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Propofol restores TRPV1 sensitivity via a TRPA1-, nitric oxide synthase-dependent activation of PKCε.

Sinharoy P, Zhang H, Sinha S, Prudner BC, Bratz IN, Damron DS - Pharmacol Res Perspect (2015)

Bottom Line: The extent to which the two pathways are directly linked or operating in parallel has not been determined.Intracellular Ca(2+) concentration was measured in individual cells via fluorescence microscopy.Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Kent State University Kent, Ohio, 44242.

ABSTRACT
We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCε)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCε activation and to elucidate the cellular mechanism by which this occurs. F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca(2+) concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCε, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing both channels, PKCε inhibition prevented the propofol- and allyl isothiocyanate (AITC)-induced restoration of TRPV1 sensitivity to agonist stimulation as well as increased phosphorylation of PKCε and TRPV1. In cells containing TRPV1 only, neither agonist induced PKCε or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCε phosphorylation, and PKCε inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. These data indicate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCε.

No MeSH data available.


Related in: MedlinePlus

(A) Representative trace depicting the effect of propofol (Pro, 10 μmol/L) alone, l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) plus propofol, S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol/L) alone, SNAP plus protein kinase C epsilon (PKCε) inhibitor peptide εV1–2 (0.5 μmol/L), l-NAME plus the PKCε activator peptide ΨεRACK (0.5 μmol/L) after capsaicin-induced (100 nmol/L) desensitization on restoration of TRPV1 sensitivity to capsaicin (Cap) in mouse DRG neurons that contain functional TRPA1 and TRPV1 receptors are depicted in Figure5A, B, C and D and E, respectively. The NOS inhibitor l-NAME was added alone for 5 min following desensitization and then propofol or the PKCε activator peptide ΨεRACK were brought on board in combination with l-NAME for an additional 5 min. Similarly, the εV1–2 inhibitor peptide was added alone for 5 min following desensitization and then SNAP was brought on board in combination with the εV1–2 inhibitor peptide for an additional 5 min. Summarized data for Figure5A–E are depicted in Figure5F. Data are expressed as a percent of the response to the final application of capsaicin in the untreated control (% of control mean value ± SEM). *P < 0.05 compared to final capsaicin in the untreated control. #P < 0.05 compared to Pro plus capsaicin. †P < 0.05 compared to SNAP plus capsaicin. n = F-11 cells from seven separate cover slips.
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fig05: (A) Representative trace depicting the effect of propofol (Pro, 10 μmol/L) alone, l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) plus propofol, S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol/L) alone, SNAP plus protein kinase C epsilon (PKCε) inhibitor peptide εV1–2 (0.5 μmol/L), l-NAME plus the PKCε activator peptide ΨεRACK (0.5 μmol/L) after capsaicin-induced (100 nmol/L) desensitization on restoration of TRPV1 sensitivity to capsaicin (Cap) in mouse DRG neurons that contain functional TRPA1 and TRPV1 receptors are depicted in Figure5A, B, C and D and E, respectively. The NOS inhibitor l-NAME was added alone for 5 min following desensitization and then propofol or the PKCε activator peptide ΨεRACK were brought on board in combination with l-NAME for an additional 5 min. Similarly, the εV1–2 inhibitor peptide was added alone for 5 min following desensitization and then SNAP was brought on board in combination with the εV1–2 inhibitor peptide for an additional 5 min. Summarized data for Figure5A–E are depicted in Figure5F. Data are expressed as a percent of the response to the final application of capsaicin in the untreated control (% of control mean value ± SEM). *P < 0.05 compared to final capsaicin in the untreated control. #P < 0.05 compared to Pro plus capsaicin. †P < 0.05 compared to SNAP plus capsaicin. n = F-11 cells from seven separate cover slips.

Mentions: In F-11 cells transfected with both TRPV1 and TRPA1, inhibition of NOS with l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) prevented the propofol-induced restoration of TRPV1 sensitivity by more than 90% (Fig.5B). Moreover, the NO donor SNAP (100 μmol/L) mimicked the effects of propofol on restoration of TRPV1 sensitivity (Fig.5C) and the restoring effect of SNAP was inhibited by the PKCε inhibitor peptide (0.5 μmol/L), εV1–2 (Fig.5D). Similarly, the PKCε activator peptide ψεRACK (0.5 μmol/L) restored TRPV1 sensitivity in the presence of NOS inhibition with l-NAME (Fig.5E). Pretreatment with l-NAME alone did not restore sensitivity of TRPV1 to capsaicin (102 ± 9% of control). Summarized data for Figure5A–E are depicted in Figure5F.


Propofol restores TRPV1 sensitivity via a TRPA1-, nitric oxide synthase-dependent activation of PKCε.

Sinharoy P, Zhang H, Sinha S, Prudner BC, Bratz IN, Damron DS - Pharmacol Res Perspect (2015)

(A) Representative trace depicting the effect of propofol (Pro, 10 μmol/L) alone, l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) plus propofol, S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol/L) alone, SNAP plus protein kinase C epsilon (PKCε) inhibitor peptide εV1–2 (0.5 μmol/L), l-NAME plus the PKCε activator peptide ΨεRACK (0.5 μmol/L) after capsaicin-induced (100 nmol/L) desensitization on restoration of TRPV1 sensitivity to capsaicin (Cap) in mouse DRG neurons that contain functional TRPA1 and TRPV1 receptors are depicted in Figure5A, B, C and D and E, respectively. The NOS inhibitor l-NAME was added alone for 5 min following desensitization and then propofol or the PKCε activator peptide ΨεRACK were brought on board in combination with l-NAME for an additional 5 min. Similarly, the εV1–2 inhibitor peptide was added alone for 5 min following desensitization and then SNAP was brought on board in combination with the εV1–2 inhibitor peptide for an additional 5 min. Summarized data for Figure5A–E are depicted in Figure5F. Data are expressed as a percent of the response to the final application of capsaicin in the untreated control (% of control mean value ± SEM). *P < 0.05 compared to final capsaicin in the untreated control. #P < 0.05 compared to Pro plus capsaicin. †P < 0.05 compared to SNAP plus capsaicin. n = F-11 cells from seven separate cover slips.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig05: (A) Representative trace depicting the effect of propofol (Pro, 10 μmol/L) alone, l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) plus propofol, S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol/L) alone, SNAP plus protein kinase C epsilon (PKCε) inhibitor peptide εV1–2 (0.5 μmol/L), l-NAME plus the PKCε activator peptide ΨεRACK (0.5 μmol/L) after capsaicin-induced (100 nmol/L) desensitization on restoration of TRPV1 sensitivity to capsaicin (Cap) in mouse DRG neurons that contain functional TRPA1 and TRPV1 receptors are depicted in Figure5A, B, C and D and E, respectively. The NOS inhibitor l-NAME was added alone for 5 min following desensitization and then propofol or the PKCε activator peptide ΨεRACK were brought on board in combination with l-NAME for an additional 5 min. Similarly, the εV1–2 inhibitor peptide was added alone for 5 min following desensitization and then SNAP was brought on board in combination with the εV1–2 inhibitor peptide for an additional 5 min. Summarized data for Figure5A–E are depicted in Figure5F. Data are expressed as a percent of the response to the final application of capsaicin in the untreated control (% of control mean value ± SEM). *P < 0.05 compared to final capsaicin in the untreated control. #P < 0.05 compared to Pro plus capsaicin. †P < 0.05 compared to SNAP plus capsaicin. n = F-11 cells from seven separate cover slips.
Mentions: In F-11 cells transfected with both TRPV1 and TRPA1, inhibition of NOS with l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) prevented the propofol-induced restoration of TRPV1 sensitivity by more than 90% (Fig.5B). Moreover, the NO donor SNAP (100 μmol/L) mimicked the effects of propofol on restoration of TRPV1 sensitivity (Fig.5C) and the restoring effect of SNAP was inhibited by the PKCε inhibitor peptide (0.5 μmol/L), εV1–2 (Fig.5D). Similarly, the PKCε activator peptide ψεRACK (0.5 μmol/L) restored TRPV1 sensitivity in the presence of NOS inhibition with l-NAME (Fig.5E). Pretreatment with l-NAME alone did not restore sensitivity of TRPV1 to capsaicin (102 ± 9% of control). Summarized data for Figure5A–E are depicted in Figure5F.

Bottom Line: The extent to which the two pathways are directly linked or operating in parallel has not been determined.Intracellular Ca(2+) concentration was measured in individual cells via fluorescence microscopy.Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Kent State University Kent, Ohio, 44242.

ABSTRACT
We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCε)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCε activation and to elucidate the cellular mechanism by which this occurs. F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca(2+) concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCε, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing both channels, PKCε inhibition prevented the propofol- and allyl isothiocyanate (AITC)-induced restoration of TRPV1 sensitivity to agonist stimulation as well as increased phosphorylation of PKCε and TRPV1. In cells containing TRPV1 only, neither agonist induced PKCε or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCε phosphorylation, and PKCε inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. These data indicate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCε.

No MeSH data available.


Related in: MedlinePlus