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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 immunoblot depicting the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol/L), propofol in the presence of the NOS inhibitor, l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) or allyl isothiocyanate (AITC, 100 μmol/L) in the presence of l-NAME on PKCε serine 729 phosphorylation (PKCεpS729) in F-11 cells transfected with both TRPV1 and TRPA1. Total PKCε was used as a loading control. (B) Summarized data for Figure6A. Data are expressed as a percent of the untreated control mean value ± SEM. *P < 0.05 compared to control. #P < 0.05 compared to AITC alone. †P < 0.05 compared to Prop alone. n = six different F-11 cell lysates.
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fig06: (A) Representative immunoblot depicting the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol/L), propofol in the presence of the NOS inhibitor, l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) or allyl isothiocyanate (AITC, 100 μmol/L) in the presence of l-NAME on PKCε serine 729 phosphorylation (PKCεpS729) in F-11 cells transfected with both TRPV1 and TRPA1. Total PKCε was used as a loading control. (B) Summarized data for Figure6A. Data are expressed as a percent of the untreated control mean value ± SEM. *P < 0.05 compared to control. #P < 0.05 compared to AITC alone. †P < 0.05 compared to Prop alone. n = six different F-11 cell lysates.

Mentions: In F-11 cells transfected with TRPA1 and TRPV1, pretreatment with the NO donor, SNAP, increased immunodetectable phosphorylation of PKCεpS729 (Fig.6A). Moreover, inhibition of NOS with l-NAME prevented the propofol- and AITC-induced increase in immunodetectable PKCεpS729. Incubation with l-NAME alone had no significant effect on basal levels of PKCεpS729 (98 ± 8% of control). Summarized data for Figure6A are depicted in Figure6B.


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 immunoblot depicting the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol/L), propofol in the presence of the NOS inhibitor, l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) or allyl isothiocyanate (AITC, 100 μmol/L) in the presence of l-NAME on PKCε serine 729 phosphorylation (PKCεpS729) in F-11 cells transfected with both TRPV1 and TRPA1. Total PKCε was used as a loading control. (B) Summarized data for Figure6A. Data are expressed as a percent of the untreated control mean value ± SEM. *P < 0.05 compared to control. #P < 0.05 compared to AITC alone. †P < 0.05 compared to Prop alone. n = six different F-11 cell lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492729&req=5

fig06: (A) Representative immunoblot depicting the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol/L), propofol in the presence of the NOS inhibitor, l-NG-nitroarginine methyl ester (l-NAME; 100 μmol/L) or allyl isothiocyanate (AITC, 100 μmol/L) in the presence of l-NAME on PKCε serine 729 phosphorylation (PKCεpS729) in F-11 cells transfected with both TRPV1 and TRPA1. Total PKCε was used as a loading control. (B) Summarized data for Figure6A. Data are expressed as a percent of the untreated control mean value ± SEM. *P < 0.05 compared to control. #P < 0.05 compared to AITC alone. †P < 0.05 compared to Prop alone. n = six different F-11 cell lysates.
Mentions: In F-11 cells transfected with TRPA1 and TRPV1, pretreatment with the NO donor, SNAP, increased immunodetectable phosphorylation of PKCεpS729 (Fig.6A). Moreover, inhibition of NOS with l-NAME prevented the propofol- and AITC-induced increase in immunodetectable PKCεpS729. Incubation with l-NAME alone had no significant effect on basal levels of PKCεpS729 (98 ± 8% of control). Summarized data for Figure6A are depicted in Figure6B.

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