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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 expression of nNOS in nontransfected F-11 cells (F-11 NT), TRPV1-transfected F-11 cells (F-11 V1), TRPA1-transfected F-11 cells (F-11 A1), and TRPV1-TRPA1 co-transfected F-11 cells (F-11 V1/A1). GAPDH was used as a loading control. (B) Represents the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, and propofol in presence of HC-030031 (0.5 μmol/L) on nNOS serine 1417 phosphorylation (nNOSS1417) in F-11 cells transfected with both TRPV1 and TRPA1. Total nNOS was used as a loading control. (C) Summarized data for Figure7B. (D) Representative fluorescence images depicting the effect of l-Arginine (NO-inducer), c-PTIO (NO scavenger), AITC (100 μmol/L), propofol (10 μmol/L), SNAP (100 μmol/L), and propofol in the presence of HC-030031(0.5 μmol/L) on NO production in TRPA1 and TRPV1 cotransfected F-11 cells. Data are expressed as a percent of the untreated control mean value ± SEM. *P < 0.05 compared to control. n = six different F-11 cell lysates.
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fig07: (A) Representative immunoblot depicting the expression of nNOS in nontransfected F-11 cells (F-11 NT), TRPV1-transfected F-11 cells (F-11 V1), TRPA1-transfected F-11 cells (F-11 A1), and TRPV1-TRPA1 co-transfected F-11 cells (F-11 V1/A1). GAPDH was used as a loading control. (B) Represents the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, and propofol in presence of HC-030031 (0.5 μmol/L) on nNOS serine 1417 phosphorylation (nNOSS1417) in F-11 cells transfected with both TRPV1 and TRPA1. Total nNOS was used as a loading control. (C) Summarized data for Figure7B. (D) Representative fluorescence images depicting the effect of l-Arginine (NO-inducer), c-PTIO (NO scavenger), AITC (100 μmol/L), propofol (10 μmol/L), SNAP (100 μmol/L), and propofol in the presence of HC-030031(0.5 μmol/L) on NO production in TRPA1 and TRPV1 cotransfected F-11 cells. Data are expressed as a percent of the untreated control mean value ± SEM. *P < 0.05 compared to control. n = six different F-11 cell lysates.

Mentions: No significant change in the expression level of nNOS was observed in nontransfected, TRPV1-trasfected, TRPA1-transfected, and TRPV1-TRPA1 cotransfected F-11 cells (Fig.7A). However, TRPV1–TRPA1 cotransfected F-11 cells pretreated with propofol (10 μmol/L) and AITC (100 μmol/L) demonstrated increased phosphorylation of nNOS at Ser1417 compared to the nontreated cells lysate, that was significantly attenuated by the TRPA1 antagonist, HC-030031 (0.5 μmol/L; Fig.7B). Summarized data for Figure7B are depicted in Figure7C. AITC (100 μmol/L) - and propofol (10 μmol/L) -induced NO production was also detected and visualized in TRPV1-TRPA1 cotransfected F-11 cells, that was mimicked by SNAP (100 μmol/L). Moreover, propofol-induced NO production was attenuated in presence of HC-030031 (0.5 μmol/L). Changes in intracellular NO production were compared to cells pretreated with l-Arginine, an NO inducer (100 μmol/L), and c-PTIO, an NO scavenger (10 μmol/L) (Fig.7).


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 expression of nNOS in nontransfected F-11 cells (F-11 NT), TRPV1-transfected F-11 cells (F-11 V1), TRPA1-transfected F-11 cells (F-11 A1), and TRPV1-TRPA1 co-transfected F-11 cells (F-11 V1/A1). GAPDH was used as a loading control. (B) Represents the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, and propofol in presence of HC-030031 (0.5 μmol/L) on nNOS serine 1417 phosphorylation (nNOSS1417) in F-11 cells transfected with both TRPV1 and TRPA1. Total nNOS was used as a loading control. (C) Summarized data for Figure7B. (D) Representative fluorescence images depicting the effect of l-Arginine (NO-inducer), c-PTIO (NO scavenger), AITC (100 μmol/L), propofol (10 μmol/L), SNAP (100 μmol/L), and propofol in the presence of HC-030031(0.5 μmol/L) on NO production in TRPA1 and TRPV1 cotransfected F-11 cells. Data are expressed as a percent of the untreated control mean value ± SEM. *P < 0.05 compared to control. n = six different F-11 cell lysates.
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Related In: Results  -  Collection

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fig07: (A) Representative immunoblot depicting the expression of nNOS in nontransfected F-11 cells (F-11 NT), TRPV1-transfected F-11 cells (F-11 V1), TRPA1-transfected F-11 cells (F-11 A1), and TRPV1-TRPA1 co-transfected F-11 cells (F-11 V1/A1). GAPDH was used as a loading control. (B) Represents the effect of allyl isothiocyanate (AITC, 100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, and propofol in presence of HC-030031 (0.5 μmol/L) on nNOS serine 1417 phosphorylation (nNOSS1417) in F-11 cells transfected with both TRPV1 and TRPA1. Total nNOS was used as a loading control. (C) Summarized data for Figure7B. (D) Representative fluorescence images depicting the effect of l-Arginine (NO-inducer), c-PTIO (NO scavenger), AITC (100 μmol/L), propofol (10 μmol/L), SNAP (100 μmol/L), and propofol in the presence of HC-030031(0.5 μmol/L) on NO production in TRPA1 and TRPV1 cotransfected F-11 cells. Data are expressed as a percent of the untreated control mean value ± SEM. *P < 0.05 compared to control. n = six different F-11 cell lysates.
Mentions: No significant change in the expression level of nNOS was observed in nontransfected, TRPV1-trasfected, TRPA1-transfected, and TRPV1-TRPA1 cotransfected F-11 cells (Fig.7A). However, TRPV1–TRPA1 cotransfected F-11 cells pretreated with propofol (10 μmol/L) and AITC (100 μmol/L) demonstrated increased phosphorylation of nNOS at Ser1417 compared to the nontreated cells lysate, that was significantly attenuated by the TRPA1 antagonist, HC-030031 (0.5 μmol/L; Fig.7B). Summarized data for Figure7B are depicted in Figure7C. AITC (100 μmol/L) - and propofol (10 μmol/L) -induced NO production was also detected and visualized in TRPV1-TRPA1 cotransfected F-11 cells, that was mimicked by SNAP (100 μmol/L). Moreover, propofol-induced NO production was attenuated in presence of HC-030031 (0.5 μmol/L). Changes in intracellular NO production were compared to cells pretreated with l-Arginine, an NO inducer (100 μmol/L), and c-PTIO, an NO scavenger (10 μmol/L) (Fig.7).

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