Capsaicin is a pungent pain-producing compound found in plants of the capsicum family; it exerts excitatory, desensitizing, and toxic effects on a subset of sensory neurons, including the polymodal nociceptor population. We have carried out a quantitative study of capsaicin-induced fluxes of sodium, guanidine, calcium, rubidium, and chloride ions in cultures of neonatal and adult rat DRG neurons, in conjunction with the use of a histochemical stain that identifies capsaicin-sensitive neurons by means of cobalt uptake. Those cells that take up cobalt in a capsaicin-dependent manner (EC50 = 0.2 microM) represent about 50% of the total neuronal population derived from neonatal DRGs on short-term culture. Overnight treatment of cultures with 2 microM capsaicin leads to the loss of the cobalt-staining subpopulation. The capsaicin-insensitive neurons contain immunoreactive neurofilament epitopes that are present in fewer than 10% of capsaicin-sensitive neurons. This observation provides indirect evidence that the sensitive cells correspond to the small, dark B-type neurons, which are negative for neurofilament immunoreactivity in vivo. A capsaicin-dependent calcium uptake (EC50 = 0.2 microM), as measured by 45Ca incorporation, is shown by a DRG neuronal subpopulation that, like the cobalt-staining population of DRG neurons, is lost after overnight capsaicin treatment (2 microM). Capsaicin application leads to the accumulation of millimolar levels of calcium within a few minutes. Cadmium and other divalent cations block capsaicin-induced calcium uptake, but little or no inhibition is seen with organic calcium channel antagonists. Mitochondria, rather than the endoplasmic reticulum, are the probable destination of the internalized calcium, because ruthenium red inhibits calcium uptake (IC50 = 0.05 microM), whereas methylxanthines are inactive. The subset of sensory neurons that takes up calcium also releases 86Rb when exposed to capsaicin (EC50 = 0.06 microM). No efflux of 36Cl ions could be induced by capsaicin. These cells also show a capsaicin-induced uptake of 22Na or 14C guanidine (EC50 = 0.06 microM). In contrast, chick DRG cells in culture showed no capsaicin-induced calcium or cobalt uptake. Primary cultures of rat superior cervical ganglion neurons and Schwann cells, and a number of neuronal cell lines, also failed to respond to capsaicin, as judged by the calcium, cobalt, or guanidine uptake assays.

1. We have studied the effect of bradykinin (Bk) on fibroblast-like satellite (FLS) cells isolated from cultures of neonatal rat dorsal root ganglia (DRG). In voltage-clamped FLS cellsBk evoked an inward current response that was concentration dependent with a half-maximal concentration of 2 nM.3. In indo-1 AM-loaded FLS cells Bk evoked a rise in intracellular Ca 2+ that was concentration dependent with a half-maximal concentration of 1 nM.4. The FLS cells still produced an inward current in response to Bk in the absence of extracellular Ca 2+ but the response was inhibited if the intracellular concentration of EGTA was increased from 0.5 to 5 mM, which suggests that the inward current was dependent on the release and subsequent rise of intracellular Ca 2+ .5. The reversal potential of the Bk-induced inward current was consistent with the current being due to an increase in Cl _ conductance and shifted in a Nernstian manner when the intracellular Cl _ concentration was reduced.6. The inward current response to Bk was blocked by the B 2 receptor antagonist HOE-140, which indicates that the response was due to activation of B 2 receptors.7. The data suggest that Bk evokes a rise in intracellular Ca 2+ and activation of a Ca 2+-activated Cl _ conductance in the FLS cells and raise the possibility that FLS cells contribute to the proinflammatory effects of Bk in DRG.Journal of Physiology (2001), 530.3, pp. 395-403 11490 395 conditions. Preliminary data have already been published (England et al. 1995). METHODS Cell cultureCell cultures were obtained following enzymatic dispersal of neonatal rat dorsal root ganglia (DRG), as described previously (Wood et al. 1988). Briefly, 1-to 3-day-old Sprague-Dawley rat pups were killed by cervical dislocation followed by decapitation, and the DRG were removed. These were collected into Ham's F14 medium containing penicillin (100 i.u. ml), streptomycin (100 µg ml ) and L-glutamine (2 mM), and supplemented with 10 % fetal calf serum. Ganglia were then transferred to F14 medium containing 0.125 % collagenase, and were incubated at 37 o C and 3 % CO 2 in air, for a period of 40 min. The partially digested ganglia were washed in enzyme-free F14 medium, and triturated in fresh medium to which had been added 50 ng m l nerve growth factor (Promega or Alamone). Cells were cultured on 35 mm Petri dishes (Nunc).After approximately 3 days in vitro the glial cells began to undergo more rapid cell division, and approached confluence at around 5-7 days after dissociation. It was therefore necessary to replate the cells in order to obtain single cells for recording. This was achieved by resuspension of the cells and gentle trituration with a fire-polished wide-bore Pasteur pipette, approximately 4 h prior to electrophysiological measurements. This procedure virtually eliminated neurones from the cultures since the dishes were not treated with polyornithine or laminin, which aids adherence of cell soma to the culture dish. The attachment and growth of the glial cells was unaffected by thi...

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