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Carvacrol inhibits the neuronal voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.3, Nav1.7, and Nav1.8 expressed in Xenopus oocytes with different potencies.
Horishita T
,
Ogata Y
,
Horishita R
,
Fukui R
,
Moriwaki K
,
Ueno S
,
Yanagihara N
,
Uezono Y
,
Sudo Y
,
Minami K
.
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Carvacrol is the predominant monoterpene in essential oils from many aromatic plants. Several animal studies showing analgesic effects of carvacrol indicate potential of carvacrol as a new medication for patients with refractory pain. Voltage-gated sodium channels (Nav) are thought to have crucial roles in the development of inflammatory and neuropathic pain, but there is limited information about whether the analgesic mechanism of carvacrol involves Nav. We used whole-cell, two-electrode, voltage-clamp techniques to examine the effects of carvacrol on sodium currents in Xenopus oocytes expressing α subunits of Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8. Carvacrol dose-dependently suppressed sodium currents at a holding potential that induced half-maximal current. The half-maximal inhibitory concentration values for Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 were 233, 526, 215, 367, and 824 μmol/L, respectively, indicating that carvacrol had more potent inhibitory effects towards Nav1.2 and Nav1.6 than Nav1.3, Nav1.7, and Nav1.8. Gating analysis showed a depolarizing shift of the activation curve and a hyperpolarizing shift of the inactivation curve in all five α subunits following carvacrol treatment. Furthermore, carvacrol exhibits a use-dependent block for all five α Nav subunits. These findings provide a better understanding of the mechanisms associated with the analgesic effect of carvacrol.
Fig. 1. (A) Effects of carvacrol on peak sodium inward currents in Xenopus oocytes expressing Nav1.2, Nav1.3, Nav1.6, Nav1.7 and Nav1.8 α subunits with β1 subunit at two holding potentials. Representative traces are shown. Sodium currents were evoked by 50 ms depolarizing pulses to −20 mV for Nav1.2 and Nav1.6, –10 mV for Nav1.3 and Nav1.7, and +10 mV for Nav1.8 from Vmax or V1/2 in both the absence and presence of 300 μmol/L of carvacrol (B) Percentage inhibition of sodium currents of carvacrol were calculated (n = 6). Open columns represent the effect at Vmax holding potential and closed columns indicate the effect at V1/2. Data are presented as means ± SEM. ***P < 0.001, ****P < 0.0001 compared to the control, based on unpaired t-test (two-tailed) (C) Concentration-response curves for inhibitory effects of carvacrol on sodium currents elicited by 50 ms depolarizing pulses to −20 mV for Nav1.2 and Nav1.6, –10 mV for Nav1.3 and Nav1.7, +10 mV for Nav1.8 α subunits with β1 subunit from V1/2 holding potential (n = 6). The peak current amplitude in the presence of carvacrol was normalized to that of the control and the effects are expressed as percentages of the control. Data are presented as means ± SEM. *P < 0.05, ****P < 0.0001 compared to the control, based on one-way analysis of variance (ANOVA) followed by Dunnett post hoc test. IC50 values and Hill coefficients were calculated using GraphPad Prism. IC50 = half maximal inhibitory concentration; Nav = voltage-gated sodium channel; Vmax holding potential = holding potential causing maximal current; V1/2 holding potential = holding potential causing half-maximal current.
Fig. 2. Effects of carvacrol on I–V curves of sodium currents in oocytes expressing Nav1.2 (A), Nav1.3 (B), Nav1.6 (C), Nav1.7 (D), and Nav1.8 (E) α subunits with β1 subunit (n = 6). Currents were elicited using 50 ms depolarizing steps between −80 and 60 mV in 10 mV increments from a Vmax holding potential (left panel) and elicited using 50 ms depolarizing steps between −60 and 60 mV in 10 mV increments from a V1/2 holding potential (right panel). Upper panel, Representative INa traces from oocytes expressing Nav1.2 (A), Nav1.3 (B), Nav1.6 (C), Nav1.7 (D), and Nav1.8 (E) α subunits with the β1 subunit in both the absence and presence of carvacrol at Vmax and V1/2 holding potential are shown. Lower panel, The effects of carvacrol on normalized I–V curves elicited from Vmax and V1/2 holding potentials are shown (closed circles, control; open circles, carvacrol). Peak currents were normalized to the maximal currents observed from −10 to +10 mV. Data are presented as means ± SEM. Nav = voltage-gated sodium channel; Vmax holding potential = holding potential causing maximal current; V1/2 holding potential = holding potential causing half-maximal current.
Fig. 3. Effects of carvacrol on channel activation in oocytes expressing Nav1.2 (A), Nav1.3 (B), Nav1.6 (C), Nav1.7 (D), and Nav1.8 (E) α subunits with β1 subunit from Vmax or V1/2 holding potentials (n = 6). Closed circles and open circles represent control and the effect of carvacrol, respectively. Data are expressed as means ± SEM. Activation curves were fitted to the Boltzmann equation; V1/2 is shown in Table 1. Nav = voltage-gated sodium channel; Vmax holding potential = holding potential causing maximal current; V1/2 holding potential = holding potential causing half-maximal current.
Fig. 4. Effects of carvacrol on inactivation curves in oocytes expressing Nav1.2 (A), Nav1.3 (B), Nav1.6 (C), Nav1.7 (D), and Nav1.8 (E) α subunits with β1 subunit (n = 6). Currents were elicited by a 50 ms test pulse to −20 mV for Nav1.2 and Nav1.6, –10 mV for Nav1.3 and Nav1.7, and +10 mV for Nav1.8 after 200 ms (500 ms for only Nav1.8) prepulses ranging from −140 mV to 0 mV in 10 mV increments from a Vmax holding potential (A) Left panel, Representative INa traces from oocytes expressing Nav1.2 (A), Nav1.3 (B), Nav1.6 (C), Nav1.7 (D), and Nav1.8 (E) α subunits with the β1 subunit in both the absence and presence of carvacrol are shown. Right panel, Effects of carvacrol on inactivation curves (closed circles, control; open circles, amitriptyline) are shown. Steady-state inactivation curves were fitted to the Boltzmann equation and the V1/2 values are shown in Table 1. Data are expressed as means ± SEM. Nav = voltage-gated sodium channel.
Fig. 5. Use-dependent blockage of sodium channels on Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 α subunits with β1 subunit by carvacrol (n = 6). Currents were elicited at 10 Hz by a 20 ms depolarizing pulse of −20 mV for Nav1.2 and Nav1.6, –10 mV for Nav1.3 and Nav1.7, and +10 mV for Nav1.8 from a V1/2 holding potential in both the absence and presence carvacrol. (A) Representative INa traces from oocytes expressing Nav1.2 α subunit with the β1 subunit in both the absence and presence of carvacrol are shown. Peak currents were measured and normalized to the first pulse and plotted against the pulse number (B, Nav1.2; C, Nav1.3; D, Nav1.6; E, Nav1.7; F, Nav1.8). Closed circles and open circles represent control and the effect of carvacrol, respectively (G) Data were fitted to the monoexponential equation and values for fractional blockage of the plateau of normalized INa are shown (open columns and closed columns represent control and the effect of carvacrol, respectively). Data are expressed as means ± SEM. ***P < 0.001, compared to the control, based on paired t-test (two-tailed). Nav = voltage-gated sodium channel.
