Time course and lag time of responses to capsaicin (CAPS), NKs [substance P (SP), [βAla⁸]-neurokinin A (4–10) (NKA), [MePhe⁷]-neurokinin B (NKB)] and phorbol 12,13-dibutyrate (PDBu) in DRG neurons from adult male rats. (A) Time course of response evoked by CAPS alone (0.5 μm) and block of response by a selective transient receptor potential vanilloid receptor 1 (TRPV1) antagonist (TRPV1-Ant, 1 μm). (B) Initial rise of CAPS response in (A) on an expanded scale showing the lag time of response from the time of application (solid horizontal line) to the start of the rising phase (dotted vertical line). (C) SP (0.5 μm) applied after block of CAPS response by the TRPV1 antagonist triggers a rapid onset, rapid desensitizing response. (D) NKA (0.5 μm) applied before CAPS also triggers a rapid onset, rapidly desensitizing inward current. The lag time between NKA application and the beginning of the rising phase was similar to that of CAPS responses (5–7 s). (E) The PKC activator PDBu (0.5 μm) did not elicit a current when administered alone. Calibration bars are shown next to currents. (F) Summary of lag times of response to CAPS, SP, NKA and NKB alone or combinations of other agents. For comparison, the lag time of response to adenosine-5′-O-(3-thiotriphosphate) (ATPγS, 50 μm)-activated P₂X currents is also shown. Comparison was considered statistically significant if P < 0.05; a one-way analysis of variance was first carried out followed by a post hoc comparison between the different groups using the Holm–Sidak test, as described in Materials and methods (**P < 0.001). (G) Inward currents in response to NKs SP, NKA and NKB. DEC, decamethrin.

Effect of a PKC inhibitor on NKs [substance P (SP), [βAla⁸]-neurokinin A (4–10) (NKA)] and a phorbol ester (phorbol 12,13-dibutyrate, PDBu) induced slowing of capsaicin (CAPS) responses in DRG neurons. (A) CAPS (0.5 μm) applied in the continuous presence of NKA (0.5 μm) followed by administration of a PKC inhibitor, bisindolylmaleimide (BIM, 0.5 μm). (B) Time course of current induced by CAPS alone (0.5 μm) and PDBu (0.5 μm) applied in the continuous presence of CAPS, after partial desensitization of the CAPS response. Dotted lines in (A) and (B) are the fit of responses to CAPS alone in 20 neurons. (C) Average traces of CAPS (0.5 μm)-evoked current densities (n = 3 neurons) in which CAPS was applied at 2–3 min after application of BIM (0.5 μm) and fitted curve of average CAPS current densities in 20 neurons in response to CAPS alone. Calibration bars for A, B and C are shown. (D) Effect of BIM on currents evoked by CAPS, PDBu, NKA and SP. Average decay time constant (desensitization of the initial rapid phase fitted by a single exponential) of CAPS (0.5 μm) alone, and currents elicited by PDBu (0.5 μm), NKA (0.5 μm) and SP (0.5 μm) in the presence of CAPS (0.5 μm). The number of experiments is shown above the graph. Bar graph data are averages of pooled data, standard error of the mean (SEM) and statistical difference from control currents (two-tailed t-test, unequal variance, **P < 0.01, NS, not significant). Comparisons in (D) were considered statistically significant if P < 0.05; a one-way analysis of variance was first carried out followed by a post hoc comparison between the different groups using the Holm–Sidak test.

Effect of NK₁ ([Sar⁹,Met¹¹]-substance P, SarMetSP) and NK₃ ([MePhe7]-neurokinin B, NKB) receptor agonists and phosphatase inhibitors [α-naphthyl acid phosphate (NAcPh), decamethrin (DEC)] on capsaicin (CAPS) responses in DRG neurons from adult male rats. (A) Time course of the response to CAPS (0.5 μm) and DEC (0.5 μm) applied in sequence. Phorbol 12,13-dibutyrate (PDBu; 0.5 μm) was applied at > 20 min after application of CAPS and DEC as shown by bars above the graph. A selective transient receptor potential vanilloid receptor 1 (TRPV1) antagonist (TRPV1-Ant, 0.5 μm) fully inhibited the current. The dotted line is the fit of average responses to CAPS alone in 20 neurons. (B) Prior application of phosphatase inhibitors NAcPh (0.5 μm) or DEC (0.5 μm) slowed the decline in the response to PDBu (n =4) or [βAla⁸]-neurokinin A (4–10) (NKA; n = 3) or substance P (SP; n = 5) that occurred during the desensitization of CAPS currents (continuous line). Continuous and dotted lines are fits shown in Fig. 4B. (C) Average time to peak (rise time) and decay time constant (initial rapid phase of desensitization fitted by a single exponential) after CAPS (0.5 μm) and SarMetSP (0.5 μm), NKB (0.5 μm), NAcPh (0.5 μm) and DEC (0.5 μm) in the presence of CAPS. (D) Peak current densities after CAPS (0.5 μm) and SarMetSP (0.5 μm), NKB (0.5 μm) NAcPh (0.5 μm) and DEC (0.5 μm) in the presence of CAPS. The number of experiments is shown as insets. Bar graph data are averages of pooled data, standard error of the mean (SEM) and statistical difference from control currents (two tailed t-test, unequal variance, *P < 0.05, **P < 0.01, NS, not significant). Comparisons in (C) and (D) were considered statistically significant if P < 0.05 (*); a one-way analysis of variance was first carried out, followed by a post hoc comparison between the different groups using the Holm–Sidak test.

Effect of NKs [substance P (SP), [βAla⁸]-neurokinin A (4–10) (NKA)] and a phorbol ester (phorbol 12,13-dibutyrate, PDBu) on capsaicin (CAPS) responses in DRG neurons from adult male rats. (A) Time course of the current induced by exposure to CAPS (0.5 μm), followed by SP (0.5 μm) and an NK₂-selective antagonist, MEN 10,376 (5 μm, continuous line). (B) Time course of the CAPS current induced by prolonged exposure to CAPS alone (0.5 μm, broken line) and later application of NKA (0.5 μm) followed by PDBu (0.5 μm) and a selective transient receptor potential vanilloid receptor 1 (TRPV1) antagonist (TRPV1-Ant, 1 μm). (C) Response to NKA alone (0.5 μm) and CAPS (0.5 μm) applied after full desensitization of NKA response. A selective TRPV1 antagonist (TRPV1-Ant, 1 μm) applied later fully inhibited the current. (D) Time course of the CAPS current induced by prolonged exposure to CAPS alone (0.5 μm) and later application of SP (0.5 μm), the NK₃ antagonist SB 235,375 (5 μm) and MEN 10,376 (5 μm, continuous line). Drugs were applied as shown by thick bars above current traces. For comparison, the broken line in (A–D), the average fit of responses to CAPS alone in 20 neurons, is also shown. (E, top) Average time from drug application to maximum response (time to peak) and time constant of desensitization (fitted by a single exponential) after CAPS alone and after application of PDBu (0.5 μm), NKA (0.5 μm) and SP (0.5 μm) in the presence of CAPS and SP after CAPS in the presence of NK₂ antagonist MEN 10,376 (MEN, 0.2 m). (E, bottom) Peak currents evoked by various agents in the presence of CAPS normalized to fitted amplitudes for responses to CAPS alone at the time the effect of various agents reached a maximum. The number of experiments is shown as insets. Bar graph data are averages of pooled data, standard error of the mean (SEM) and statistical difference from control currents (two tailed t-test, unequal variance, *P < 0.05, **P < 0.01, NS, not significant). Comparisons in (E) were considered statistically significant if P < 0.05 (*); a one-way analysis of variance was first carried out, followed by post hoc comparisons between the different groups using the Holm–Sidak test. NAcPh, α-naphthyl acid phosphate.

The enhancement of capsaicin (CAPS) currents by agents that promote phosphorylation or inhibit dephosphorylation. The peak magnitude of the currents declines with time in the continuous presence of CAPS. (A) Average response to CAPS alone (X, n = 20 cells) normalized to maximum amplitude and after applications of agents that are presumed to promote PKC-mediated TRPV1 phosphorylation (filled symbols) or to inhibit dephosphorylation (empty symbols). The response to CAPS alone was fitted by a sum of one exponential rising and two exponential decaying curves (dotted line, equation shown above graph, also see Materials and methods). (B) The increase of CAPS currents in response to agents that facilitated the currents. The extent of facilitation decayed exponentially as the CAPS responses desensitize. Data in (A) for effects of various agents that either promoted phosphorylation (filled circles) or inhibited dephosphorylation (empty circles) were grouped in early, intermediary and late time segments and averaged as shown. DEC, decamethrin; NAcPh, α-naphthyl acid phosphate; NKA, [βAla⁸]-neurokinin A (4–10); PDBu, phorbol 12,13-dibutyrate; SP, substance P.

Effect of substance P (SP) on tachyphylaxis of capsaicin (CAPS)-induced increases in intracellular Ca²⁺ in cultured DRG neurons. (A) Example from a DRG neuron responding with increases in intracellular Ca²⁺ to six consecutive applications of CAPS (0.2 μm, 3–5-s duration; arrows) every 6–8 min. (B, C) Examples from two DRG neurons responding with increases in intracellular Ca²⁺ to six consecutive applications of CAPS (0.2 μm, 3–5-s duration), every 6–8 min before (first three applications) and after (last three application) SP treatment (0.2 μm, 2 min). (D, F) Summary of the area under the curve (D), response amplitude (F) from cells responding to six consecutive CAPS stimuli (S1–S6; 0.2 μm, 3–5-s duration; n = 12 cells) in control conditions. (E, G) Summary of the area under the curve (E), response amplitude (G) from cells responding to six consecutive CAPS stimuli (0.2 μm, 3–5-s duration; n = 15 cells) in control conditions S1–S3 and after SP (0.2 μm, 2 min) treatment S4–S6. Statistical significance was tested using anova followed by Tukey–Kramer post hoc test. Asterisks indicate statistical significance (*P < 0.05) in between S1 and S2, S3, S4, S5, S6. S indicates statistical significance (P < 0.05) in between S3 and S4; s indicates statistical significance (P < 0.05) between S1 and S4; ns, NS indicate no statistical significant differences (P > 0.05).