Limits...
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.

Show MeSH

Related in: MedlinePlus

Pharmacological characterisation of the receptor mediating potentiation of RA currentsA, potentiation of RA currents by the indicated substances. For better comparability, data for UTP was re-plotted from Fig. 1. Drugs were applied and potentiation (% of control) was calculated as described in Fig. 1. When antagonists were used, PPADS+ATP and suramin+UTP, they were also present before and after application of ATP and UTP, respectively. GDP-βS was applied intracellularly via the patch electrode; recordings were started 5 min after membrane rupture to allow GDP-βS to sufficiently dialyse the cytosol. (**P < 0.01; n.s., P > 0.5). B, the concentration–response curve of UTP induced potentiation fitted with the Hill equation (EC50= 1.6 μm). Data points were normalised to the potentiation induced by 10 μm UTP in the very same neurone (error bars represent s.e.m.). Abbreviations: PPADS, pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonate; GDP-βS, guanosine-5′-O-(2-thio-diphosphate).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2742277&req=5

fig03: Pharmacological characterisation of the receptor mediating potentiation of RA currentsA, potentiation of RA currents by the indicated substances. For better comparability, data for UTP was re-plotted from Fig. 1. Drugs were applied and potentiation (% of control) was calculated as described in Fig. 1. When antagonists were used, PPADS+ATP and suramin+UTP, they were also present before and after application of ATP and UTP, respectively. GDP-βS was applied intracellularly via the patch electrode; recordings were started 5 min after membrane rupture to allow GDP-βS to sufficiently dialyse the cytosol. (**P < 0.01; n.s., P > 0.5). B, the concentration–response curve of UTP induced potentiation fitted with the Hill equation (EC50= 1.6 μm). Data points were normalised to the potentiation induced by 10 μm UTP in the very same neurone (error bars represent s.e.m.). Abbreviations: PPADS, pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonate; GDP-βS, guanosine-5′-O-(2-thio-diphosphate).

Mentions: RA currents recorded in mechanoreceptors, which are pharmacologically indistinguishable from RA currents in nociceptors (Hu & Lewin, 2006), were not potentiated by UTP (Fig. 1B). This suggests that the effect of UTP is mediated by binding to a purine receptor which may not be present in mechanoreceptive neurones. Uridine nucleotides have very low affinities for P2X receptors (Khakh et al. 2001), and thus we first investigated the possible role of G-protein-coupled P2Y receptors. Replacement of intracellular GTP with the G-protein inhibitor guanosine-5′-O-(2-thio-diphosphate) (GDP-βS, 2 mm), completely abolished UTP induced potentiation of RA currents (110.2 ± 3.8%; n= 11; Fig. 2A), indicating the involvement of metabotropic P2Y receptors. UTP is a selective agonist for P2Y2 and P2Y4 receptors, which are both equally activated by ATP (von Kugelgen & Wetter, 2000; Suarez-Huerta et al. 2001). When applied alone, ATP elicited large inward currents in many of the neurones recorded (4/7, data not shown), most likely due to the activation of P2X receptors. In order to isolate the effect of ATP on RA currents, P2X currents were blocked with the non-selective P2X antagonist pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS, 25 μm). PPADS alone did not affect RA currents (107.9 ± 9.9% of control; n= 5, Student's paired t test, P > 0.05); however, ATP applied in the continuous presence of PPADS potentiated RA currents to 174.3 ± 11.8% of control (n= 7, Student's paired t test, P < 0.01). Since PPADS not only blocks P2X receptors but also antagonises P2Y4 mediated effects (von Kugelgen & Wetter, 2000; Suarez-Huerta et al. 2001) these results strongly suggest that the potentiation of RA currents is mediated by P2Y2 receptors. A P2Y2 mechanism is further supported by the fact that suramin (100 μm), an antagonist at P2Y1 and P2Y2 receptors, completely abolishes UTP induced potentiation of RA currents (98.8 ± 7.7%, n= 7; Fig. 3A). Moreover UDP, which is produced by enzymatic degradation of UTP by ectonucleotidases and preferentially activates P2Y6, did not mimic the effect of UTP. Finally, the concentration dependence of UTP induced potentiation (EC50= 1.6 μm; Fig. 3B) is comparable to published EC50 values for P2Y2 receptors (Lustig et al. 1993). Taken together, the pharmacology of the potentiation of RA currents by UTP and ATP is highly suggestive of a P2Y2 subtype.


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

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

Pharmacological characterisation of the receptor mediating potentiation of RA currentsA, potentiation of RA currents by the indicated substances. For better comparability, data for UTP was re-plotted from Fig. 1. Drugs were applied and potentiation (% of control) was calculated as described in Fig. 1. When antagonists were used, PPADS+ATP and suramin+UTP, they were also present before and after application of ATP and UTP, respectively. GDP-βS was applied intracellularly via the patch electrode; recordings were started 5 min after membrane rupture to allow GDP-βS to sufficiently dialyse the cytosol. (**P < 0.01; n.s., P > 0.5). B, the concentration–response curve of UTP induced potentiation fitted with the Hill equation (EC50= 1.6 μm). Data points were normalised to the potentiation induced by 10 μm UTP in the very same neurone (error bars represent s.e.m.). Abbreviations: PPADS, pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonate; GDP-βS, guanosine-5′-O-(2-thio-diphosphate).
© Copyright Policy
Related In: Results  -  Collection

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

fig03: Pharmacological characterisation of the receptor mediating potentiation of RA currentsA, potentiation of RA currents by the indicated substances. For better comparability, data for UTP was re-plotted from Fig. 1. Drugs were applied and potentiation (% of control) was calculated as described in Fig. 1. When antagonists were used, PPADS+ATP and suramin+UTP, they were also present before and after application of ATP and UTP, respectively. GDP-βS was applied intracellularly via the patch electrode; recordings were started 5 min after membrane rupture to allow GDP-βS to sufficiently dialyse the cytosol. (**P < 0.01; n.s., P > 0.5). B, the concentration–response curve of UTP induced potentiation fitted with the Hill equation (EC50= 1.6 μm). Data points were normalised to the potentiation induced by 10 μm UTP in the very same neurone (error bars represent s.e.m.). Abbreviations: PPADS, pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonate; GDP-βS, guanosine-5′-O-(2-thio-diphosphate).
Mentions: RA currents recorded in mechanoreceptors, which are pharmacologically indistinguishable from RA currents in nociceptors (Hu & Lewin, 2006), were not potentiated by UTP (Fig. 1B). This suggests that the effect of UTP is mediated by binding to a purine receptor which may not be present in mechanoreceptive neurones. Uridine nucleotides have very low affinities for P2X receptors (Khakh et al. 2001), and thus we first investigated the possible role of G-protein-coupled P2Y receptors. Replacement of intracellular GTP with the G-protein inhibitor guanosine-5′-O-(2-thio-diphosphate) (GDP-βS, 2 mm), completely abolished UTP induced potentiation of RA currents (110.2 ± 3.8%; n= 11; Fig. 2A), indicating the involvement of metabotropic P2Y receptors. UTP is a selective agonist for P2Y2 and P2Y4 receptors, which are both equally activated by ATP (von Kugelgen & Wetter, 2000; Suarez-Huerta et al. 2001). When applied alone, ATP elicited large inward currents in many of the neurones recorded (4/7, data not shown), most likely due to the activation of P2X receptors. In order to isolate the effect of ATP on RA currents, P2X currents were blocked with the non-selective P2X antagonist pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS, 25 μm). PPADS alone did not affect RA currents (107.9 ± 9.9% of control; n= 5, Student's paired t test, P > 0.05); however, ATP applied in the continuous presence of PPADS potentiated RA currents to 174.3 ± 11.8% of control (n= 7, Student's paired t test, P < 0.01). Since PPADS not only blocks P2X receptors but also antagonises P2Y4 mediated effects (von Kugelgen & Wetter, 2000; Suarez-Huerta et al. 2001) these results strongly suggest that the potentiation of RA currents is mediated by P2Y2 receptors. A P2Y2 mechanism is further supported by the fact that suramin (100 μm), an antagonist at P2Y1 and P2Y2 receptors, completely abolishes UTP induced potentiation of RA currents (98.8 ± 7.7%, n= 7; Fig. 3A). Moreover UDP, which is produced by enzymatic degradation of UTP by ectonucleotidases and preferentially activates P2Y6, did not mimic the effect of UTP. Finally, the concentration dependence of UTP induced potentiation (EC50= 1.6 μm; Fig. 3B) is comparable to published EC50 values for P2Y2 receptors (Lustig et al. 1993). Taken together, the pharmacology of the potentiation of RA currents by UTP and ATP is highly suggestive of a P2Y2 subtype.

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
Related in: MedlinePlus