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

Funk K, Woitecki A, Franjic-Würtz C, Gensch T, Möhrlen F, Frings S - Mol Pain (2008)

Bottom Line: 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.In contrast, the mRNA levels of the two transporters did not change markedly during this time.Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Physiology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany. katha.funk@gmx.de

ABSTRACT

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 microM ATP, 0.9 microM bradykinin, and 1.4 microM 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.

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.

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(A) Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intact DRGs loaded with the chloride indicator dye MQAE. The colour code indicates the fluorescence lifetime (τ) which is inversely proportional to the Cl- concentration. Warm colours represent high intracellular Cl- concentration (small τ values). (B) Quantitative evaluation of 2P-FLIM data show a highly significant increase of the inverse lifetime (LT = 1/τ) two hours after the start of treament with inflammatory mediators. Mean LT values were determined in control DRGs (LTctr) and in contralateral test DRGs (LTtest), and the percent increase of LT is depicted as ΔLT = (LTtest-LTctr)/LTctr·100 [%] for each treatment time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**).
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Figure 2: (A) Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intact DRGs loaded with the chloride indicator dye MQAE. The colour code indicates the fluorescence lifetime (τ) which is inversely proportional to the Cl- concentration. Warm colours represent high intracellular Cl- concentration (small τ values). (B) Quantitative evaluation of 2P-FLIM data show a highly significant increase of the inverse lifetime (LT = 1/τ) two hours after the start of treament with inflammatory mediators. Mean LT values were determined in control DRGs (LTctr) and in contralateral test DRGs (LTtest), and the percent increase of LT is depicted as ΔLT = (LTtest-LTctr)/LTctr·100 [%] for each treatment time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**).

Mentions: To look for changes of the intracellular Cl- concentration during exposure to inflammatory mediators, we loaded the DRG neurons with the Cl--sensitive fluorescent dye MQAE. MQAE fluorescence is quenched by Cl- and can therefore be used to monitor time-dependent changes of the intracellular Cl- level. To obtain an experimental parameter which depends only on changes of intracellular Cl-, we analyzed the fluorescence lifetime of MQAE [39-41]. Recordings were taken from intact DRGs (T7 – T9) by focusing the light of a two-photon microscope through the dura mater into cell layers 20 – 100 μm deep in the DRG. The recorded fluorescence lifetime was colour-coded for the microscopic images such that warmer colours represent higher Cl- concentrations (Fig. 2A). We did not attempt to calibrate the 2P-FLIM signals in terms of absolute Cl- concentrations (see Discussion). We used the inverse of the fluorescence lifetime τ (LT = 1/τ) as a parameter that is proportional to the Cl- concentration, according to 1/τ = 1/τ0 + (KSV [Cl-]i)/τ0, where τ0 is the value of τ in Cl--free solution, and KSV is the Stern-Volmer constant. Fig. 2A illustrates that the Cl- levels rise in most DRG neurons 2 hr after addition of the inflammatory mediators, and show a further increase after another hour. For the 2P-FLIM analysis, 18 DRGs (9 treated, 9 control) from 6 animals (in total, 365 control cells and 361 test cells) were evaluated. For each treatment time, the mean inverse lifetime, LT, was determined for all neurons in a control DRG (LTctr) and the contralateral test DRG (LTtest). Corresponding to our evaluation of immunostains, we determined the percent increase of LT as ΔLT = (LTtest-LTctr)/LTctr·100 [%]. The data in Fig. 2B demonstrate that Cl- increased significantly within 2 hr of treatment with inflammatory mediators. ΔLT (± SD; 3 animals) was -0.3 ± 1.2% (1 hr), 7.7 ± 0.9% (2 hr), and 10.3 ± 5.3% (3 hr). Moreover, the 2P-FLIM measurements revealed that virtually all DRG neurons visible in the images increase their intracellular Cl- concentrations in response to the inflammatory stimulus. Thus, DRG neurons respond to inflammatory mediators more or less uniformly by boosting the driving force for depolarizing Cl- efflux.


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

Funk K, Woitecki A, Franjic-Würtz C, Gensch T, Möhrlen F, Frings S - Mol Pain (2008)

(A) Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intact DRGs loaded with the chloride indicator dye MQAE. The colour code indicates the fluorescence lifetime (τ) which is inversely proportional to the Cl- concentration. Warm colours represent high intracellular Cl- concentration (small τ values). (B) Quantitative evaluation of 2P-FLIM data show a highly significant increase of the inverse lifetime (LT = 1/τ) two hours after the start of treament with inflammatory mediators. Mean LT values were determined in control DRGs (LTctr) and in contralateral test DRGs (LTtest), and the percent increase of LT is depicted as ΔLT = (LTtest-LTctr)/LTctr·100 [%] for each treatment time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2526990&req=5

Figure 2: (A) Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intact DRGs loaded with the chloride indicator dye MQAE. The colour code indicates the fluorescence lifetime (τ) which is inversely proportional to the Cl- concentration. Warm colours represent high intracellular Cl- concentration (small τ values). (B) Quantitative evaluation of 2P-FLIM data show a highly significant increase of the inverse lifetime (LT = 1/τ) two hours after the start of treament with inflammatory mediators. Mean LT values were determined in control DRGs (LTctr) and in contralateral test DRGs (LTtest), and the percent increase of LT is depicted as ΔLT = (LTtest-LTctr)/LTctr·100 [%] for each treatment time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**).
Mentions: To look for changes of the intracellular Cl- concentration during exposure to inflammatory mediators, we loaded the DRG neurons with the Cl--sensitive fluorescent dye MQAE. MQAE fluorescence is quenched by Cl- and can therefore be used to monitor time-dependent changes of the intracellular Cl- level. To obtain an experimental parameter which depends only on changes of intracellular Cl-, we analyzed the fluorescence lifetime of MQAE [39-41]. Recordings were taken from intact DRGs (T7 – T9) by focusing the light of a two-photon microscope through the dura mater into cell layers 20 – 100 μm deep in the DRG. The recorded fluorescence lifetime was colour-coded for the microscopic images such that warmer colours represent higher Cl- concentrations (Fig. 2A). We did not attempt to calibrate the 2P-FLIM signals in terms of absolute Cl- concentrations (see Discussion). We used the inverse of the fluorescence lifetime τ (LT = 1/τ) as a parameter that is proportional to the Cl- concentration, according to 1/τ = 1/τ0 + (KSV [Cl-]i)/τ0, where τ0 is the value of τ in Cl--free solution, and KSV is the Stern-Volmer constant. Fig. 2A illustrates that the Cl- levels rise in most DRG neurons 2 hr after addition of the inflammatory mediators, and show a further increase after another hour. For the 2P-FLIM analysis, 18 DRGs (9 treated, 9 control) from 6 animals (in total, 365 control cells and 361 test cells) were evaluated. For each treatment time, the mean inverse lifetime, LT, was determined for all neurons in a control DRG (LTctr) and the contralateral test DRG (LTtest). Corresponding to our evaluation of immunostains, we determined the percent increase of LT as ΔLT = (LTtest-LTctr)/LTctr·100 [%]. The data in Fig. 2B demonstrate that Cl- increased significantly within 2 hr of treatment with inflammatory mediators. ΔLT (± SD; 3 animals) was -0.3 ± 1.2% (1 hr), 7.7 ± 0.9% (2 hr), and 10.3 ± 5.3% (3 hr). Moreover, the 2P-FLIM measurements revealed that virtually all DRG neurons visible in the images increase their intracellular Cl- concentrations in response to the inflammatory stimulus. Thus, DRG neurons respond to inflammatory mediators more or less uniformly by boosting the driving force for depolarizing Cl- efflux.

Bottom Line: 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.In contrast, the mRNA levels of the two transporters did not change markedly during this time.Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Physiology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany. katha.funk@gmx.de

ABSTRACT

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 microM ATP, 0.9 microM bradykinin, and 1.4 microM 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.

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.

Show MeSH