Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons

BACKGROUND: Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl(- )accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl(- )currents. The intracellular Cl(- )concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl(- )currents to signal generation differs between individual afferent neurons, and whether the specific Cl(- )levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl(- )homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl(- )concentrations and on the expression levels of Cl(- )transporters in rat DRG neurons. RESULTS: We developed an in vitro assay for testing how inflammatory mediators influence Cl(- )concentration and the expression of Cl(- )transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, and 1.4 μM PGE(2 )for 1–3 hours. Two-photon fluorescence lifetime imaging with the Cl(-)-sensitive dye MQAE revealed an increase of the intracellular Cl(- )concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na(+)-K(+)-2Cl(- )cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl(- )levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl(- )importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl(- )homeostasis toward a state of augmented Cl(- )accumulation, which is induced by a 1–3 hour treatment with inflammatory mediators. CONCLUSION: Our findings indicate that inflammatory mediators impact on Cl(- )homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl(- )levels and, hence, the driving force for depolarizing Cl(- )efflux. These findings corroborate current concepts for the role of Cl(- )regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl(- )concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl(- )currents in the presynaptic terminals. Moreover, excitatory Cl(- )currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.