Figure 5.

GsMTx-4 also markedly reduces nociceptive flinching in response to hypotonic stimulation. A, Rats treated with PGE₂ and 5-HT, carrageenan, or paclitaxel have, respectively, a 4-, 3.6-, and 3.2-fold increase in the number of nociceptive flinches in response to an intradermal injection of hypotonic solution (Hypo) (10 μl of deionized water, 17 mOsm) compared with control rats. GsMTx-4 markedly reduced the number of flinches in PGE₂ and 5-HT-treated (16 ± 2, n = 12 before and 8 ± 1, n = 6 after GsMTx-4), carrageenan-treated (17 ± 1, n = 12 before and 6 ± 1, n = 8 after GsMTx-4), and paclitaxel-treated (13 ± 2, n = 10 before and 7 ± 1, n = 6 after GsMTx-4) rats. All asterisks, p < 0.05, Tukey's post hoc multiple comparison test. Twenty-four hours after the administration of GsMTx-4, its effect was no longer present, and there was no significant difference in the number of flinches between the different groups of rats (p > 0.05, Tukey's multiple comparison test). B, Small-diameter (≤25 μm) DRG neurons from TRPV4^+/+ and TRPV4^−/− mice were first challenged with a 30% hypotonic solution containing PGE₂ and 5-HT (10 μm each) for 3 min and then challenged with a 30% hypotonic solution containing PGE₂, 5-HT, and GsMTx-4 (500 nm) for 3 min. The increase in [Ca²⁺]_i induced by the hypotonic solution containing PGE₂ and 5-HT is reduced in the presence of GsMTx-4 in TRPV4^+/+ mice (2.4 ± 0.2 μm before and 1.7 ± 0.2 μm after GsMTx-4; n = 24; p = 0.007, paired Student's t test) as well as in TRPV4^−/− mice (1.7 ± 0.1 μm before and 1.4 ± 0.1 μm after GsMTx-4; n = 14; p = 0.03, paired Student's t test). C, Dissociated lumbar DRG neurons were cultured for 2 d. Cells were scraped in homogenization buffer, and subcellular fractionation was performed. The membrane fraction was separated on an electrophoresis gel and transferred to a polyvinylidene difluoride membrane. The membrane was then probed in parallel with anti-TRPV4 (1:500), anti-TRPC1 (1:500), and anti-TRPC6 (1:500) antibodies. The expected bands at 120 kDa for TRPC1, 110 kDa for TRPC6, and the doublet bands at 98 and 107 kDa for TRPV4 were detected. D, The cytoplasm of 47 DRG neurons was harvested after 2 d in culture, and multiplex single-cell RT-PCR was performed. Neurons expressed from none to all three of the mRNAs of interest; here we show the example of neurons expressing mRNA for TRPC1 (168 bp) or TRPC1 and TRPC6 (228 bp) or TRPC1, TRPC6, and TRPV4 (221 bp).