Inhibition and potentiation of the exercise pressor reflex by pharmacological modulation of TRPC6 in male rats

Abstract

We determined the role played by the transient receptor potential canonical 6 (TRPC6) channel in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague-Dawley rats. TRPC6 channels were identified by quadruple-labelled (DiI, TRPC6, neurofilament-200 and peripherin) immunohistochemistry in dorsal root ganglion (DRG) cells innervating the triceps surae muscles (n = 12). The exercise pressor reflex was evoked by statically contracting the triceps surae muscles before and after injection of the TRPC6 antagonist BI-749327 (n = 11; 12 μg kg^-1 ) or SAR7334 (n = 11; 7 μg kg^-1 ) or the TRPC6 positive modulator C20 (n = 11; 18 μg kg^-1 ). Similar experiments were conducted while the muscles were passively stretched (n = 8-12), a manoeuvre that isolated the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded. Of the DRG cells innervating the triceps surae muscles, 85% stained positive for the TRPC6 antigen, and 45% of those cells co-expressed neurofilament-200. Both TRPC6 antagonists decreased the reflex pressor responses to static contraction (-32 to -42%; P < 0.05) and to passive stretch (-35 to -52%; P < 0.05), whereas C20 increased these responses (55-65%; P < 0.05). In addition, BI-749327 decreased the peak and integrated RSNA responses to both static contraction (-39 to -43%; P < 0.05) and passive stretch (-56 to -62%; P < 0.05), whereas C20 increased the RSNA to passive stretch only. The onset latency of the decrease or increase in RSNA occurred within 2 s of the onset of the manoeuvres (P < 0.05). Collectively, our results show that TRPC6 plays a key role in evoking the mechanical component of the exercise pressor reflex. KEY POINTS: The exercise pressor reflex plays a key role in the sympathetic and haemodynamic responses to exercise. This reflex is composed of two components, namely the mechanoreflex and the metaboreflex. The receptors responsible for evoking the mechanoreflex are poorly documented. A good candidate for this function is the transient receptor potential canonical 6 (TRPC6) channel, which is activated by mechanical stimuli and expressed in dorsal root ganglia of rats. Using two TRPC6 antagonists and one positive modulator, we investigated the role played by TRPC6 in evoking the mechanoreflex in decerebrated rats. Blocking TRPC6 decreased the renal sympathetic and the pressor responses to both contraction and stretch, the latter being a manoeuvre that isolates the mechanoreflex. In contrast, the positive modulator increased the pressor reflex to contraction and stretch, in addition to the sympathetic response to stretch. Our results provide strong support for a role played by the TRPC6 channel in evoking the mechanoreflex.

Keywords: exercise pressor reflex; immunohistochemistry; mechanoreflex; neural control of autonomic circulation; sympathetic nervous system; transient receptor potential canonical 6.

Figure 1.. Co-localization of TRPC6 and Nf200 in L4-L5 dorsal root ganglion cells innervating the triceps surae muscles.

A, Representative images of the quadruple-labelled immunohistochemistry staining of the drosal root ganlion cells. The neurons were stained with the retrolabelled DiI, and TRPC6, Nf200 and peripherin antigen. The merged image shows that the majority of cells that were positive to TRPC6 were also positive to Nf200. These cells had a bigger diameter compared to the cells that wre positive to peripherin. Three representative cells that are positive for DiI, TRPC6 and Nf200 are indicated by a big white arrowhead. A neuron positive for DiI, TRPC6 and peripherin is indicated by a small white arrow. A neuron positive for DiI, TRPC6, Nf200 and peripherin is indicated by a small white arrowhead. B, percent of DiI-labelled cells that co-localized TRPC6 and percent of TRPC6 and DiI positive cells that co-localized Nf200 and/or peripherin. Data are plotted as group mean (open bars) and individual data (open circles). A total of 12 dorsal root ganglia were collected from four animals (3 sections per animals).

Figure 2. Dose response effect of each drug on the peak and integrated pressor response to passive stretch

Data are presented as mean ± SEM for clarity. Passive stretches were evoked before and after (10 min) injecting the TRPC6 blockers BI-749327 (n = 7) or SAR7334 (n = 9) or after (4 min) injecting the TRPC6 postive modulator C20 (n = 8). Control experiment (Con) were performed by two passive stretches without injecting the active compounds. Note that the x-axis follows a logarithmic scale. BPi, Blood pressure index calculated as the integrated blood pressor response to stretch. *, P < 0.05 vs control; **, P< 0.01; ***, P < 0.001.

Figure 3.. Effect of TRPC6 blockade with BI-749327 (12μg.kg⁻¹) on the pressor, tension, renal sympathetic, blood flow, and vascular conductance responses to static contraction of the triceps surae muscles.

Data are presented as the mean ± SD changes over time in blood pressure, tension, RSNA, blood flow and vascular conductance induced by static contraction. The averaged time course includes 2 s of baseline, 30 s of contraction, and 2 s after the end of contraction. Contractions were evoked before, 10min, and 20min following the blockade. P10, post-10 min; P20, post-20 min; RSNA, renal sympathetic nerve activity; MAP, mean arterial pressure. †, P < 0.05 between pre blockade and post-10min blockade; ‡, P < 0.05 between pre blockade and post-20 min blockade.

Fig. 4.. Effect of TRPC6 blockade with BI-749327 (12μg.kg⁻¹) on the peak and integrated pressor, and renal sympathetic nerve activity (RSNA) responses to static contraction of the triceps surae muscles.

Data are presented as individual (open dots) and group means (open bars) for the peak and integrated changes evoked by static contraction. Contractions were evoked before, 10min, and 20min following the blockade. P10, post-10 minutes; P20, post-20 minutes; BPi, blood pressure index calculated as the integrated blood pressor response to contraction; RSNAi, renal sympathetic nerve activity integrated; MAP, mean arterial pressure. *, P < 0.05 between the corresponding data points; ***, P < 0.001;

Fig. 5.

Representative traces of the first s of renal sympathetic nerve activity (RSNA) responses to static contraction and stretch before and after TRPC6 blockade.

Fig. 6.. Effect of TRPC6 blockade with SAR7334 (7μg.kg⁻¹) on the pressor, tension, blood flow and vascular conductance responses to static contraction of the triceps surae muscles.

Data are presented as the mean ± SD changes over time in blood pressure, tension, blood flow and vascular conductance induced by static contraction (upper panels) or as individual (open dots) and group means (open bars) for the peak and integrated changes in blood pressure evoked by static contraction (lower panels). The averaged time courses include 2 s of baseline, 30 s of contraction, and 2 s after the end of contraction. Contractions were evoked before, 10min, and 20min following the blockade. Renal sympathetic nerve activity was not measured during these experiments. BPi, blood pressure index calculated the integrated blood pressor response to contraction; P10, post-10 min; P20, post-20 min; MAP, mean arterial pressure. †, P < 0.05 between pre blockade and post-10min blockade; ‡, P < 0.05 between pre blockade and post-20 min blockade; **, P < 0.01 between the corresponding data points; ***, P < 0.001

Fig. 7.. Effect of TRPC6 sensitization with C20 (18μg.kg⁻¹) and subsequent TRPC6 blockade with BI-749327 (12μg.kg⁻¹) on the pressor, tension, renal sympathetic, blood flow, and vascular conductance responses to static contraction of the triceps surae muscles.

Data are presented as the mean ± SD changes over time in blood pressure, tension, RSNA, blood flow, and vascular conductance induced by static contraction. The averaged time courses include 2 s of baseline, 30 s of contraction, and 2 s after the end of contraction. Contractions were evoked before, 4 min after the sensitization, and 10 min after the blockade. P4, post-4 min; P10, post-10 min; RSNA, renal sympathetic nerve activity; MAP, mean arterial pressure. †, P < 0.05 between baseline and post-4min sensitization; ‡, P < 0.05 between post-4min sensitization and post-10 min blockade; $, P < 0.05 between baseline and post-10 min blockade.

Fig. 8.. Effect of TRPC6 sensitization with C20 (18μg.kg⁻¹) and subsequent TRPC6 blockade with BI-749327 (12μg.kg⁻¹) on the peak and integrated pressor, and renal sympathetic nerve activity responses to static contraction of the triceps surae muscles.

Data are presented as individual (open dots) and group means (open bars) for the peak and integrated changes evoked by static contraction. Contractions were evoked before, 4min after the sensitization, and 10min after the blockade. P4, post-4 minutes; P10, post-10 minutes; BPi, blood pressure index calculated the integrated blood pressor response to contraction; RSNAi, renal sympathetic nerve activity integrated; MAP, mean arterial pressure. *, P < 0.05 between the corresponding data points;

Figure 9.. Effect of TRPC6 blockade with BI-749327 (12μg.kg⁻¹) on the pressor, tension, renal sympathetic, blood flow, and vascular conductance responses to passive stretch of the triceps surae muscles.

Data are presented as the mean ± SD changes over time in blood pressure, tension, RSNA, blood flow and vascular conductance induced by passive stretch. The averaged time courses include 2 s of baseline, 30 s of stretch, and 2 s after the end of the stretch. Stretches were evoked before, 10min, and 20min following the blockade. P10, post-10 min; P20, post-20 min; RSNA, renal sympathetic nerve activity; MAP, mean arterial pressure. †, P < 0.05 between pre blockade and post-10min blockade; ‡, P < 0.05 between pre blockade and post-20 min blockade.

Fig. 10.. Effect of TRPC6 blockade with BI-749327 (12μg.kg⁻¹) on the peak and integrated pressor, and renal sympathetic nerve activity (RSNA) responses to pasive stretch of the triceps surae muscles.

Data are presented as individual (open dots) and group means (open bars) for the peak and integrated changes evoked by passive stretch. Stretches were evoked before, 10min, and 20min following the blockade. P10, post-10 minutes; P20, post-20 minutes; BPi, blood pressure index calculated as the integrated blood pressor response to stretch; RSNAi, renal sympathetic nerve activity integrated; MAP, mean arterial pressure. *, P < 0.05 between the corresponding data points; **, P < 0.01;

Fig. 11.. Effect of TRPC6 blockade with SAR7334 (7μg.kg⁻¹) on the pressor, tension, blood flow and vascular conductance responses to passive stretch of the triceps surae muscles.

Data are presented as the mean ± SD changes over time in blood pressure, tension, blood flow and vascular conductance induced by passive stretch (upper panels) or as individual (open dots) and group means (open bars) for the peak and integrated changes in blood pressure evoked by passive stretch (lower panels). The averaged time courses include 2 s of baseline, 30 s of stretch, and 2 s after the end of stretch. Stretches were evoked before, 10min, and 20min following the blockade. Renal sympathetic nerve activity was not measured during these experiments. BPi, blood pressure index calculated the integrated blood pressor response to contraction; P10, post-10 min; P20, post-20 min; MAP, mean arterial pressure. †, P < 0.05 between pre blockade and post-10min blockade; ‡, P < 0.05 between pre blockade and post-20 min blockade; *, P < 0.05 between the corresponding data points;

Fig. 12.. Effect of TRPC6 sensitization with C20 (18μg.kg⁻¹) and subsequent TRPC6 blockade with BI-749327 (12μg.kg⁻¹) on the pressor, tension, renal sympathetic, blood flow, and vascular conductance responses to passive stretch of the triceps surae muscles.

Data are presented as the mean ± SD changes over time in blood pressure, tension, RSNA, blood flow, and vascular conductance induced by passive stretch. The averaged time courses include 2 s of baseline, 30 s of stretch, and 2 s after the end of stretch. Stretches were evoked before, 4 min after the sensitization, and 10 min after the blockade. P4, post-4 min; P10, post-10 min; RSNA, renal sympathetic nerve activity; MAP, mean arterial pressure. †, P < 0.05 between baseline and post-4min sensitization; ‡, P < 0.05 between post-4min sensitization and post-10 min blockade.

Fig. 13.. Effect of TRPC6 sensitization with C20 (18μg.kg⁻¹) and subsequent TRPC6 blockade with BI-749327 (12μg.kg⁻¹) on the peak and integrated pressor, and renal sympathetic nerve activity responses to passive stretch of the triceps surae muscles.

Data are presented as individual (open dots) and group means (open bars) for the peak and integrated changes evoked by passive stretch. Stretches were evoked before, 4min after the sensitization, and 10min after the blockade. P4, post-4 minutes; P10, post-10 minutes; BPi, blood pressure index calculated as the integrated blood pressor response to stretch; RSNAi, renal sympathetic nerve activity integrated; MAP, mean arterial pressure. *, P < 0.05 between the corresponding data points;

Fig. 14.. Effect of intravenous injection of C20 (18μg.kg⁻¹) on the pressor, and renal sympathetic responses to static contraction or passive stretch of the triceps surae muscles.

Data are presented as the mean ± SD changes over time in blood pressure, and RSNA induced by static contraction (left panel) and passive stretch (right panel). The averaged time courses include 2 s of baseline, 30 s of contraction, and 2 s after the end of contraction. The maneuvers were evoked before, 4min, and 14min after the i.v. injection. i.v. intravenous; P4, post-4 min; P14, post-14 min; MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity . †, P < 0.05 between baseline and post-4min i.v. injection; ‡, P < 0.05 between baseline and post-14 min i.v. injection;

Figure 15.. Effect of TRPC6 antagonists or positive modulator on the pressor response to capsaicin and lactic acid.

Data are presented as individual (open dots) and group means (open bars) for the peak pressor response to capsaicin (upper panels) and lactic acid (lower panels) before and after injecting the TRPC6 drugs. Capsaicin, lactic acid, the TRPC6 blockers BI-749327 (n = 9 for capsaicin; n = 7 for lactic acid) and SAR7334 (n = 8 for capsaicin and lactic acid), and the TRPC6 postive modulator C20 (n = 9 for capsaicin; n = 7 for lactic acid) for injected into the superficial epigastric artery. Mean ± SD for baseline blood pressure is presented below each open bar. MAP, mean arterial pressure