Migraine is a frequent and often disabling disease. Treatment is unsatisfactory in many patients. A disturbed dynamic balance between excitatory and inhibitory signal processing with enhanced cortical activity probably underlies common forms of migraine. Presynaptic voltage-gated Ca2+ channels are critical determinants of neurotransmitter release and also contribute to trigeminovascular signal transduction. Because clinical evidence exists for migraine-prophylactic actions of Petasites hybridus extracts, we investigated whether petasins comprising the main constituents of the extract inhibit currents through presynaptic Cav2.1 channels expressed in Xenopus laevis oocytes. P. hybridus extract (0.02 mg/ml), petasin, neopetasin, isopetasin, S-petasin, and iso-S-petasin (50 μM) were weak tonic blockers of Cav2.1-mediated barium currents (IBa) during infrequent depolarizations (0.1 Hz), but their inhibitory potency increased at higher stimulation rates (1 Hz), indicating preferential block of open and/or inactivated channels. Sulfur-containing compounds (S-petasin, Iso-S-petasin) were the most potent significantly promoting the accumulation of Cav2.1 channel in inactivated states during pulse trains (IBa decrease during 1-Hz pulse trains: control, 45%, S-petasin, 79%; iso-S-petasin, 80%). For the Eucalyptus williamsiania sesquiterpenes α- and γ-eudesmol, a comparable use-dependent inhibition was found in addition to a tonic block component. α-Eudesmol and petasins accelerated the voltage-dependent inactivation of Cav2.1 channels during depolarizations. We demonstrate that S-petasin, iso-S-petasin, and eudesmol are Cav2.1 channel inhibitors preferentially acting as use-dependent channel blockers and with the sulfur-containing substituent in position 3 of the petasins serving as important functional feature. The Cav2.1-inhibitory properties of these petasins may contribute to migraine-prophylactic properties described for P. hybridus extracts.
Depolarization-induced Ca2+ ion influx through voltage-gated Ca2+ channels (VGCCs) supports many physiological processes in electrically excitable tissues. This includes muscle contraction, cardiac pacemaking, neurotransmitter and hormone release, synaptic plasticity, and sensory cell function (Koschak and Striessnig, 2008). Five different types of VGCCs with different pharmacological properties evolved to support these diverse functions. So far, only the pharmacological block of L-type (Cav1) Ca2+ channels (LTCCs) is exploited for therapy (treatment of hypertension and myocardial ischemia). Clinically used LTCC blockers belong to different chemical classes distinguished by their mechanism of channel block. The affinity for clinically used LTCC blockers is determined by the different conformational states (resting, open, inactivated) of the channel (Bean, 1984; Hering et al., 1998). Phenylalkylamines (like verapamil) and benzothiazepines [like (+)-cis-diltiazem] exhibit high potency when channels are depolarized more frequently (use-dependent block). This is explained by a model in which these drugs gain intracellular access to their binding site through the open channel and then promote channel inactivation (Hering et al., 1998; Beyl et al., 2007). In contrast, block by dihydropyridines (like isradipine) shows no use dependence but increases at more depolarized membrane potentials by preferentially interacting with inactivated channel states (voltage-dependent block; Bean, 1984; Berjukow and Hering, 2001). These differences in state-dependent inhibition result in different in vivo pharmacological effects and therapeutic indications (Striessnig, 1999; Triggle, 2007).
The pharmacological inhibition of non-LTCCs also appears as an attractive therapeutic concept. For example, the N-type (Cav2.2) channel blocker ziconotide is licensed to treat neuropathic pain. Unfortunately, therapy is limited by neurological side effects. These may be caused by its state-independent (Feng et al., 2003) inhibitory effects blocking both slowly and rapidly firing neurons. Use-dependent N-type channel blockers are currently developed that are likely to exhibit a more favorable side-effect profile by preferential inhibition of rapidly firing neurons in the pain pathway.
Migraine headache represents a frequent pain disorder in which primary central nervous system dysfunction leads to trigeminovascular activation, central sensitization, and enhanced pain sensation (Pietrobon and Striessnig, 2003; Burstein et al., 2004; Goadsby, 2007). Although the neurobiology and genetics of common migraine are not fully understood, rare inherited forms of migraine indicate that changes in signal transduction that increase synaptic glutamatergic activity can favor cortical spreading depression known to underlie migraine aura (Pietrobon, 2005; Goadsby, 2007). Although other mechanisms leading to disturbances in the fine-tuning of the dynamic balance between excitatory and inhibitory signal processing probably underlie common forms of migraine with and without aura (Pietrobon and Striessnig, 2003; Coppola et al., 2007; Brighina et al., 2009), experimental evidence exists suggesting that presynaptic P/Q-(Cav2.1) and N-type Ca2+ channels contribute to nociceptive synaptic transmission of trigeminovascular neurons (Akerman et al., 2003; Shields et al., 2005; Xiao et al., 2008). Moreover, excitatory and inhibitory neurotransmitter release in the brain critically depends on the activity of these presynaptic Ca2+ channels. Therefore, it is possible that P/Q- and/or N-type channel inhibitors reduce nociceptive signaling of the trigeminovascular neurons and perhaps also counteract enhanced cortical activity observed in patients with common forms of migraine, thus exerting antimigraine effects.
Because of the frequent lack of efficacy and adverse events of existing migraine medications, alternative treatments, including the identification of natural compounds, are sought. Although data about their efficacy and safety from long-term clinical studies are limited, some evidence points to short-term migraine-preventive effects of herbal preparations from Tanacetum parthenium (feverfew; Murphy et al., 1988; Pfaffenrath et al., 2002) and from Petasites hybridus (butterbur; Grossman and Schmidramsl, 2001; Diener et al., 2004; Lipton et al., 2004). Petasins comprise at least 20% of the constituents in the preparations of P. hybridus used in clinical trials, suggesting that these sesquiterpenes represent the active principle. Low micromolar concentrations of petasins have been shown to inhibit L-type VGCCs in smooth and cardiac muscle (Wang et al., 2001, 2002, 2004; Esberg et al., 2003). Therefore, it is possible that they are also inhibitors of neuronal Cav2.1 channels. In this case, this pharmacological effect may contribute to the antimigraine activity and may justify further clinical development of pure petasins rather than plant extracts. α-Eudesmol is an example for a natural compound inhibiting P/Q-type currents in rat cerebellar Purkinje neurons (Asakura et al., 1999).
Here, we characterize the state-dependent inhibitory effect of a P. hybridus extract and five different petasins on P/Q-type VGCCs. Recombinant Cav2.1 channels were used for these studies to directly prove the pharmacological modulation of this channel isoform by these natural compounds. Their effects were compared with the natural sesquiterpene α-eudesmol and its β- and γ-isomers.
Materials and Methods
Materials. HPLC-grade solvent (acetonitrile) was purchased from Acros Organics (Fairlawn, NJ) and Merck (Darmstadt, Germany), HPLC-grade water was obtained from a Nanopure system (Dubuque, IA), and organic solvents were distilled according to standard procedures. Salts for the oocyte culture media, recording solution, and microelectrode filling solution were purchased from Sigma-Aldrich (St. Louis, MO).
Plant Material.P. hybridus compounds were isolated from a fluid CO2 extract of P. hybridus foliage free of pyrrolizidine alkaloids and rich in petasins and S-petasins obtained from a standardized, commercially available source. α-, β-, and γ-Eudesmols were isolated from Eucalyptus williamsiania essential oil (eudesmols accounting for approximately 70% of the oil), a kind gift from Dr. J. Brophy (University of New South Wales, Sydney, NSW, Australia). Pure compounds were isolated as described previously (Debrunner and Neuenschwander, 1994). Purity of all isolated compounds was determined by HPLC or gas chromatography and was >90% for isopetasins, >95% for petasins and S-petasins, and >98% for β- and γ-eudesmol. Purity of α-eudesmol was 86% and contained 10% γ-eudesmol.
Expression of Cav2.1 VGCCs in Xenopus laevis Oocytes. Capped run-off poly(A)+ cRNA transcripts from XbaI-, HindIII-, and NotI-linearized cDNA templates, respectively, were synthesized according to Krieg and Melton (1984). Oocytes were harvested from an MS 222-anesthetized female X. laevis. One- to 2-day-old oocytes were injected (Microinjector 510-X and Nanoject II; Drummond Scientific, Broomall, PA) with cRNAs encoding human Cav2.1 α1 subunits (5–10 ng) together with β3 (1–3 ng) and α2δ1 (3–6 ng) subunits as described previously (Kraus et al., 1998; Wappl et al., 2002). Oocytes were incubated at 18°C in ND96 solution (1.8 mM CaCl2, 1 mM MgCl2, 2 mM KCl, 96 mM NaCl, 5 mM HEPES, pH 7.5, with NaOH) before recording.
Electrophysiological Recordings. Two-electrode voltage-clamp experiments were performed as described previously (Kraus et al., 1998; Wappl et al., 2002). One to 2 days after cRNA injection, a Ba2+ inward current through CaV2.1 channel complexes (IBa) was recorded at room temperature using a Turbo Tec 01C amplifier (NPI Electronic GmbH, Tamm, Germany) and Digidata 1322 digitizer (Molecular Devices, Sunnyvale, CA). Data acquisition and analysis were performed with the pClamp software package version 9.2 (Axon Instruments). Contribution of endogenous IBa was quantified using β3/α2δ1-injected oocytes. Cav2.1α1/β3/α2δ1-injected oocytes were only used for analysis if peak IBa were at least 10 times larger than endogenous currents and did not exceed 1.9 μA. Recording electrodes had resistances between 0.5 and 2 MΩ and were filled with solution containing 2.8 M CsCl, 0.2 M CsOH, 10 mM HEPES, and 10 mM EGTA (pH adjusted to 7.3 with HCl). The recording solution contained 10 mM Ba(OH)2, 95 mM NaOH, 2 mM CsOH, and 5 mM HEPES, pH 7.4 (adjusted with methane-sulfonic acid; 229 mOsm).
Stock solutions of the purified compounds were prepared in dimethylsulfoxide (DMSO) and diluted to the indicated final concentrations in recording solution. Two milligrams of plant extract was dissolved in 0.1 ml of DMSO and diluted 100-fold in recording solution. The highest concentration of DMSO employed in our assay [1% (v/v)] did not affect channel currents.
The voltage dependence of activation was determined by 150-ms step depolarization from a holding potential of -80 mV to various test potentials (10-mV increments, every 15 s). I-V curves were fitted to equation: I = Gmax (V - Vrev)/(1 + exp [(V - V0.5,act)/kact]) + C, where Vrev is the extrapolated reversal potential of IBa, V is the membrane potential, I is the peak current, Gmax is the maximal conductance, V0.5,act is the voltage for half-maximal activation, kact is the slope factor of the Boltzmann equation, and C is an offset factor.
Voltage-dependent inactivation was calculated during 3-s pulses from a holding potential of -80 mV to the voltage of maximal current influx (Vmax). Traces were best fitted to a biexponential decay yielding time constants for a fast (τfast) and slowly (τslow) inactivating component. Drug effects were measured either during 0.1-Hz depolarizations of 100-ms pulses applied from a holding potential of -80 mV to Vmax (preferential block of resting channels) or during 1-Hz pulse trains of 15 consecutive 100-ms pulses to Vmax from a holding potential of -60 mV (inducing additional use-dependent block; Kraus et al., 1998). IBa was stable during 0.1-Hz depolarizations. Drug effects were quantified from IBa amplitudes at the end of 100-ms test pulses, which also take into account drug effects on IBa inactivation during depolarization. In some experiments, 1-Hz pulse trains were applied in the absence of drug after stabilization of IBa during preceding 0.1-Hz depolarizations. Cells were then again stimulated at 0.1 Hz to allow IBa recovery before the drug was added; after inhibition at 0.1 Hz was complete, another 1-Hz pulse train was applied.
Statistics. All data are presented as means ± S.E. Statistical calculations (statistical tests are indicated in the figure legends or in the text) were performed in Prism 4.03 (GraphPad Software Inc., San Diego, CA). Statistical significance was set at p < 0.05.
The inhibition of a P/Q-type current component by 15 and 45 μM α-eudesmol has been reported previously in rat cerebellar Purkinje cells. However, α-eudesmol also inhibits ω-agatoxin IVA-insensitive N- and L-type current components in neuronal NG108-15 cells (Asakura et al., 1999). To demonstrate unequivocally the modulation of Cav2.1 currents by α-eudesmol and by different petasins isolated from a plant extract with antimigraine efficacy (Fig. 1), we heterologously expressed Cav2.1 channel complexes in X. laevis oocytes and quantified drug effects using the two-electrode voltage-clamp technique (Kraus et al., 1998). As illustrated in Fig. 2, 50 μM α-eudesmol caused an approximately 50% inhibition of IBa when channels were stimulated infrequently (0.1 Hz) from -80 mV to Vmax. Inhibition by α-eudesmol occurred over the whole voltage range without shifting the current-voltage relationship (Fig. 2A, inset) and was approximately complete after 20 sweeps (Fig. 2B). This inhibition must reflect mostly tonic inhibition of resting Cav2.1 channels, although some drug-induced acceleration of current inactivation (Fig. 2B, inset) also indicates interaction with open or inactivated channel states (see below). γ-Eudesmol was of similar potency, whereas β-eudesmol caused significantly less inhibition (p < 0.05; see legend to Fig. 2A). P. hybridus extract (0.02 mg/ml), petasin, neopetasin, isopetasin, S-petasin, and iso-S-petasin (all 50 μM) caused significantly smaller tonic block than α-eudesmol (for statistics, see legend to Fig. 2A). Like for α-eudesmol, all compounds reduced IBa at all voltages examined without significant changes of Vmax values compared with control (data not shown). Tonic inhibition of Cav2.1 currents by α-eudesmol was concentration-dependent and significant at concentrations ≥ 10 μM (for statistics, see Fig. 2C). These data demonstrate that α-eudesmol causes a tonic inhibition of Cav2.1 channels in a stereoselective manner and that P. hybridus sesquiterpenes are less potent inhibitors of resting Cav2.1 channels.
Next, we tested whether these natural compounds exert an additional use-dependent inhibitory component unmasked during more frequent stimulations from a slightly more depolarized holding potential (-60 mV) as described previously (Kraus et al., 1998). This protocol favors the availability of open and/or inactivated channels. A typical experimental time course is illustrated in Fig. 3A for α-eudesmol. After stable IBa was obtained (0.1-Hz depolarizations), drug was applied during the 0.1-Hz protocol yielding the expected tonic inhibition. After 20 sweeps, a 1-Hz pulse train was applied, causing further fast and strong reduction in IBa. Note that in the absence of drug, the same protocol caused only an approximately 50% decrease of the IBa remaining at the end of the 0.1-Hz protocol, as evident from the representative current traces in Fig. 3B (left) and the statistical data illustrated in Fig. 3, C and D. To quantify the additional drug-induced use-dependent inhibitory effect, current amplitudes during the 1-Hz train were normalized to the IBa amplitude obtained at the end of the 0.1-Hz protocol in the absence (control) or presence of drugs (Fig. 3, C and D). In the absence of drug, IBa decreased by approximately 50% during the 1-Hz train. Instead, 50 μM α-, β-, and γ-eudesmol induced 70 to 80% inhibition of IBa (Fig. 3C) in addition to their tonic block (as quantified in Fig. 2). The sulfur-containing compounds S-petasin and iso-S-petasin, which were only weak tonic blockers (Fig. 2A), were strong use-dependent inhibitors, similar to the eudesmols and significantly more potent than petasin (Fig. 3D). In contrast, petasin, P. hybridus extract, neopetasin, and isopetasin were significantly (p < 0.001) less potent use-dependent blockers than α-eudesmol (for statistics, see legend to Fig. 3, C and D).
The concentration dependence of the use-dependent component for α-eudesmol and petasin (which was commonly used to standardize P. hybridus extracts) is illustrated in Fig. 3E. With no α-eudesmol added, IBa declined to 55.5 ± 1% during the pulse train (Fig. 3E). In the presence of 3 μM α-eudesmol, which caused no tonic inhibition (Fig. 2C), significant channel inhibition was induced (for statistics, see legend to Fig. 3E). α-Eudesmol (10 μM), which caused 15% tonic block, inhibited approximately half of the current that remained after tonic block (remaining IBa, 10 μM: 31.8 ± 2%). Use-dependent inhibition by 50 μM α-eudesmol (Fig. 3E) was also larger than tonic block (50% inhibition, Fig. 2, A and C), clearly indicating that drug action occurred in a state-dependent manner. Considering tonic and use-dependent block together (as illustrated in Fig. 3A), it can be calculated that mean IBa decreased to 27% (10 μM) and 8% (50 μM) in the presence of α-eudesmol compared with 55% in the absence of drug. The same calculation revealed an overall IBa decrease by iso-S-petasin to 19.3% (tonic, 3.5 ± 2.7% inhibition, followed by 79.6 ± 1.9% use-dependent inhibition) and to 35% by the less potent petasin (50 μM).
Figure 4 illustrates that both drugs (50 μM) also significantly accelerated IBa inactivation during prolonged (3-s) depolarizations to Vmax. α-Eudesmol significantly decreased the time constants for the slow (τslow, control, 2015 ± 237 ms, n = 11; +α-eudesmol, 730 ± 74 ms, n = 8, p = 0.0003; unpaired Student's t test) and fast (τfast, control, 221 ± 9 ms, n = 11; +α-eudesmol, 103 ± 15 ms, n = 8, p = 0.0001) components of the biexponential time course but did not change their relative contribution to the inactivation process (% τslow, control, 13.1 ± 0.79%, n = 11; +α-eudesmol, 12.2 ± 1.31%, n = 8, p = 0.56; % τfast, control, 82.2 ± 1.59%, n = 11; +α-eudesmol, 84.9 ± 1.67%, n = 8, p = 0.27). A smaller but consistent acceleration of both inactivation time constants was also observed for petasin (Fig. 4B; τslow, 1133 ± 84 ms, p < 0.05; τfast, 157 ± 20 ms, p = 0.005; n = 4). These effects are also in agreement with interaction of both natural compounds with open and/or inactivated channel states.
Here, we demonstrate that the sesquiterpene α-eudesmol and its β- and γ-isomers block recombinant Cav2.1 channels at micromolar concentrations. Inhibition was observed during infrequent stimulation, which mostly reflects drug action on resting channels. However, an additional use-dependent component was revealed when 1-Hz pulse trains were elicited from a slightly more depolarized membrane potential that both favors availability of open and/or inactivated channels. We also found that petasins, the main constituents of the antimigraine herb P. hybridus, block Cav2.1 channels. They were only weak blockers of Cav2.1 channels at low stimulation rates. However, their inhibitory effects were unmasked during the 1-Hz protocol. We identified the sulfur-containing derivatives, S-petasin and iso-S-petasin, as the most active compounds. Although tonic block was almost absent, they were potent use-dependent inhibitors, comparable with α-eudesmol when Cav2.1 channels were depolarized frequently. The more pronounced decrease of IBa during 1-Hz pulse trains can be explained by enhanced inactivation during pulses or by slowing of recovery from inactivation between pulses or both (Kraus et al., 1998). We have observed an acceleration of inactivation during depolarizing pulses, suggesting that this contributed to inhibition by α-eudesmol and petasins during frequent stimulation.
S-Petasin (Wang et al., 2001) and iso-S-petasin (Wang et al., 2002) were found recently to inhibit smooth and cardiac muscle contraction by inhibiting L-type VGCCs (presumably of the Cav1.2 isoform, which is predominant in these tissues; Sinnegger-Brauns et al., 2004) within a concentration range similar to the Cav2.1 inhibition reported here. This indicates that these petasins are not isoform-selective blockers of VGCCs. This broader pharmacological profile of S-petasin and iso-S-petasin resembles the calcium channel blocker flunarizine, a piperazine derivative inhibiting ω-agatoxin IVA-sensitive P/Q-type channels (Geer et al., 1993) and N-type (Tytgat et al., 1991), L-type, and T-type VGCCs (Tytgat et al., 1996). Flunarizine is widely used as effective treatment for the prophylaxis of migraine attacks (Goadsby et al., 2002). Although the precise mechanism of its antimigraine activity is unknown, it is likely that its ability to inhibit several ion channels controlling neuronal excitability contributes to its antimigraine effects. It is thought that P/Q-, N-, and L-type VGCCs mediate calcitonin gene-related peptide release from trigeminal nerve fibers (and subsequent blood vessel dilation as observed during migraine attacks; Akerman et al., 2003) and nociceptive transmission to central neurons (in the trigeminovascular complex; Shields et al., 2005). Therefore, the combined block of both P/Q- and L-type channels by sulfur-containing petasins could represent an advantage for inhibition of processes involved in triggering and/or sustaining migraine attacks. If preferential activity of S-petasins on channels opening frequently and from slightly depolarized resting potentials would be beneficial for therapy cannot be answered because the pharmacological effects of systemically applied nonpeptide P/Q-type channel blockers have not yet been reported. However, like for N-type channel block in neuropathic pain (see Introduction), use-dependent properties of P/Q-type channel blockers may facilitate the inhibition of neurotransmitter release in rapidly firing trigeminal neurons.
Our findings prompt further electrophysiological studies investigating the modulatory capacity of these compounds on other ion channels implicated in migraine pathophysiology, such as voltage-gated Na+ channels (Dichgans et al., 2005). This may encourage future preclinical studies to evaluate the pharmacokinetic and therapeutic actions of sulfur-containing petasins in migraine.
In our P. hybridus preparation, petasin, isopetasin, and neopetasin comprise approximately 35% of the extract, and only approximately 6% are sulfur-containing petasins. Therefore, the calculated approximate concentration of petasins in the diluted extract (20 μg/ml) is approximately 30 μM. The extent of tonic and use-dependent Cav2.1 inhibition by the extract was similar to the more abundant petasins. Therefore, the pharmacological effect of the extract can be explained by the combined effect of petasins and sulfur-containing petasins. It appears unlikely that other potent Cav2.1 channel inhibitors of reasonable abundance are present in the extract.
In contrast to previous studies on L-type VGCCs, we compared different isomers of the sesquiterpenes eudesmol and petasin. Neither the structural differences between the eudesmol isomers nor between petasin and isopetasin (Fig. 1) caused major differences in their channel-inhibitory properties. However, replacement of the angeloyl group of petasin and isopetasin with a 3-methylthiopropanoyl moiety significantly increased the potency as use-dependent blockers of Cav2.1 VGCCs. This suggest that a sulfur-containing substituent in position 3 of the petasins (Fig. 1) is an important feature for binding of these compounds to the open and/or inactivated state of the Cav2.1 channel. Based on these observations, a more systematic analysis of the structure activity relationship of substituents in position 3 is justified to investigate whether compounds with higher potency blockers can be obtained or perhaps derivatives that could serve as lead compounds for the development of isoform-selective inhibitors of VGCCs.
We thank Elisabeth Kaltenegger and Sonja Sturm for providing purified eudesmols and petasins, Stefan Schwaiger for helping with HPLC methods, Katrin Watschinger for discussions, and Gilda Pelster and Jasmin Aldrian for expert technical assistance.
This work was supported by the Austrian Science Fund [Grant FWF P17159]; and the University of Innsbruck and the Tiroler Wissenschaftsfonds [UNI-0404/353].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: VGCC, voltage-gated Ca2+ channel; LTCC, L-type (Cav1) Ca2+ channel; HPLC, high-performance liquid chromatography; IBa, inward Ba2+ current; DMSO, dimethyl sulfoxide; τfast, τslow, time constants of fast and slowly inactivating current components; MS-222, tricaine (ethyl 3-aminobenzoate) methanesulfonate.
- Received January 20, 2009.
- Accepted April 14, 2009.
- The American Society for Pharmacology and Experimental Therapeutics