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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, the protein kinase C epsilon (PKCε) activator peptide ΨεRACK (0.5 μmol/L) alone, and AITC or propofol in the presence of the PKCε inhibitor peptide εV1–2 (0.5 μmol/L) on PKCε serine 729 phosphorylation (PKCεpS729) in F-11 cells transfected with transient receptor potential vanilloid receptor type 1 (TRPV1) channels only. Total PKCε was used as a loading control. (B) Summarized data for Figure3A. (C and D) Same as Figure3A and B, respectively, except in -11 cells transfected with both TRPV1 and transient receptor potential ankyrin receptor subtype-1 (TRPA1) channels. 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 propofol alone. n = six different F-11 cell lysates.
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fig03: (A) Representative immunoblot depicting the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, the protein kinase C epsilon (PKCε) activator peptide ΨεRACK (0.5 μmol/L) alone, and AITC or propofol in the presence of the PKCε inhibitor peptide εV1–2 (0.5 μmol/L) on PKCε serine 729 phosphorylation (PKCεpS729) in F-11 cells transfected with transient receptor potential vanilloid receptor type 1 (TRPV1) channels only. Total PKCε was used as a loading control. (B) Summarized data for Figure3A. (C and D) Same as Figure3A and B, respectively, except in -11 cells transfected with both TRPV1 and transient receptor potential ankyrin receptor subtype-1 (TRPA1) channels. 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 propofol alone. n = six different F-11 cell lysates.

Mentions: To determine if the presence of TRPA1 receptors is required for propofol or AITC to activate PKCε at S729, immunoblot analysis of PKCεpS729 was performed before and after treatment of cells with either propofol (10 μmol/L; 10 min), AITC (100 μmol/L), or ψεRACK (0.5 μmol/L). We also assessed the effect of the PKCε inhibitor peptide, εV1–2 (0.5 μmol/L), on the propofol- and AITC-induced phosphorylation of S729. PKCεpS729 levels were normalized to total PKCε protein levels detected in the lysate. In TRPV1 transfectedF-11 cells, neither AITC (100 μmol/L) nor propofol (10 μmol/L) increased PKCεpS729 levels as compared to untreated control (Fig.3A). However, treatment with the PKCε activator peptide, ψεRACK (0.5 μmol/L), increased PKCεpS729 levels compared to control. In TRPV1-TRPA1 cotransfected F-11 cells, AITC, propofol, and ψεRACK all increased PKCεpS729 levels compared to control (Fig.3C). Moreover, the AITC- and propofol-induced increases in PKCεpS729 levels were prevented by pretreatment with the PKCε inhibitory peptide, εV1–2 in TRPV1-TRPA1 cotransfected F-11 cells (Fig.3C). In addition, following treatment with propofol (10 μmol/L) or AITC (100 μmol/L) in either cell type, the amount of immunodetectable PKCε in the membrane fraction was unchanged as compared to untreated control (Fig.3A and C). The Intralipid (amount equivalent to 10 μmol/L propofol) increased PKCεpS729 levels to 127 ± 8% of control similar to our previous findings (Wickley et al. 2010). Summarized data for Figure3A and C are depicted in Figure3B and D, respectively.


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, the protein kinase C epsilon (PKCε) activator peptide ΨεRACK (0.5 μmol/L) alone, and AITC or propofol in the presence of the PKCε inhibitor peptide εV1–2 (0.5 μmol/L) on PKCε serine 729 phosphorylation (PKCεpS729) in F-11 cells transfected with transient receptor potential vanilloid receptor type 1 (TRPV1) channels only. Total PKCε was used as a loading control. (B) Summarized data for Figure3A. (C and D) Same as Figure3A and B, respectively, except in -11 cells transfected with both TRPV1 and transient receptor potential ankyrin receptor subtype-1 (TRPA1) channels. 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 propofol alone. n = six different F-11 cell lysates.
© Copyright Policy - open-access
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fig03: (A) Representative immunoblot depicting the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, the protein kinase C epsilon (PKCε) activator peptide ΨεRACK (0.5 μmol/L) alone, and AITC or propofol in the presence of the PKCε inhibitor peptide εV1–2 (0.5 μmol/L) on PKCε serine 729 phosphorylation (PKCεpS729) in F-11 cells transfected with transient receptor potential vanilloid receptor type 1 (TRPV1) channels only. Total PKCε was used as a loading control. (B) Summarized data for Figure3A. (C and D) Same as Figure3A and B, respectively, except in -11 cells transfected with both TRPV1 and transient receptor potential ankyrin receptor subtype-1 (TRPA1) channels. 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 propofol alone. n = six different F-11 cell lysates.
Mentions: To determine if the presence of TRPA1 receptors is required for propofol or AITC to activate PKCε at S729, immunoblot analysis of PKCεpS729 was performed before and after treatment of cells with either propofol (10 μmol/L; 10 min), AITC (100 μmol/L), or ψεRACK (0.5 μmol/L). We also assessed the effect of the PKCε inhibitor peptide, εV1–2 (0.5 μmol/L), on the propofol- and AITC-induced phosphorylation of S729. PKCεpS729 levels were normalized to total PKCε protein levels detected in the lysate. In TRPV1 transfectedF-11 cells, neither AITC (100 μmol/L) nor propofol (10 μmol/L) increased PKCεpS729 levels as compared to untreated control (Fig.3A). However, treatment with the PKCε activator peptide, ψεRACK (0.5 μmol/L), increased PKCεpS729 levels compared to control. In TRPV1-TRPA1 cotransfected F-11 cells, AITC, propofol, and ψεRACK all increased PKCεpS729 levels compared to control (Fig.3C). Moreover, the AITC- and propofol-induced increases in PKCεpS729 levels were prevented by pretreatment with the PKCε inhibitory peptide, εV1–2 in TRPV1-TRPA1 cotransfected F-11 cells (Fig.3C). In addition, following treatment with propofol (10 μmol/L) or AITC (100 μmol/L) in either cell type, the amount of immunodetectable PKCε in the membrane fraction was unchanged as compared to untreated control (Fig.3A and C). The Intralipid (amount equivalent to 10 μmol/L propofol) increased PKCεpS729 levels to 127 ± 8% of control similar to our previous findings (Wickley et al. 2010). Summarized data for Figure3A and C are depicted in Figure3B and D, respectively.

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