Propofol restores TRPV 1 sensitivity via a TRPA 1‐, nitric oxide synthase‐dependent activation of PKC ε

We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype‐1 ( TRPV 1) receptors via a protein kinase C epsilon ( PKC ε )‐dependent and transient receptor potential ankyrin channel subtype‐1 ( TRPA 1)‐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 TRPA 1 activation directly results in PKC ε activation and to elucidate the cellular mechanism by which this occurs. F‐11 cells were transfected with complimentary DNA ( cDNA ) for TRPV 1 only or both TRPV 1 and TRPA 1. Intracellular Ca 2+ concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKC ε , nitric oxide synthase ( nNOS ), and TRPV 1 was also performed. In F‐11 cells containing both channels, PKC ε inhibition prevented the propofol‐ and allyl isothiocyanate ( AITC )‐induced restoration of TRPV 1 sensitivity to agonist stimulation as well as increased phosphorylation of PKC ε and TRPV 1. In cells containing TRPV 1 only, neither agonist induced PKC ε or TRPV 1 phosphorylation. Moreover, NOS inhibition blocked propofol‐and AITC ‐induced restoration of TRPV 1 sensitivity and PKC ε phosphorylation, and PKC ε inhibition prevented the nitric oxide donor, SNAP , from restoring TRPV 1 sensitivity. Also, propofol‐and AITC ‐induced phosphorylation of nNOS and nitric oxide ( NO ) production were blocked with the TRPA 1‐antagonist, HC ‐030031. These data indicate that the AITC ‐ and propofol‐induced restoration of TRPV 1 sensitivity is mediated by a TRPA 1‐dependent, nitric oxide synthase‐dependent activation of PKC ε .