ABSTRACT
The effect of small G-proteins of the Rho family on sodium current conducted by cardiac isoform NaV1.5 of voltage-gated sodium channels was studied in heterologous expression system, CHO-K1 cell line transfected with a plasmid containing the NaV1.5 gene. The influence of cotransfection with genes of wild-type, constitutively-active, and dominant-negative small G-proteins RhoA, Rac1, and Cdc2 on the parameters of sodium current and its noninactivating component (INa,late) was estimated. Among three studied small G-proteins, only RhoA (wild-type and constitutively-active type) strongly affected sodium current reducing its peak amplitude, but not the value of INa,late. Cotransfection with wild-type Rac1 resulted in a minor decrease in sodium current. Thus, small G-protein RhoA has potential capability for suppression of sodium current, although physiological relevance of this property has to be verified.
Subject(s)
Gene Expression Regulation , Membrane Potentials/physiology , NAV1.5 Voltage-Gated Sodium Channel/genetics , cdc42 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/genetics , Animals , CHO Cells , Cnidarian Venoms/pharmacology , Cricetulus , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Membrane Potentials/drug effects , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Neurotoxins/pharmacology , Patch-Clamp Techniques , Plasmids/chemistry , Plasmids/metabolism , Transfection , Transgenes , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
Relevant cell model is essential to study pathogenesis of muscle disorders. However, in the field of muscle research there is no ultimate cell line considered as a standard for studying muscular and neuromuscular diseases. Standard cell line claimed to be well differentiated in muscle lineage, be morphological and physiological similar to mature muscle cells and be easily genetically modified. Therefore, the goal of our study was to pick up available and fruitful cell model of muscle differentiation, that could be further applied for examination of muscular disorder pathogenesis in vitro. We characterized human mesenchymal stem cells (MSC), mature murine muscle fibers and primary murine satellite cells. It has been shown that MSC have very small capacity to myogenic differentiation; moreover, they were able to differentiate only in presence of C2C12 cells. Lentiviral transduction exhibited rather high toxic effect on primary myofibers, and positively transduced cells were not able to response to electrical stimulation, i. e. were functionally inactive. Satellite cells turned out to be the most fruitful cell model since they were easily transduced via lentiviruses and rapidly formed myotubes in differentiation media. Functional analysis of obtained myotubes has confirmed their ability to react to electrical and chemical stimulations; besides, potassium and calcium channels availability has been also demonstrated via patch-clump technique. Taken together, these results imply that satellite cells are the most promising cell line for further experiments aimed at exploring the molecular pathways of muscle pathologies.
Subject(s)
Mesenchymal Stem Cells/cytology , Muscle Fibers, Skeletal/cytology , Satellite Cells, Skeletal Muscle/cytology , Action Potentials/physiology , Animals , Calcium Channels/metabolism , Cell Differentiation , Coculture Techniques , Humans , Lentivirus/genetics , Mesenchymal Stem Cells/physiology , Mice , Muscle Fibers, Skeletal/physiology , Patch-Clamp Techniques , Potassium Channels/metabolism , Primary Cell Culture , Satellite Cells, Skeletal Muscle/physiology , Transduction, GeneticABSTRACT
The location of centromeric protein CENP-B and telomeric protein TRF2/MTBP in the mouse spermatogenic line has been studied using indirect immunofluorescent and immunoelectron microscopy. CENP-B localized to the heterochromatic parts of the nuclei at meiotic stages. A clearly distinct chromocenter forms in the nucleus at stages 3-4 of spermatid maturation; CENP-B localizes in it and in the area adjacent to the future acrosome. CENP-B localization in the subacrosomal area and in the chromocenters' periphery demonstrates that centromeres are organized in two groups in mouse spermatozoa, unlike human centromeres. TRF2/MTBP concentrates around the forming chromocenter at spermiogenesis early stages. The TRF2/MTBP main signal migrates into the area of acrosomal membrane at the course of spermatozoon maturation. TRF2/MTBP never localizes inside the synaptonemal complex but can be found in the areas where the synaptonemal complex attaches to the nuclear envelope. At the pachytene and diplotene stages when chromosomes separate from the nuclear envelope, some amount of the protein remains bound to the nuclear membrane while the other part reveals itself in chromosomes. TRF2/MTBP accumulates in the future acrosome from the very beginning of its formation. In the mature spermatozoon TRF2/MTBP decorates the acrosomal membrane as well as spreads in condensed chromatin.
Subject(s)
Carrier Proteins/metabolism , Cell Nucleus/metabolism , Centromere Protein B/metabolism , Spermatozoa/metabolism , Telomeric Repeat Binding Protein 2/metabolism , Animals , Male , Mice , Mice, Inbred C3H , Microscopy, Fluorescence , Microscopy, Immunoelectron , Spermatogenesis , Spermatozoa/ultrastructureABSTRACT
Histamine produced either a bronchodilation or a bronchoconstriction in rats. In a 0.01-1.0 mcg/ml concentration histamine augmented the contractions amplitude in electrical stimulation of the trachea, in a 10-100 mcg/ml concentration histamine enhances the muscle tone thus decreasing the induced contractions. The histamine effects seems to be connected with its prevailing influence on different structures of the airways depending on the concentration. Its high concentrations act directly on the smooth muscle whereas its lower concentration affects receptors signaling the functional modules of the metasympathetic nervous system within the intramural ganglia of the trachea.
