Nociception, neurogenic inflammation and thermoregulation in TRPV1 knockdown transgenic mice.

Transgenic mice with a small hairpin RNA construct interfering with the expression of transient receptor potential vanilloid 1 (TRPV1) were created by lentiviral transgenesis. TRPV1 expression level in transgenic mice was reduced to 8% while the expression of ankyrin repeat domain 1 (TRPA1) was unchanged. Ear oedema induced by topical application of TRPV1 agonist capsaicin was completely absent in TRPV1 knockdown mice. Thermoregulatory behaviour in relation to environmental thermopreference (30 vs. 35°C) was slightly impaired in male knockdown mice, but the reduction of TRPV1 function was not associated with enhanced hyperthermia. TRPV1 agonist resiniferatoxin induced hypothermia and tail vasodilatation was markedly inhibited in knockdown mice. In conclusion, shRNA-mediated knock down of the TRPV1 receptor in mice induced robust inhibition of the responses to TRPV1 agonists without altering the expression, gating function or neurogenic oedema provoked by TRPA1 activation. Thermoregulatory behaviour in response to heat was inhibited, but enhanced hyperthermia was not observed.

Effect of lipid raft disruption on TRPV1 receptor activation of trigeminal sensory neurons and transfected cell line.

The transient receptor potential vanilloid 1 (TRPV1) is a noxious heat-sensitive, chemonociceptive cation channel which is expressed in primary sensory neurons of polymodal nociceptors. The present study is devoted to analyse the role of lipid raft constituents in calcium influx evoked by various TRPV1 agonists on sensory neurons and on rTRPV1-transfected CHO cell line. Depletion of cholesterol by methyl beta-cyclodextrin (MCD, 1-10mM) diminished the percent of the calcium uptake response of cultured trigeminal neurons to capsaicin (100nM) or resiniferatoxin (RTX, 3nM). In contrast, in TRPV1-transfected cells the inhibition was observed only when capsaicin or N-oleoyldopamine (OLDA, 10microM) was applied, but not when RTX, anandamide (AEA, 10microM) or pH 5.5 was used for gating. The magnitude of Ca(2+)-transients evoked by capsaicin (330nM) was also inhibited in both cell types. Treatment of rTRPV1-expressing cells with sphinomyelinase inhibited the capsaicin-evoked (45)Ca-uptake leaving the RTX-induced response unchanged. On the other hand, in trigeminal neurons the effect of both compounds was inhibited by sphingomyelinase treatment. Inhibition of ganglioside biosynthesis by d-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP, 10-20microM) or myriocyn (5-50nM) diminished similarly capsaicin- or RTX-evoked calcium uptake in both cultured trigeminal neurons and rTRPV1-expressing cells. The present study revealed that depletion of different constituents of lipid raft inhibited gating the TRPV1 cation channel by various vanilloid and non-vanilloid agents. Evidence for a supporting role of cholesterol, sphingomyelin and gangliosides were obtained both in native and TRPV1-transfected cells. Differential modulation of responses to capsaicin and RTX was often observed.