We have previously shown that ATP and α,β-meATP evoke rapid inward currents via P2X3 receptors in dissociated neurones of the rat DRG (Robertson et al. 1996; Rae et al. 1998). The aim of this study was to characterise the rise in cytoplasmic [Ca2+]i elicited by ATP and α,β-meATP in these neurones using the fluorescent dyes fura-2 and fluo-4. The responses were also compared with those evoked by capsaicin.
Sprague-Dawley rats of either sex (3-7 days) were humanely killed. DRG from all spinal levels were dissected aseptically and dissociated enzymatically. Cells were perfused at 8 ml min-1 with a Hepes-based buffer. Drugs were applied in the perfusate for 20 s. Only cells that responded to KCl (50 mM) were included in the analysis.
Initially, we used fura-2 to quantify the agonist-induced rise in cytoplasmic [Ca2+] in single cells. KCl (50 mM) evoked a reversible rise with a peak of 207 ± 11 nM (mean ± S.E.M.) (n = 108). An increase was also evoked by ATP (100 µM) in 29/44 cells (peak = 75 ± 9 nM), by capsaicin (10 µM) in 16/36 cells (peak = 143 ± 26 nM) and by α,β-meATP (30 µM) in 11/28 cells (peak = 45 ± 6 nM).
Subsequently, we used fluo-4 to measure the responsiveness of populations of cells. Here, the mean peak increase in fluorescence evoked by KCl (50 mM) was 2.0 ± 0.1 ΔF/F0 (n = 174). An increase in fluorescence was evoked by ATP (100 µM) in 95/174 cells (peak = 1.4 ± 0.1 ΔF/F0), by capsaicin (10 µM) in 156/174 cells (peak = 2.5 ± 0.2 ΔF/F0) and by α,β-meATP (30 µM) in 102/174 cells (peak = 0.9 ± 0.1 ΔF/F0).
Subsequently, we determined the role of extracellular [Ca2+] in these responses. Under zero [Ca2+]o conditions all KCl- (n = 154) and α,β-meATP- (n = 49) evoked changes were abolished. In contrast, an increase in fluorescence was seen in 23 % of ATP-sensitive cells and 30 % of capsaicin-sensitive cells. For both drugs, the peak rise was smaller than in the presence of [Ca2+]o (0.7 ± 0.1 and 0.18 ± 0.08ΔF/F0 respectively).
Thus, these results show that the depolarising currents induced by ATP, α,β-meATP and capsaicin are associated with a rise in intracellular [Ca2+]. For α,β-meATP the rise is entirely dependent upon Ca2+ influx, whereas ATP and capsaicin induce both Ca2+ influx and release.
A.J.C. holds an MRC sudentship