The present study was planned to investigate the action of pregabalin on voltage-dependent Ca2+ channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca2+ current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca2+ channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca2+ current charge blockade were obtained when pregabalin was tested in human or bovine chromaffin cells, which express very different percentages of VDCC types with respect to mouse chromaffin cells. These results demonstrate that the inhibitory action of pregabalin on VDCCs and exocytosis does not depend on α1 Ca2+ channel subunit types. Carbon fiber amperometric recordings of digitonin-permeabilized cells showed that neither the fusion pore nor the exocytotic machinery were targeted by pregabalin. Mitochondrial Ca2+ measurements performed with mitochondrial ratiometric pericam demonstrated that Ca2+ uptake or release from mitochondria were not affected by the drug. The selectivity of action of pregabalin might explain its safety, good tolerability, and reduced adverse effects. In addition, the inhibition of the exocytotic process in chromaffin cells might have relevant clinical consequences.
(S)-3-(aminomethyl)-5-methylhexanoic acid (pregabalin; PGB) is a drug indicated in the treatment of central and peripheral neuropathic pain and generalized anxiety disorder and in the adjunctive therapy of partial seizures in adults. Its mechanism of action has not been completely clarified. The inhibitory effect of pregabalin on voltage-dependent Ca2+ channels (VDCCs) after acute application of the drug has been reported previously (Dooley et al., 2002; Fink et al., 2002; McClelland et al., 2004). Pregabalin preferentially blocks Cav2.1 channels in rat neocortical slices (Dooley et al., 2002), human neocortical synaptosomes (Fink et al., 2002), and mice neurons of the calyx of Held (Di Guilmi et al., 2011).
In addition, many studies have documented the effect of pregabalin on neurotransmitter release. Pregabalin inhibits the release of glutamate in rat entorhinal synapses in vitro (Cunningham et al., 2004) and rat neocortical and hippocampal slices (Dooley et al., 2000a), noradrenaline (Dooley et al., 2000b), acetylcholine and serotonine (Dooley et al., 2000a,b: Brawek et al., 2008) in human and rat neocortical slices, glutamate in rodent neocortical slices (Quintero et al., 2011), GABA in human neocortical synaptosomes (Brawek et al., 2009), and capsaicin-evoked substance P and calcitonin gene-related peptide in rat spinal cord slices (Fehrenbacher et al., 2003). Pregabalin is a potent and selective ligand for α2δ-1 and α2δ-2 Ca2+ channel subunits (Li et al., 2011). Indeed, α2δ-1 subunits of VDCCs have been identified as the molecular target for the analgesic action exerted by pregabalin (Field et al., 2006), as well as for its inhibitory effect on glutamate release in rodent neocortical slices (Quintero et al., 2011). In addition to the action of pregabalin on α2δ subunits to inhibit release (Joshi and Taylor, 2006; Micheva et al., 2006; Quintero et al., 2011), it has been postulated that an independent mechanism to Ca2+ entry through VDCCs might be involved (Cunningham et al., 2004; McClelland et al., 2004; Micheva et al., 2006).
In fact, pregabalin uses the system L of amino acid transport across the plasma membrane to access the cytosol (Jezyk et al., 1999; Su et al., 2005), where it might act on different targets to generate its neuronal effects. In this sense, pregabalin could reach the axoplasm, act on the exocytotic machinery that controls neurotransmitter release, or accumulate into intracellular organelles such as mitochondria. Indeed, antiepileptic drugs such as topiramate have been shown to affect the SNARE-associated monoamine exocytotic mechanism (Okada et al., 2005). In addition, there has been increasing evidence supporting the association between mitochondrial oxidative stress and epilepsy (Waldbaum and Patel, 2010a,b; Folbergrová and Kunz, 2012). Several mutations in the electron transport chain associated with epilepsy have been described previously (Shoffner et al., 1990; Tryoen-Tóth et al., 2003; Kudin et al., 2009). Given that mitochondria can reach Ca2+ transients of millimolar concentration (Montero et al., 2000), the consequence of those mutations would be the inefficiency to buffer Ca2+ and, therefore, the uncontrolled elevation of cytosolic Ca2+. This increment in the cytosolic Ca2+ concentration will enhance neurotransmitter release, triggering epileptogenic action potentials at the postsynaptic level.
The goal of the present study was to further investigate the effect of pregabalin on VDCCs and also on additional targets such as the fusion pore formed between the plasma membrane and the secretory vesicle, the exocytotic apparatus, or the mitochondria. To this purpose, we used chromaffin cells of the adrenal gland, modified postganglionic sympathetic neurons innervated by the splanchnic nerve that mainly control the release of adrenaline to the bloodstream, to prepare muscle and cardiovascular systems to a situation of stress. Our study shows that pregabalin unselectively inhibited Cav1, Cav2.1, and Cav2.2 channels. This inhibitory effect was halved by l-Ile, which binds to the α2δ-auxiliary subunit of VDCCs. Furthermore, similar percentages of blockade were exerted by pregabalin on the VDCCs of chromaffin cells from murine, human, and bovine species, which express very different percentages of VDCC types. This means that the blockade exerted by pregabalin on VDCCs does not depend on the α1 subunit type of Ca2+ channels, but most probably on their α2δ auxiliary subunits. In addition, no intracellular effects on the fusion pore, the exocytotic machinery, or the mitochondria were observed here, which might explain the selectivity of action of this drug, its reduced adverse effects respect to other antiepileptic drugs, and its safety and tolerability.
Materials and Methods
Isolation and Culture of Murine, Human, and Bovine Chromaffin Cells.
Mice from 2 to 3 months old were used to obtain the adrenal glands. The mice were housed in the animal facility of the Universidad Autónoma de Madrid (UAM), Madrid Registry number ES-280790000097. The procedure of isolation and culture of cells was performed as reported previously (Pérez-Alvarez et al., 2011). The study protocol for the use of human chromaffin cells was approved by the Ethics Committees of the Hospital Ramón y Cajal and Universidad Autónoma de Madrid. Adrenal glands were harvested from two organ donors who had died of cerebral hemorrhage. The method of isolation and culture of the human chromaffin cells was performed as reported previously (Pérez-Alvarez and Albillos, 2007). Bovine chromaffin cells from the adrenal glands of adult cows were isolated according to the method described previously (Moro et al., 1990).
For the perforated-patch whole-cell recordings, the external solution was 5 mM CaCl2, 100 mM NaCl, 45 mM tetraethylammonium-Cl, 5.5 mM KCl, 1 mM MgCl2, 0.2 mM d-tubocurarine, 0.002 mM tetrodotoxin, 0.0002 mM apamin, 10 mM HEPES, and 10 mM glucose, pH 7.4 adjusted with NaOH. The intracellular solution composition was 145 mM Cs-glutamate, 8 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 0.5 mM amphotericin B (Sigma-Aldrich, Madrid, Spain), and the pH was adjusted to 7.2 with CsOH. An amphotericin B stock solution was prepared every day at a concentration of 50 mg/ml in dimethyl sulfoxide and kept protected from light. The final concentration of amphotericin B was prepared by ultrasonicating 10 μl of stock amphotericin B in 1 ml of Cs-glutamate internal solution in the dark. Pipettes were tip-dipped in amphotericin-free solution for several seconds and back-filled with freshly mixed intracellular amphotericin solution.
The perfusion system for drug application consisted of a multibarrelled glass pipette positioned close to the cell under study, which allowed for the complete exchange of solutions near the cell within 100 ms. The level of the bath fluid was continuously controlled by a home-made fiber optics system coupled to a pump that removed excess fluid.
Electrophysiological measurements were made by using an EPC-10 amplifier and Pulse software (HEKA, Lambrecht/Pfalz, Germany) running on a computer. Pipettes of 2- to 3-MΩ resistance were pulled from borosilicate glass capillary tubes, partially coated with wax, and fire-polished. Only recordings in which the leak current and access resistance were lower than 20 pA and 20 MΩ, respectively, were accepted. Cell membrane capacitance (Cm) changes as an index of exocytosis were estimated by the Lindau-Neher technique implemented in the Sine+DC feature of the Pulse lock-in software. A 1-kHz, 70-mV peak-to-peak amplitude sinewave was applied at a holding potential (Vh) of −80 mV.
All toxins used to pharmacologically characterize the Ca2+ channels were purchased from Peptide Institute Inc. (Osaka, Japan), except the dihidropyridine nifedipine (Nife), which was purchased from Sigma-Aldrich. Pregabalin was always perfused for at least 15 min in every experiment.
Experiments were performed at room temperature (22–24°C). Analysis of data was conducted with IGOR Pro software (Wavemetrics, Lake Oswego, OR). The nonspecific background current and Cm recorded under 200 μM CdCl2 were subtracted off-line from Ca2+ current and Cm traces. Unless otherwise stated, data are given as the mean ± S.E.M. Data were compared by using paired or unpaired Student's t test.
Carbon fiber electrodes were prepared by cannulating a 10-μm-diameter carbon fiber in polyethylene tubing (o.d., 1 mm; i.d., 0.5 mm). The carbon fiber tip was glued into a glass capillary for mounting on a patch-clamp headstage and back-filled with 3 M KCl to connect to the Ag/AgCl wire, which was kept at +700 mV. Amperometric currents were recorded by using an EPC-10 amplifier and Pulse software running on a computer. The sampling rate was 14.5 kHz. Samples were digitally filtered at 2 kHz. The sensitivity of the electrodes was routinely monitored before and after the experiments by using 50 μM adrenaline as standard solution. Only fibers that rendered 200 to 300 pA of current increment after 50-μM adrenaline pulse were used for the experiments. The tip of the fiber was recut for each experiment and calibrated again.
The bath solution was composed of 139 mM K-glutamate, 0.2 mM EGTA, 20 mM PIPES (1,4-piperazinediethanesulfonic acid), 2 mM ATP, and 2 mM MgCl2, pH 6.5, and the permeabilitation solution contained 139 mM K-glutamate, 5 mM EGTA, 20 mM PIPES, 2 mM ATP, 2 mM MgCl2, 20 μM digitonin, and 10 μM free Ca2+, pH 6.5.
Analysis of Amperometric Data.
Spike analysis was performed by using IGOR Pro software and macros that allow the analysis of single events and the rejection of overlapping spikes (Segura et al., 2000; Mosharov and Sulzer, 2005). The macros from those references were used to analyze the amperometric spikes and “foot” of the spikes, respectively. A threshold of 4.5 times the first derivative of the noise standard deviation was calculated to clearly detect amperometric events. Then, among the events whose first derivative was above this threshold, only those showing one peak and one rising and falling phase were considered as single spikes. To minimize variability among cells, the overall mean of average spike values recorded in several single cells was used. Unpaired Student's t test was used to compare the data.
Mitochondrial Ca2+ Measurements.
Mitochondrial Ca2+ measurements were performed by using mit-r-pericam (Nagai et al., 2001), transduced in chromaffin cells using the pHSVmit-pericam amplicon vector (VAN4), derived from herpes simplex virus type 1. Packaging (Lim et al., 1996) and titering of the amplicon with a titer of 1,02 × 107 infectious vector units × ml−1 was performed as described previously (Chamero et al., 2008). Five microliters of the virus suspension was added to each well containing 600 μl of Dulbecco's modified Eagle's medium (DMEM). After gentle shaking, the plate was introduced into the culture chamber for 90 min, and later on 1 ml of DMEM was added to each well. Experiments were performed 12 to 24 h after this procedure.
Cells expressing mit-r-pericam were placed in the perfusion chamber in standard medium containing 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4. Standard medium containing 0.5 mM EGTA instead of CaCl2 was then perfused for 1 min, followed by a 1-min perfusion of intracellular medium (130 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM K3PO4, 0.2 mM EGTA, 1 mM ATP, 20 μM ADP, 2 mM succinate, and 20 mM HEPES, pH 7) containing 20 μM digitonin. Intracellular medium without digitonin but with 30 μM free Ca2+ was perfused for 5 min. In the experiments where the effect of pregabalin was evaluated, all solutions contained 30 μM of this drug.
To measure mitochondrial Ca2+ transients, MetaFluor software (Molecular Devices, Sunnyvale, CA), a Nikon (Tokyo, Japan) Eclipse TE2000-S microscope with a Nikon S-Fluor objective (40×, NA 1.30), coupled to a monochromator (Cairn Research, Kent, UK), and a cooled Orca ER digital camera (Hamamatsu Photonics, Hamamatsu City, Japan) were used. A 506 dicroic mirror and a 536/40 emission filter were used. After appropriate regions of interest were selected, changes in mitochondrial Ca2+ signal were monitored by taking alternative epifluorescence images at 415- and 485-nm excitation wavelengths and calculating the 485/415 ratio. The rate of image acquisition was 2 Hz.
Pregabalin Inhibits VDCCs and Exocytosis in Mouse Chromaffin Cells.
To investigate the possible effect of the acute application of pregabalin on VDCCs and exocytosis in mouse chromaffin cells of the adrenal gland, simultaneous measurements of Ca2+ currents and Cm were performed in the perforated-patch configuration of the patch-clamp technique. Mouse chromaffin cells have been shown to possess all of the VDCCs reported in neurons and Cav1, Cav2.1, Cav2.2, and Cav2.3 channels (Albillos et al., 2000; Aldea et al., 2002), as well as the transcript for α2δ subunits (García-Palomero et al., 2000). We started testing the effect of increasing concentrations of pregabalin, from 3 to 300 μM, on the Ca2+ currents elicited by 200-ms depolarizing pulses to the voltage peak current. Pregabalin inhibited VDCCs in a dose-dependent manner (Fig. 1A). Because the plasma membrane concentration reached by a single therapeutic dose of 600 mg of pregabalin is approximately 30 μM (Arroyo et al., 2003; Beydoun et al., 2005) this concentration was chosen for the experiments of this study.
When 30 μM pregabalin was perfused on top of voltage-clamped mouse chromaffin cells the inhibition of Ca2+ currents and exocytosis developed gradually, reaching a stable value after 10 to 15 min. The percentage of inhibition exerted by pregabalin on the Ca2+ charge density and Cm was 33.4 ± 2.4% (n = 38) and 39 ± 4% (n = 36), respectively, in mouse chromaffin cells. Figure 1 shows the time course of Ca2+ charge blockade and exocytosis induced by pregabalin (B) and the original recordings of Ca2+ currents (C) and exocytosis (D) under control conditions, pregabalin, and CdCl2 perfusion.
The Inhibition of Pregabalin on VDCCs Is Mediated by α2δ Auxiliary Subunits in Mouse Chromaffin Cells.
Next, experiments were conducted to investigate whether the observed inhibitory effect of pregabalin on VDCCs in mouse chromaffin cells was exerted through its interaction with α2δ subunits, as reported previously in other cell systems (Thurlow et al., 1993; Cunningham et al., 2004; Joshi and Taylor, 2006; Micheva et al., 2006; Quintero et al., 2011). To achieve that purpose, l-Ile, which binds to the α2δ subunit of VDCCs (Brown et al., 1998; Dooley et al., 2007; Brawek et al., 2009), was tested. The time course of Ca2+ charge blockade and the original recordings of the Ca2+ currents after perfusion of l-Ile or l-Ile in the presence of pregabalin are shown in Fig. 2, A and B, respectively. l-Ile diminished the Ca2+ charge density by 30 ± 4.8% (n = 11). The subsequent addition of pregabalin in the presence of l-Ile exerted still an additional blockade of 15.3 ± 1.1% (n = 8), which might be caused by an α2δ-independent inhibitory mechanism. However, the effect of pregabalin on VDCCs after l-Ile treatment was significantly diminished with respect to its action in the absence of l-Ile (33.4 ± 2.4%; data from Fig. 1A), showing that pregabalin inhibits VDCCs in chromaffin cells through α2δ-dependent and -independent mechanisms.
Pregabalin Blocks Cav1, Cav2.1, and Cav2.2 Channels in Mouse Chromaffin Cells.
To assess whether pregabalin exhibits any specificity to inhibit a certain Ca2+ channel type and to analyze the interaction of pregabalin with α2δ subunits, different selective Ca2+ channel blockers were perfused before pregabalin. Nife (3 μM), 200 nM ω-agatoxin IVA (ω-Aga IVA), and 1 μM ω-conotoxin GVIA (ω-Ctx GVIA) were used to block Cav1, Cav2.1, and Cav2.2 channels, respectively. The action of pregabalin on Cav2.3 channels could not be investigated, because SNX-482, the selective Ca2+ channel blocker available to inhibit Cav2.3 channels, also inhibits Cav2.1 channels in chromaffin cells (Arroyo et al., 2003).
After Ca2+ currents inhibited by the blockers reached the steady state, pregabalin was added to the perfusion solution to evaluate whether the inhibitory effect of the drug on Ca2+ currents and exocytosis had been modified by the corresponding blocker. The effect of pregabalin after the perfusion of the Ca2+ channel blocker was compared with its effect when it was first perfused in a different set of experiments (data from Fig. 1). Figure 3 shows the original traces of Ca2+ currents (A–C) and Cm (D–F) obtained under each condition. After perfusion of Nife, ω-Ctx GVIA, and ω-Aga IVA, the additional inhibition exerted by pregabalin on Ca2+ currents and exocytosis was 10 ± 1% (n = 9) and 12.4 ± 2% (n = 3), 10 ± 2% (n = 8) and 8.6 ± 4% (n = 4), and 20.5 ± 4% (n = 9) and 28.6 ± 4.6% (n = 7), respectively, which notably differ from the inhibition achieved by pregabalin when it was first applied (33.4 ± 2.4 and 39 ± 4% for Ca2+ currents and exocytosis, respectively).
Identical Percentages of Ca2+ Charge Density and Exocytosis Blockade Are Achieved by Pregabalin in Human and Bovine Chromaffin Cells with Respect to Mouse Chromaffin Cells.
Martin et al. (2002) reported that the sensitivity of gabapentin, an structural and functional analog of pregabalin, to Ca2+ currents depends on the relative abundance of accessory Ca2+ channel subunits, in particular β2 and α2δ-2 subunits, expressed in dorsal root ganglion (DRG) neurons. α1 Ca2+ channel subunits did not seem to influence the inhibition of Ca2+ currents by gabapentin in these cells. Gabapentin exerted an unselective inhibition on VDCC types in DRG neurons (Sutton et al., 2002), similarly as it was obtained with pregabalin in the present study. Thus, we found it interesting to investigate whether pregabalin also inhibited Ca2+ channels in chromaffin cells independently of the α1 Ca2+ channel type. Therefore, we analyzed the inhibitory effect of pregabalin on chromaffin cells that express different percentages of Ca2+ channel types to those of the murine species, such as human or bovine species. Whereas mouse chromaffin cells express 45, 14, 25, and 16% for Cav1, Cav2.1, Cav2.2, and Cav2.3 channels, respectively (Pérez-Alvarez et al., 2011), human chromaffin cells, which could be sorted into two groups of similar size according to the predominance of either Cav2.1 or Cav2.2 channels, express 14.5 and 17.7% for Cav1 and Cav2.3 channels, respectively, and 46 and 20% or 18 and 51% for Ca2.1 and Cav2.2 channels, in cells with predominance of Cav2.1 and Cav2.2, respectively (Pérez-Alvarez et al., 2008). Bovine chromaffin cells show percentages of Ca2+ channel types that approach more like the human species, accounting for approximately 15, 30, 40, and 15% for Cav1, Cav2.1, Cav2.2, and Cav2.3 channels, respectively (Albillos et al., 1993, 1996; García-Palomero et al., 2000).
The time course of Ca2+ charge blockade exerted by 30 μM pregabalin and the original traces of the Ca2+ currents under control and pregabalin conditions in human and bovine chromaffin cells are shown in Fig. 4, A and B, respectively. The blockades of the Ca2+ charge density obtained were similar to those achieved in mouse, amounting to 33.6 ± 7 and 32.6 ± 4%, respectively, in human (n = 7) and bovine (n = 9) chromaffin cells. In addition, the exocytotic process was blocked at a similar extent in human (36.7 ± 12.5%; n = 7) and bovine (40 ± 7%; n = 6) chromaffin cells. These data suggest that the inhibitory action of pregabalin on VDCCs does not depend on α1 Ca2+ channel types, as reported previously for gabapentin in DRG neurons (Martin et al., 2002).
Pregabalin Does Not Act on the Fusion Pore or the Exocytotic Machinery in Mouse Chromaffin Cells.
Carbon fiber amperometric recordings were performed to assess the action of pregabalin on the fusion pore or intracellular exocytotic machinery. Chromaffin cells possess all of the same elements that build the secretory apparatus in neurons (Neher, 1998), and the released catecholamines can be detected at the single level by using the carbon fiber amperometric technique (Wightman et al., 1991). Each vesicle that exhibits exocytosis generates a spike with kinetic properties that can be determined. The release of neurotransmitter molecules through the narrow fusion pore formed after the fusion of a chromaffin vesicle with the plasma membrane appears as a foot signal that precedes the main body of the amperometric spikes (Chow et al., 1992; Albillos et al., 1997). If pregabalin interacts with some element of the exocytotic apparatus, this would be reflected in the number or the kinetic parameters of the spikes or foot signals.
To avoid any effect derived from the blockade of pregabalin on VDCCs, cells were permeabilized with digitonin in a 10-μM free Ca2+ solution to allow Ca2+ entry into the cytosol. Original recordings of the spikes obtained under control conditions and after incubation with 30 μM pregabalin for 1 h are shown in Fig. 5, A and B, respectively. Typical spikes recorded under both conditions (marked by *) are displayed at the right of the figure.
The average number of foot signals obtained in control and pregabalin-treated cells was similar (Table 1). The following parameters were determined for each foot signal: Imax (maximal amplitude), t (duration), Q (charge, expressed as pC or number of molecules), and n foot (number of foot signals). The parameters of the amperometric foot signals of the spikes obtained in the digitonin-treated cells were unchanged in the presence of pregabalin, as shown in Table 1. This means that pregabalin does not act on the fusion pore formed between the secretory vesicle and the plasma membrane to release the neurotransmitter content stored at the chromaffin vesicle.
The average number of spikes obtained in control or pregabalin-treated cells was identical. The kinetic parameters of the individual amperometric spikes were not modified after pregabalin treatment (Table 2). The following parameters were determined: Imax (peak amplitude), Q (charge), m (ascending slope, calculated from the linear portion of the trace between 25 and 75% of the Imax), t1/2 (half-width or duration of the amperometric signal at 50% of its peak amplitude), and tp (time to peak, time from the start of the spike until the peak in seconds) (Fig. 6A). In addition, the frequency histograms of the different parameters under both conditions did not vary after the treatment with the drug, showing that pregabalin did not interfere with any component of the exocytotic machinery to modulate neurotransmitter release (Fig. 6B).
Positive control experiments of the negative effect of pregabalin on the exocytotic apparatus were performed by using the anti-SNARE tetanus toxin. This toxin has been shown to inhibit exocytosis in chromaffin cells (Penner et al., 1986; Bittner and Holz, 1988; Xu et al., 1998). Digitonin-permeabilized cells were incubated for 3 min with 300 nM tetanus toxin, resulting in a marked reduction of the number of amperometric spikes (227 spikes in control versus 96 spikes in tetanus toxin-treated cells; n = 6 cells; p = 0.002) (Supplemental Fig. 1).
Pregabalin Does Not Interfere with the Mitochondrial Ca2+ Fluxes in Mouse Chromaffin Cells .
Pregabalin uses the system L of amino acid transport across the plasma membrane to generate its neuronal effects (Jezyk et al., 1999; Su et al., 2005). Thus, pregabalin might enter the cell and interact with mitochondria, an organelle implied in epilepsy. Mitochondrial Ca2+ transients were monitored by using mit-r-pericam. Digitonin-permeabilized cells were perfused with a solution containing 30 μM Ca2+ to analyze how mitochondria uptakes and releases Ca2+ under control conditions (n = 14 cells) and after 20-min pretreatment with pregabalin (n = 15 cells) (Fig. 7).
The following kinetic parameters of the mitochondrial Ca2+ signal were determined: tpeak (time to peak, time from the start of the rise until the maximal value of the fluorescence ratio in seconds), increase (increment of the fluorescence ratio from the basal line to the maximal value), decrease at 150 s (decrement of the fluorescence ratio from the maximal value until 150 s later), and τ (time constant) (Fig. 7A).
The release of Ca2+ from mitochondria could be well fitted by a double exponential. None of the kinetic parameters of the mitochondrial Ca2+ signal were modified after pregabalin treatment (Table 3), reflecting that pregabalin does not affect either mitochondrial uptake or release. Thus, mitochondria is not affected by pregabalin in inhibiting neurotransmitter release.
Positive control experiments of the negative effect of pregabalin on the mitochondrial Ca2+ fluxes were performed by using Ru-360, a selective inhibitor of the mitochondrial Ca2+ uniporter (Matlib et al., 1998; Santo-Domingo and Demaurex, 2010). Digitonin-permeabilized cells that expressed mit-r-Pericam were perfused for 15 min with 1 μM Ru-360 (n = 7), which completely abolished the mitochondrial Ca2+ uptake observed in control cells (n = 4) (Supplemental Fig. 2).
The mechanism of action of pregabalin to inhibit Ca2+ channels and consequently neurotransmitter release has been reported to be mediated by the α2δ-1 subunit of VDCCs (Field et al., 2006; Quintero et al., 2011). Other targets different from these auxiliary subunits have been posed (Cunningham et al., 2004; McClelland et al., 2004; Micheva et al., 2006). Indeed, some intracellular additional mechanism might explain pregabalin's inhibitory action on neurotransmitter release, because pregabalin uses the system L of amino acid transport across the plasma membrane to enter into the cytosol (Su et al., 2005). Therefore, it is plausible that pregabalin interacts with cellular structures such as the fusion pore, the exocytotic machinery, or intracellular organelles such as the mitochondria, involved in the epileptogenic processes, to reduce neurotransmitter release.
In the present study, high-resolution techniques were used to investigate possible functional interactions of pregabalin with VDCCs, fusion pore, exocytotic machinery, and mitochondria. The main findings in the present study are: 1) pregabalin inhibited VDCCs and exocytosis in murine, human, and bovine chromaffin cells of the adrenal gland, thus limiting the release of catecholamines to the bloodstream; 2) the inhibition of pregabalin on VDCCs is partially mediated by α2δ auxiliary subunits of Ca2+ channels; 3) pregabalin inhibition of VDCCs does not depend on α1 Ca2+ channel types; and 4) pregabalin does not interfere with the fusion pore, the exocytotic machinery, or the handling of Ca2+ by mitochondria.
In relation to the inhibitory effect of pregabalin on VDCCs, we first investigated whether it was mediated through α2δ auxiliary subunits. Transcripts for α2δ auxiliary subunits have been reported in bovine chromaffin cells (García-Palomero et al., 2000). The partial retrieval of blockade achieved with l-Ile in mouse chromaffin cells, also reported in other cell systems (McClelland et al., 2004; Di Guilmi et al., 2011), shows that pregabalin acts through α2δ-dependent and -independent mechanisms to regulate Ca2+ channels and neurotransmitter release.
Extensive research has been performed on the VDCC type targeted by gabapentin. This drug was found to preferentially inhibit Cav1 (Stefani et al., 2001), Cav2.1 (Bayer et al., 2004), and Cav2.2 (Sutton et al., 2002) channels. Pregabalin has been reported to inhibit Cav2.1 channels (Dooley et al., 2002; Fink et al., 2002; Di Guilmi et al., 2011). In the present study we found that pregabalin inhibits Cav1, Cav2.1, and Cav2.2 channel types. However, Martin et al. (2002) reported that the inhibition of gabapentin in DRG neurons depended on the expression of β2 and α2δ-2 subunits, but not α1 subunits. The unselective action of pregabalin in chromaffin cells prompted us to investigate whether, indeed, the effect of this drug did not depend on the α1 types of Ca2+ channels. Therefore, we evaluated the effect of pregabalin on species of chromaffin cells that express very different percentages of Ca2+ channel types. Murine, human, and bovine chromaffin cells, where Cav1, Cav2.1, or Cav2.2, and Cav2.2 channels, respectively, predominate, were challenged with 30 μM pregabalin and exhibited identical amounts of Ca2+ charge density blockade. Therefore, the data obtained in the present study further support the idea that pregabalin action depends primarily on the amount and type of expressed α2δ, but not α1, subunits.
The experiments designed to investigate the Ca2+ channel type targeted by pregabalin, perfusing the Ca2+ channel blocker first, reflect that once the α1 subunit is targeted by the Ca2+ antagonist the action of the α2δ subunit ligand is mostly prevented. In the case of Cav1.2 channels, this idea is supported by previous data showing that α2δ subunits bind to the binding site for dihydropyridines in loop S5–S6 of the α1Cav1.2 channel (Gurnett et al., 1997). Thus, the blockade of the channel by Nife would further prevent the regulatory action of an α2δ subunit already bound to pregabalin.
On the other hand, the possibility that pregabalin may be acting on different targets to Ca2+ channels has been proposed previously (Cunningham et al., 2004; McClelland et al., 2004; Micheva et al., 2006). In the present study, the action of pregabalin on the fusion pore formed between the plasma membrane and the secretory vesicle, the exocytotic apparatus, or the mitochondria were also evaluated. Indeed, the anticonvulsant topiramate has been shown to affect the SNARE-associated monoamine exocytotic mechanism (Okada et al., 2005). To investigate the action of pregabalin on the fusion pore and the exocytotic machinery, cells were treated with digitonin to avoid the effect of pregabalin on VDCCs, so that Ca2+ would access the cytosol through pores formed by the detergent in the plasma membrane. If the fusion pore or any protein of the exocytotic machinery would be affected by the drug, a significant change in the number or the kinetic parameters of foot signals or spikes, recorded with the carbon fiber amperometric technique in single cells, would be detected. However, this was not the case, reflecting that pregabalin does not act on these cellular structures.
It has been reported that mitochondria is largely involved in epilepsy (Folbergrová and Kunz, 2012). This organelle can transiently store high Ca2+ concentrations (Montero et al., 2000), and therefore, its dysfunction would trigger an increase of cytosolic Ca2+ and the enhancement of neurotransmitter release. This idea prompted us to investigate the possible functional interaction between pregabalin and the mitochondria, which might be affecting the mitochondrial handling of Ca2+. However, the Ca2+ uptake or release by mitochondria, measured with mit-r-Pericam in permeabilized cells challenged with 30 μM free Ca2+, was identical, showing that pregabalin does not act on the mitochondrial Ca2+ uniporter or the Na+/Ca2+ exchanger.
Our study reports the effect of pregabalin on VDCCs of chromaffin cells of the adrenal gland, the main source of adrenaline released to the bloodstream, an effect that might have clinical consequences. Indeed, it has been reported that pregabalin treatment improved heart rate variability in patients with painful diabetic neuropathy (Jiang et al., 2011). An increased resting heart rate is frequently observed in diabetic patients, most likely because of vagal cardiac neuropathy that results in increased cardiac sympathetic activity. The tachycardia may be followed by a decrease in heart rate and, ultimately, by a fixed heart rate caused by progressive dysfunction of the cardiac sympathetic nervous system. Therefore, pregabalin, by decreasing the exocytotic process, and consequently the adrenaline release, would initially diminish reflex tachycardia, thus stabilizing heart rate.
On the other hand, a decompensation of chronic heart failure associated with pregabalin in patients with neuropathic pain has been observed, probably caused not only by the effect of pregabalin on VDCC of myopathic ventricles (Murphy et al., 2007), but also, as shown in the present study, by the decrease of the exocytotic process, and consequently of adrenaline release, yielded by the drug in human chromaffin cells.
In conclusion, our data show that pregabalin inhibits exocytosis by blocking Cav1, Cav2.1, and Cav2.2 channels through α2δ-dependent and -independent pathways. These mechanisms lead to the inhibition of Ca2+ channels to a certain extent, independently of the amount and type of α1 Ca2+ channel types. The inhibition of Ca2+ channels by pregabalin provokes the inhibition of the exocytotic process, which might possess clinical relevance. Finally, pregabalin does not act on other targets related with exocytosis or Ca2+ homeostasis such as the fusion pore, the exocytotic machinery, and the mitochondria. This selective mechanism of action of the drug may contribute to its safety, good tolerability, and lack of adverse effects.
Participated in research design: Albillos.
Conducted experiments: Hernández-Vivanco, Pérez-Alvarez, Caba-González, Moreno-Ortega, Cano-Abad, Ruiz-Nuño, and Carmona-Hidalgo.
Contributed new reagents or analytic tools: Alonso.
Performed data analysis: Hernández-Vivanco.
Wrote or contributed to the writing of the manuscript: Albillos.
We thank Dr. Antonio G. García for the supply of bovine chromaffin cells and Dr. María Pérez Páramo and Dr. Luis Miguel Gutiérrez for comments on the manuscript.
This work was supported by the Ministerio de Ciencia e Innovación [Grants BFU2008-01382, BFU2011-27690 (to A.A.), BFU2010-17379 (to M.T.A.)]; and Pfizer S.L.U. (to A.A.). A.H.-V. holds a fellowship from the Universidad Autónoma de Madrid, and A.J.M.-O. holds a fellowship from the Ministerio de Educación.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- (S)-3-(aminomethyl)-5-methylhexanoic acid (pregabalin)
- voltage-dependent calcium channel
- mitochondrial ratiometric pericam
- soluble N-ethylmaleimide-sensitive factor attachment protein receptor
- ω-Aga IVA
- ω-agatoxin IVA
- ω-Ctx GVIA
- ω-conotoxin GVIA
- dorsal root ganglion.
- Received December 5, 2011.
- Accepted April 24, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics