Simultaneous Intracellular Calcium and Sodium Flux Imaging in Human Vanilloid Receptor 1 (VR1)-Transfected Human Embryonic Kidney Cells: A Method to Resolve Ionic Dependence of VR1-Mediated Cell Death

Abstract

The vanilloid receptor 1 (VR1) is a ligand-gated, nonselective cation channel important for the sensory processing of painful stimuli. Activation of VR1 leads to increases in intracellular concentrations of calcium and sodium. Prolonged activation of VR1 in mammalian expression systems leads to cell death. The mechanism of VR1-mediated toxicity may have relevance to pathophysiological processes that can occur in neurons. Therefore, we have evaluated the relative contributions of intracellular calcium and sodium changes to VR1-mediated toxicity in human embryonic kidney 293 cells stably transfected with the human VR1 channel. The data demonstrate that VR1 receptor agonists capsaicin and resiniferatoxin lead to a sustained increase in intracellular calcium and sodium in a concentration-dependent manner, followed by cell death. Pretreatment with VR1 receptor antagonists capsazepine or ruthenium red block both the calcium and sodium responses to agonists, and block agonist-induced cell death in a concentration-dependent manner. However, addition of antagonists several minutes after agonists selectively reverses the agonist-induced increase in intracellular calcium, but does not reverse the elevated intracellular sodium concentration. Nonetheless, antagonists retain protective efficacy against capsaicin toxicity when added several minutes after capsaicin, conditions in which the cells still manifest elevated intracellular sodium, but not elevated intracellular calcium. In addition, a transient VR1-mediated increase in intracellular calcium that returns to baseline within minutes, induced by a rapid drop in pH, from pH 7.5 to pH 6.3, also does not lead to cell death. Collectively, these data demonstrate that the most important intracellular ionic change for mediating VR1-dependent toxicity is a sustained increase of calcium.The VR1 receptor is a ligand-gated, nonspecific cation channel activated by vanilloid compounds, low pH, and noxious heat (Tominaga et al., 1998). The best-known natural vanilloids are capsaicin, which is the pungent ingredient in hot chili peppers, and resiniferatoxin, synthesized by a cactus-like plant called Euphorbia resinifera . High temperatures, Ͼ42°C, protons, and high concentrations of anandamide gate the VR1 receptor directly (Tominaga et al., 1998). Because the VR1 receptor is permeable to numerous cations, including Ca 2ϩ

Regulation of CD20 expression by radiation-induced changes in intracellular redox status

“…This molecule passively diffuses into cells and its chloromethyl group reacts with intra-cellular thiols [22][23][24]. Briefly, cells were treated with either H 2 O 2 (10 μM, 1 h at 37°C as positive control) or PEG-catalase (100 U/ml, 2 h at 37°C), followed by washing and treatment with H 2 O 2 (10 μM, 1 h at 37°C).…”

Distinct thymocyte subsets express the vanilloid receptor VR1 that mediates capsaicin-induced apoptotic cell death

“…Our results are consistent with previous evidence showing that stimulation of VR1 with low mM dose of CPS resulted in Ca 2 þ influx, which was inhibited by CPZ in a number of different neuronal and non-neuronal cell types including DRG and sensory neurons, 27,35 C6 rat glioma cells, 23 human epidermal keratinocytes, 14,37 bone marrowderived mast cells 15 and VR1 HEK293 cell transfectants. 32 Calcium has been implicated in the regulation of apoptosis in several cell types. Elevation of Ca 2 þ following CD3 engagement, glucocorticoids and Ca 2 þ ionophores can induce apoptosis of thymocytes.…”

“…20,21,23,29 Excessive Ca 2 þ influx triggered by vanilloid exposure can promote necrotic cell death rather than apoptosis in primary sensory neurons in vivo, 30 and in vitro 31 and in cell transfectants expressing VR1 vanilloid receptor. 9,32 In regard to the vanilloid receptor mediating CPS-induced cell death, VR1 has been implicated in the triggering of apoptosis, paraptosis and necrosis in response to low doses of CPS in a variety of cell types. 9,22,32 However, VR1-independent cell death has also been described in a number of studies using high doses of CPS and/or long-term exposure, experimental conditions that markedly affect the integrity of plasma membrane.…”

“…9,32 In regard to the vanilloid receptor mediating CPS-induced cell death, VR1 has been implicated in the triggering of apoptosis, paraptosis and necrosis in response to low doses of CPS in a variety of cell types. 9,22,32 However, VR1-independent cell death has also been described in a number of studies using high doses of CPS and/or long-term exposure, experimental conditions that markedly affect the integrity of plasma membrane. [19][20][21][22][23]25,28,29,33,34 We have previously reported that neonatal CPS treatment results in marked reduction of thymus weight and cellularity, impaired thymocyte proliferation in response to T-cell mitogens and apoptotic cell death.…”

“…9,32 Differences in pharmacological profiles (sensitivity), regulatory mechanisms associated with vanilloid receptors or accessory multimeric subunits, receptor membrane localization, VR1 ability of forming supramolecular complexes with different receptors and signalling molecules 10 among native and recombinant VR1 channels could be responsible for the differences in cell death pathways triggered by CPS through VR1 activation.…”