Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunohistochemical analysis of NKCC1 phosphorylation. (A) A cryosection of a T9 DRG was co-stained with antisera raised against NKCC1 and p-NKCC1 after a 3-hr treatment with inflammatory mediators. The two antibodies showed an almost perfect co-staining. The calibration bar indicates 100 μm. (B) In contrast to the NKCC1 immunosignal, the p-NKCC1 signal increased already 1 hr after the start of the treatment. The change of p-NKCC1 immune fluorescence in treated DRGs (FItest) compared to the respective contralateral control DRGs (FIctr) is expressed as ΔFI = (FItest-FIctr)/FIctr·100 [%]. ΔFI (± SD; 3 animals) was 18 ± 5.9% (1 hr), 45 ± 3.5% (2 hr), and 54 ± 3.3% (3 hr). Indicated significance levels are p ≤ 0.05 (*) and p ≤ 0.001 (***). Numbers in parentheses indicate cells evaluated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2526990&req=5

Figure 4: Immunohistochemical analysis of NKCC1 phosphorylation. (A) A cryosection of a T9 DRG was co-stained with antisera raised against NKCC1 and p-NKCC1 after a 3-hr treatment with inflammatory mediators. The two antibodies showed an almost perfect co-staining. The calibration bar indicates 100 μm. (B) In contrast to the NKCC1 immunosignal, the p-NKCC1 signal increased already 1 hr after the start of the treatment. The change of p-NKCC1 immune fluorescence in treated DRGs (FItest) compared to the respective contralateral control DRGs (FIctr) is expressed as ΔFI = (FItest-FIctr)/FIctr·100 [%]. ΔFI (± SD; 3 animals) was 18 ± 5.9% (1 hr), 45 ± 3.5% (2 hr), and 54 ± 3.3% (3 hr). Indicated significance levels are p ≤ 0.05 (*) and p ≤ 0.001 (***). Numbers in parentheses indicate cells evaluated.

Mentions: The activity of NKCC1 and KCC2 can be modulated by phosphorylation [26,27]. To test whether NKCC1 phosphorylation may contribute to the early rise of Cl- levels (< 3 hr), we stained 9 DRG sections from 3 animals with an antibody raised specifically against the phosphorylated form of NKCC1 (p-NKCC1). Preadsorption with a peptide corresponding to the non-phosphorylated epitope ensured the specificity of that immunosignal. The antisera directed against NKCC1 (C-14 antibody) and p-NKCC1 showed a virtually perfect match in the DRG sections (Fig. 4A) indicating that both non-phosphorylated and phosphorylated forms of NKCC1 are present in most neurons. However, in contrast to the NKCC1 immunosignal, the p-NKCC1 signal responded rapidly and robustly to the inflammatory mediators. Already after 1 hr, a significant rise of the p-NKCC1 signal (+18% ± 5.9%; p ≤ 0.05) was observed (Fig. 4B). Two hours after addition of the inflammatory mediators, the p-NKCC1 signal was 45% (± 3.5%; p ≤ 0.001) above control, while the total expression level of NKCC1 showed no response (Fig. 3). After 3 hours, the p-NKCC1 signal was increased by 54% (± 3.3%; p ≤ 0.001) relative to control DRGs. These data demonstrate that the inflammatory mediators induce the phosphorylation of NKCC1 in DRG neurons and suggest that this phosphorylation causes the early (< 3 hr) phase of enhanced Cl- accumulation observed by 2P-FLIM in response to the stimulus.


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)

Immunohistochemical analysis of NKCC1 phosphorylation. (A) A cryosection of a T9 DRG was co-stained with antisera raised against NKCC1 and p-NKCC1 after a 3-hr treatment with inflammatory mediators. The two antibodies showed an almost perfect co-staining. The calibration bar indicates 100 μm. (B) In contrast to the NKCC1 immunosignal, the p-NKCC1 signal increased already 1 hr after the start of the treatment. The change of p-NKCC1 immune fluorescence in treated DRGs (FItest) compared to the respective contralateral control DRGs (FIctr) is expressed as ΔFI = (FItest-FIctr)/FIctr·100 [%]. ΔFI (± SD; 3 animals) was 18 ± 5.9% (1 hr), 45 ± 3.5% (2 hr), and 54 ± 3.3% (3 hr). Indicated significance levels are p ≤ 0.05 (*) and p ≤ 0.001 (***). Numbers in parentheses indicate cells evaluated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Immunohistochemical analysis of NKCC1 phosphorylation. (A) A cryosection of a T9 DRG was co-stained with antisera raised against NKCC1 and p-NKCC1 after a 3-hr treatment with inflammatory mediators. The two antibodies showed an almost perfect co-staining. The calibration bar indicates 100 μm. (B) In contrast to the NKCC1 immunosignal, the p-NKCC1 signal increased already 1 hr after the start of the treatment. The change of p-NKCC1 immune fluorescence in treated DRGs (FItest) compared to the respective contralateral control DRGs (FIctr) is expressed as ΔFI = (FItest-FIctr)/FIctr·100 [%]. ΔFI (± SD; 3 animals) was 18 ± 5.9% (1 hr), 45 ± 3.5% (2 hr), and 54 ± 3.3% (3 hr). Indicated significance levels are p ≤ 0.05 (*) and p ≤ 0.001 (***). Numbers in parentheses indicate cells evaluated.
Mentions: The activity of NKCC1 and KCC2 can be modulated by phosphorylation [26,27]. To test whether NKCC1 phosphorylation may contribute to the early rise of Cl- levels (< 3 hr), we stained 9 DRG sections from 3 animals with an antibody raised specifically against the phosphorylated form of NKCC1 (p-NKCC1). Preadsorption with a peptide corresponding to the non-phosphorylated epitope ensured the specificity of that immunosignal. The antisera directed against NKCC1 (C-14 antibody) and p-NKCC1 showed a virtually perfect match in the DRG sections (Fig. 4A) indicating that both non-phosphorylated and phosphorylated forms of NKCC1 are present in most neurons. However, in contrast to the NKCC1 immunosignal, the p-NKCC1 signal responded rapidly and robustly to the inflammatory mediators. Already after 1 hr, a significant rise of the p-NKCC1 signal (+18% ± 5.9%; p ≤ 0.05) was observed (Fig. 4B). Two hours after addition of the inflammatory mediators, the p-NKCC1 signal was 45% (± 3.5%; p ≤ 0.001) above control, while the total expression level of NKCC1 showed no response (Fig. 3). After 3 hours, the p-NKCC1 signal was increased by 54% (± 3.3%; p ≤ 0.001) relative to control DRGs. These data demonstrate that the inflammatory mediators induce the phosphorylation of NKCC1 in DRG neurons and suggest that this phosphorylation causes the early (< 3 hr) phase of enhanced Cl- accumulation observed by 2P-FLIM in response to the stimulus.

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
Related in: MedlinePlus