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Role of anoctamin-1 and bestrophin-1 in spinal nerve ligation-induced neuropathic pain in rats.

Pineda-Farias JB, Barragán-Iglesias P, Loeza-Alcocer E, Torres-López JE, Rocha-González HI, Pérez-Severiano F, Delgado-Lezama R, Granados-Soto V - Mol Pain (2015)

Bottom Line: Intrathecal administration of non-selective CaCCs inhibitors (NPPB, 9-AC and NFA) dose-dependently reduced tactile allodynia.Blockade of these CaCCs suppresses compound action potential generation in putative C fibers and lessens established tactile allodynia.As CaCCs activity contributes to neuropathic pain maintenance, selective inhibition of their activity may function as a tool to generate analgesia in nerve injury pain states.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology of Pain Laboratory, Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados (Cinvestav), Sede Sur, Calzada de los Tenorios 235, Colonia Granjas Coapa, 14330, México, D.F., México. jorgepinedafarias@yahoo.com.mx.

ABSTRACT

Background: Calcium-activated chloride channels (CaCCs) activation induces membrane depolarization by increasing chloride efflux in primary sensory neurons that can facilitate action potential generation. Previous studies suggest that CaCCs family members bestrophin-1 and anoctamin-1 are involved in inflammatory pain. However, their role in neuropathic pain is unclear. In this investigation we assessed the involvement of these CaCCs family members in rats subjected to the L5/L6 spinal nerve ligation. In addition, anoctamin-1 and bestrophin-1 mRNA and protein expression in dorsal root ganglion (DRG) and spinal cord was also determined in the presence and absence of selective inhibitors.

Results: L5/L6 spinal nerve ligation induced mechanical tactile allodynia. Intrathecal administration of non-selective CaCCs inhibitors (NPPB, 9-AC and NFA) dose-dependently reduced tactile allodynia. Intrathecal administration of selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) also dose-dependently diminished tactile allodynia and thermal hyperalgesia. Anoctamin-1 and bestrophin-1 mRNA and protein were expressed in the dorsal spinal cord and DRG of naïve, sham and neuropathic rats. L5/L6 spinal nerve ligation rose mRNA and protein expression of anoctamin-1, but not bestrophin-1, in the dorsal spinal cord and DRG from day 1 to day 14 after nerve ligation. In addition, repeated administration of CaCCs inhibitors (T16Ainh-A01, CaCCinh-A01 or NFA) or anti-anoctamin-1 antibody prevented spinal nerve ligation-induced rises in anoctamin-1 mRNA and protein expression. Following spinal nerve ligation, the compound action potential generation of putative C fibers increased while selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) attenuated such increase.

Conclusions: There is functional anoctamin-1 and bestrophin-1 expression in rats at sites related to nociceptive processing. Blockade of these CaCCs suppresses compound action potential generation in putative C fibers and lessens established tactile allodynia. As CaCCs activity contributes to neuropathic pain maintenance, selective inhibition of their activity may function as a tool to generate analgesia in nerve injury pain states.

No MeSH data available.


Related in: MedlinePlus

Spinal nerve injury increases anoctamin-1 expression in the spinal cord and DRG. RT-PCR (a, b) and western blot (c, d) analysis of anoctamin-1 (Ano-1) at the ipsilateral dorsal portion of the spinal cord (SC) and DRG obtained from naïve (Na), sham (S) and spinal nerve ligated (SNL) rats. Data were normalized against β-actin and are expressed as the mean ± SEM of three independent rats. Insets show a representative band of the PCR product (upper panels) and immunoblot (lower panels) obtained with the specific anoctamin-1 and β-actin primers or antibodies, respectively. *Significantly different from the S group (P < 0.05), #significantly different from the Na group (P < 0.05), as determined by one-way ANOVA, followed by the Student–Newman–Keuls test. Note that spinal nerve injury enhanced Ano-1 mRNA and protein expression in spinal cord and DRG whereas that surgery increased Ano-1 expression in sham (S) animals in spinal cord but not DRG.
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Fig5: Spinal nerve injury increases anoctamin-1 expression in the spinal cord and DRG. RT-PCR (a, b) and western blot (c, d) analysis of anoctamin-1 (Ano-1) at the ipsilateral dorsal portion of the spinal cord (SC) and DRG obtained from naïve (Na), sham (S) and spinal nerve ligated (SNL) rats. Data were normalized against β-actin and are expressed as the mean ± SEM of three independent rats. Insets show a representative band of the PCR product (upper panels) and immunoblot (lower panels) obtained with the specific anoctamin-1 and β-actin primers or antibodies, respectively. *Significantly different from the S group (P < 0.05), #significantly different from the Na group (P < 0.05), as determined by one-way ANOVA, followed by the Student–Newman–Keuls test. Note that spinal nerve injury enhanced Ano-1 mRNA and protein expression in spinal cord and DRG whereas that surgery increased Ano-1 expression in sham (S) animals in spinal cord but not DRG.

Mentions: PCR and western blot analysis of the ipsilateral dorsal spinal cord and DRG demonstrated bestrophin-1 (Figure 4) and anoctamin-1 (Figure 5) mRNA and protein expression, in naïve, sham and ligated rats. Western blots resolved bands of about 68 and 90 kDa for bestrophin-1 (Additional file 3: Figure S3A) and anoctamin-1 (Additional file 3: Figure S3B), respectively. On the other hand, immunoreactive bands for both proteins were absent when the primary antibodies were pre-adsorbed with the corresponding antigenic peptides (Additional file 3: Figure S3).Figure 4


Role of anoctamin-1 and bestrophin-1 in spinal nerve ligation-induced neuropathic pain in rats.

Pineda-Farias JB, Barragán-Iglesias P, Loeza-Alcocer E, Torres-López JE, Rocha-González HI, Pérez-Severiano F, Delgado-Lezama R, Granados-Soto V - Mol Pain (2015)

Spinal nerve injury increases anoctamin-1 expression in the spinal cord and DRG. RT-PCR (a, b) and western blot (c, d) analysis of anoctamin-1 (Ano-1) at the ipsilateral dorsal portion of the spinal cord (SC) and DRG obtained from naïve (Na), sham (S) and spinal nerve ligated (SNL) rats. Data were normalized against β-actin and are expressed as the mean ± SEM of three independent rats. Insets show a representative band of the PCR product (upper panels) and immunoblot (lower panels) obtained with the specific anoctamin-1 and β-actin primers or antibodies, respectively. *Significantly different from the S group (P < 0.05), #significantly different from the Na group (P < 0.05), as determined by one-way ANOVA, followed by the Student–Newman–Keuls test. Note that spinal nerve injury enhanced Ano-1 mRNA and protein expression in spinal cord and DRG whereas that surgery increased Ano-1 expression in sham (S) animals in spinal cord but not DRG.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487556&req=5

Fig5: Spinal nerve injury increases anoctamin-1 expression in the spinal cord and DRG. RT-PCR (a, b) and western blot (c, d) analysis of anoctamin-1 (Ano-1) at the ipsilateral dorsal portion of the spinal cord (SC) and DRG obtained from naïve (Na), sham (S) and spinal nerve ligated (SNL) rats. Data were normalized against β-actin and are expressed as the mean ± SEM of three independent rats. Insets show a representative band of the PCR product (upper panels) and immunoblot (lower panels) obtained with the specific anoctamin-1 and β-actin primers or antibodies, respectively. *Significantly different from the S group (P < 0.05), #significantly different from the Na group (P < 0.05), as determined by one-way ANOVA, followed by the Student–Newman–Keuls test. Note that spinal nerve injury enhanced Ano-1 mRNA and protein expression in spinal cord and DRG whereas that surgery increased Ano-1 expression in sham (S) animals in spinal cord but not DRG.
Mentions: PCR and western blot analysis of the ipsilateral dorsal spinal cord and DRG demonstrated bestrophin-1 (Figure 4) and anoctamin-1 (Figure 5) mRNA and protein expression, in naïve, sham and ligated rats. Western blots resolved bands of about 68 and 90 kDa for bestrophin-1 (Additional file 3: Figure S3A) and anoctamin-1 (Additional file 3: Figure S3B), respectively. On the other hand, immunoreactive bands for both proteins were absent when the primary antibodies were pre-adsorbed with the corresponding antigenic peptides (Additional file 3: Figure S3).Figure 4

Bottom Line: Intrathecal administration of non-selective CaCCs inhibitors (NPPB, 9-AC and NFA) dose-dependently reduced tactile allodynia.Blockade of these CaCCs suppresses compound action potential generation in putative C fibers and lessens established tactile allodynia.As CaCCs activity contributes to neuropathic pain maintenance, selective inhibition of their activity may function as a tool to generate analgesia in nerve injury pain states.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology of Pain Laboratory, Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados (Cinvestav), Sede Sur, Calzada de los Tenorios 235, Colonia Granjas Coapa, 14330, México, D.F., México. jorgepinedafarias@yahoo.com.mx.

ABSTRACT

Background: Calcium-activated chloride channels (CaCCs) activation induces membrane depolarization by increasing chloride efflux in primary sensory neurons that can facilitate action potential generation. Previous studies suggest that CaCCs family members bestrophin-1 and anoctamin-1 are involved in inflammatory pain. However, their role in neuropathic pain is unclear. In this investigation we assessed the involvement of these CaCCs family members in rats subjected to the L5/L6 spinal nerve ligation. In addition, anoctamin-1 and bestrophin-1 mRNA and protein expression in dorsal root ganglion (DRG) and spinal cord was also determined in the presence and absence of selective inhibitors.

Results: L5/L6 spinal nerve ligation induced mechanical tactile allodynia. Intrathecal administration of non-selective CaCCs inhibitors (NPPB, 9-AC and NFA) dose-dependently reduced tactile allodynia. Intrathecal administration of selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) also dose-dependently diminished tactile allodynia and thermal hyperalgesia. Anoctamin-1 and bestrophin-1 mRNA and protein were expressed in the dorsal spinal cord and DRG of naïve, sham and neuropathic rats. L5/L6 spinal nerve ligation rose mRNA and protein expression of anoctamin-1, but not bestrophin-1, in the dorsal spinal cord and DRG from day 1 to day 14 after nerve ligation. In addition, repeated administration of CaCCs inhibitors (T16Ainh-A01, CaCCinh-A01 or NFA) or anti-anoctamin-1 antibody prevented spinal nerve ligation-induced rises in anoctamin-1 mRNA and protein expression. Following spinal nerve ligation, the compound action potential generation of putative C fibers increased while selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) attenuated such increase.

Conclusions: There is functional anoctamin-1 and bestrophin-1 expression in rats at sites related to nociceptive processing. Blockade of these CaCCs suppresses compound action potential generation in putative C fibers and lessens established tactile allodynia. As CaCCs activity contributes to neuropathic pain maintenance, selective inhibition of their activity may function as a tool to generate analgesia in nerve injury pain states.

No MeSH data available.


Related in: MedlinePlus