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Peripheral sensitisation of nociceptors via G-protein-dependent potentiation of mechanotransduction currents.

Lechner SG, Lewin GR - J. Physiol. (Lond.) (2009)

Bottom Line: Here we show that the algogens UTP and ATP potentiate mechanosensitive RA currents in peptidergic nociceptive DRG neurons and reduce thresholds for mechanically induced action potential firing in these neurones.Pharmacological characterisation suggests that this effect is mediated by the Gq-coupled P2Y(2) nucleotide receptor.Together our findings suggest that UTP sensitises a subpopulation of cutaneous C-fibre nociceptors via a previously undescribed G-protein-dependent potentiation of mechanically activated RA-type currents.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Max-Delbrück-Center for Molecular Medicine, Robert Rössle Str. 10, 3125 Berlin, Germany.

ABSTRACT
Mechanical stimuli impinging on the skin are converted into electrical signals by mechanically gated ion channels located at the peripheral nerve endings of dorsal root ganglion (DRG) neurons. Under inflammatory conditions sensory neurons are commonly sensitised to mechanical stimuli; a putative mechanism that may contribute to such sensitisation of sensory neurons is enhanced responsiveness of mechanotransduction ion channels. Here we show that the algogens UTP and ATP potentiate mechanosensitive RA currents in peptidergic nociceptive DRG neurons and reduce thresholds for mechanically induced action potential firing in these neurones. Pharmacological characterisation suggests that this effect is mediated by the Gq-coupled P2Y(2) nucleotide receptor. Moreover, using the in vitro skin nerve technique, we show that UTP also increases action potential firing rates in response to mechanical stimuli in a subpopulation of skin C-fibre nociceptors. Together our findings suggest that UTP sensitises a subpopulation of cutaneous C-fibre nociceptors via a previously undescribed G-protein-dependent potentiation of mechanically activated RA-type currents.

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SA currents are insensitive to modulation by UTPA, example traces showing the effect of 100 μm UTP on SA currents. Neurones were clamped to −60 mV and currents were evoked by mechanical stimuli (500 ms duration) applied at 3 s intervals (black trace). UTP was present as indicated. All cells with an SA current had action potentials characteristic of nociceptors (inflection in the falling phase). B, bars represent mean SA current amplitudes measured before (control), during (UTP) and after (wash) application of UTP. Note, SA currents in the presence of UTP did not differ from those recorded under control/wash conditions (n.s., P > 0.4, Student's paired t test, n= 8).
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fig02: SA currents are insensitive to modulation by UTPA, example traces showing the effect of 100 μm UTP on SA currents. Neurones were clamped to −60 mV and currents were evoked by mechanical stimuli (500 ms duration) applied at 3 s intervals (black trace). UTP was present as indicated. All cells with an SA current had action potentials characteristic of nociceptors (inflection in the falling phase). B, bars represent mean SA current amplitudes measured before (control), during (UTP) and after (wash) application of UTP. Note, SA currents in the presence of UTP did not differ from those recorded under control/wash conditions (n.s., P > 0.4, Student's paired t test, n= 8).

Mentions: To test whether UTP augments the responses of DRG neurones to mechanical stimuli, whole-cell, voltage-clamp recordings were performed and mechanically gated currents were evoked by repetitive mechanical stimulation (duration 500 ms) in the presence and absence of 100 μm UTP (Fig. 1A). Three types of mechanically activated currents have been described in DRG neurones that can readily be distinguished by their inactivation time constants (Hu & Lewin, 2006; Wetzel et al. 2007): rapidly adapting (RA, inactivation times < 5 ms), intermediately adapting (IA, inactivation time 5–50 ms) and slowly adapting (SA, little or no inactivation, >50 ms). Cells were classified as mechanoreceptors or nociceptors based on their characteristic action potential configuration (Fig. 1A, inset) (Koerber et al. 1988). Only nociceptors have humped action potentials with the exception of low threshold C-fibres, which are rare in rodents (Traub & Mendell, 1988; Lewin & Mendell, 1994). Strikingly, UTP rapidly and reversibly potentiated RA current amplitude and slowed inactivation kinetics in 58% (n= 53) of the tested nociceptors (mean cell diameter 19.7 ± 1.4 μm; mean half-peak duration 2.84 ± 0.37 ms), but the RA current in large diameter low threshold mechanoreceptors was completely unaffected (mean cell diameter 27.3 ± 2.1 μm; mean half-peak duration 1.03 ± 0.21 ms) (Fig. 1A and B). Potentiation of peak current amplitudes amounted to 206 ± 16% (n= 31, Student's paired t test, P < 0.01, Fig. 1B) of control and inactivation time constants – derived from a single exponential fit – increased significantly from 1.3 ± 0.15 ms under control conditions to 10.8 ± 2.02 ms (n= 31, Student's paired t test, P < 0.01, Fig. 1C) in the presence of UTP. Thus the potentiation effect in terms of total charge transfer was much larger than 200% increase in the peak current amplitude. A small number of nociceptors possess an IA mechanosensitive current and we could also show that this current is modulated by UTP (Fig. 1A, C and D green trace). In contrast to the modulation of the RA current the peak amplitude of the IA current was not increased by UTP but the inactivation time constant was slowed from 16.4 ± 3.33 ms to 25.1 ± 5.4 ms (n= 9, Student's paired t test, P < 0.05, Fig. 1C), which resulted in an increase of the total charge transfer to 219 ± 43% of control (n= 9, P < 0.05, Fig. 1D). We next asked whether potentiation of RA currents is confined to a molecularly defined subset of nociceptive sensory neurones, as only 58% of all tested nociceptors with an RA current were sensitive to UTP. There are two major populations of cutaneous nociceptors in mice. One population expresses TrkA, the receptor for nerve growth factor (NGF), and calcitonin gene-related peptide (CGRP) and is thus often referred to as the population of peptidergic neurones. The other population, the non-peptidergic nociceptors, expresses the tyrosine kinase Ret, the receptor for glial-derived neurotrophic factor (GDNF) and is characterised by its ability to bind the isolectin B4 (IB4). Strikingly, potentiation of RA currents was significantly larger in IB4-negative neurones (226 ± 51% of control) than in neurones that were labelled by IB4 (111 ± 6% of control, n= 5–6, Mann–Whitney-test, P < 0.05, Fig. 1E). SA currents, which are present in around 30% of all nociceptors (Hu & Lewin, 2006; Lechner et al. 2009), were not affected by application of UTP (Fig. 2A and B).


Peripheral sensitisation of nociceptors via G-protein-dependent potentiation of mechanotransduction currents.

Lechner SG, Lewin GR - J. Physiol. (Lond.) (2009)

SA currents are insensitive to modulation by UTPA, example traces showing the effect of 100 μm UTP on SA currents. Neurones were clamped to −60 mV and currents were evoked by mechanical stimuli (500 ms duration) applied at 3 s intervals (black trace). UTP was present as indicated. All cells with an SA current had action potentials characteristic of nociceptors (inflection in the falling phase). B, bars represent mean SA current amplitudes measured before (control), during (UTP) and after (wash) application of UTP. Note, SA currents in the presence of UTP did not differ from those recorded under control/wash conditions (n.s., P > 0.4, Student's paired t test, n= 8).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742277&req=5

fig02: SA currents are insensitive to modulation by UTPA, example traces showing the effect of 100 μm UTP on SA currents. Neurones were clamped to −60 mV and currents were evoked by mechanical stimuli (500 ms duration) applied at 3 s intervals (black trace). UTP was present as indicated. All cells with an SA current had action potentials characteristic of nociceptors (inflection in the falling phase). B, bars represent mean SA current amplitudes measured before (control), during (UTP) and after (wash) application of UTP. Note, SA currents in the presence of UTP did not differ from those recorded under control/wash conditions (n.s., P > 0.4, Student's paired t test, n= 8).
Mentions: To test whether UTP augments the responses of DRG neurones to mechanical stimuli, whole-cell, voltage-clamp recordings were performed and mechanically gated currents were evoked by repetitive mechanical stimulation (duration 500 ms) in the presence and absence of 100 μm UTP (Fig. 1A). Three types of mechanically activated currents have been described in DRG neurones that can readily be distinguished by their inactivation time constants (Hu & Lewin, 2006; Wetzel et al. 2007): rapidly adapting (RA, inactivation times < 5 ms), intermediately adapting (IA, inactivation time 5–50 ms) and slowly adapting (SA, little or no inactivation, >50 ms). Cells were classified as mechanoreceptors or nociceptors based on their characteristic action potential configuration (Fig. 1A, inset) (Koerber et al. 1988). Only nociceptors have humped action potentials with the exception of low threshold C-fibres, which are rare in rodents (Traub & Mendell, 1988; Lewin & Mendell, 1994). Strikingly, UTP rapidly and reversibly potentiated RA current amplitude and slowed inactivation kinetics in 58% (n= 53) of the tested nociceptors (mean cell diameter 19.7 ± 1.4 μm; mean half-peak duration 2.84 ± 0.37 ms), but the RA current in large diameter low threshold mechanoreceptors was completely unaffected (mean cell diameter 27.3 ± 2.1 μm; mean half-peak duration 1.03 ± 0.21 ms) (Fig. 1A and B). Potentiation of peak current amplitudes amounted to 206 ± 16% (n= 31, Student's paired t test, P < 0.01, Fig. 1B) of control and inactivation time constants – derived from a single exponential fit – increased significantly from 1.3 ± 0.15 ms under control conditions to 10.8 ± 2.02 ms (n= 31, Student's paired t test, P < 0.01, Fig. 1C) in the presence of UTP. Thus the potentiation effect in terms of total charge transfer was much larger than 200% increase in the peak current amplitude. A small number of nociceptors possess an IA mechanosensitive current and we could also show that this current is modulated by UTP (Fig. 1A, C and D green trace). In contrast to the modulation of the RA current the peak amplitude of the IA current was not increased by UTP but the inactivation time constant was slowed from 16.4 ± 3.33 ms to 25.1 ± 5.4 ms (n= 9, Student's paired t test, P < 0.05, Fig. 1C), which resulted in an increase of the total charge transfer to 219 ± 43% of control (n= 9, P < 0.05, Fig. 1D). We next asked whether potentiation of RA currents is confined to a molecularly defined subset of nociceptive sensory neurones, as only 58% of all tested nociceptors with an RA current were sensitive to UTP. There are two major populations of cutaneous nociceptors in mice. One population expresses TrkA, the receptor for nerve growth factor (NGF), and calcitonin gene-related peptide (CGRP) and is thus often referred to as the population of peptidergic neurones. The other population, the non-peptidergic nociceptors, expresses the tyrosine kinase Ret, the receptor for glial-derived neurotrophic factor (GDNF) and is characterised by its ability to bind the isolectin B4 (IB4). Strikingly, potentiation of RA currents was significantly larger in IB4-negative neurones (226 ± 51% of control) than in neurones that were labelled by IB4 (111 ± 6% of control, n= 5–6, Mann–Whitney-test, P < 0.05, Fig. 1E). SA currents, which are present in around 30% of all nociceptors (Hu & Lewin, 2006; Lechner et al. 2009), were not affected by application of UTP (Fig. 2A and B).

Bottom Line: Here we show that the algogens UTP and ATP potentiate mechanosensitive RA currents in peptidergic nociceptive DRG neurons and reduce thresholds for mechanically induced action potential firing in these neurones.Pharmacological characterisation suggests that this effect is mediated by the Gq-coupled P2Y(2) nucleotide receptor.Together our findings suggest that UTP sensitises a subpopulation of cutaneous C-fibre nociceptors via a previously undescribed G-protein-dependent potentiation of mechanically activated RA-type currents.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Max-Delbrück-Center for Molecular Medicine, Robert Rössle Str. 10, 3125 Berlin, Germany.

ABSTRACT
Mechanical stimuli impinging on the skin are converted into electrical signals by mechanically gated ion channels located at the peripheral nerve endings of dorsal root ganglion (DRG) neurons. Under inflammatory conditions sensory neurons are commonly sensitised to mechanical stimuli; a putative mechanism that may contribute to such sensitisation of sensory neurons is enhanced responsiveness of mechanotransduction ion channels. Here we show that the algogens UTP and ATP potentiate mechanosensitive RA currents in peptidergic nociceptive DRG neurons and reduce thresholds for mechanically induced action potential firing in these neurones. Pharmacological characterisation suggests that this effect is mediated by the Gq-coupled P2Y(2) nucleotide receptor. Moreover, using the in vitro skin nerve technique, we show that UTP also increases action potential firing rates in response to mechanical stimuli in a subpopulation of skin C-fibre nociceptors. Together our findings suggest that UTP sensitises a subpopulation of cutaneous C-fibre nociceptors via a previously undescribed G-protein-dependent potentiation of mechanically activated RA-type currents.

Show MeSH