We have confirmed previously that bradykinin (Bk) activates an inward current in neonatal rat dorsal root ganglion (DRG) neurones (I_Bk) and shown that I_Bk is dependent on a close association between the DRG neurones and their satellite cells (SCs) in culture (Heblich et al., 2001). Since DRG SCs are sensitive to Bk this raises the possibility that the neuronal I_Bk is caused by the release of one or more unidentified mediators from the SCs that act on ion channel targets in the DRG neurones. It has recently been shown that Bk receptors in DRG neurones are coupled to phospholipase C such that Bk causes breakdown of phosphatidyl inositol (4,5)-bis- phosphate (PIP2) and this leads to sensitization of TRPV1 receptors due partly to relief of PIP2-mediated block of the ion channels (Chuang et al., 2001) and partly to activation of protein kinase C which phosphorylates them (Premkumar and Ahern, 2000). Thus TRPV1 channels are clearly implicated in the mechanism of Bk-induced sensitization of sensory neurones. In the present experiments we have tested whether TRPV1 might also serve as a target for the putative mediators of I_Bk that are released from the DRG SCs. We used published methods (Heblich et al., 2001) to make cultures of DRG cells from 1 or 2 day post-natal rat pups. After 24 hours (to allow the SCs to separate from the neurones) the cultures were re-plated and left for a further 24 hours whilst the dissociated SCs proliferated. Meanwhile, COS-7 cells were transiently co-transfected with TRPV1 and green fluorescent protein (GFP) and after 14-16 hours the transfected (COS7-TRPV1-GFP) cells were harvested and applied over the top of the DRG SC cultures. The co-cultures were used after 24 hours when the COS7-TRPV1-GFP cells had associated closely with the underlying SCs. Coverslips of cells were placed on the stage of an inverted stage microscope where cells were identified visually that were GFP positive and associated with SCs and these cells were whole-cell voltage-clamped using patch electrodes (Heblich et al., 2001). We used this protocol on three different occasions (i.e. 3 separate DRG SC cultures, COS-7 transfections etc.) and 10 COS7-TRPV1-GFP cells were identified that responded to capsaicin (500 nM) with an inward current (0.67 ± 0.11 nA, n = 10). Bk (100 nM) was applied to the same 10 cells prior to capsaicin and 8 of these responded to Bk with an inward current (0.24 ± 0.08 nA, n = 8). Untransfected COS-7 cells or COS-7 cells that were transfected with TRPV1 alone (in the absence of DRG SCs) did not respond to Bk. In the DRG and COS7-TRPV1-GFP co-cultures, GFP positive cells that did not respond to capsaicin did not respond to Bk either (n = 2). Since the Bk receptors are on the DRG SCs and the response is recorded from the COS7-TRPV1-GFP cells these data suggest that Bk provokes release of a factor from the SCs that then acts on the TRPV1 receptors in the COS7-TRPV1-GFP cells. If the mechanism is the same as the mechanism of interaction of SCs with sensory neurones on which I_Bk depends then this result has wide-ranging and important implications for SC-neurone signalling in DRG.

Chuang HH et al., (2001) Nature 411, 957-962.
Heblich F et al., (2001). J Physiol. 536, 111-121.
Premkumar LS et al., (2000) Nature 408, 985-990.