Abstract
Intracellular Ca2+ is critical for regulating airway smooth muscle (ASM) tension. A rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) of ASM cells is crucial for modulating the intensity and length of the ASM contraction. Because this rapid increase in [Ca2+]i largely depends on the balance between Ca2+ released from intracellular Ca2+ stores and extracellular Ca2+ entry, exploring the mechanisms mediating Ca2+ transport is critical for understanding ASM contractility and the pathogenesis of bronchial contraction disorders. Transient receptor potential vanilloid 4 (TRPV4) is a highly Ca2+-permeable non-selective cation channel that mediates Ca2+ influx to increase [Ca2+]i, which then directly or indirectly regulates the contraction and relaxation of ASM. The [Ca2+]i returns to basal levels through several uptake and extrusion pumps, such as the sarco(endo)plasmic reticulum Ca2+ ATPase and inositol 1,4,5-trisphosphate receptors (IP3Rs), the plasmalemmal Ca2+ ATPase, and the plasma membrane Na+/Ca2+ exchanger (NCX). Thus, to further understand ASM tension regulation in normal and diseased tissue, the present study examined whether an interaction exists among TRPV4, IP3Rs, and NCX. The TRPV4-specific and potent agonist GSK1016790A increased [Ca2+]i in mouse ASM cells, an effect that was completely blocked by the TRPV4-specific antagonist HC067047. However, GSK1016790A induced relaxation in mouse tracheal rings precontracted with carbachol in vitro. To determine the mechanism underlying this TRPV4-induced relaxation of ASM, we blocked specific downstream molecules. We found that the GSK1016790A-induced relaxation was abolished by the NCX inhibitors KB-R7943 and LiCl but not by specific inhibitors of the Ca2+-activated large-, intermediate-, or small-conductance K+ channels (BKCa, IK, and SK3, respectively). The results of co-immunoprecipitation (co-IP) assays showed an interaction of TRPV4 and IP3R1 with NCXs. Taken together, these findings support a physical and functional interaction of TRPV4 and IP3R1 with NCXs as a novel TRPV4-mediated Ca2+ signaling mechanism and suggest a potential target for regulation of ASM tension and treatment of respiratory diseases, especially tracheal spasm.
Highlights
Control of the intracellular calcium concentration ([Ca2+]i) is critical to the regulation of airway smooth muscle (ASM)function and mediates many processes, including contraction, proliferation, and gene expression (Hirota and Janssen, 2007).Contractility in ASM is largely mediated by the interaction of ligands with specific G protein-coupled receptors (GPCRs), leading to Ca2+ release from intracellular stores as well as Ca2+ influx from the extracellular medium
Activation of transient receptor potential vanilloid 4 (TRPV4) resulted in concentration-dependent relaxation in mouse tracheal rings precontracted by 1 mM carbachol, a muscarinic receptor agonist; the relaxation induced by a final cumulative concentration of 10 nM GSK1016790A was about 40%, and which was significantly weakened by pretreatment of 10 μM HC067047 (Figure 1F)
We found that an anti-TRPV4 antibody could pull down NCX1,2,3, and the anti-NCX1,2,3 antibody could pull down TRPV4 (Figures 5A,B)
Summary
Contractility in ASM is largely mediated by the interaction of ligands with specific G protein-coupled receptors (GPCRs), leading to Ca2+ release from intracellular stores as well as Ca2+ influx from the extracellular medium. The TRPV4 channel can be activated by various stimulating factors, including moderate heat (>24◦C), osmotic pressure changes, shear stress, cell swelling, and chemical stimulation (Watanabe et al, 2003; Grace et al, 2017). TRPV4 channels are widely expressed in various tissues, including ASM (Jia et al, 2004). In the ASM of humans and guinea pigs, activation of TRPV4 by relevant stimulators mediates Ca2+ influx and increases [Ca2+]i (Jia et al, 2004; Allen et al, 2014; Buday et al, 2017). Junctional membrane complexes between the plasma membrane and the ER/SR are needed to provide effective mechanisms for cross talk between Ca2+ channels/transporters in the plasma membrane and Ca2+-
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