Lembo et al. (2002) identified a novel family of G-protein coupled receptors localised in rat sensory neurones, and named them sensory neurone-specific receptors (SNSRs). The 8-22 fragment of bovine adrenal medulla peptide 22 (BAM (8-22)) was identified as a selective agonist of SNSRs. The aim of the current study was to establish whether BAM (8-22) sensitised the response of sensory neurones to capsaicin.

Neonatal rats (Wistar, P3-P7) were killed by cervical dislocation followed by decapitation. DRG neurones were prepared essentially as described in Cesare & McNaughton (1996) and were cultured for 1 - 3 days in the presence of 50ng ml^-1 NGF. For calcium imaging experiments, neurones were loaded with the fluorescent indicator Fluo-4, and fluorescence was monitored using a confocal scanning microscope. Neurones were continuously perfused with Hank's Balanced Salt Solution (HBSS) at room temperature and were exposed to 100nM capsaicin for 15s, every four minutes. BAM (8-22) (5µM) was applied for 4 minutes between the fifth and sixth capsaicin additions and ratios of the responses immediately before and after application were monitored. Those neurones with ratios above the upper 95% confidence interval obtained from control experiments were deemed to have been sensitised. BAM (8-22) caused sensitisation in 14.7% ± 2.8 neurones (mean ± SEM; p<0.05 compared with control). We next explored the signalling pathways involved. The kinase inhibitor staurosporine (200nM) reduced the percentage of sensitised neurones following exposure to BAM (8-22) to 5.3% ± 3.7, while the more selective PKC inhibitor Ro-31-8220 (500nM) reduced it to 4.1% ± 1.6 (p<0.05 compared with BAM (8-22) only). A selective PKA inhibitor, H-89 (500nM), did not have a significant effect (10.5% ± 3.2 sensitised). These experiments suggest that the signalling pathway involves PKC. Patch clamp experiments were next carried out to confirm the results obtained with calcium imaging. Neurones were perfused with HBSS and clamped in the whole cell configuration using pipettes filled with (in mM) 135 KCl, 1.6 MgCl₂, 10 HEPES, 2 EGTA, 2.5 Mg-ATP, and 0.2 Li-GTP (pH 7.3). Neurones were exposed to 100nM capsaicin for 1s every 30s and the peak inward current was monitored. BAM (8-22) (5µM) was applied after 5 exposures for the next 10 exposures. Neurones were counted as sensitised if the peak inward current activated by capsaicin was significantly enhanced (p<0.01) by exposure to BAM (8-22) as compared to control data (n=4). We found that the inward current activated by capsaicin was significantly enhanced in 2 out of 17 neurones. This is a similar percentage to that found in calcium imaging experiments.

In conclusion, BAM (8-22) sensitises the capsaicin receptor, TRPV1, in a subset of nociceptive neurones via a pathway involving PKC.

Cesare, P. & McNaughton, P.A. (1996) Proc. Natl. Acad. Sci. USA, 93, 15435-15439.
Lembo, P.M. et al. (2002) Nat. Neurosci., 5, 201-209.