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1.
Article in English | WPRIM | ID: wpr-162255

ABSTRACT

During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.


Subject(s)
Animals , Calcium/metabolism , Calcium Channels/metabolism , Calcium Channels, L-Type/genetics , Cations/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , Excitation Contraction Coupling , Gene Knockdown Techniques , Membrane Potentials , Mice , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/metabolism , Myoblasts, Skeletal/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/physiology , Synaptophysin/metabolism , TRPC Cation Channels/genetics , Transient Receptor Potential Channels/metabolism
2.
Braz. j. med. biol. res ; 39(6): 739-748, June 2006. graf
Article in English | LILACS | ID: lil-428284

ABSTRACT

Store-operated Ca2+ entry plays an important role in Ca2+ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca2+ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca2+ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca2+ transient was inhibited by at least 50-70 percent by several inhibitors of the RyR, including ryanodine (10 µM), dantrolene (10 µM), and ruthenium red (10 µM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8-Br-cADPR (100 µM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80 percent. Pre-incubation of cells with 100 µM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 µM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca2+ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca2+ transient by 50-80 percent. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca2+ entry in human smooth muscle cells.


Subject(s)
Female , Humans , Calcium/metabolism , Cyclic ADP-Ribose/metabolism , Myocytes, Smooth Muscle/metabolism , Myometrium/cytology , Blotting, Western , Calcium/pharmacology , Cyclic ADP-Ribose/antagonists & inhibitors , Cyclic ADP-Ribose/pharmacology , Immunohistochemistry , Immunoprecipitation , Myocytes, Smooth Muscle/drug effects , Myometrium/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Signal Transduction , Transient Receptor Potential Channels/metabolism
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