The complex actions of sumatriptan on rat dural afferents

Animals

Adult female Sprague Dawley rats (Harlan, Indianapolis, IN) weighing between 180 and 290 g were used for all experiments. Rats were housed two per cage at the University of Pittsburgh animal facility on a 12:12 light: dark schedule with food and water freely available. Before all procedures, animals were deeply anesthetized with an i.p. injection (1 ml/kg) of rat cocktail containing ketamine (55 mg/kg), xylazine (5.5 mg/kg), and acepromazine (1.1 mg/kg). Experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in accordance with National Institutes of Health guidelines for the use of laboratory animals in research. All efforts were made to minimize the total number of animals used.

Retrograde labeling

Afferents innervating the dura were identified as previously described following labeling with the retrograde tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Invitrogen, Carlsbad, CA) (8). Immediately post-operatively, animals received a single i.m. injection of penicillin G (10,000 units/kg) and a single injection of buprenorphine (0.03 mg/kg) to minimize infection and discomfort. Subsequent administration of ketoprofen was provided if evidence of hypersensitivity persisted over subsequent days after labeling.

Tissue preparation

Ten to fourteen days after DiI application, trigeminal ganglia (TG) were removed, enzymatically treated and mechanically dissociated as previously described (7). Acutely dissociated cells were plated on laminin/ornithine coated glass coverslips. Changes in current and excitability were measured 2-8 hours after cells were plated.

Electrophysiology

All whole cell and perforated patch-clamp recordings were performed with a HEKA EPC10 amplifier (HEKA Electronik, Lambrecht/Rhineland-Pfalz, Germany). Data were low-pass filtered at 5–10 kHz with a four-pole Bessel filter and digitally sampled at 25–100 kHz.

Drugs

All salts and reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless indicated below. Bradykinin was dissolved in 1% acetic acid (23.58 mM stock concentration), PGE₂ was dissolved in 100% ethanol (10 mM stock concentration), and histamine was dissolved in water (100 mM stock concentration). All stock solutions were stored at −20°C until the day of use. IM-vehicle bath containing the final concentration of ethanol (0.01%) and acetic acid (0.001%) was used as a control. NFA was dissolved in 100% ethanol. Sumatriptan was a gift from Glaxo SmithKline. Sumatriptan was dissolved as a 10 mM stock solution in water and subsequently diluted in bath solution. The 5-HT_1D receptor antagonist BRL 15572 was obtained from Tocris Biosciences (R & D Systems, Minneapolis, MN), was dissolved as a 10 mM stock in 100% ethanol and diluted in bath solution.

Data analysis

Data were analyzed with PulseFit (HEKA), Sigma Plot and Sigma Stat software (Systat Software Inc., Richmond, CA). Conductance-voltage (G-V) curves were constructed from I-V curves by dividing the evoked current by the driving force on the current, such that G = I/(V_m – V_rev), where V_m is the potential at which current was evoked and V_rev is the reversal potential for the current was measured directly (for K and Ca²⁺). Instantaneous I-V data was obtained from the tail currents measured following activation of voltage-gated Ca²⁺ currents.

Statistical analysis

For comparisons of data collected before and after IM application, either a paired t-test or repeated-measures ANOVA was used if data were parametric. Otherwise, a Wilcoxon or Friedman test was used for nonparametric analysis. For unpaired comparisons, Student’s t-test, one- and two-way ANOVA were used for parametric data and a Mann Whitney U test for nonparametric analysis. Data were considered statistically significant when p < 0.05. All data are represented as mean ± standard error.

Sumatriptan dose-dependently inhibits voltage-gated calcium currents

Previous data suggests a primary mechanism of triptan action is a G-protein-mediated inhibition of voltage-gated Ca²⁺ currents (VGCC) (17, 18). To determine whether such a mechanism exists in dural afferents and to determine the appropriate concentration of sumatriptan for subsequent experiments, VGCC in dural afferents (n = 7) were studied with increasing concentrations (0.001–10 μM) of sumatriptan. Currents were evoked with 50 ms pulses from −60 to +80 mV following a 100 ms pre-pulse to −100 mV.

A concentration-dependent inhibition of VGCC was observed in seven of seven dural afferents studied (Figure 1A, B). Inhibition of peak current evoked at 10 mV was converted to percent inhibition and data from all seven neurons were pooled, plotted as a function of the concentration of sumatriptan and fitted with a modified Hill equation (Figure 1C). The IC₅₀ for sumatriptan-induced inhibition of VGCC was 142 nM, with a maximal fractional inhibition of 20 ± 2%. Interestingly, there was no evidence of a low threshold VGCC in any of the dural afferents studied. OPEN IN VIEWER

To determine whether a membrane-delimited displacement of the N-type Ca²⁺ channel β-subunit by the G-protein βγ subunits (19) could also be mediating the decrease in VGCC with sumatriptan, Ca²⁺ currents were elicited with a two-pulse protocol in which a test pulse to +10 mV was preceded by a conditioning pulse to either −60 mV or +80 mV (20). A 50 ms step to −60 mV between the conditioning and test pulses was used to enable channel deactivation following the step to +80 mV (n = 5). Consistent with the absence of a detectable shift in the VGCC instantaneous I-V curve, there was no evidence of pre-pulse potentiation, as the ratio of the currents elicited before sumatriptan application (1.07 ± 0.03) were comparable to that after application (1.12 ± 0.07, Figure 1D). To confirm that sumatriptan-induced inhibition of voltage-gated Ca²⁺ currents in dural afferents was mediated by the 5-HT_1D receptor, sumatriptan (1 μM) was co-applied to three dural afferents with the 5-HT_1D receptor selective antagonist BRL 15572 (1 μM). Two minutes after the application of the combination of sumatriptan and BRL 15572, the decrease in maximal conductance (7.0 ± 0.1% of baseline) was significantly (p < 0.01, Student’s t-test) less than that observed with sumatriptan alone.

Acute sumatriptan increases baseline dural afferent excitability

Acute (2 min) application of 1 µM sumatriptan alone produced a significant increase in excitability of dural afferents (n = 7) as evidenced by changes in rheobase (p < 0.01, Figure 2A), AP threshold (p < 0.01, Figure 2B), and the response to suprathreshold current injection (p < 0.01, Figure 2C). These changes were associated with a significant (p < 0.01, paired t-test) depolarization of E_m from −71.3 ± 1.6 mV to −54.0 ± 3.6 mV. These sumatriptan-induced changes in excitability were blocked by the co-application of the 5-HT_1D receptor antagonist BRL 15572 (1 μM, n = 5, Figure 2A, B and C). OPEN IN VIEWER

To determine whether sensitization of dural afferents via sumatriptan or IM involve comparable mechanisms, IM were applied following sumatriptan. No further increase in excitability was detected in these neurons (Figure 2). These results suggest that either sumatriptan-induced sensitization shares common mechanisms with those of IM, or this drug has blocked the actions of IM.

Prolonged sumatriptan exposure has no influence on baseline excitability and attenuates IM-induced sensitization of dural afferents

There is evidence that triptan analgesia does not occur immediately after administration. Instead, pain relief is experienced typically 20–30 min after taking the drug (21, 22). Therefore, we examined the possibility that with a longer exposure time, sumatriptan may switch from being excitatory to inhibitory.

Following a 30 min pre-incubation with sumatriptan, in which neurons (n = 8) were incubated in sumatriptan prior to recording, there was no significant (p > 0.05, Student’s t-test) difference in rheobase compared with that in control (vehicle) neurons (n = 8), indicating that the decrease in rheobase following acute sumatriptan application returns to baseline levels with longer incubation times: rheobase normalized to membrane capacitance was 8.4 ± 1.3 pA/pF and 9.5 ± 1.5 pA/pF in neurons from vehicle- and sumatriptan-treated groups, respectively. Similarly, sumatriptan pre-incubation produced no significant (p > 0.05, Student’s t-test) changes in AP threshold compared with control: AP threshold was −29.5 ± 1.6 mV and −23.6 ± 4.3 mV in neurons from vehicle- and sumatriptan-treated groups, respectively. There was also no significant (p < 0.05, two-way repeated measure ANOVA) influence of sumatriptan pre-incubation on the response to suprathreshold current injection (Figure 3C). Furthermore, pre-incubation of sumatriptan with BRL 15572 (n = 5) had no detectable influence on rheobase (which was 7.3 ± 2.1 pA/pF), the AP threshold (which was −25.4 ± 4.0), or the response to suprathreshold current injection (Figure 3C). OPEN IN VIEWER

To determine the effects of sumatriptan pre-incubation on IM-induced sensitization of dural afferents, changes in excitability were recorded with IM in the presence of sumatriptan. In contrast to our previous observations in which application of IM to dural afferents resulted in a significant decrease in rheobase and a leftward shift in the response to suprathreshold stimulation (6), IM had no significant influence on rheobase (Figure 3A) or the response to suprathreshold stimulation (Figure 3C) in dural afferents pre-incubated with sumatriptan as compared with vehicle treated dural afferents. However, pre-incubation with sumatriptan did not prevent IM-induced hyperpolarization of AP threshold (Figure 3B). The suppressive effects of sumatriptan pre-incubation on IM-induced changes in rheobase and the response to suprathreshold current injection were blocked by the presence of BRL 15572 during the 30 min pre-incubation (n = 5, Figure 3A and C).

Sumatriptan modulates active and passive electrophysiological properties

To begin to determine the basis for the sumatriptan-induced inhibition of IM-induced sensitization, changes in passive and active electrophysiological properties were examined. A 30 min sumatriptan pre-incubation had no effect on baseline passive electrophysiological properties as assessed by the resting membrane potential and input resistance, as these values, −69.0 ± 1.7 mV and 635 ± 146 MΩ (n = 8), respectively, were comparable to values previously reported (−71.3 ± 1.6 mV and 473 ± 57.3 MΩ (6)).

We previously demonstrated that IM produce significant changes in passive and active electrophysiological properties of dural afferents (6). These IM-induced changes included a ∼10 mV membrane depolarization that was accompanied by a decrease in R_in subsequent to activation of I_IM-Cl (6). Although sumatriptan pre-incubation did not prevent the IM-induced decrease in R_in, it blocked the IM-induced membrane depolarization, as the IM-induced depolarization in neurons pre-incubated with the combination of sumatriptan and BRL 15572 (n = 5) was significantly (p < 0.01) larger than that in neurons pre-incubated with sumatriptan alone (n = 8, Table 1). We also previously demonstrated an IM-induced increase the AP overshoot subsequent to modulation of voltage-gated Na⁺ currents (VGSC) in dural afferents (6). Sumatriptan pre-incubation did not prevent the IM-induced increase in AP overshoot either, suggesting that sumatriptan did not prevent IM modulation of VGSC.

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Table 1.

Sumatriptan blocks IM-induced changes in passive and active electrophysiological properties.

Group	N	Δ Em (mV)	%Δ Rin (MΩ)	Δ AP duration (ms)	Δ AP overshoot (mV)	Δ AHP magnitude (mV)	Δ τ AHP (ms)
30 min Suma + IM	8	1.0 ± 0.9	41.2 ± 14.6	0.38 ± 0.4	2.9 ± 5.8	2.3 ± 1.6	17.9 ± 26.5
30 min Suma + Antag + IM	5	7.5 ± 1.7	42.3 ± 9.0	0.34 ± 0.5	2.6 ± 1.4	3.1 ± 1.6	24.5 ± 11.4

Sumatriptan does not prevent IM-induced activation of I_IM-Cl

Although data from the AP waveform suggest that VGSC are probably not a convergent target of sumatriptan, the observation that sumatriptan was able to block the IM-induced depolarization suggests that these drugs may block IM-induced activation of I_IM-Cl. To test this possibility, IM-induced changes in I_IM-Cl were monitored in dural afferents (n = 7) with a protocol in which I_IM-Cl was evoked with 100 ms test pulses from −70 mV to +50 mV following a 40 ms pre-pulse to 0 mV to evoke Ca²⁺ currents (Figure 4A). The currents reversed at −30 mV close to the predicted reversal potential for Cl⁻ (−34 mV) based on the composition of our intracellular and extracellular solutions. Pre-incubation with sumatriptan had no detectable influence on the peak density or rectification of I_IM-Cl (Figure 4A). OPEN IN VIEWER

Given the influence of both sumatriptan and IM on VGCC in dural afferents (7), to rule out a potential interaction between changes in Ca²⁺ influx and I_IM-Cl activation, this experiment was repeated in the presence of Cd²⁺ to block VGCC and low intracellular EGTA (1.2 mM) to buffer intracellular Ca²⁺ at a high concentration (622 nM). I_IM-Cl was again recorded with test pulses from −70 mV to +50 mV. Sumatriptan did not produce any change in the amplitude of I_IM-Cl (n = 5) at any potential under these conditions (Figure 4B). Furthermore, I_IM-Cl recorded in the presence of sumatriptan was blocked by the Cl⁻ channel blocker NFA (100 μM, Figure 4B), as previously demonstrated (7).

Because the excitatory influence of IM-induced activation of I_IM-Cl on dural afferents seems to reflect a depolarized Cl⁻ equilibrium potential (E_Cl) in these neurons (7), we also examined the effects of sumatriptan on the reversal potential of I_IM-Cl. Cl⁻ currents were recorded in response to a ramp voltage protocol from +50 mV to −100 mV using gramicidin perforated patch to prevent dialysis of intracellular Cl⁻ (23). Sumatriptan pre-incubation (n = 5) did not shift the reversal potential of I_IM-Cl (Figure 4C).

Sumatriptan modulates K⁺ currents and inhibits IM-induced suppression of K⁺ currents

The decrease in R_in observed in the absence of an IM-induced depolarization of E_m suggests that pre-incubation with sumatriptan may result in the activation of a K⁺ current (I_K) that counters the depolarization driven by the activation of I_IM-Cl. To test this possibility, I_K was evoked with voltage protocols described in the Materials and methods section, in the absence (n = 7) and presence (n = 6) of 30 min pre-incubation with sumatriptan and IM (Figure 5A). From these data, changes in the voltage dependence of activation and maximal conductance (G_max) were determined. Consistent with the decrease in R_in in the absence of E_m depolarization, 30 min sumatriptan pre-incubation resulted in a dramatic leftward shift in the voltage dependence of I_K activation. There was a significant (p < 0.01) hyperpolarization of the V_0.05 of activation (Figure 5B) following sumatriptan pre-incubation (−27.3 ± 4.7 mV) as compared with control (−11.5 ± 2.4 mV). IM application alone produced no change in the voltage dependence of activation of I_K (Figure 5B). However, IM (n = 7) significantly reduced the maximal conductance (Figure 5C). This effect was completely blocked by sumatriptan (n = 8) pre-incubation (Figure 5C). OPEN IN VIEWER

Because the shift in I_K activation should have attenuated the initial sumatriptan-induced sensitization of dural afferents, our current clamp results suggested that this shift takes time to develop. To begin to test this suggestion, we recorded I_K in dural afferents before and after the application of sumatriptan. The results of this analysis confirmed that this shift takes time to develop, as the change in the V_0.05 of activation was −2.5 ± 1.8 mV after 5 minutes of incubation and −3.7 ± 3.7 mV after 10 minutes (n = 3).

Sumatriptan-mediated inhibition of VGCC

Our data demonstrate that sumatriptan concentration-dependently inhibits VGCC in dural afferents. One of the most dramatic mechanisms of G-protein-coupled-receptor-mediated inhibition of VGCC involves a rapid displacement of the VGCC β subunit via the G-protein βγ subunit (19). A unique feature of this form of inhibition is that it can be overcome with a strong depolarizing pre-pulse (20). However, following a depolarizing pre-pulse to +80 mV, Ca²⁺ currents did not recover from sumatriptan inhibition. Furthermore, a lack of depolarizing shift in the voltage dependence of activation with increasing concentrations of sumatriptan suggests that this inhibition is via an as-yet unidentified intracellular second messenger. These conclusions are consistent with previous results from a study of Xenopus larvae spinal neurons indicating that 5-HT_1B/1D receptor agonists (L694 247) reduce high-voltage-activated N- and P/Q-type currents by a G-protein-activated diffusible second messenger pathway (18). More relevantly, these data are also consistent with previous reports that zolmitriptan can block P/Q- and possibly R-type currents in dissociated TG neurons. This effect was pertussis toxin sensitive, indicating the activation of Gi/Go class of G proteins (17). Such a mechanism was recently suggested to account for the sumatriptan-induced suppression of capsaicin-evoked currents in dural afferents (24).

Acute sumatriptan application increases dural afferent excitability

Acute application of sumatriptan produced an increase in dural afferent excitability. These data may explain the clinical observation that triptans transiently aggravate headache. Within 5-15 min of taking sumatriptan, approximately 50% of patients experience exacerbated pain that lasts for about 10–15 min (25) before the onset of pain relief. Our data are also consistent with previous observations that sumatriptan can drive a Ca²⁺-dependent discharge (26), increase the firing rate of C and Aδ meningeal nociceptors, and increase their mechanical sensitivity (25).

Multiple mechanisms are probably involved in this sumatriptan-induced transient increase in dural afferent excitability. However, the only change in active or passive electrophysiological properties observed in this study following acute sumatriptan was a significant depolarization in the membrane potential from −71.3 mV to −54.0 mV. The fact that the depolarization was not accompanied by a significant change in R_in suggests that there was no net change in the number of open channels, only a shift in the proportion of the various types of channels that were open.

Pre-incubation with sumatriptan has no influence on dural afferent excitability

There was no significant influence of pre-incubation with sumatriptan on baseline dural afferent excitability. This is somewhat surprising, in retrospect, given the dramatic leftward shift in the activation of I_K. Multiple K⁺ currents are expressed in sensory neurons and are critically involved in regulating their excitability (27). K⁺ channels regulate the timing between APs and therefore affect AP frequency. Thus, the dramatic shift in the voltage dependence of activation of K⁺ currents should have resulted in an increase in rheobase and/or a decrease in the response to suprathreshold current injection. The failure to detect such changes in excitability suggests that the shift in the voltage dependence of K⁺ current activation is compensated for, at least in part, by excitatory changes that persist following acute application of summatriptan. One such mechanism would include a suppression of Ca²⁺-dependent K⁺ channels secondary to the sumatriptan-induced inhibition of VGCC. We have recently demonstrated that such a channel is present in a subpopulation of cutaneous neurons, where it plays a significant role in the regulation of afferent excitability (28) and appears to be tightly coupled to the Ca²⁺ influx via VGCC (29). Given that we have also demonstrated that a Ca²⁺-dependent K⁺ channel is present in dural afferents and suppressed following IM application (7), sumatriptan-induced suppression of such a current could also account for the apparent block of the IM-induced suppression of total K⁺ current following sumatriptan pre-incubation. Such an explanation would suggest that the shift in the voltage dependence of K⁺ current activation is associated with an increase in K⁺ channel density. That is, an increase in one K⁺ channel type associated with the shift in the voltage-dependence of activation would compensate for a decrease in Ca²⁺-dependent K⁺ channel activity, resulting in the observed no net change in peak K⁺ conductance. K⁺ channel subunits present in sensory neurons that could undergo such dramatic shifts in the voltage-dependence of activation include Kv2.1 (30), which can undergo a ∼26 mV hyperpolarizing shift in the G-V following Ca²⁺/calcineurin dependent dephosphorylation (31). Future studies will be needed to identify the K⁺ channel subunit(s) that underlie the actions of sumatriptan in dural afferents.

Sumatriptan selectivity

The observation that both the sumatriptan-induced acute sensitization and the subsequent inhibition of IM-induced sensitization of dural afferents were blocked by BRL 15572 indicates that both processes are mediated by the 5-HT_1D receptor. This is consistent with previous data suggesting that although both 5-HT_1B and 5-HT_1D receptors are present on trigeminal ganglion neurons (32,33), the vasoconstrictive effects of triptans are due to the 5-HT_1B receptors on the dural vasculature (34), whereas the selective therapeutic efficacy of triptans for migraine is due to 5-HT_1D receptors in dural afferents (35). However, evidence that the 5-HT_1D receptor is present on subpopulations of afferents throughout the body (9) and that triptans have analgesic efficacy in other preclinical pain models (12,13) still leaves a question as to the basis of the selective clinical profile of this class of drugs. Our recent observation of the higher density of the 5-HT_1DR in nerve fibers preferentially involved in signaling migraine pain may partially explain the selectivity of these drugs (8). However, in light of the fact that a receptor for these drugs is present in other afferent populations, albeit at lower densities, we proposed that other mechanisms probably contribute to efficacy and selectivity. Given evidence that IM-induced activation of I_IM-Cl appears to be a relatively unique mechanism underlying the sensitization of dural afferents, sumatriptan-induced inhibition of I_IM-Cl would provide another mechanism to account for the therapeutic selectivity of this compound. The observations that sumatriptan neither blocked the activation of I_IM-Cl nor shifted the equilibrium potential for Cl⁻ indicates that this channel cannot account for the therapeutic actions of triptans. However, the modulation of I_K could account for the therapeutic selectivity of this compound if data from subsequent studies confirm that this modulation is observed only in dural afferents.

The complex actions of sumatriptan on dural afferents raise at least three questions. One question is how a decrease in VGCC could contribute to the antinociceptive efficacy of triptans at the same time triptans have increased excitability of dural afferents. VGCCs are largely responsible for the influx of Ca²⁺ necessary to enable transmitter release from pre-synaptic terminals. The suppression of VGCC on the central terminals of dural afferents should contribute to the antinociceptive efficacy of triptans and account for the normalization of dural stimulation-induced activity in trigeminal dorsal horn neurons following IM-induced sensitization (36). While there is evidence that low threshold or T-type VGCC may contribute to afferent sensitization (37), the high-threshold channels described in the present study that mediate transmitter release have a minimal direct contribution to AP generation (38). However, Ca²⁺ influx through these channels may contribute to the activation and/or modulation of a number of channels, including two-pore K⁺ channels (39) and Ca²⁺-modulated K⁺ channels (28). Thus, as noted above, although the triptan-induced suppression of VGCC in dural afferents may occur in parallel with the increase in excitability, the two may be causally linked.

A second question pertains to the differential time course of the sumatriptan-induced excitation and inhibition of IM-induced sensitization, particularly if both processes are mediated by the same receptor. Although pharmacokinetics could explain the relatively slow onset of triptan-induced pain relief observed clinically, the present results suggest an alternative possibility: distinct cellular processes underlie excitatory and inhibitory actions of the drug, in which those underlying inhibition develop far more slowly than those underlying excitation. Additional work will be needed to tease apart the specific mechanisms underlying the actions of sumatriptan in dural afferents, but the literature is now full of examples of receptor-mediated processes, in particular those, like the 5-HT_1D receptors that are coupled to G-proteins, that develop over very different time scales. For example, the membrane-delimited form of G-protein-mediated suppression of VGCC can occur within tens of milliseconds in sensory neurons (40), while there is evidence that metabotropic-glutamate-receptor-mediated decrease in membrane ionotropic glutamate receptors develops over tens of minutes (41).

A third question is why triptans fail to alleviate migraine pain once it is already established. Our results indicating that pre-incubation with sumatriptan blocked IM-induced sensitization are consistent with the evidence that triptans administered before the development of migraine pain can abort a migraine. However, the leftward shift in the activation of K⁺ currents should enable sumatriptan to reverse afferent sensitization even after it is established. The observation that triptans fail to reverse IM-induced sensitization of dural afferents (36) suggests that the second messenger pathways activated by IM block the actions of sumatriptan, at least those underlying the modulation of K⁺ currents. Ongoing experiments are designed to identify the point(s) of convergence of the underlying second messenger pathways.