P2X7 receptor and NLRP3 inflammasome activation in head and neck cancer P2X7 receptor and NLRP3 inflammasome activation in head and neck cancer

Salivary gland epithelial cells (SGEC) release several cytokines that play important roles in the inflammatory process. In this study, we examined whether capsaicin can modulate cytokine release in SGEC. After cells were stimulated with polyinosinic-polycytidylic acid [poly(I:C)] or lipopolysaccharide (LPS), mRNA transcript and protein levels were detected by reverse-transcriptase-polymerase chain-reaction (RT-PCR), real-time PCR, and enzyme-linked immunosorbent assay (ELISA). These findings demonstrated that the increases in TNFα and IL-6 mRNA transcripts were highest at 3 hrs and 1 hr after incubation with poly(I:C) and LPS, respectively. Pre-treatment of the cells with 10 μµ capsaicin, however, significantly inhibited mRNA transcripts and its protein levels. The simultaneous application of 10 μµ capsazepine with capsaicin did not block the inhibitory effect of capsaicin. Furthermore, the inhibitory effect of capsaicin was also shown in primary cultured cells from TRPV1^−/− mice. We found that both poly(I:C) and LPS induced IκB-α degradation and phosphorylation, which resulted in NF-κB activation, and capsaicin inhibited this NF-κB pathway. These results demonstrate that SGEC release pro-inflammatory cytokines mediated by TLR, and capsaicin inhibits this process through the NF-κB pathway. This study suggests that capsaicin could potentially alleviate inflammation in salivary glands.

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca²⁺-induced acidification and subsequent recovery of intracellular pH (pH_i) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μm) and capsaicin (1 μm) increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) with a following decrease in pH_i. The recovery of [Ca²⁺]_i to the prestimulus level was inhibited by LaCl₃ (1 mm) and o-vanadate (10 mm), a plasma membrane Ca²⁺/ATPase (PMCA) inhibitor. Removal of extracellular Ca²⁺ also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na⁺/H⁺ exchanger type 1 (NHE1), pancreatic Na⁺-HCO₃⁻ cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1–3 were detected by RT-PCR. pH_i recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH_i. Application of the NHE1 inhibitor 5′-(N-ethyl-N-isopropyl) amiloride (5 μm) or the pNBC1 inhibitor 4′,4′-di-isothiocyanostilbene-2′,2′-sulfonic acid (500 μm) delayed pH_i recovery and decreased AP frequency. Simultaneous application of 5′-(N-ethyl-N-isopropyl) amiloride and 4′,4′-di-isothiocyanostilbene-2′,2′-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca²⁺]_i in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH_i recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.