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Excess nitric oxide activates TRPV1-Ca2+-calpain signaling

Researchers from the National Yang-Ming University in Taipei, Taiwan have found that excess nitric oxide (NO) activates TRPV1-Ca2+-calpain signaling and promotes PEST (Pro-Glu-Ser-Thr)-dependent degradation of liver X receptor α (LXRα). The aim of their study was to delineate the mechanism by which NO could ultimately downregulate LXRα and calpain-modulated ATP-binding cassette transporter A1 (ABCA1) in macrophages, by considering the roles of several other ions and proteins that may or may not serve as secondary messengers. Characterizing this new regulatory mechanism in cholesterol management could lead to discoveries that are applicable to developing treatments for cardiovascular system diseases.

The researchers first confirmed that the calcium-dependent calpain was a key player in the NO donor SNAP-induced effects on cholesterol metabolism. These effects included downregulation of ABCA1, the main reverse cholesterol transporter, which lead to unfavorable oxidized low-density lipoprotein (oxLDL) -induced lipid accumulation in macrophage-foam cells. After proving their hypothesis on calpain's participation in LXRα/ABCA1 dynamics, they also used a Fluo-8® Calcium Assay Kit to examine the effect of calcium influx through TRPV1 on SNAP-activated calpain. This allowed them to quickly gauge calcium responses of several macrophage samples treated with gain-and-loss of function approaches, such as removing extracellular calcium by EGTA, or treating with TRPV1 inhibitor CPZ or SB366791. Both of these strategies prevented calcium influx, even in SNAP-treated cells, implying that TRPV1-calcium signals are also critical to NO-induced calpain/LXRα/ABCA1 interaction. The final proposed model of the signaling pathway was defined to be as follows: NO triggers a calcium influx through the TRPV1 channel; the calcium activates calpain, which results in LXRα degradation and downregulation of both LXRα and ABCA1; less ABCA1 impairs reverse cholesterol efflux, causing accumulation of lipids within the macrophage.

For other calcium-essential pathways requiring analysis, the Fluo-8® Calcium Assay Kit is a valuable asset. The proprietary dye Fluo-8® can provide swift results in detecting calcium levels, as shown in the study which observed changes "as early as 1 min after treatment". Aside from Fluo-8®'s rapid kinetics, the dye's convenient green signal (Ex/Em = ~490/520 nm) also exhibits high intensity and sensitivity, while the Fluo-8® kit provides all the necessary reagents and written protocols prepared for easy calcium monitoring anywhere within a project.

 

References


  1. Zhao, Song-Kun Shyue, and Tzong-Shyuan Lee. Excess Nitric Oxide Activates TRPV1-Ca2+-Calpain Signaling and Promotes PEST-dependent Degradation of Liver X Receptor α. Int J Biol Sci. 2016; 12(1): 18-29. doi: 10.7150/ijbs.13549.
  2. Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit. AAT Bioquest, n.d. Web. 12 July 2016


Original created on January 4, 2017, last updated on January 4, 2017
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