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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 a single application of capsaicin (100 nmol/L) and allyl isothiocyanate (AITC, 100 μmol/L) on intracellular free calcium concentration inF-11 cells transfected with only functional transient receptor potential vanilloid receptor type 1 (TRPV1) receptors c-DNA. Representative traces depicting the effect of time and the protein kinase C epsilon (PKCε) activator peptide (ΨεRACK, 0.5 μmol/L) on restoration of TRPV1 sensitivity in F-11 cells containing only functional TRPV1 receptors (B and C). Summarized data for Figure2C are depicted in Figure2D. *P < 0.05 compared to capsaicin-treated TRPV1-transfected F-11 cells. n = seven different cover slips containing TRPV1 transfected F-11 cells.
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fig02: (A) Representative trace depicting the effect of a single application of capsaicin (100 nmol/L) and allyl isothiocyanate (AITC, 100 μmol/L) on intracellular free calcium concentration inF-11 cells transfected with only functional transient receptor potential vanilloid receptor type 1 (TRPV1) receptors c-DNA. Representative traces depicting the effect of time and the protein kinase C epsilon (PKCε) activator peptide (ΨεRACK, 0.5 μmol/L) on restoration of TRPV1 sensitivity in F-11 cells containing only functional TRPV1 receptors (B and C). Summarized data for Figure2C are depicted in Figure2D. *P < 0.05 compared to capsaicin-treated TRPV1-transfected F-11 cells. n = seven different cover slips containing TRPV1 transfected F-11 cells.

Mentions: F-11 cells containing only TRPV1 receptors were first identified by treating the cells with single applications of capsaicin (100 nmol/L) and AITC (100 μmol/L). Cells that responded with a transient increase in [Ca2+]i subsequent only to capsaicin were selected for the experiment (Fig.2A). Repetitive stimulation of F-11 cells with capsaicin resulted in a progressive decrease (desensitization) in peak [Ca2+]i that was maintained following a 10-min pause in capsaicin stimulation as indicated by the lack of any response to capsaicin after reapplication of capsaicin to the bath (Fig.2B). In contrast, when the PKCε activator peptide ψεRACK (0.5 μmol/L) was added to the bath during the 10-min pause in stimulation, subsequent reapplication of capsaicin resulted in a robust transient increase in [Ca2+]i (resensitization) (Fig.2C). Summarized data depicting the effect of ψεRACK (0.5 μmol/L) on TRPV1 resensitization are depicted in Figure2D. Summarized results are expressed as a percent of the response to the final application of capsaicin during the initial desensitization of TRPV1.


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 a single application of capsaicin (100 nmol/L) and allyl isothiocyanate (AITC, 100 μmol/L) on intracellular free calcium concentration inF-11 cells transfected with only functional transient receptor potential vanilloid receptor type 1 (TRPV1) receptors c-DNA. Representative traces depicting the effect of time and the protein kinase C epsilon (PKCε) activator peptide (ΨεRACK, 0.5 μmol/L) on restoration of TRPV1 sensitivity in F-11 cells containing only functional TRPV1 receptors (B and C). Summarized data for Figure2C are depicted in Figure2D. *P < 0.05 compared to capsaicin-treated TRPV1-transfected F-11 cells. n = seven different cover slips containing TRPV1 transfected F-11 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: (A) Representative trace depicting the effect of a single application of capsaicin (100 nmol/L) and allyl isothiocyanate (AITC, 100 μmol/L) on intracellular free calcium concentration inF-11 cells transfected with only functional transient receptor potential vanilloid receptor type 1 (TRPV1) receptors c-DNA. Representative traces depicting the effect of time and the protein kinase C epsilon (PKCε) activator peptide (ΨεRACK, 0.5 μmol/L) on restoration of TRPV1 sensitivity in F-11 cells containing only functional TRPV1 receptors (B and C). Summarized data for Figure2C are depicted in Figure2D. *P < 0.05 compared to capsaicin-treated TRPV1-transfected F-11 cells. n = seven different cover slips containing TRPV1 transfected F-11 cells.
Mentions: F-11 cells containing only TRPV1 receptors were first identified by treating the cells with single applications of capsaicin (100 nmol/L) and AITC (100 μmol/L). Cells that responded with a transient increase in [Ca2+]i subsequent only to capsaicin were selected for the experiment (Fig.2A). Repetitive stimulation of F-11 cells with capsaicin resulted in a progressive decrease (desensitization) in peak [Ca2+]i that was maintained following a 10-min pause in capsaicin stimulation as indicated by the lack of any response to capsaicin after reapplication of capsaicin to the bath (Fig.2B). In contrast, when the PKCε activator peptide ψεRACK (0.5 μmol/L) was added to the bath during the 10-min pause in stimulation, subsequent reapplication of capsaicin resulted in a robust transient increase in [Ca2+]i (resensitization) (Fig.2C). Summarized data depicting the effect of ψεRACK (0.5 μmol/L) on TRPV1 resensitization are depicted in Figure2D. Summarized results are expressed as a percent of the response to the final application of capsaicin during the initial desensitization of TRPV1.

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