Limits...
A role for Piezo2 in EPAC1-dependent mechanical allodynia.

Eijkelkamp N, Linley JE, Torres JM, Bee L, Dickenson AH, Gringhuis M, Minett MS, Hong GS, Lee E, Oh U, Ishikawa Y, Zwartkuis FJ, Cox JJ, Wood JN - Nat Commun (2013)

Bottom Line: Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton.Piezo2 knockdown also enhanced thresholds for light touch.Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres.

View Article: PubMed Central - PubMed

Affiliation: Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK. N.Eijkelkamp@umcutrecht.nl

ABSTRACT
Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1-/- mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1-Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.

Show MeSH

Related in: MedlinePlus

The selective Epac activator 8-pCPT induces an Epac1-dependent long-lasting allodynia in vivo.Different doses of 8-pCPT (specific Epac activator; n=4−8) or 6-Bnz-cAMP (specific PKA activator; n=4−8) were injected intraplantarly and (a) 50% threshold to von Frey was determined 30 min after administration of drug. (b) Duration of 6-Bnz-cAMP or 8-pCPT-induced mechanical allodynia determined as the earliest time point in which 50% threshold is equal or higher than the average baseline 50% threshold±2x standard deviation (n=4–8). Time course of (c) mechanical hypersensitivity after intraplantar injection of 8-pCPT or 6-Bnz-cAMP (12.5 nmol per paw, n=8). (d–e) Epac1 antisense or mismatch antisense ODN were administered intrathecally once every two days for three times. (d) Two days after the last injection L2-L5 DRG Epac1 protein levels were determined by western Blot (n=4). (e) Time course of 8-pCPT-induced allodynia using von Frey in antisense ODN-treated animals (n=8). Repeated measures one-way analysis of variance: ODN treatment: P<0.001; time: P<0.001; interaction: P<0.001. (f) Time course of intraplantar 8-pCPT-induced allodynia (12.5 nmol per paw) in WT (n=12), Epac1+/− (n=8), and Epac1−/− (n=8) mice. Statistical analysis showed a genotype effect (F(2, 25)=13,053, P<0.01) and post hoc analysis shows that Epac1−/− (P<0.001) as well as Epac1+/− (P<0.05) significantly differed from WT mice. (g) Time course of intraplantar 6-Bnz-cAMP-induced mechanical allodynia (12.5 nmol per paw) in WT (n=8), Epac1+/− (n=6), Epac1−/− (n=8) mice. (h) Mice received intraplantar injection of 8-pCPT or vehicle and sensitivity of the mechanical stimulation was determined after 2 h. At this time point mice received an injection of FM1-43, or vehicle and threshold to mechanical stimulation was determined 1 and 2 h after injection. Data are expressed as mean±s.e.m. Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. (d) Data are analysed by t-test *P<0.05, **P<0.01, ***P<0.001. In f ‘#’ indicates P<0.05 compared to WT mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3644070&req=5

f5: The selective Epac activator 8-pCPT induces an Epac1-dependent long-lasting allodynia in vivo.Different doses of 8-pCPT (specific Epac activator; n=4−8) or 6-Bnz-cAMP (specific PKA activator; n=4−8) were injected intraplantarly and (a) 50% threshold to von Frey was determined 30 min after administration of drug. (b) Duration of 6-Bnz-cAMP or 8-pCPT-induced mechanical allodynia determined as the earliest time point in which 50% threshold is equal or higher than the average baseline 50% threshold±2x standard deviation (n=4–8). Time course of (c) mechanical hypersensitivity after intraplantar injection of 8-pCPT or 6-Bnz-cAMP (12.5 nmol per paw, n=8). (d–e) Epac1 antisense or mismatch antisense ODN were administered intrathecally once every two days for three times. (d) Two days after the last injection L2-L5 DRG Epac1 protein levels were determined by western Blot (n=4). (e) Time course of 8-pCPT-induced allodynia using von Frey in antisense ODN-treated animals (n=8). Repeated measures one-way analysis of variance: ODN treatment: P<0.001; time: P<0.001; interaction: P<0.001. (f) Time course of intraplantar 8-pCPT-induced allodynia (12.5 nmol per paw) in WT (n=12), Epac1+/− (n=8), and Epac1−/− (n=8) mice. Statistical analysis showed a genotype effect (F(2, 25)=13,053, P<0.01) and post hoc analysis shows that Epac1−/− (P<0.001) as well as Epac1+/− (P<0.05) significantly differed from WT mice. (g) Time course of intraplantar 6-Bnz-cAMP-induced mechanical allodynia (12.5 nmol per paw) in WT (n=8), Epac1+/− (n=6), Epac1−/− (n=8) mice. (h) Mice received intraplantar injection of 8-pCPT or vehicle and sensitivity of the mechanical stimulation was determined after 2 h. At this time point mice received an injection of FM1-43, or vehicle and threshold to mechanical stimulation was determined 1 and 2 h after injection. Data are expressed as mean±s.e.m. Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. (d) Data are analysed by t-test *P<0.05, **P<0.01, ***P<0.001. In f ‘#’ indicates P<0.05 compared to WT mice.

Mentions: 8-pCPT has been shown to cause increased sensitivity to noxious mechanical stimuli (hyperalgesia)23. Activation of the cAMP-sensor PKA, induces hyperalgesia through effects on excitability, but not through sensitizing mechanotransduction1. We tested whether selective activation of Epac increased sensitivity to touch and compared this with the development of mechanical hypersensitivity induced by a PKA-selective cAMP analogue (6-Bnz-cAMP). Intraplantar injection of either 6-Bnz-cAMP or 8-pCPT dose-dependently (12.5 pmol per paw–12.5 nmol per paw) induced mechanical hypersensitivity that increased in magnitude and duration with increasing doses (Fig. 5a). At every dose tested, the magnitude of 6-Bnz-cAMP and 8-pCPT-induced mechanical hypersensitivity was statistically indistinguishable (Fig. 5a). Importantly, however, 8-pCPT-induced mechanical hypersensitivity lasted significantly longer than 6-Bnz-cAMP-induced mechanical hypersensitivity (Fig. 5b). At the highest dose tested (12.5 nmol per paw), 8-pCPT-induced sensitization lasted ~3 days while 6-Bnz-cAMP-induced mechanical hypersensitivity only lasted ~1 day (Fig. 5c).


A role for Piezo2 in EPAC1-dependent mechanical allodynia.

Eijkelkamp N, Linley JE, Torres JM, Bee L, Dickenson AH, Gringhuis M, Minett MS, Hong GS, Lee E, Oh U, Ishikawa Y, Zwartkuis FJ, Cox JJ, Wood JN - Nat Commun (2013)

The selective Epac activator 8-pCPT induces an Epac1-dependent long-lasting allodynia in vivo.Different doses of 8-pCPT (specific Epac activator; n=4−8) or 6-Bnz-cAMP (specific PKA activator; n=4−8) were injected intraplantarly and (a) 50% threshold to von Frey was determined 30 min after administration of drug. (b) Duration of 6-Bnz-cAMP or 8-pCPT-induced mechanical allodynia determined as the earliest time point in which 50% threshold is equal or higher than the average baseline 50% threshold±2x standard deviation (n=4–8). Time course of (c) mechanical hypersensitivity after intraplantar injection of 8-pCPT or 6-Bnz-cAMP (12.5 nmol per paw, n=8). (d–e) Epac1 antisense or mismatch antisense ODN were administered intrathecally once every two days for three times. (d) Two days after the last injection L2-L5 DRG Epac1 protein levels were determined by western Blot (n=4). (e) Time course of 8-pCPT-induced allodynia using von Frey in antisense ODN-treated animals (n=8). Repeated measures one-way analysis of variance: ODN treatment: P<0.001; time: P<0.001; interaction: P<0.001. (f) Time course of intraplantar 8-pCPT-induced allodynia (12.5 nmol per paw) in WT (n=12), Epac1+/− (n=8), and Epac1−/− (n=8) mice. Statistical analysis showed a genotype effect (F(2, 25)=13,053, P<0.01) and post hoc analysis shows that Epac1−/− (P<0.001) as well as Epac1+/− (P<0.05) significantly differed from WT mice. (g) Time course of intraplantar 6-Bnz-cAMP-induced mechanical allodynia (12.5 nmol per paw) in WT (n=8), Epac1+/− (n=6), Epac1−/− (n=8) mice. (h) Mice received intraplantar injection of 8-pCPT or vehicle and sensitivity of the mechanical stimulation was determined after 2 h. At this time point mice received an injection of FM1-43, or vehicle and threshold to mechanical stimulation was determined 1 and 2 h after injection. Data are expressed as mean±s.e.m. Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. (d) Data are analysed by t-test *P<0.05, **P<0.01, ***P<0.001. In f ‘#’ indicates P<0.05 compared to WT mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3644070&req=5

f5: The selective Epac activator 8-pCPT induces an Epac1-dependent long-lasting allodynia in vivo.Different doses of 8-pCPT (specific Epac activator; n=4−8) or 6-Bnz-cAMP (specific PKA activator; n=4−8) were injected intraplantarly and (a) 50% threshold to von Frey was determined 30 min after administration of drug. (b) Duration of 6-Bnz-cAMP or 8-pCPT-induced mechanical allodynia determined as the earliest time point in which 50% threshold is equal or higher than the average baseline 50% threshold±2x standard deviation (n=4–8). Time course of (c) mechanical hypersensitivity after intraplantar injection of 8-pCPT or 6-Bnz-cAMP (12.5 nmol per paw, n=8). (d–e) Epac1 antisense or mismatch antisense ODN were administered intrathecally once every two days for three times. (d) Two days after the last injection L2-L5 DRG Epac1 protein levels were determined by western Blot (n=4). (e) Time course of 8-pCPT-induced allodynia using von Frey in antisense ODN-treated animals (n=8). Repeated measures one-way analysis of variance: ODN treatment: P<0.001; time: P<0.001; interaction: P<0.001. (f) Time course of intraplantar 8-pCPT-induced allodynia (12.5 nmol per paw) in WT (n=12), Epac1+/− (n=8), and Epac1−/− (n=8) mice. Statistical analysis showed a genotype effect (F(2, 25)=13,053, P<0.01) and post hoc analysis shows that Epac1−/− (P<0.001) as well as Epac1+/− (P<0.05) significantly differed from WT mice. (g) Time course of intraplantar 6-Bnz-cAMP-induced mechanical allodynia (12.5 nmol per paw) in WT (n=8), Epac1+/− (n=6), Epac1−/− (n=8) mice. (h) Mice received intraplantar injection of 8-pCPT or vehicle and sensitivity of the mechanical stimulation was determined after 2 h. At this time point mice received an injection of FM1-43, or vehicle and threshold to mechanical stimulation was determined 1 and 2 h after injection. Data are expressed as mean±s.e.m. Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. (d) Data are analysed by t-test *P<0.05, **P<0.01, ***P<0.001. In f ‘#’ indicates P<0.05 compared to WT mice.
Mentions: 8-pCPT has been shown to cause increased sensitivity to noxious mechanical stimuli (hyperalgesia)23. Activation of the cAMP-sensor PKA, induces hyperalgesia through effects on excitability, but not through sensitizing mechanotransduction1. We tested whether selective activation of Epac increased sensitivity to touch and compared this with the development of mechanical hypersensitivity induced by a PKA-selective cAMP analogue (6-Bnz-cAMP). Intraplantar injection of either 6-Bnz-cAMP or 8-pCPT dose-dependently (12.5 pmol per paw–12.5 nmol per paw) induced mechanical hypersensitivity that increased in magnitude and duration with increasing doses (Fig. 5a). At every dose tested, the magnitude of 6-Bnz-cAMP and 8-pCPT-induced mechanical hypersensitivity was statistically indistinguishable (Fig. 5a). Importantly, however, 8-pCPT-induced mechanical hypersensitivity lasted significantly longer than 6-Bnz-cAMP-induced mechanical hypersensitivity (Fig. 5b). At the highest dose tested (12.5 nmol per paw), 8-pCPT-induced sensitization lasted ~3 days while 6-Bnz-cAMP-induced mechanical hypersensitivity only lasted ~1 day (Fig. 5c).

Bottom Line: Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton.Piezo2 knockdown also enhanced thresholds for light touch.Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres.

View Article: PubMed Central - PubMed

Affiliation: Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK. N.Eijkelkamp@umcutrecht.nl

ABSTRACT
Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1-/- mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1-Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.

Show MeSH
Related in: MedlinePlus