1.Store-operated calcium entry suppressed the TGF-β1/Smad3 signaling pathway in glomerular mesangial cells.
Chaudhari S;Li W;Wang Y;Jiang H;Ma Y;Davis ME;Zuckerman JE;Ma R Am J Physiol Renal Physiol. 2017 Sep 1;313(3):F729-F739. doi: 10.1152/ajprenal.00483.2016. Epub 2017 Jun 21.
Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca;2+; entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca;2+; channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La;3+;; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca;2+; channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca;2+; channels, significantly augmented TGF-β1-induced Smad3 phosphorylation.
2.Ca
Drumm BT;Rembetski BE;Cobine CA;Baker SA;Sergeant GP;Hollywood MA;Thornbury KD;Sanders KM J Physiol. 2018 Apr 15;596(8):1433-1466. doi: 10.1113/JP275719.
KEY POINTS: ;Contraction of urethral smooth muscle cells (USMCs) contributes to urinary continence. Ca;2+; signalling in USMCs was investigated in intact urethral muscles using a genetically encoded Ca;2+; sensor, GCaMP3, expressed selectively in USMCs. USMCs were spontaneously active in situ, firing intracellular Ca;2+; waves that were asynchronous at different sites within cells and between adjacent cells. Spontaneous Ca;2+; waves in USMCs were myogenic but enhanced by adrenergic or purinergic agonists and decreased by nitric oxide. Ca;2+; waves arose from inositol trisphosphate type 1 receptors and ryanodine receptors, and Ca;2+; influx by store-operated calcium entry was required to maintain Ca;2+; release events. Ca;2+; release and development of Ca;2+; waves appear to be the primary source of Ca;2+; for excitation-contraction coupling in the mouse urethra, and no evidence was found that voltage-dependent Ca;2+; entry via L-type or T-type channels was required for responses to α adrenergic responses.;ABSTRACT: ;Urethral smooth muscle cells (USMCs) generate myogenic tone and contribute to urinary continence. Currently, little is known about Ca;2+; signalling in USMCs in situ, and therefore little is known about the source(s) of Ca;2+; required for excitation-contraction coupling.
3.Characterization of selective Calcium-Release Activated Calcium channel blockers in mast cells and T-cells from human, rat, mouse and guinea-pig preparations.
Rice LV;Bax HJ;Russell LJ;Barrett VJ;Walton SE;Deakin AM;Thomson SA;Lucas F;Solari R;House D;Begg M Eur J Pharmacol. 2013 Mar 15;704(1-3):49-57. doi: 10.1016/j.ejphar.2013.02.022. Epub 2013 Feb 27.
Loss of function mutations in the two key proteins which constitute Calcium-Release Activated Calcium (CRAC) channels demonstrate the critical role of this ion channel in immune cell function. The aim of this study was to demonstrate that inhibition of immune cell activation could be achieved with highly selective inhibitors of CRAC channels in vitro using cell preparations from human, rat, mouse and guinea-pig. Two selective small molecule blockers of CRAC channels; GSK-5498A and GSK-7975A were tested to demonstrate their ability to inhibit mediator release from mast cells, and pro-inflammatory cytokine release from T-cells in a variety of species. Both GSK-5498A and GSK-7975A completely inhibited calcium influx through CRAC channels. This led to inhibition of the release of mast cell mediators and T-cell cytokines from multiple human and rat preparations. Mast cells from guinea-pig and mouse preparations were not inhibited by GSK-5498A or GSK-7975A; however cytokine release was fully blocked from T-cells in a mouse preparation. GSK-5498A and GSK-7975A confirm the critical role of CRAC channels in human mast cell and T-cell function, and that inhibition can be achieved in vitro. The rat displays a similar pharmacology to human, promoting this species for future in vivo research with this series of molecules.