To evaluate the interaction of the vanilloid receptor (TRPV1) with endogenous Ca2+ signalling mechanisms, TRPV1 was heterologously expressed in insect Sf9 and HEK cells. In the absence of extracellular Ca2+, stimulation of TRPV1 with agonists capsaicin and resiniferatoxin (RTX) caused a release of Ca2+ from internal stores. This release was not blocked by U73122 suggesting phospholipase C was not involved. Substantial overlap occurred between the TRPV1- and thapsigargin-sensitive Ca2+ pools, and TRPV1 immunofluorescence colocalized with the endoplasmic reticulum targeting motif "…KDEL…". To determine if TRPV1-induced release of Ca2+ from internal stores activated endogenous store-operated Ca2+ entry, the effect of 2-APB on Ba2+ was evaluated. 2-ABP blocked thapsigargin-induced Ba2+ influx, but not RTX-induced Ba2+ influx. In the combined presence of thapisgargin and RTX, the 2-APB sensitive component was essentially identical to the thapsigargin-induced component. These results indicate that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca2+ from internal stores but fail to activate store-operated Ca2+ entry.
Maitotoxin (MTX), a potent marine toxin, is a useful tool for the study of a Ca2+-overload induced necrotic/oncotic cell death. Upon stimulation with MTX, bovine aortic endothelial cells (BAEC) undergo three sequential changes in plasmalemmal permeability. Initially, MTX activates CaNSC leading to a massive increase in [Ca2+]i. Second is the activation of large pores known as cytolytic/oncotic pores (COP) that allows ions and small molecules (< 800Da) to enter the cell. Finally, the cells lyse, not by plasma membrane rupture but through the activation of a "death" channel. Concurrent with COP activation is the formation of plasma membrane blebs, which dilate as the cell undergoes lysis. Previous studies have shown that removal of extracellular Ca2+ or blockade of the MTX-induced rise in [Ca2+]i block all subsequent steps in the cell death cascade suggesting one or more of the steps is dependent upon or modulated by the rise in [Ca2+]i. The purpose of the study is to evaluate the role that Ca2+ plays in the regulation of the each phase of the MTX-induced cell death cascade. The results indicate that Ca2+ influences the initiation of COP, the time to cell lysis, and membrane blebbing.