniformly low. We describe a simple method for the propagation of large numbers of DC from rat BM and document cell yield with the rat DC marker, OX-62. After depletion of plastic-adherent and Fc+ cells by panning on dishes coated with normal serum, residual BM cells were cultured in gelatin coated flasks using murine rGM-CSF supplemented medium. Prior to analysis, non-adherent cells were re-depleted of contaminating Fc+ cells. Propagation of DC was monitored by double staining for FACS analysis (major histocompatibility complex (MHC) class II+ OX-62+, OX-19-). Functional assay, morphological analysis and evaluation of homing patterns of cultured cells revealed typical DC characteristics. MHC class II and OX-62 antigen expression increased with time in culture and correlated with allostimulatory ability. DC yield increased until day 7, when 3.3 × 106 DC were obtained from an initial 3 × 108 unfractionated BM cells. Significant numbers of DC can be generated from rat BM using these simple methods. This should permit analysis and manipulation of rat DC functions in vivo and in vitro. © 1995

Oxidação da pirita e seus efeitos em argamassas de cimento Portland sujeitas ao ataque por sulfatos de origem interna

RESUMO O ataque por sulfatos de origem interna é resultante da reação química entre os íons Fe2+ e SO4 2-, provenientes da oxidação de agregados sulfetados, e os compostos da pasta cimentícia, e tende a promover a degradação do concreto devido formação de produtos expansivos que geram fissuração e desagregação do material. Neste trabalho, a morfologia da pirita, presente em alguns tipos de agregado, foi avaliada. O mineral foi utilizado em substituição parcial à areia (10%, em massa) na dosagem de argamassas, para estudo das modificações microestruturais e da variação dimensional linear sob envelhecimento natural durante 42 dias. A caracterização microestrutural foi executada em equipamento FEG/SEM com sonda analítica de EDS. Verificou-se, na superfície da pirita oxidada, a formação de óxidos de ferro, os quais afetaram a interface entre a pasta de cimento e o agregado nos materiais produzidos com a pirita. Quanto a variação dimensional, a argamassa apresentou apenas contrações durante o período de estudo. Verificou-se a ocorrência de fissuração e a predominância na formação de cristais aciculares de etringita aos 42 dias enquanto aos 90 dias a presença de gipsita predomina. Óxidos de ferro oriundos da oxidação da pirita são evidentes nas argamassas aos 90 dias