Modulation of Chloride Homeostasis by Inflammatory Mediators in Dorsal Root Ganglion Neurons

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Abstract
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. 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 PGE2 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. 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.