The complex actions of sumatriptan on rat dural afferents

Abstract

Aim: To test the hypothesis that the clinical efficacy of triptans reflects convergent modulation of ion channels also involved in inflammatory mediator (IM)-induced sensitization of dural afferents.

Methods: Acutely dissociated retrogradely labeled rat dural afferents were studied with whole cell and perforated patch techniques in the absence and presence of sumatriptan and/or IM (prostaglandin E2, bradykinin, and histamine).

Results: Sumatriptan dose-dependently suppressed voltage-gated Ca²⁺ currents. Acute (2 min) sumatriptan application increased dural afferent excitability and occluded further IM-induced sensitization. In contrast, pre-incubation (30 min) with sumatriptan had no influence on dural afferent excitability and partially prevented IM-induced sensitization of dural afferents. The sumatriptan-induced suppression of voltage-gated Ca²⁺ currents and acute sensitization and pre-incubation-induced block of IM-induced sensitization were blocked by the 5-HT(1D) antagonist BRL 15572. Pre-incubation with sumatriptan failed to suppress the IM-induced decrease in action potential threshold and overshoot (which results from modulation of voltage-gated Na⁺ currents) and activation of Cl⁻ current, and had no influence on the Cl⁻ reversal potential. However, pre-incubation with sumatriptan caused a dramatic hyperpolarizing shift in the voltage dependence of K⁺ current activation.

Discussion: These results indicate that although the actions of sumatriptan on dural afferents are complex, at least two distinct mechanisms underlie the antinociceptive actions of this compound. One of these mechanisms, the shift in the voltage dependence of K⁺ channel activation, may suggest a novel strategy for future development of anti-migraine agents.

Figure 1. Sumatriptan inhibits high threshold voltage gated Ca²⁺ currents (I_Ca) in dural afferents

A. Example of sumatriptan mediated inhibition of I_Ca. B. Bath application of increasing concentrations of sumatriptan from 0.001 to 10μM suppressed I_Ca amplitude recorded with a single pulse to 10mV in 7 of 7 dural afferents studied. C. The IC₅₀ for sumatriptan was determined with percent inhibition plotted against sumatriptan concentration. D. To examine the voltage dependence of inhibition, currents were elicited with a test pulse to +10mV following pre-pulses to −60 and +80mV before (Baseline) and after sumatriptan (10 μM Suma) application (n=5). The ratio of the current amplitude following a prepulse to +80 divided by the current amplitude following a prepulse to −60 was determined before and after sumatriptan application. Following sumatriptan application, there was no significant difference in the current ratio. E. Instantaneous I–V data were plotted from tail currents. Sumatriptan decreased the amplitude of the tail currents but did not produce a shift in their voltage dependence of activation.

Figure 2. Acute sumatriptan increases dural afferent excitability

A. Acute bath application of 1μM sumatriptan resulted in a significant reduction in rheobase in dural afferents (n=7). This effect was blocked when sumatriptan was co-applied with the 5-HT_1D receptor antagonist BRL 15572 (Antag, 1 μM, n = 6). When IM were applied to dural afferents in the presence of sumatriptan, there was no further decrease in rheobase. B. Acute sumatriptan application also significantly hyperpolarized the AP threshold. This change was also blocked when sumatriptan was co-applied with BRL 15572. There was no further change in AP threshold following IM application. Data in A and B were analyzed with a one way ANOVA with a Holm-Sidak test used for post-hoc analysis. The most relevant comparisons are illustrated for clarity where * is p < 0.05. C. The stimulus response function data in C for neurons treated with sumatriptan alone (Suma) or sumatriptan + BRL 15572 (Suma + Antag) were analyzed with a Fisher Exact test. The proportion of neurons treated with Suma alone (7 of 7) with a left shift in the stimulus response function (relative to baseline) was significantly (p < 0.05) greater than that for the Suma + Antag group (2 of 6). There was no further shift in the stimulus response function in the Suma group (0 of 7) following application of IM. Baseline data are plotted for comparison.

Figure 3. Prolonged sumatriptan exposure has no influence on excitability and attenuates IM-induced sensitization of dural afferents

A. Following 30 minute pre-incubation with sumatriptan, IM application (Suma + IM) had little influence on rheobase (expressed as a % of baseline determined prior to the application of IM for each neuron). However, the IM-induced decrease in rheobase in neurons pre-incubated with the combination of sumatriptan and BRL 15572 (Suma + antag + IM) was significantly (Student's t test) greater than the change in observed in neurons treated with sumatriptan alone. B. In contrast to rheobase, application of IM resulted in a decrease in AP threshold in neurons preincubated with sumatriptan alone as well as the combination of sumatriptan and BRL 15572. There was no significant difference between these groups. C. Sumatriptan pre-incubation (Suma) had no significant influence on the baseline response to suprathreshold stimulation. Nor was there an influence of pre-incubating neurons with the combination of sumatriptan and BRL 15572 (Suma + Antag). Data were analyzed with a two-way repeated measures ANOVA and compared to control neurons incubated in vehicle for 30 minutes (Vehicle). Furthermore, the application of IM to neurons pre-incubated for 30 minutes with sumatriptan (Suma + IM) had no significant influence on the stimulus response function as determined with a one-way repeated measures ANOVA. However, application of IM to neurons pre-incubated with the combination of sumatriptan and BRL 15572 (Suma + Antag +IM) resulted in a significant leftward shift in the stimulus response function as determined by both the increase in the number of evoked action potentials at 2× and 3× rheobase (relative to the response prior to the application of IM), as well as the proportion of neurons in which IM produced a change (4 of 4) relative to the Suma + IM group (0 of 7, p < 0.01, Fisher Exact test). * Indicates a significant difference between groups in A and before and after IM application in C where p<0.05.

Figure 4. Sumatriptan does not prevent IM-induced activation of I_IM-Cl

I_IM-Cl was activated by IM application and elicited with 100 ms test pulses from −70mV to +50mV following a 40ms pre-pulse to 0mV to evoke Ca²⁺ currents (n=7) and isolated as the difference between current evoked before and after application of IM (I_IM-Cl Difference Current). A. Pre-incubation with sumatriptan had no significant (p > 0.05, two-way repeated measures ANOVA) influence on peak I_IM-Cl density (at any voltage tested). B. To determine if sumatriptan may change the sensitivity of I_IM-Cl to high intracellular Ca²⁺, I_IM-Cl was recorded in the presence of Cd²⁺ and low intracellular EGTA to buffer intracellular Ca²⁺ at 622nM (n=5). Sumatriptan had no significant (p > 0.05, two-way repeated measures ANOVA) influence on the amplitude of I_IM-Cl at any potential under these conditions. Currents were blocked with 100μM niflumic acid (NFA). C. The reversal potential for Cl- was recorded in response to a ramp voltage protocol from +50mV to −100mV using the gramicidin perforated patch configuration (n=5). Sumatriptan pre-incubation had no significant (p > 0.05, Student's t test) influence on the reversal potential of the IM-induced current.

Figure 5. Sumatriptan both modulates K⁺ currents and blocks IM-induced suppression of K⁺ currents

A. The voltage-dependence of K⁺ current activation was determined with current-voltage protocols consisting of 10mV, 500ms voltage-steps between −60 and +60mV following a 500ms pre-pulse to −120mV. B. 30 minutes of sumatriptan (Suma) pre-incubation (n=6) resulted in a significant (p < 0.01, Student's t test) left shift in the voltage dependence of K⁺ current activation compared to vehicle (V) control (n=7): the V0.5 of current activation was shifted from −11.5± 2.4mV to −27.3 ± 4.7mV. IM application produced no significant (p > 0.05, one way ANOVA with Holm-Sidak post-hoc) change in the voltage dependence of K⁺ current activation in the presence or absence of sumatriptan. C. IM resulted in a significant (p > 0.05, one-way ANOVA with Holm-Sidak post-hoc test) reduction maximal K⁺ conductance (normalized by membrane capacitance), compared to vehicle treated neurons. However, there was no significant influence of IM on the maximal K⁺ conductance when applied to neurons pre-incubated with sumatriptan. Inset: When analyzed as a change from baseline, the IM-induced decrease in K⁺ conductance observed in vehicle treated neurons (V) was significantly greater (p < 0.05, Student's t test) than that observed in neurons pre-incubated with sumatriptan (Suma).