We have demonstrated that IBk can be evoked in neonatal rat DRG neurones only when there is a close association between the neurones and fibroblast-like satellite cells (Heblich et al. 2001). In this study we have determined the subtype of Bk receptor that mediates IBk and investigated the possible involvement of vanilloids in the response, since Bk has been shown to induce vanilloid receptor activity in DRG neurones (Premkumar & Ahern, 2000).
Animals were killed humanely. DRG cells were maintained in culture for 1-5 days. Recordings were made using whole-cell patch clamp (Vh = -80 mV). Intracellular and extracellular solutions were the same as those in Heblich et al. (2001). All control and test groups of cells were matched, containing cells from the same culture and age. Statistical comparisons were made using Student’s unpaired t test, with P < 0.05 taken as significant. Data are expressed as means ± S.E.M.
Bk (100 nM) failed to evoke IBk in neurones that were pre-treated with the selective B2 receptor antagonist HOE 140 (10 nM, n = 5). After 5 min wash, to remove HOE 140, there was some recovery of IBk. By contrast, application of the B1 receptor antagonist Des Arg10 HOE 140 (100 nM) for 3 min prior to and throughout Bk (100 nM) application, failed to inhibit IBk, which was 408 ± 120 pA (n = 5) in control and 239 ± 80 pA (n = 3) in the test group. These data demonstrate that IBk is mediated by B2 receptors.
The effect of ruthenium red (RR, 10 µM) on IBk was measured using two different protocols. In the first protocol RR was applied 3 min before and then throughout the application of Bk (100 nM). In the second protocol Bk was applied first and then RR was applied at the peak of IBk. The effect of RR on capsaicin (0.5 µM)-induced inward currents (ICAPS) was measured for comparison. Application of RR 3 min prior to and during Bk significantly reduced IBk from 430 ± 103 pA in controls (n = 10) to 63 ± 50 pA in test neurones (n = 10). ICAPS was reduced from 684 ± 210 pA (n = 7) to 15 ± 8 pA (n = 6) using this protocol. By contrast, application of RR (with Bk) at the peak of IBk failed to inhibit IBk or to reduce the duration of current decay (158 ± 25 s, n = 10) compared with control neurones (140 ± 11 s, n = 10). Using this protocol RR significantly reduced the decay of ICAPS from 117 ± 56 s (n = 7) to 8.6 ± 0.5 s (n = 7). These data suggest that IBk is not mediated by vanilloid receptors, since RR does not reduce IBk decay duration even though that of ICAPS is reduced. IBk inhibition by prior exposure to RR may be due to blockade of intracellular Ca2+ release by RR.This work was supported by The Wellcome Trust (053230/Z/97/A/JHW/KB/RH).
- Heblich, F., England, S. & Docherty, R.J. (2001). J. Physiol. 536, 111-121. abstract
Premkumar, L.S. & Ahern, G.P. (2000). Nature 408, 985-990.