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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 the specific transient receptor potential vanilloid subtype-1 (TRPV1) agonist capsaicin (Cap; 100 nmol/L) and the specific transient receptor potential ankyrin receptor subtype-1 (TRPA1) activator allyl isothiocyanate (AITC, 100 μmol/L) on intracellular free calcium concentration in F11 cells transfected with both TRPA1 and TRPV1 complementary DNA (cDNA). Representative traces depicting the effect of AITC (100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, AITC in the presence of specific PKCε inhibitor εV1–2 (0.5 μmol/L), propofol in the presence of specific PKCε inhibitor εV1–2 after capsaicin-induced (100 nmol/L) desensitization on restoration of TRPV1 sensitivity in F-11 cells transfected with both TRPA1 and TRPV1 cDNA are depicted in Figure1B, C, D, and E, respectively. The εV1–2 inhibitor peptide was added alone for 5 min following desensitization and then AITC or propofol was brought on board in combination with the εV1–2 inhibitor peptide for an additional 5 min. Summarized data for Figure1B–E are depicted in Figure1F. Data are expressed as a percent of the response to the final application of capsaicin in the untreated control (% of control mean value ± SEM). *P < 0.05 compared to final capsaicin in the untreated control. #P < 0.05 compared to AITC plus capsaicin. †P < 0.05 compared to propofol plus capsaicin. n = seven separate coverslips of F-11 cells were used.
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fig01: (A) Representative trace depicting the effect of a single application of the specific transient receptor potential vanilloid subtype-1 (TRPV1) agonist capsaicin (Cap; 100 nmol/L) and the specific transient receptor potential ankyrin receptor subtype-1 (TRPA1) activator allyl isothiocyanate (AITC, 100 μmol/L) on intracellular free calcium concentration in F11 cells transfected with both TRPA1 and TRPV1 complementary DNA (cDNA). Representative traces depicting the effect of AITC (100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, AITC in the presence of specific PKCε inhibitor εV1–2 (0.5 μmol/L), propofol in the presence of specific PKCε inhibitor εV1–2 after capsaicin-induced (100 nmol/L) desensitization on restoration of TRPV1 sensitivity in F-11 cells transfected with both TRPA1 and TRPV1 cDNA are depicted in Figure1B, C, D, and E, respectively. The εV1–2 inhibitor peptide was added alone for 5 min following desensitization and then AITC or propofol was brought on board in combination with the εV1–2 inhibitor peptide for an additional 5 min. Summarized data for Figure1B–E are depicted in Figure1F. Data are expressed as a percent of the response to the final application of capsaicin in the untreated control (% of control mean value ± SEM). *P < 0.05 compared to final capsaicin in the untreated control. #P < 0.05 compared to AITC plus capsaicin. †P < 0.05 compared to propofol plus capsaicin. n = seven separate coverslips of F-11 cells were used.

Mentions: Individual F-11 cells containing both functional TRPV1 and TRPA1 receptors were first identified by treating the cells with single applications of capsaicin (100 nmol/L) and AITC (100 μmol/L) a (Fig.1A). Repetitive stimulation of F-11 cells with capsaicin resulted in a progressive decrease (de-sensitization) in peak [Ca2+]i (Fig.1B). When AITC (100 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.1B). A similar resensitizing effect was observed with propofol (Fig.1C). Pretreatment with the PKCε-specific inhibitory peptide εV1–2 (0.5 μmol/L) following desensitization inhibited the AITC- and propofol-induced restoration of TRPV1 sensitivity to capsaicin compared with F-11 cells treated only with propofol or AITC (Fig.1D and E). Summarized data depicting the effect of AITC and propofol alone or in the presence of εV1–2 on restoration of TRPV1 sensitivity to capsaicin in TRPV1-TRPA1 cotransfected F-11 cells are depicted in Figure1F. Summarized results are expressed as a percent of the response to the final application of capsaicin in untreated cells (control).


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 the specific transient receptor potential vanilloid subtype-1 (TRPV1) agonist capsaicin (Cap; 100 nmol/L) and the specific transient receptor potential ankyrin receptor subtype-1 (TRPA1) activator allyl isothiocyanate (AITC, 100 μmol/L) on intracellular free calcium concentration in F11 cells transfected with both TRPA1 and TRPV1 complementary DNA (cDNA). Representative traces depicting the effect of AITC (100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, AITC in the presence of specific PKCε inhibitor εV1–2 (0.5 μmol/L), propofol in the presence of specific PKCε inhibitor εV1–2 after capsaicin-induced (100 nmol/L) desensitization on restoration of TRPV1 sensitivity in F-11 cells transfected with both TRPA1 and TRPV1 cDNA are depicted in Figure1B, C, D, and E, respectively. The εV1–2 inhibitor peptide was added alone for 5 min following desensitization and then AITC or propofol was brought on board in combination with the εV1–2 inhibitor peptide for an additional 5 min. Summarized data for Figure1B–E are depicted in Figure1F. Data are expressed as a percent of the response to the final application of capsaicin in the untreated control (% of control mean value ± SEM). *P < 0.05 compared to final capsaicin in the untreated control. #P < 0.05 compared to AITC plus capsaicin. †P < 0.05 compared to propofol plus capsaicin. n = seven separate coverslips of F-11 cells were used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: (A) Representative trace depicting the effect of a single application of the specific transient receptor potential vanilloid subtype-1 (TRPV1) agonist capsaicin (Cap; 100 nmol/L) and the specific transient receptor potential ankyrin receptor subtype-1 (TRPA1) activator allyl isothiocyanate (AITC, 100 μmol/L) on intracellular free calcium concentration in F11 cells transfected with both TRPA1 and TRPV1 complementary DNA (cDNA). Representative traces depicting the effect of AITC (100 μmol/L) alone, propofol (Prop, 10 μmol/L) alone, AITC in the presence of specific PKCε inhibitor εV1–2 (0.5 μmol/L), propofol in the presence of specific PKCε inhibitor εV1–2 after capsaicin-induced (100 nmol/L) desensitization on restoration of TRPV1 sensitivity in F-11 cells transfected with both TRPA1 and TRPV1 cDNA are depicted in Figure1B, C, D, and E, respectively. The εV1–2 inhibitor peptide was added alone for 5 min following desensitization and then AITC or propofol was brought on board in combination with the εV1–2 inhibitor peptide for an additional 5 min. Summarized data for Figure1B–E are depicted in Figure1F. Data are expressed as a percent of the response to the final application of capsaicin in the untreated control (% of control mean value ± SEM). *P < 0.05 compared to final capsaicin in the untreated control. #P < 0.05 compared to AITC plus capsaicin. †P < 0.05 compared to propofol plus capsaicin. n = seven separate coverslips of F-11 cells were used.
Mentions: Individual F-11 cells containing both functional TRPV1 and TRPA1 receptors were first identified by treating the cells with single applications of capsaicin (100 nmol/L) and AITC (100 μmol/L) a (Fig.1A). Repetitive stimulation of F-11 cells with capsaicin resulted in a progressive decrease (de-sensitization) in peak [Ca2+]i (Fig.1B). When AITC (100 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.1B). A similar resensitizing effect was observed with propofol (Fig.1C). Pretreatment with the PKCε-specific inhibitory peptide εV1–2 (0.5 μmol/L) following desensitization inhibited the AITC- and propofol-induced restoration of TRPV1 sensitivity to capsaicin compared with F-11 cells treated only with propofol or AITC (Fig.1D and E). Summarized data depicting the effect of AITC and propofol alone or in the presence of εV1–2 on restoration of TRPV1 sensitivity to capsaicin in TRPV1-TRPA1 cotransfected F-11 cells are depicted in Figure1F. Summarized results are expressed as a percent of the response to the final application of capsaicin in untreated cells (control).

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