Natural products have many health benefits, and their application can improve the quality of life. Recently, the diterpene (+)-larixol and its acetylated congeners demonstrated selective inhibition of the second-messenger-gated cation channel transient receptor potential canonical 6 (TRPC6) over its close isoforms TRPC3 and TRPC7. Building on this knowledge, we expanded these findings by chemical diversification of (+)-larixol mostly at position C6. Implementing high-throughput Ca FLIPR screening assays and electrophysiological patch-clamp recordings, we showcase larixyl N-methylcarbamate, termed SH045, as a compound with nanomolar affinity and 13-fold subtype selectivity over TRPC3 in stably expressing HEK293 cells. Expanding on this finding, TRPC6 inhibition was also observed in rat pulmonary smooth muscle cells. Furthermore, treatment of isolated perfused lung preparations with SH045 led to a decrease in lung ischemia-reperfusion edema (LIRE), a life-threatening condition associated with TRPC6 that may occur after organ transplantation. Taken together, and given the inexpensive, straightforward, and scalable preparation of SH045, we report a TRPC6 blocker that holds promise for the translational treatment of LIRE.
Ischemia/reperfusion-induced edema (IRE), one of the most significant causes of mortality after lung transplantation, can be mimicked ex vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel studied in endothelium; however, its role in the lung epithelium remains elusive. Here, we show enhanced IRE in TRPV4-deficient (TRPV4 –/– ) IPL compared with that of WT controls, indicating a protective role of TRPV4 in maintenance of the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling, and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar epithelial type I (ATI), and alveolar epithelial type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water-conducting aquaporin-5 (AQP-5) channel in ATI cells. Migration of TRPV4 –/– ATI cells was reduced, and cell barrier function was impaired. Analysis of isolated primary TRPV4 –/– ATII cells revealed a reduced expression of surfactant protein C, and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4 –/– lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared with WT lungs. Therefore, our data illustrate essential functions of TRPV4 channels in alveolar epithelial cells and in protection from edema formation.
In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca2+ concentration [Ca2+]i. Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG-sensitive classical transient receptor channels 3, 6, and 7 (TRPC3/6/7), initiating Ca2+ influx from the extracellular space. Patients with focal segmental glomerulosclerosis (FSGS) express gain-of-function mutants of TRPC6, while others carry loss-of-function mutants of PLCε, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLCβ and PLCγ isozymes was extensively studied, the role of PLCε in TRPC6 activation remains elusive. TRPC6 was co-immunoprecipitated with PLCε in a heterologous overexpression system in HEK293 cells as well as in freshly isolated murine podocytes. Receptor-operated TRPC6 currents in HEK293 cells expressing TRPC6 were reduced by a specific PLCε siRNA and by a PLCε loss-of-function mutant isolated from a patient with FSGS. PLCε-induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking Gαq/11 proteins. Further analysis of the signal transduction pathway revealed a Gα12/13 Rho-GEF activation which induced Rho-mediated PLCε stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLCε. PLCε-/- podocytes however, were undistinguishable from WT podocytes in their angiotensin II-induced formation of actin stress fibers and their GTPγS-induced TRPC6 activation, pointing to a redundant role of PLCε-mediated TRPC6 activation at least in podocytes.
Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca ([Ca]) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1-/-) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca] levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1-/- controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1-/- mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca] levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.
Competing interests:The authors declare that no competing interests exist. ischemia and reperfusion, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. TRPV4 was detected in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells by immunohistochemistry or mRNA profiling. Genetic ablation of TRPV4 resulted in reduced expression and plasma membrane insertion of water conducting aquaporin-5 (AQP-5) channels in ATI cells compared to WT mice.Analysis of isolated primary TRPV4-/-ATII cells revealed a reduced expression of pro surfactant protein-C (pSP-C) a precursor of a protein important for decreasing surface tension and for alveolar fluid homeostasis. Moreover, the TRPV4 activator GSK1016790A induced increases in current densities only in WT but not in TRPV4-/-ATII cells. On a molecular level ablation of TRPV4 induced less Ca 2+ -mediated nuclear translocation of nuclear factor of activated T-cells (NFAT) to the nucleus, which may be responsible for reduced expression of the identified proteins. Although the ability of TRPV4-/-ATII to differentiate to ATI cells was unchanged, migration of TRPV4deficient ATI cells was reduced and cell barrier function was impaired. Moreover, TRPV4-/-lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared to WT lungs. The findings of Weber et al. highlights novel essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation. Akazawa Y, Yuki T, Yoshida H, Sugiyama Y, Inoue, S. 2013. Activation of TRPV4 strengthens the tight-junction barrier in human epidermal keratinocytes. Skin Pharmacol Physiol 26: 15-21. Alpizar YA, Boonen B, Sanchez A, Jung C, Lopez-Requena A, Naert R, Steelant B, Luyts K, Plata C, De Vooght V et al. 2017. TRPV4 activation triggers protective responses to bacterial lipopolysaccharides in airway epithelial cells. Nature communications 8: 1059. Alvarez DF, King JA, Weber D, Addison E, Liedtke W, Townsley MI 2006. Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury. Circ Res 99: 988-995. Balakrishna S, Song W, Achanta S, Doran SF, Liu B, Kaelberer MM, Yu Z, Sui A, Cheung M, Leishman E et al. 2014. TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury. American journal of physiology Lung cellular and molecular physiology 307: L158-172. Corti M, Brody AR, and Harrison JH 1996. Isolation and primary culture of murine alveolar type II cells. American journal of respiratory cell and molecular biology 14: 309-315. Curcic S, Schober R, Schindl R, Groschner K 2019. TRPC-mediated Ca(2+) signaling and control of cellular functions. Semin Cell Dev Biol. in press de Perrot M, Liu M, Waddell TK, and Keshavjee S 2003. Ischemia-reperfusion-induced lung injury. American journal of respiratory and critical care medicine 167: 490-511. Desai TJ, Brownfield DG, Krasnow ...
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