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Research ArticleNEUROPHARMACOLOGY

Vinpocetine Is a Potent Blocker of Rat NaV1.8 Tetrodotoxin-Resistant Sodium Channels

Xiaoping Zhou, Xiao-Wei Dong, James Crona, Maureen Maguire and Tony Priestley
Journal of Pharmacology and Experimental Therapeutics August 2003, 306 (2) 498-504; DOI: https://doi.org/10.1124/jpet.103.051086

Abstract

Vinpocetine is a clinically used synthetic vincamine derivative with a diverse pharmacological profile that includes action at several ion channels, principally “generic” populations of sodium channels that give rise to tetrodotoxin-sensitive conductances. A number of cell types are known to express tetrodotoxin-resistant (TTXr) sodium conductances, the molecular bases of which have remained elusive until recently. One such TTXr channel, termed NaV1.8, is of particular interest because of its prominent and selective expression in peripheral afferent nerves. The effects of vinpocetine on TTXr channels specifically, are unknown. We have assessed the effects of the drug on cloned rat NaV1.8 channels expressed in a dorsal root ganglion-derived cell line, ND7/23. Vinpocetine produced a concentration- and state-dependent inhibition of NaV1.8 sodium channel activity. Voltage-clamp experiments revealed an ∼3-fold increase in vinpocetine potency when whole-cell NaV1.8 conductances were elicited from relatively depolarized potentials (–35 mV; IC50 = 3.5 μM) compared with hyperpolarized holding potentials (–90 mV; IC50 = 10.4 μM). Vinpocetine also produced an ∼22 mV leftward shift in the voltage dependence of NaV1.8 channel inactivation but did not affect the voltage range of channel activation. These properties are reminiscent of several other known sodium channel blockers and suggested that vinpocetine may exhibit frequency-dependent block. Accordingly, tonic block of NaV1.8 channels by vinpocetine (3 μM) increased proportionally with increasing depolarizing commands over the frequency range 0.1 to 1Hz. In summary, the present data demonstrate that vinpocetine is capable of blocking NaV1.8 sodium channel activity and suggest a potential additional utility in various sensory abnormalities arising from abnormal peripheral nerve activity.

Vinpocetine (Fig. 1) is a synthetic ethyl ester of the Vinca minor alkaloid apovincamine. A therapeutic formulation of vinpocetine, marketed under the proprietary name Cavinton has been used for more than 20 years in certain European countries and Japan as a cerebroprotective agent to reduce brain damage resulting from an ischemic infarct. The vincamine derivative, in common with many other plant-derived alkaloids, has a diverse pharmacological profile that includes phosphodiesterase inhibition, antioxidant properties, and an action at several ion channels (Bonoczk et al., 2000). This somewhat promiscuous pharmacology has led to the justifiable classification of vinpocetine as a “nootropic”, an aptly vague terminology given the lack of a clear mechanism of action.

Despite the somewhat nonselective pharmacological profile, vinpocetine is claimed to be remarkably devoid of major side effects at clinically used dosages (Anonymous, 2002). A number of studies have indicated, clearly, that vinpocetine achieves good brain exposure after systemic administration (Bonoczk et al., 2002; Gulyas et al., 2002), and it is reasonable to assume that all of the above-mentioned actions may contribute to a therapeutic neuroprotective effect. The significance of the ion channel blocking effect of vinpocetine, particularly in respect to a sodium channel action, is supported by an anticonvulsant action in rodent seizure models (Schmidt, 1990) and from a comparative study using the established anticonvulsant drug phenytoin (Molnar and Erdo, 1995). In addition to their clinical utility in the prevention of epileptiform seizures, anticonvulsants have found favor as a therapeutic approach to the management of certain pain states (Hunter and Loughhead, 1999; Dickenson et al., 2002). There is mounting evidence (Hunter and Loughhead, 1999; Devor, 2001) to support a fundamental role for ectopic, epileptiform-like burst firing in peripheral nerves as a substrate for paroxysmal pains often described as “stabbing” or “electric-shock-like.” Peripheral nerve axons express a mixed complement of sodium channel isoforms, one of which is a TTX-insensitive conductance mediated by NaV1.8 (Hunter and Loughhead, 1999). Although there is an ongoing debate over the relevant roles of various sodium channel activities in generating and sustaining chronic pain states, several studies have indicated a prominent role for NaV1.8 (Lai et al., 2000; Lai et al., 2002; Gold et al., 2003). This study was designed to address the effects of vinpocetine on recombinant rat NaV1.8 sodium channels expressed in a clonal cell line (ND7/23, derived from a dorsal root ganglion progenitor; Wood et al., 1990), the data suggest a potential additional utility of the drug as an antinociceptive for use in neuropathic pain states.

Materials and Methods

Cloning and Expression of NaV1.8 Sodium Channels in the ND7/23 Cell Line. Total RNA was isolated from rat dorsal root ganglia with RNA STAT-60 (Iso-Tex Diagnostics, Friendswood, TX). Random primed cDNA was reverse transcribed from 3 μg of total RNA using SuperScript II RT (Invitrogen, Carlsbad, CA). Full-length polymerase chain reaction primers were designed based on the published rat NaV1.8 (PN3) sequence U53833 (Sangameswaran et al., 1996). The oligonucleotide sequences were as follows: 5′-ATGGAGCTCCCCTTTGCGTCC and 5′-TCACTGAGGTCCAGGGCTGTT. Clones were sequence verified on an ABI3700 DNA sequencer with Big Dye Terminators version 2.0 (Applied Biosystems, Foster City, CA). The full-length coding sequence was inserted into the retroviral expression vector pMX for cloning.

For all whole-cell voltage-clamp experiments, a transient expression strategy was adopted that involved the cotransfection of rNaV1.8 and green fluorescent protein (GFP) cDNAs, the latter was used as a marker gene to identify successfully transfected cells within a given population. ND7/23 cells were bulk cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 1% penicillin and 1% streptomycin. Transfections were performed weekly, cells were seeded into six-well culture plates at a density of 500 K/well and allowed 24 h to attach. A transfection mixture (100 μl) containing FuGENE-6 (3 ml in 100 ml of serum-free DMEM; Invitrogen) and cDNA vectors (pcDNA3.1) containing coding sequences for GFP (0.5 μg) and rNaV1.8 (0.5 μg), was added to each well. After 24-h incubation, the cells were resuspended in fresh DMEM and seeded, at a density of 50 K, on poly-l-lysine-coated coverslips for subsequent electrophysiological recordings.

Whole-Cell Voltage-Clamp Recordings. All whole-cell patch-clamp recordings were carried out 48 to 72 h post-transfection; experiments were conducted at room temperature (19–21°C). At the time of the experiment, an individual coverslip was removed from the culture plate and placed into a perfusion chamber mounted on the stage of an inverted phase-contrast microscope equipped with fluorescence optics. The cells were continuously perfused with a salt solution of the following composition: 129 mM NaCl, 3.2 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 11 mM d-glucose, and 20 mM tetraethylammonium-Cl, pH 7.4, 345 mOsM. Conventional whole-cell voltage-clamp electrophysiology was used to record voltage-activated currents; patch electrodes contained a solution of the following composition: 120 mM CsF, 10 mM NaCl, 10 mM HEPES, 11 mM EGTA, 10 mM tetraethylammonium-Cl, 1 mM CaCl2, and 1 mM MgCl2, pH 7.3, 325 mOsM. Cells were viewed briefly using a fluorescence microscope (fluorescein isothiocyanate filter set) and were selected on the basis of their relative fluorescence intensity. In all experiments, membrane potential was held at –90 mV between voltage command protocols. Drugs were applied by bath perfusion. Data were acquired online (sampling frequency 10 kHz, filtered at 2 kHz) using an Axopatch 200B amplifier and pCLAMP 8.0 software (Axon instruments, Inc., Foster City, CA). Series resistance (80%) and capacitance currents were electronically compensated.

Results

Characterization of Sodium Channel Conductances in Wild-Type and Transfected ND7/23 Cells. Nontransfected (i.e., wt) ND7/23 cells express a prominent sodium conductance (Fig. 2A; Table 1) that activates rapidly upon depolarization and is highly sensitive to TTX (IC50 = 3 nM). The molecular identity of the channel(s) responsible for the wt TTX-sensitive (TTXs) conductance is unknown but it is presumed that this is likely to arise from the activity of a mixed population of sodium channels. Regardless, the wt conductance was almost completely abolished by the perfusion of 300 nM TTX (Fig. 2A), the remaining sodium current under these conditions represented only 1.2% of the peak current recorded prior to TTX perfusion (Table 1), indicating that ND7/23 cells do not constitutively express any appreciable TTX-resistant (TTXr) sodium channel isoforms. Transfection of rNaV1.8 into this background (successful in approximately 85% of GFP-fluorescent cells; Table 1) introduced a prominent conductance that persisted in the presence of 30 μM TTX (Fig. 2B), a 100-fold excess of that required to completely suppress wt currents. By definition, the residual current is TTXr and must be considered to be due to the functional expression of the recombinant rNaV1.8 protein. The amplitude of the TTXr currents varied considerably between sampled cells but was usually several hundred pico-Amps (see Table 1 for a representative data set). Transfection of GFP alone did not seem to change the phenotype of the ND7/23 cells, either morphologically or electrophysiologically and these control experiments found no evidence of a TTXr current induced by GFP transfection (Table 1). All subsequent experiments were performed in the continuous presence of 300 nM TTX to effectively isolate the conductance attributed to rNaV1.8. Characterization of the rNaV1.8 current under these conditions revealed a kinetic profile that was clearly distinct from that attributed to wt channels but very reminiscent of previously published data for rNaV1.8. Although resistant to TTX, rNaV1.8 currents were susceptible to very high concentrations of the toxin (∼23% inhibition at 30 μM, n = 6, comparable with data obtained by Akopian et al., 1996), the fact that we never observed similar TTXr currents in wt cells strongly suggests that the TTXr conductance was, indeed, sodium channel-mediated.

Fig. 2.
Fig. 2.

Macroscopic sodium currents recorded from wt and rNaV1.8-transfected ND7/23 cells. A, typical rapidly activating and inactivating sodium current (black trace) elicited in a wt ND7/23 cell by a 50-ms voltage step from –90 to 10 mV. The current was completely abolished by the addition of TTX (300 nM) to the perfusate (gray trace). B, sodium currents recorded from a transfected ND7/23 cell. The larger of the three superimposed currents (black trace, solid line) represents total sodium current evoked by the same voltage step as in A. TTX (300 nM) was then added to the perfusate resulting, in this case, an incomplete (∼50%) reduction in current amplitude (dotted line). Perfusion of a 10-fold higher concentration of TTX (30 μM) produced a small additional decrement in the conductance (gray trace) confirming the extreme resistance of rNaV1.8 to the toxin. Scale bar, 2.5 nA and 25 ms, applies to both A and B.

View this table:
TABLE 1

TTXr currents are not present in wt or GFP-transfected ND7/23 cells

TTXr defined as current remaining in presence of 300 nM TTX.

Effect of Vinpocetine on rNaV1.8 Activation. Voltage-current relationships were established for rNaV1.8 channels by measuring peak current amplitude at various destination command voltages from a holding voltage of –90 mV. Perfusion of vinpocetine (30 μM) reduced maximal current amplitude (command potential, 10 mV) by approximately 50% (Fig. 3A). A plot of the corresponding fractional conductance (G/Gmax) elicited at each command voltage (Fig. 3B) revealed a negligible effect of vinpocetine on the voltage dependence of rNaV1.8 activation (V0.5 control = 1.0 ± 0.6 mV; V0.5 vinpocetine = –1.4 ± 0.9 mV).

Fig. 3.
Fig. 3.

A, current-voltage plots for rNaV1.8 macroscopic currents obtained in the absence and presence of vinpocetine. Whole-cell currents were evoked using a series of depolarizing steps from a holding potential of –90 mV, in 10-mV increments, to a final command potential of 80 mV. Mean data (± S.E.M., n = 7) are shown for control (•) and vinpocetine (30 μM; ▪) conditions. Vinpocetine reduced peak current amplitude but did not change in the shape of the current-voltage relationship. B, activation curves (mean ± S.E.M., n = 7) for rNaV1.8 conductance in the absence (•) and presence (▪) of vinpocetine (30 μM). Vinpocetine produced a marginal, although significant (P < 0.05) modification of activation gating only over the negative activation-voltage range.

Effect of Vinpocetine on rNaV1.8 Inactivation. In common with all other voltage-gated sodium channels, the number of rNaV1.8 channels available for activation is dependent upon the resting membrane potential. Proportionally fewer channels are available for activation as the resting membrane potential is moved progressively more depolarized, due to the accumulation of channels in an inactivated, nonconducting state. The extent to which this phenomenon occurs is a complex function of both time and voltage parameters. Experimentally, this relationship can be examined, at least empirically, and quantified by imposing constant duration prepulses to sequentially depolarized potentials and then stepping to a common destination voltage (Fig. 4A). A plot of prepulse potential versus normalized peak current amplitude yielded a typical steady-state inactivation relationship from which the half-inactivation voltage (V0.5,inact) was determined by curve fitting (Boltzman function). Consistent with previously published data (Sangameswaran et al., 1996), rNaV1.8 channels were found to have a relatively positive V0.5,inact value that was moved ∼22 mV in the hyperpolarizing direction (V0.5,inact control = –34.1 ± 0.7 mV, n = 8; V0.5,inact vinpocetine = –56.4 ± 1.0 mV, n = 8) by vinpocetine (30 μM; Fig. 4B).

Fig. 4.
Fig. 4.

A, standard steady-state inactivation voltage command protocol (insert) and the corresponding superimposed macroscopic rNaV1.8 currents. Holding potential was –90 mV, prepulse command potential range was –120 to 10 mV (10-mV increments, 100-ms duration), and test command potential was 10 mV. The current traces have been cropped to the initial 50 ms for clarity; scale bar, 50 pA, 10 ms. B, steady-state inactivation curves (mean ± S.E.M., n = 8) for macroscopic rNaV1.8 currents before (•) and during (▪) vinpocetine (30 μM) using the protocol described in A. Vinpocetine produced a significant hyperpolarizing shift (ΔV0.5, inact = 22.3 mV) of the steady-state inactivation curve.

State-Dependent Inhibition of Sodium Currents by Vinpocetine. Preliminary experiments revealed that the potency of vinpocetine was dependent upon the membrane holding potential even when the destination command voltage was held constant. Thus, vinpocetine (10 μM) produced a greater attenuation (∼90%) of available current elicited by a step command to 10 mV from a holding potential of –35 mV (corresponding to the V0.5,inact for rNaV1.8) (Fig. 5B) compared with similar experiments performed from a more hyperpolarized holding potential of –90 mV (∼50% attenuation) (Fig. 5A). This is a characteristic feature of “local anesthetic” sodium channel blockers (e.g., lidocaine and mexilitine) and suggested a higher affinity interaction for vinpocetine with the inactivated channel state. More detailed experiments revealed the concentration-inhibition curves for vinpocetine sodium channel block were left-shifted when holding potential was clamped at the relatively more depolarized –35 mV compared with –90 mV (Fig. 5C). Vinpocetine was approximately 3-fold more potent (IC50(–90 mV) = 10.4 μM; IC50(–35 mV) = 3.5 μM) when currents were evoked from a holding potential of –35 mV.

Fig. 5.
Fig. 5.

A, tonic block of macroscopic rNaV1.8 currents by vinpocetine. Discrete whole-cell currents were evoked at 30-s intervals by 50-ms command pulses to 10 mV from a holding potential of –90 mV (left trace) or –35 mV (approximately half inactivation voltage, right trace) before (black lines) and after equilibration (gray lines) with vinpocetine (10 μM). The superimposed traces represent the final control current and first currents recorded in vinpocetine, the attenuation of current amplitude (gray trace) represents tonic block by the drug. Note the significantly greater inhibition (∼85% versus ∼40%) of the current by the drug under depolarized conditions. Scale bar, 500 pA, 20 ms. B, vinpocetine concentration-response curves for tonic and inactivated state block (mean ± S.E.M., n = 8) obtained from transfected ND7/23 cells subjected to the protocols described in A and B. Vinpocetine exhibits higher affinity (∼3-fold) for the inactivated state of the rNaV1.8 channel (▪) compared with tonic block from the resting state (•).

Although we do not know the identity of the sodium channels responsible for the background TTXs conductance in the ND7/23 cell line, we considered it of interest to determine the influence of vinpocetine on this conductance. Experiments similar to those described above were conducted using the wt cell line, and revealed a similar potency of vinpocetine and a similar state dependence of the drug on the wt TTXs current. Thus, vinpocetine was ∼6-fold more potent at blocking currents elicited from the half-inactivation voltage for TTXs currents (IC50(–65 mV) = 3.8 μM) than from the full-activation voltage (IC50(–120 mV) = 24.5 μM).

Vinpocetine Block of rNaV1.8 Sodium Channels Is Frequency-Dependent. Voltage-gated sodium channels require a period of membrane repolarization to reprime after depolarization-induced activation. In the case of particular native TTXr channels in dorsal root ganglion neurons, repriming has been previously reported to be rapid (Elliott and Elliott, 1993; Rush et al., 1998). We have found a similar property for rNaV1.8 in ND7/23 cells (Dong et al., 2001); in other words, these channels seem to require only brief periods of membrane repolarization to reestablish their availability and, hence, can sustain relatively high-frequency duty cycles. Repriming rates are usually studied using a series of paired pulse experiments in which two depolarizing pulses (designed to achieve full activation) are separated by a series of variable but precise interpulse intervals. A comparison of peak current amplitude elicited by the second pulse with respect to the first provides an index of channel availability at the specified interpulse interval. However, similar information can be derived from frequency-response relationships of the sort depicted in Fig. 7. A comparison of the fractional availability of rNaV1.8 channels is provided by the amplitude of each second pulse in the series in relation to the initial amplitude, the pulse interval being the reciprocal of the stimulation frequency. These experiments revealed approximately 80% of the rNaV1.8 channel population to be available for activation by the second stimulus at the shortest interval (50 ms) tested (Fig. 6). However, the same pulse interval in the presence of vinpocetine (30 μM) resulted in a significant reduction in channel availability (∼20%), suggesting that the majority of channels had failed to reprime during the 50-ms interval (Fig. 6). As the interval between successive paired pulses was increased, progressively more channels recovered, despite the presence of vinpocetine, although an interval of ∼10 s was required to ensure a degree of repriming (80%) comparable with that seen in the absence of the drug (Fig. 6).

Fig. 7.
Fig. 7.

A, frequency-response relationships for macroscopic rNaV1.8 currents recorded in the absence (A, control) and presence (B) of vinpocetine (10 μM). Whole-cell currents were evoked by step depolarization commands (holding potential, –90 mV; destination potential, 10 mV; pulse duration, 50 ms) delivered at the following frequencies: 0.1 Hz (▪), 0.3Hz (•), 1 Hz (▴), 3 Hz (▾), and 10 Hz (♦). Peak-current amplitudes (mean ± S.E.M., n = 6–8) were normalized to that of the first response at each frequency and are shown plotted against pulse number. The additional fractional block elicited at each stimulation frequency is shown in C to better illustrate the use-dependent sodium channel block by vinpocetine.

Fig. 6.
Fig. 6.

Vinpocetine delays rNaV1.8 channel repriming. Histogram plot showing the fractional recovery of rNaV1.8 sodium current as a function of time interval between two successive voltage pulses. The holding potential was –90 mV and voltage commands to 10 mV were delivered at varying intervals, the amplitude of the current response to the second pulse was expressed as a function of that elicited to the first to derive the fractional recovery of channels from the inactivated state (i.e., repriming). The control data (open columns) show that rNaV1.8 channels were capable of following instantaneous stimulation frequencies up to 10 Hz (50-ms interval) with little decrement (∼80% recovery), shorter intervals were not examined. Vinpocetine (30 μM) produced a dramatic reduction in repriming rate (filled columns) such that intervals up to almost 10 s were required to achieve the same ∼80% recovery between successive pulses.

A corollary to the slowing of repriming by vinpocetine is that the drug should promote an accumulation of blocked channels during periods of high-frequency stimulation. Figure 7 provides some indication of accumulation, particularly at higher stimulation frequencies. The control experiments (Fig. 7A) reveal that there is no loss of channel availability at 0.1-Hz stimulation, whereas at 10 Hz approximately 60% of the total channel population remains available at the end of a 25-sequence pulse train. The data obtained from similar experiments repeated in the presence of vinpocetine (10 μM) revealed clear evidence of frequency-dependent channel block by the drug (Fig. 7B). At the lowest stimulation frequency (0.1Hz) approximately 20% of the available channels were rapidly taken out of commission by vinpocetine, and there is a small but noticeable accumulation of blocked channels throughout the remaining stimuli. Increasing the stimulation frequency revealed additional, rapidly equilibrating channel blockade that seemed to saturate at frequencies above 1 Hz (Fig. 7B). The frequency dependence is better appreciated by considering the extra fractional block elicited at each stimulation frequency (Fig. 7C). Analyzing the data in this way revealed clear evidence of frequency-dependent block by vinpocetine up to stimulation frequencies of 1 Hz, approximately 50% additional TTXr conductance is eliminated by the drug at this frequency.

Discussion

Molecular cloning has led to the identification of nine functionally characterized sodium channel transcripts (Goldin, 2000), although the possibility of hitherto unidentified splice variants may add to this complexity. The majority of sodium channels are highly sensitive to channel block by TTX, yet two family members, NaV1.8 and NaV1.9, stand out as being distinct in their relative resistance to TTX, whereas a third family member, NaV1.5, occupies an intermediate TTX sensitivity (Goldin, 2000). Several previous studies have shown vinpocetine to be a sodium channel blocker, these studies have used rat cultured cerebrocortical neurons (Molnar and Erdo, 1995) or guinea pig cortical synaptosomes (Tretter and Adam-Vizi, 1998). Although the precise details of the sodium channel isoforms expressed in these preparations are unknown, it is reasonable to assume that the sodium conductances were likely to be predominantly, if not exclusively, mediated by TTX-sensitive channels. Whole-cell voltage-clamp analysis of the effects of vinpocetine on sodium currents recorded from cerebrocortical neurons (Molnar and Erdo, 1995) revealed a potency (IC50 = 44 μM) comparable with that of the established sodium channel blocker phenytoin (IC50 = 50 μM). A subsequent study revealed a similar blocking effect of vinpocetine on sodium currents recorded from rat cardiac ventricular myocytes (Wei et al., 1997). Although, once again, the precise molecular identity of the conductance was not established, this study is of interest because cardiac myocytes are known to express the TTX-insensitive NaV1.5 channel isoform. Our data suggest that vinpocetine is appreciably (>10-fold) more potent (IC50(–65 mV) = 3.8 μM) at blocking TTXs than Molnar and Erdo (1995) found in their studies. The difference may be due, at least in part, to methodological differences, although the latter studies used a similar holding potential (–60 mV), a key factor governing apparent potency. We consider a more likely explanation may be biological (channel isoform) differences.

The present study was undertaken as a result of the continued clinical use of vinpocetine and, specifically, sought to explore the action of the alkaloid derivative on a molecularly identified sodium channel isoform, rNaV1.8. This sodium channel is of particular interest in view of its selective expression pattern. In the rat, the protein is only found in a subpopulation of peripheral sensory afferent nerves (Akopian et al., 1996; Novakovic et al., 1998). Moreover, NaV1.8 displays gating characteristics that are different to the other sodium family members and, as mentioned previously, is pharmacologically novel in terms of its resistance to site I toxins. The possibility exists, therefore, for additional pharmacological divergence between NaV1.8 and other sodium channel isoforms although, at least to date, there have been no reports of small molecules that are capable of discriminating between the various members of the sodium channel family.

Expression analysis, using the whole-cell voltage-clamp approach, revealed the clonal ND7/23 cell line to be a suitable heterologous host cell line capable of sustaining robust rNaV1.8 expression that could be studied, in isolation, by the simple addition of TTX to the extracellular perfusate (Dong et al., 2001).

Vinpocetine had negligible effect on the voltage characteristics of rNaV1.8 activation but produced a clear hyperpolarizing shift in the inactivation curve. This profile is consistent with vinpocetine binding selectively to the inactivated state of the channel, the functional consequence of this effect would be to limit the dynamic range of membrane potentials over which rNaV1.8 would likely operate. These data suggested that the binding of vinpocetine was state-dependent, and this was confirmed by determining the potency of the drug at different resting membrane potentials. Although these experiments revealed vinpocetine to be more effective during sustained depolarized states the difference in affinity is marginal (∼3-fold) compared with other known sodium channel blockers. For example, similar but more marked differences in state-dependent affinity have been described previously for other sodium channel blockers, notably the local anesthetics (e.g., lidocaine) (Balser et al., 1996) and certain anticonvulsants (phenytoin, carbamazepine, and lamotrigine; Catterall, 1999), observations that are consistent with the concept of the modulated receptor model (Hille, 1992). It must be considered likely that vinpocetine acts in a similar manner and that the higher apparent affinity of the drug for the inactivated state reflects a slower dissociation rate from that state. Also consistent with this mechanism is the phenomenon of use dependence. Repetitive and brief membrane depolarizations force sodium channels to cycle through activation, inactivation, and repriming states. The ability of a given channel to follow such activity at high cycle rates is, primarily, a function of the rate of recovery from inactivation, the repriming rate. Native TTXr channels have been shown previously to display rapid repriming kinetics. The use-dependent block exerted by vinpocetine is likely to reflect the slow rate of dissociation of the drug from blocked channels during repolarization, effectively slowing the repriming rate. At low-frequency stimulations (e.g., 0.1 Hz) the drug off-rate is presumably fast enough to ensure recovery of the majority of the available channels before the next depolarizing pulse in the cycle. As the stimulation frequency is increased, the interpulse interval decreases accordingly and the result is manifest as a proportional increase in the drug-bound channels at the onset of the subsequent depolarization. The frequency-dependent component of vinpocetine's effects on rNaV1.8, although clear, is modest compared with that observed for other sodium channel blockers studied under the same conditions (X. W. Dong, unpublished observations). A previous study (Wei et al., 1997) that examined the effect of vinpocetine (40 μM) on cardiac myocyte sodium channels found no evidence of use-dependent block. This apparent contradiction may represent a genuine difference attributed to different channel isoforms or may reflect different experimental conditions. The manifestation of use-dependent block is a complex interaction between channel and drug kinetics, a simple change in drug concentration can result in a profoundly different use-dependent profile.

We describe vinpocetine as a potent (IC50 = ∼3 μM) rNaV1.8 channel blocker, although it is more meaningful to consider whether the affinity of the drug for the NaV1.8 channel is relevant in relation to the plasma levels attained in typical clinical settings. The reported Cmax values, achieved in human subjects after a 10-mg dose of vinpocetine, are around 30 to 60 ng ml–1 (Vereczkey et al., 1979; Lohmann et al., 1992), corresponding to around 100 to 200 nM. However, the drug as a very high volume of distribution and readily partitions into the brain (Gulyas et al., 1999) and, presumably, other fatty tissues that are likely to include the peripheral nerves. Hence, true tissue exposure levels may be somewhat higher than the circulating plasma level and, in any event, are within the range that might be expected to produce some attenuation of sodium channel function. It is somewhat harder to predict, with respect to therapeutic efficacy, the relative contribution of sodium channel block compared with the many other pharmacological actions of the drug. However, similar potencies have been claimed for an antioxidant action (Horvath et al., 2002) and phosphodiesterase E-1 inhibition (Chiu et al., 1988), suggesting that sodium channel block may impart a significant contribution to overall efficacy (Bonoczk et al., 2000).

In summary, the present data demonstrate that vinpocetine is capable of blocking rNaV1.8 sodium channels at concentrations that may be therapeutically relevant. The nature of these experiments reveal this to be a direct effect on the channel (probably the “local anesthetic” pharmacophore) and not secondary to an action on Ca2+ or K+ channels, although an action at either of these additional targets could contribute to an overall effect on neuronal excitability. The continued clinical use of the drug in the neurotrauma arena and the absence of reported serious side effects may warrant expansion of vinpocetine's clinical utility to examine a potential benefit in the control of neuropathic pain (Lai et al., 2000) and bladder hyperreflexia (Yoshimura et al., 2002) conditions that have been linked to inappropriate NaV1.8 sodium channel activity.

Footnotes

  • DOI: 10.1124/jpet.103.051086.

  • ABBREVIATIONS: TTX, tetrodotoxin; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; TTXs, tetrodotoxin sensitive; TTXr, tetrodotoxin resistant, wt, wild-type.

    • Received February 28, 2003.
    • Accepted April 22, 2003.

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Research ArticleNEUROPHARMACOLOGY

Vinpocetine Is a Potent Blocker of Rat NaV1.8 Tetrodotoxin-Resistant Sodium Channels

Xiaoping Zhou, Xiao-Wei Dong, James Crona, Maureen Maguire and Tony Priestley
Journal of Pharmacology and Experimental Therapeutics August 1, 2003, 306 (2) 498-504; DOI: https://doi.org/10.1124/jpet.103.051086
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