Cytotoxicity of capsaicin in monkey kidney cells: lack of antagonistic effects of capsazepine and Ruthenium red.

Capsaicin is a natural product of Capsicum peppers, excitatory effects of which have been shown to be mediated by the recently cloned vanilloid receptor 1 (VR1). Since previous studies have shown that capsaicin inhibits protein synthesis, experiments were performed to investigate whether this effect is mediated by VR1 receptor on cultured monkey kidney cells (Vero cells). The capsaicin uptake was assessed in cellular homogenate and in medium by high-performance liquid chromatography (HPLC) separation and quantification on C18 reverse-phase column and fluorescence detection. Toxic effects were assessed by incorporation of [3H]L-leucine into cellular proteins in the presence of capsazepine, the VR1 vanilloid receptor antagonist and Ruthenium red or tyrosine or calcium. Capsazepine (1 to 256 microM) did not modify the uptake rate of capsaicin for incubation times up to 24 h and did not antagonize capsaicin-induced protein synthesis inhibition. It rather inhibited protein synthesis per se from 100 to 256 microM. Ruthenium red which blocks mitochondrial calcium uptake, inhibited protein synthesis and did not antagonise or increase synergistically the effects of capsaicin. Interestingly in a medium deprived of calcium and supplemented by calcium chloride (10-50 microM) the protein synthesis inhibition induced by capsaicin is antagonised somehow. There was no prevention of capsaicin diffusion into the cells. Tyrosine, which seems to be the best preventive agent of capsaicin inhibitory effects, prevents its metabolism but not its diffusion. Capsaicin might enter cells by diffusion and interfere with protein synthesis machinery by competition with tyrosine which in turn prevents the metabolism of capsaicin. The results of the present study suggest that cell responses to capsaicin may be transduced through at least two molecular pathways, one involving VR1, since the receptor antagonist capsazepine fails to prevent the inhibitory effect of capsaicin in Vero cells of renal origin.

Tyrosine prevents capsaicin-induced protein synthesis inhibition in cultured cells.

Our previous studies have shown that capsaicin competitively inhibits the fixation of tyrosine on its specific tRNA catalysed by the tyrosyl-tRNA synthetase in a cell-free system. These results suggested a probable protective effect of tyrosine versus capsaicin cytotoxicity. The experiments were performed on Vero cells originating from monkey kidney and on primocultures of hippocampal astrocytes. Firstly, the toxicity of capsaicin was determined on these cells by assessing the protein synthesis followed by [3H]L-leucine incorporation after 24 h of incubation with different concentrations of capsaicin (17, 34, 68, 136, 340 and 680 microM). The concentration required to inhibit 50% of the protein synthesis (IC50) was found to be 97 microM. Then, both cell types were cultured with capsaicin at IC50 concentration and supplemented with different concentrations of the amino acid (ratio [capsaicin]/[tyrosine]: 1:1, 1:2, 1:4 and 1:8). The inhibition of protein synthesis induced by capsaicin was prevented in a concentration-dependent manner by tyrosine. The inhibition was completely prevented by a tyrosine concentration four times higher than capsaicin concentration. Provided that this competitive action of tyrosine on the molecular mode of action of capsaicin is confirmed in other cells such as neurones, it could be speculated that capsaicin could disturb the synthesis of the catecholamines, neurotransmitters of the central nervous system deriving from tyrosine.

Capsaicin, a structural analog of tyrosine, inhibits the aminoacylation of tRNA(Tyr).

Because of its structural homology to capsaicin, the amino acid tyrosine appeared as a potential analog of the toxin and, consequently, could be a tool that could be used to clarify the mechanism of action of capsaicin. The in vitro experimental system was used to determine whether tyrosine and capsaicin effectively compete for tyrosyl-tRNA synthetase (extracted from mouse liver). The conditions for an efficient aminoacylation reaction were determined by kinetic tests and tests with different amounts of enzyme (in the absence of capsaicin). The reaction was fast and 40 microliters of the enzyme solution in a final reaction mixture volume of 100 microliters gave satisfactory results. The effect of different concentrations of capsaicin on the charging of [14C]tyrosine on its specific tRNA was tested for two concentrations of tyrosine (10 and 20 microM). The results obtained were represented in a Dixon plot and showed that capsaicin acts as a competitive and efficient inhibitor of tyrosyl-tRNA synthetase catalyzed reaction (Ki = 41.7 microM). The consequence of this inhibition in the whole animal and in cell culture remains to be determined and quantified. However, these results were supported by the fact that the capsaicin concentration that induced 50% of cell death was 48 microM for the toxin alone, whereas it was 96 microM when an equimolar concentration of tyrosine was added to capsaicin in the culture medium of hippocampal astrocytes.