The neurosteroids allopregnanolone and pregnanolone are potent positive modulators of γ-aminobutyric acid type A receptors. Antinociceptive effects of allopregnanolone have attracted much attention because recent reports have indicated the potential of allopregnanolone as a therapeutic agent for refractory pain. However, the analgesic mechanisms of allopregnanolone are still unclear. Voltage-gated sodium channels (Nav) are thought to play important roles in inflammatory and neuropathic pain, but there have been few investigations on the effects of allopregnanolone on sodium channels.
Using voltage-clamp techniques, the effects of allopregnanolone sulfate (APAS) and pregnanolone sulfate (PAS) on sodium current were examined in Xenopus oocytes expressing Nav1.2, Nav1.6, Nav1.7, and Nav1.8 α subunits.
APAS suppressed sodium currents of Nav1.2, Nav1.6, and Nav1.7 at a holding potential causing half-maximal current in a concentration-dependent manner, whereas it markedly enhanced sodium current of Nav1.8 at a holding potential causing maximal current. Half-maximal inhibitory concentration values for Nav1.2, Nav1.6, and Nav1.7 were 12 ± 4 (n = 6), 41 ± 2 (n = 7), and 131 ± 15 (n = 5) μmol/l (mean ± SEM), respectively. The effects of PAS were lower than those of APAS. From gating analysis, two compounds increased inactivation of all α subunits, while they showed different actions on activation of each α subunit. Moreover, two compounds showed a use-dependent block on Nav1.2, Nav1.6, and Nav1.7.
APAS and PAS have diverse effects on sodium currents in oocytes expressing four α subunits. APAS inhibited the sodium currents of Nav1.2 most strongly.
Sodium channels are important targets for analgesic actions in the spinal cord, but their role in neurosteroid analgesia is unclear
The effects of two sulfated neurosteroids with analgesic and anesthetic properties were tested on heterologously expressed rat voltage-gated sodium channel function
The neurosteroids tested produced voltage and use-dependent block of all the subtypes tested, with more potent effects on Nav1.2
Inhibition of Nav1.2 in the spinal cord by allopregnanolone is a plausible mechanism for its analgesic effects if confirmed in neuronal preparations and pain models
NEUROSTEROIDS are neuroactive steroids synthesized from cholesterol in both central and peripheral nervous systems, and they accumulate in the nervous system.1 They rapidly alter neuronal excitability by mediating actions through ion-gated neurotransmitter receptors, but not through classic steroid hormone nuclear receptors.2 Many of them are converted to sulfated metabolites by hydroxysteroid sulfotransferases, and neurosteroid sulfates are also known to regulate physiological processes. They are thought to be potentially therapeutic because of their many pharmacological properties.3,4
Two 3α-hydroxylated metabolites of progesterone, allopregnanolone (3α-hydroxy-5α-pregnane-20-one) and pregnanolone (3α-hydroxy-5β-pregnane-20-one), are known to be positive modulators at γ-aminobutyric acid type A (GABAA) receptors with high potency.5 These neurosteroids have been shown to have greater anesthetic potencies than those of other intravenous anesthetics that are clinically used, and not to cause acute tolerance that are observed in other anesthetics, suggesting usefulness of these neurosteroids as general anesthetics.6,7 On the contrary, allopregnanolone was shown to have the most potent analgesic effects among all neurosteroids in pain models.8 Recent studies demonstrated its analgesic effects in neuropathic pain models. Allopregnanolone alleviates thermal and mechanical hyperalgesia by ligation of the sciatic nerve in rats,9 produces analgesic effects on formalin-induced pain in rats,10 and prevents anticancer drug oxaliplatin-induced cold and mechanical allodynia and hyperalgesia.11 In addition, it was suggested that stimulation of allopregnanolone synthesis might be involved in the antinociceptive effects of several analgesic drugs in neuropathic pain models.12–14 Its effect on GABAA receptors may be important for its antinociceptive properties because GABA is involved in pain pathways in the nervous systems, and drugs targeting subtypes of GABA receptors have analgesic effects in chronic pain.15 However, these two neurosteroids, allopregnanolone and pregnanolone, also act on other ion channels in pain signaling pathways, including T-type calcium channels16 and N-methyl-d-aspartate receptors.17
Voltage-gated sodium channels (Nav) have an important role in action potential initiation and propagation in excitable nerve and muscle cells. Nine α subunits (Nav1.1 to Nav1.9) and four auxiliary β subunits have been identified in mammals.18,19 Each pore-forming α subunit has a different pattern of development and localization and has distinct physiological and pathophysiological roles. Sodium channel α subunits expressed in the dorsal root ganglion are considered possible targets for analgesics for inflammatory and neuropathic pain.20–22 However, there has been little investigation on the effects of allopregnanolone on sodium channel function. It is important to examine these effects because they may be useful in clarifying the mechanisms of the analgesic effects of allopregnanolone and developing natural and safe neurosteroid-based analgesics for refractory pain. In addition, our recent report demonstrated the importance of neurosteroid sulfonation for regulation of ion channels because of more potent effects of sulfated steroid than those of nonsulfated steroids.23 Here, we investigate the effects of two sulfated neurosteroids, allopregnanolone sulfate (APAS) and pregnanolone sulfate (PAS) (fig. 1), on several sodium channel α subunits, including Nav1.2, which is expressed in the central nervous system; Nav1.6, which is expressed in the central nervous system and dorsal root ganglion neurons; and Nav1.7 and Nav1.8, which are expressed in dorsal root ganglion neurons.
Materials and Methods
This study was approved by the Animal Research Committee of the University of Occupational and Environmental Health, Kitakyushu, Japan.
Allopregnanolone sulfate and PAS were purchased from Steraloids, Inc. (Newport, RI).
Rat Nav1.2 α subunit complementary DNA (cDNA) was a gift from Dr. William A. Catterall, Ph.D. (Professor, Department of Pharmacology, University of Washington, Seattle, Washington). Rat Nav1.6 α subunit cDNA was a gift from Dr. Alan L. Goldin, M.D., Ph.D. (Professor, Department of Anatomy and Neurobiology, University of California, Irvine, California). Rat Nav1.7 α subunit cDNA was a gift from Gail Mandel, Ph.D. (Professor, Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon). Rat Nav1.8 α subunit cDNA was a gift from Dr. Armen N. Akopian, Ph.D. (Assistant Professor, University of Texas Health Science Center, San Antonio, Texas), and human β1 subunit cDNA was a gift from Dr. Alfred L. George, Jr., M.D. (Professor, Department of Pharmacology, Vanderbilt University, Nashville, Tennessee). The percentages of homology between rat and human protein of Nav1.2, Nav1.6, Nav1.7, and Nav1.8 are 98, 99, 93, and 83%, respectively, suggesting the possible limitations imposed by using rat α subunit for only Nav1.8 to make conclusions in humans.
Complementary RNA (cRNA) Preparation and Oocyte Injection
After linearization of cDNA with ClaI (Nav1.2 α subunit), NotI (Nav1.6, 1.7 α subunits), XbaI (Nav1.8 α subunit), and EcoRI (β1 subunit), cRNAs were transcribed using SP6 (Nav 1.8 α, β1 subunits) or T7 (Nav1.2, 1.6, and 1.7 α subunits) RNA polymerase from the mMESSAGE mMACHINE kit (Ambion, Austin, TX). Adult female Xenopus laevis frogs were obtained from Kyudo Co., Ltd. (Saga, Japan). X. laevis oocytes and cRNA microinjection were prepared as described previously.24 Nav α subunit cRNAs were coinjected with β1 subunit cRNA at a ratio of 1:10 (total volume was 20 to 40 ng/50 nl) into Xenopus oocytes (all α subunits were coinjected with the β1 subunit) that were randomly assigned to four α subunit groups for injection. Injected oocytes were incubated at 19°C in incubation medium, and 2 to 6 days after injection, the cells were used for electrophysiological recordings.
All electrical recordings were performed at room temperature (23°C). Oocytes were placed in a 100-μl recording chamber and perfused at 2 ml/min with Frog Ringer’s solution containing 115 mmol/l NaCl, 2.5 mmol/l KCl, 10 mmol/l HEPES, 1.8 mmol/l CaCl2, pH 7.2, using a peristaltic pump (World Precision Instruments Inc., Sarasota, FL). Recording electrodes were prepared, and the whole-cell voltage clamp and recordings were achieved as described previously.24 Transients and leak currents were subtracted using the P/N procedure, in which N subsweeps each 1/Nth of the amplitude of the main stimulus waveform (P) are applied. APAS and PAS stocks were prepared in dimethylsulfoxide and diluted in Frog Ringer’s solution to a final dimethylsulfoxide concentration not exceeding 0.05%. APAS and PAS were perfused for 3 min to reach equilibrium. All recordings were performed by the experimenters who were blind to the type of compound.
The voltage dependence of activation was determined using 50-ms depolarizing pulses from a holding potential causing maximal current (Vmax) (−90 mV for Nav1.2 and Nav1.6, −100 mV for Nav1.7 and Nav1.8) and from a holding potential causing half-maximal current (V1/2) (from approximately −40 mV to −70 mV) to 60 mV in 10-mV increments. Vmax and V1/2 holding potentials induce resting and inactivated states of sodium channels. Because the effects of many analgesics in the inactivated state are known to be important for analgesic action,25 we used these two different holding potentials to compare the effects of compounds in the resting and inactivated states. Normalized activation curves were fitted to the Boltzmann equation as described previously24 : briefly, G/Gmax = 1/(1 + exp(V1/2 − V)/k), where G is the voltage-dependent sodium conductance, Gmax is the maximal sodium conductance, G/Gmax is the normalized fractional conductance, V1/2 is the potential at which activation is half maximal, and k is the slope factor. To measure steady-state inactivation, currents were elicited by a 50-ms test pulse to −20 mV for Nav1.2 and Nav1.6, −10 mV for Nav1.7, and +10 mV for Nav1.8 after 200 ms (500 ms for only Nav1.8) prepulses ranging from −140 to 0 mV in 10-mV increments from a holding potential of Vmax. Steady-state inactivation curves were fitted to the Boltzmann equation: I/Imax = 1/(1 + exp(V1/2 − V)/k), where Imax is the maximal sodium current, I/Imax is the normalized current, V1/2 is the voltage of half-maximal inactivation, and k is the slope factor. To investigate a use-dependent sodium channel block, currents were elicited at 10 Hz by a 20-ms depolarizing pulse of −20 mV for Nav1.2 and Nav1.6, −10 mV for Nav1.7, and +10 mV for Nav1.8 from a V1/2 holding potential in both the absence and presence of 100 μmol/l APAS and PAS. Peak currents were measured and normalized to the first pulse and plotted against the pulse number. Data were fitted to the monoexponential equation INa = exp(−τuse·n) + C, where n is pulse number, C is the plateau INa, and τuse is the time constant of use-dependent decay.
The GraphPad Prism software (GraphPad Software, Inc., San Diego, CA) was used to perform the statistical analysis, and a statistical power analysis was performed using G*Power software. All values are presented as means ± SEM. The n values refer to the number of oocytes examined. Each experiment was performed with oocytes taken from at least two frogs. Data were statistically evaluated by paired t test (two-tailed). We assessed the inhibitory effects at different APAS concentrations in the concentration–response curve, using one-way ANOVA followed by Dunnet post hoc test for multiple comparisons. Hill slope, half-maximal inhibitory concentration (IC50), and half-maximal effective concentration (EC50) values were also calculated. P value less than 0.05 was considered to indicate a significant difference.
Effects of APAS and PAS on Peak Na+ Inward Currents Elicited from Two Different Holding Potentials
Currents were elicited using a 50-ms depolarizing pulse to −20 mV for Nav1.2 and Nav1.6, −10 mV for Nav1.7, and +10 mV for Nav1.8 applied every 10 s from a Vmax or V1/2 holding potential in both the absence and presence of 100 μmol/l APAS and PAS (fig. 2). The amplitude of expressed sodium currents was typically 2 to 15 μA, and oocytes that showed a maximal current greater than 20 μA were not included in the data collection in all the following experiments. APAS had dual effects on sodium currents depending on the holding potential and α subunit (figs. 2 and 3). At V1/2, APAS reduced the peak INa (sodium current) induced by Nav1.2, Nav1.6, and Nav1.7 by 79 ± 1%, 71 ± 2%, and 49 ± 3%, respectively. At Vmax, APAS also reduced INa induced by Nav1.2 by 60 ± 4%, whereas it enhanced INa induced by Nav1.6 and Nav1.7 by 15 ± 6% and 14 ± 1%, respectively, although these effects were small. In contrast, APAS greatly enhanced INa induced by Nav1.8 at both V1/2 and Vmax by 112 ± 34% and 202 ± 14%, respectively (fig. 3A). PAS reduced INa induced by Nav1.2, Nav1.6, and Nav1.7 at V1/2 by 54 ± 4%, 71 ± 1%, and 48 ± 2%, respectively. Effects of PAS on INa at Vmax were smaller than those at V1/2, and the magnitudes of inhibitory effects on Nav1.2, Nav1.6, and Nav1.7 were 31 ± 5%, 10 ± 1%, and 6 ± 1%, respectively. While PAS enhanced INa induced by Nav1.8 at Vmax by 39 ± 6%, it did not affect INa induced by Nav1.8 at V1/2 (fig. 3B). In summary, PAS inhibited INa induced by Nav1.2, Nav1.6, and Nav1.7 at both V/1/2 and Vmax holding potentials. APAS had inverse effects on Nav1.6 and Nav1.7 according to the different holding potentials, whereas it suppressed INa induced by Nav1.2 at both V/1/2 and Vmax. Moreover, APAS markedly enhanced INa induced by Nav1.8 at both V/1/2 and Vmax.
Next, we examined the concentration–response relationship for suppression of the peak INa induced through Nav1.2, Nav1.6, and Nav1.7 by APAS and PAS at V1/2 holding potential because suppression by both neurosteroids of these α subunits at V1/2 was more potent than that at Vmax (fig. 4, A and B). In addition, we investigated the concentration–response relationship for potentiation of the peak INa of Nav1.8 by APAS and PAS at Vmax, because both neurosteroids showed potent enhancement of INa at Vmax compared with that at V1/2 (fig. 4C). IC50 values, EC50 values, and Hill slopes calculated from nonlinear regression analyses of the dose–response curves are shown in table 1. From these analyses, the effect of APAS on Nav1.2 was the most potent among the two neurosteroids and four α subunits.
Effects of APAS and PAS on Activation of Sodium Currents
We examined the effects of APAS and PAS on four α subunits in sodium current activation. Voltage dependence of activation was determined using 50-ms depolarizing pulses from a holding potential of Vmax to 50 mV in 10-mV increments or from a holding potential of V1/2 to 60 mV in 10-mV increments for Nav1.2, Nav1.6, Nav1.7, and Nav1.8 in both the absence and presence of 100 μmol/l APAS and PAS (fig. 5). Activation curves were derived from the I–V curves (see Electrophysiological Recordings under Materials and Methods). At Vmax, APAS greatly reduced the peak INa induced by Nav1.2, whereas it greatly enhanced the peak INa induced by Nav1.8 in the depolarizing region where channel opening begins. It also enhanced the peak INa induced by Nav1.6 and Nav1.7, similar to its effects on Nav1.8, although both effects were small. At V1/2, APAS greatly suppressed the peak INa induced by Nav1.2, Nav1.6, and Nav1.7, but it enhanced the peak INa induced by Nav1.8, similar to its effects on Nav1.8 at Vmax. PAS reduced INa induced by Nav1.2, Nav1.6, and Nav1.7 at both V/1/2 and Vmax, whereas it enhanced INa induced by Nav1.8 in the depolarizing region at Vmax, but had no effect at V1/2.
At Vmax holding potential, APAS significantly shifted the midpoint of the steady-state activation (V1/2) in a depolarizing direction for Nav1.2, but it significantly shifted V1/2 in a hyperpolarizing direction for Nav1.6, Nav1.7, and Nav1.8. At V1/2, APAS also shifted V1/2 in a similar direction as the shift at Vmax, although the shift was small and not significant, except for Nav1.8. The shifts of V1/2 by PAS were smaller than those by APAS. PAS significantly shifted V1/2 in a depolarizing direction for Nav1.2 and Nav1.6 at V1/2, but it had no or slight effects on all α subunits at Vmax, and on Nav1.7 and Nav1.8 at V1/2 (fig. 6 and tables 2 and 3).
Effects of APAS and PAS on Inactivation of Sodium Currents
We also investigated the effects of APAS and PAS on steady-state inactivation. Currents were elicited by a 50-ms test pulse to −20 mV for Nav1.2 and Nav1.6, −10 mV for 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 Vmax holding potential. Steady-state inactivation curves were fitted to the Boltzmann equation (see Electrophysiological Recordings under Materials and Methods). APAS and PAS significantly shifted the midpoint of steady-state inactivation (V1/2) in the hyperpolarizing direction for all α subunits; APAS shifted by 8.0, 8.9, 6.7, and 8.9 mV and PAS shifted by 4.5, 8.0, 6.6, and 10.2 mV for Nav1.2, Nav1.6, Nav1.7, and Nav1.8, respectively (fig. 7 and tables 2 and 3). The effects of APAS and PAS in the hyperpolarizing range were consistent with the effects of these two neurosteroids on the peak INa at Vmax and their effects on the I–V curves in the hyperpolarizing range at Vmax.
Use-dependent Block of Sodium Currents by APAS and PAS
The use-dependent block of sodium currents by APAS and PAS was also investigated. Currents were elicited at 10 Hz by a 20-ms depolarizing pulse of −20 mV for Nav1.2 and Nav1.6 and −10 mV for Nav1.7 from a V1/2 holding potential in both the absence and presence of 100 μmol/l APAS and PAS. Peak currents were measured and normalized to the first pulse and plotted against the pulse number (fig. 8, A–D). Data were fitted by the monoexponential equation (see Electrophysiological Recordings under Materials and Methods). APAS significantly reduced the plateau INa amplitude of Nav1.2, Nav1.6, and Nav1.7 from 0.80 ± 0.03 to 0.57 ± 0.03, 0.89 ± 0.01 to 0.49 ± 0.07, and 0.89 ± 0.02 to 0.62 ± 0.06, respectively (fig. 8E). PAS also reduced the plateau INa amplitudes of Nav1.2, Nav1.6, and Nav1.7 from 0.81 ± 0.2 to 0.70 ± 0.03, 0.94 ± 0.01 to 0.73 ± 0.02, and 0.91 ± 0.02 to 0.75 ± 0.01, respectively, and the reductions were significant except for Nav1.2 (fig. 8F). These results demonstrated a use-dependent block of APAS and PAS on sodium channels, and the block by APAS was more potent than that by PAS.
In the current study, we demonstrated that APAS and PAS differentially affected INa induced by four α subunits at both Vmax and V1/2 holding potentials. Moreover, we found that both neurosteroids suppress Nav1.2, Nav1.6, and Nav1.7 at V1/2 in a concentration-dependent manner. IC50 values indicated that the effect of APAS on Nav1.2 was most potent among the two compounds and three α subunits. To the best of our knowledge, this is the first direct evidence of the various effects of these two neurosteroids on neuronal sodium channel α subunits. It is thought that APAS is synthesized from allopregnanolone by 3α-hydroxysteroid sulfotransferase in vivo, because 3α-hydroxysteroid sulfotransferase has been isolated in vivo.26 Therefore, allopregnanolone likely exerts a portion of its effects through APAS, which is its metabolite.
It was reported that the level of endogenous allopregnanolone changes in many physiological and pathological situations within a serum concentration range of 1 to 10 nmol/l.27,28 However, it is not clear whether allopregnanolone has an analgesic effect in physiological concentrations. A recent study demonstrated that 1 and 10 μmol/l of allopregnanolone reduced mechanical allodynia and thermal heat hyperalgesia in normal and neuropathic pain models in rats after 10-μl intrathecal injection.29 Another investigator reported that intrathecal administration of 10 μmol/l of allopregnanolone showed antihyperalgesic effects in hyperalgesic rats after spinal nerve ligation.30 From these previous studies, concentrations approximately 1 μmol/l allopregnanolone at receptive fields are estimated to have an analgesic effect. In the current study, APAS tended to, albeit not significantly, suppress the INa of Nav1.2 at 0.3 μmol/l by 8% and significantly (P < 0.01) inhibited it at 1 μmol/l by 19 ± 2%. The IC50 value of Nav1.2 inhibition by APAS was 12 μmol/l. It was reported that relatively small degrees of sodium channel inhibition could have profound effects on the neuronal firing rate because a 10% inhibition of sodium current reduces the number of action potentials to 10 from a control response of 21 in 750 ms.24 Therefore, APAS may reduce neuronal firing for Nav1.2 at a concentration exhibiting the antinociceptive effects of allopregnanolone in animal models, whereas the effects of APAS and PAS on another three α and four α subunits, respectively, may not be pharmacologically relevant because these effects were observed at concentrations over 10 μmol/l. In addition, the effects of highly hydrophobic compounds—such as neurosteroids—we used tend to be attenuated in the voltage-clamp techniques with Xenopus oocytes, compared with the whole-cell voltage-clamp methods using mammalian cells. Indeed, it was reported that the enhancing effect by allopregnanolone on GABAA receptor combination (α1β2γ2L) was more potent in the human embryonic kidney 293 cells system (EC50; 41 ± 2 nmol/l)31 than that in the Xenopus oocyte system (EC50; 177 ± 2 nmol/l).32 This may be a limitation of experiments using the Xenopus oocyte expression system; this limitation indicates that APAS might inhibit function of Nav1.2 more potently in a mammalian cell system than in the oocyte system, however, it also could potentiate Nav1.8 function more potently in a mammalian cell. Therefore, further investigation is needed to consider the roles of these α subunits in humans.
Analysis of gating revealed common characteristics but also some differences in the effects of APAS and PAS on different α subunits. A common effect on all α subunits was enhancement of inactivation. Because of this enhancement effect, the inhibitions by two compounds at V1/2 holding potentials could be interpreted as stronger effects because they shift inactivation curve to the hyperpolarizing direction, which makes the channel into further inactivated state. In contrast, APAS enhanced peak INa at Vmax, shifted activation in the hyperpolarizing direction, and increased sodium currents in the hyperpolarizing range of the inactivation curves for Nav1.6, Nav1.7, and Nav1.8. These changes indicate that APAS shifts channel gating equilibrium toward the open channel state and activates sodium channels. This action might attenuate the effects on the inactivated state and, especially, lead to enhancement of INa even in the inactivated state (V1/2 holding potential) for Nav1.8 in spite of the great enhancement of inactivation. However, for Nav1.2, APAS profoundly suppressed peak INa at Vmax, shifted activation in the depolarizing direction at Vmax, and greatly decreased sodium currents in the hyperpolarizing range of the inactivation curve, indicating that resting channel block is an important mechanism of APAS inhibition for only Nav1.2. Both compounds demonstrated use-dependency for inhibition of Nav1.2, Nav1.6, and Nav1.7, suggesting the ability to slow the recovery time from inactivation.33 Many investigators have shown that sodium channel blockers, including local anesthetics, tricyclic antidepressants, and volatile anesthetics, enhance steady-state inactivation with no effect on activation and exhibit use-dependent block.34–36 We demonstrated that APAS enhances inactivation and shows use-dependent block similar to other sodium channel blockers, yet it also has diverse effects on activation according to differences in α subunits. These actions suggest that APAS may have different binding sites or allosteric conformational mechanisms to change sodium channel function, although further investigation with site-directed mutagenesis is needed to rule out nonspecific membrane effects. PAS may have common binding sites with APAS, because it shows similar effects, although these changes were small.
The α subunit consists of four homologous domains (I to IV) containing six transmembrane segments (S1 to S6), and one reentrant P-region connecting S5 to S6 (SS1/SS2). Tetrodotoxin-sensitive α subunits, Nav1.2, Nav1.6, and Nav1.7, are phylogenetically related and show 70 to 80% amino acid sequence identity. In contrast, tetrodotoxin-resistant α subunits, Nav1.8, are phylogenetically distant and show only 55 to 56% sequence identity to the other three α subunits. In addition, the lengths of amino acid sequences of four α subunits differed within the range of 1957 to 2005 residues. Therefore, these differences would result in the diversity in neurosteroid action, especially in the effects on channel activation. Indeed, the longest extracellular regions in the α subunit (IS5 to SS1) are 93, 77, 73, and 66 amino acid residues in Nav1.2, Nav1.6, Nav1.7, and Nav1.8, respectively. The diversity in sequence and differences in the effects on activation according to α subunit may be important for clarifying binding sites and the mechanism of Nav1.2 inhibition by APAS in further investigations.
γ-Aminobutyric acid type A receptors have been considered to be important for the analgesic effects of allopregnanolone because it has high potency as a positive GABAA modulator compared with other neurosteroids. Pregnanolone also affects GABAA receptors in a manner similar to that of allopregnanolone; nevertheless, its analgesic effect is weak. In fact, pregnanolone was shown to reduce mechanical allodynia without reduction of thermal heat hyperalgesia in a neuropathic pain model in contrast to attenuation of both by allopregnanolone.28 The investigators suggested that the partial analgesic effects of pregnanolone are caused by suppression of glycine receptors by demonstrating that pregnanolone had a significant analgesic effect only in animals displaying a strychnine-induced allodynia in two types of allodynia models induced by bicuculline and strychnine.28 Moreover, a recent report demonstrated that allopregnanolone shows analgesic effects in rats through suppression of T-type Ca2+ currents and potentiation of GABAA currents.16 These previous reports indicate several mechanisms underlying the analgesic effect of allopregnanolone likely exist, as well as potentiation of GABAA receptors.
Sodium channel α subunits expressed in the dorsal root ganglion (Nav1.7, Nav1.8, and Nav1.9) are thought to be involved in the pathogenesis of inflammatory and neuropathic pain. A recent study reported that Nav1.2 also plays an important role in pain signaling. It was reported that Nav1.2 and Nav1.3 predominantly compose functional sodium channel currents within lamina I/II (dorsal horn) neurons, which mediate acute and chronic nociceptive signals from peripheral nociceptors to pain-processing regions in the brain.37 Another recent report showed that mutations in Nav1.2 are associated with seizures and pain characterized by headaches and back pain.38 A disubstituted succinamide, a potent sodium channel blocker, was reported to attenuate nociceptive behavior in a rat model of tonic pain and was demonstrated to potently block Nav1.2, as well as Nav1.7 and Nav1.8, with a potency two orders of magnitude higher than anticonvulsant and antiarrhythmic sodium channel blockers currently used to treat neuropathic pain.39 Other investigators demonstrated that four sodium channel blockers, including lidocaine, mexiletine, benzocaine, and ambroxol, which are used clinically to treat pain, suppressed recombinant Nav1.2 currents as well as tetrodotoxin-resistant Na+ channel currents in rat sensory neurons, which comprised mostly Nav1.8 currents. The authors suggested that these sodium channel blockers would induce analgesia according to the amount of sodium channel blocking, including Nav1.2 and Nav1.8.40 These recent reports support that suppression of Nav1.2 function by APAS might be a mechanism underlying the analgesic effects of allopregnanolone.
In conclusion, APAS and PAS have diverse effects on Nav1.2, Nav1.6, Nav1.7, and Nav1.8 α subunits expressed in Xenopus oocytes, with differences in the effects on sodium channel gating. In particular, only APAS inhibited sodium currents of Nav1.2 at pharmacologically relevant concentrations. These results raise the possibility that suppression of Nav1.2 by APAS may be important for pain relief by allopregnanolone and provide a better understanding of the mechanisms underlying the analgesic effects of allopregnanolone. However, further studies are needed to clarify the relevance of sodium channel inhibition by APAS.
This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Tokyo, Japan (grant no. 21791480 to Dr. Horishita).
The authors declare no competing interests.