Limits...
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 transient receptor potential vanilloid receptor type 1 (TRPV1) serine 800 phosphorylation (TRPV1pS800) in F-11 cells transfected with TRPV1 channels only. Total TRPV1 was used as a loading control. (B) Summarized data for Figure4A. (C and D) Same as Figure4A and B, respectively, except in F-11 cells transfected with both TRPV1 channels 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 Prop alone. n = six different F-11 cell lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492729&req=5

fig04: (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 transient receptor potential vanilloid receptor type 1 (TRPV1) serine 800 phosphorylation (TRPV1pS800) in F-11 cells transfected with TRPV1 channels only. Total TRPV1 was used as a loading control. (B) Summarized data for Figure4A. (C and D) Same as Figure4A and B, respectively, except in F-11 cells transfected with both TRPV1 channels 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 Prop alone. n = six different F-11 cell lysates.

Mentions: To determine if the presence of TRPA1 receptors is required for propofol or AITC to phosphorylate TRPV1 at S800, immunoblot analysis of TRPV1pS800 was performed. TRPV1pS800 levels were normalized to total TRPV1 protein detected in the lysate. In TRPV1 transfected F-11 cells, neither AITC (100 μmol/L) nor propofol (10 μmol/L) increased TRPV1pS800 levels as compared to untreated control (Fig.4A). However, treatment with the PKCε activator peptide, ψεRACK (0.5 μmol/L), increased TRPV1pS800 levels compared to control. In TRPV1-TRPA1 cotrasfected F-11 cells, AITC, propofol, and ψεRACK all increased TRPV1pS800 levels compared to control (Fig.4B). Moreover, the AITC- and propofol-induced increases in TRPV1pS800 levels were prevented by pretreatment with the PKCε inhibitory peptide, εV1–2. In addition, following treatment with propofol (10 μmol/L) or AITC (100 μmol/L) in either cell type, the amount of immunodetectable TRPV1 in the cell lysate was unchanged as compared to untreated control (Fig.4A and C). The Intralipid vehicle had no detectable effect on TRPV1pS800 levels (103 ± 5% of control). Summarized data for Figure4A and C are depicted in Figure4B 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 transient receptor potential vanilloid receptor type 1 (TRPV1) serine 800 phosphorylation (TRPV1pS800) in F-11 cells transfected with TRPV1 channels only. Total TRPV1 was used as a loading control. (B) Summarized data for Figure4A. (C and D) Same as Figure4A and B, respectively, except in F-11 cells transfected with both TRPV1 channels 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 Prop alone. n = six different F-11 cell lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: (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 transient receptor potential vanilloid receptor type 1 (TRPV1) serine 800 phosphorylation (TRPV1pS800) in F-11 cells transfected with TRPV1 channels only. Total TRPV1 was used as a loading control. (B) Summarized data for Figure4A. (C and D) Same as Figure4A and B, respectively, except in F-11 cells transfected with both TRPV1 channels 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 Prop alone. n = six different F-11 cell lysates.
Mentions: To determine if the presence of TRPA1 receptors is required for propofol or AITC to phosphorylate TRPV1 at S800, immunoblot analysis of TRPV1pS800 was performed. TRPV1pS800 levels were normalized to total TRPV1 protein detected in the lysate. In TRPV1 transfected F-11 cells, neither AITC (100 μmol/L) nor propofol (10 μmol/L) increased TRPV1pS800 levels as compared to untreated control (Fig.4A). However, treatment with the PKCε activator peptide, ψεRACK (0.5 μmol/L), increased TRPV1pS800 levels compared to control. In TRPV1-TRPA1 cotrasfected F-11 cells, AITC, propofol, and ψεRACK all increased TRPV1pS800 levels compared to control (Fig.4B). Moreover, the AITC- and propofol-induced increases in TRPV1pS800 levels were prevented by pretreatment with the PKCε inhibitory peptide, εV1–2. In addition, following treatment with propofol (10 μmol/L) or AITC (100 μmol/L) in either cell type, the amount of immunodetectable TRPV1 in the cell lysate was unchanged as compared to untreated control (Fig.4A and C). The Intralipid vehicle had no detectable effect on TRPV1pS800 levels (103 ± 5% of control). Summarized data for Figure4A and C are depicted in Figure4B 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