Gabapentin inhibits catecholamine release from adrenal chromaffin cells

Abstract

Background: Gabapentin is most commonly prescribed for chronic pain, but acute perioperative effects, including preemptive analgesia and hemodynamic stabilization, have been reported. Adrenal chromaffin cells are a widely used model to investigate neurosecretion, and adrenal catecholamines play important physiologic roles and contribute to the acute stress response. However, the effects of gabapentin on adrenal catecholamine release have never been tested.

Methods: Primary cultures of bovine adrenal chromaffin cells were treated with gabapentin or vehicle for 18-24 h. The authors quantified catecholamine secretion from dishes of cells using high-performance liquid chromatography and resolved exocytosis of individual secretory vesicles from single cells using carbon fiber amperometry. Voltage-gated calcium channel currents were recorded using patch clamp electrophysiology and intracellular [Ca2+] using fluorescent imaging.

Results: Gabapentin produced statistically significant reductions in catecholamine secretion evoked by cholinergic agonists (24 ± 3%, n = 12) or KCl (16 ± 4%, n = 8) (mean ± SEM) but did not inhibit Ca2+ entry or calcium channel currents. Amperometry (n = 51 cells) revealed that gabapentin inhibited the number of vesicles released upon stimulation, with no change in quantal size or kinetics of these unitary events.

Conclusions: The authors show Ca2+ entry was not inhibited by gabapentin but was less effective at triggering vesicle fusion. The work also demonstrates that chromaffin cells are a useful model for additional investigation of the cellular mechanism(s) by which gabapentin controls neurosecretion. In addition, it identifies altered adrenal catecholamine release as a potential contributor to some of the beneficial perioperative effects of gabapentin.

Figure 1. Gabapentin reduces catecholamine secretion but not calcium entry evoked by cholinergic stimulation

(A) Chromaffin cells were seeded on 24-well plates and treated with 1mM gabapentin (GBP) or vehicle (control) for 18–24 h. The amount of catecholamines released under basal conditions (in the absence of secretagogue) or during a 5-min stimulation with carbachol (100 μM) was determined using high performance liquid chromatography (HPLC) and expressed as a percentage of total cellular content (mean ± SEM). Gabapentin significantly reduced carbachol-evoked secretion (** p = 0.00023) but not basal release (p = 0.15). (B) Gabapentin treatment (GBP) did not alter the total cellular content of norepinephrine (p = 0.43) or epinephrine (p = 0.16) compared to control cells (mean ± SEM). (C) Secretion evoked by carbachol in gabapentin treated cells (GBP) was normalized to controls (CTL). Gabapentin reduced both epinephrine (epi) (p = 0.0007) and norepinephrine (norepi) (p = 0.0001) secretion to a similar extent (mean ± SEM). (D) Same layout as in panel C except using a 5-min stimulation with 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (30 μM), a selective nicotinic receptor agonist. Gabapentin significantly reduced both epinephrine (epi) (p = 0.042) and norepinephrine (norepi) (p = 0.026) secretion. (E) Representative experiment showing the [Ca²⁺]_i increase evoked by a 5-min application of 100μM carbachol in FURA-2 loaded chromaffin cells (n = 8 cells, mean ± SEM - for clarity error bars are only shown for a few data points). (F) Pooled data from multiple experiments like that shown in panel E. The peak increase in [Ca²⁺]_i evoked by carbachol was not significantly altered in gabapentin treated cells compared to control cells (mean ± SEM; p = 0.10).

Figure 2. Gabapentin reduces KCl-evoked catecholamine secretion even though calcium entry was increased

(A) Chromaffin cells were seeded on 24-well plates and treated with 1mM gabapentin (GBP) or vehicle (control) for 18–24 h. The amount of catecholamines released under basal conditions (in the absence of secretagogue) or during a 5-min stimulation with 30 mM KCl was determined using high performance liquid chromatography and expressed as a percentage of total cellular content (mean ± SEM). Gabapentin treatment significantly reduced KCl-evoked secretion (* p < 0.019; n = 8) but not basal release (p = 0.42; n = 8). (B) Secretion evoked by KCl in gabapentin treated cells (GBP) was normalized to controls from the same plate (mean ± SEM). There was a statistically significant inhibition of both epinephrine (p = 0.002; n = 8) and norepinephrine (p = 0.006; n = 8) secretion by gabapentin. (C) Representative experiment showing the [Ca²⁺]_i increase evoked by a 5-min application of 30mM KCl in FURA-2 loaded chromaffin cells (n = 18 cells, mean ± SEM - for clarity error bars are only shown for a few data points). (D) Pooled data from multiple experiments like that shown in panel C. The peak increase in [Ca²⁺]_i (peak) and the sustained increase at the end of the KCl application (end) were significantly greater in gabapentin treated cells (n = 97) compared to controls (mean ± SEM; n = 128) (** p < 0.0001).

Figure 3. The inhibition of catecholamine secretion by gabapentin is concentration-dependent

(A) Chromaffin cells were seeded on 24-well plates and treated with vehicle (control) or gabapentin (0.1mM GBP or 1mM GBP) for 18–24 h. Evoked catecholamine secretion was determined using high performance liquid chromatography (HPLC) and expressed as a percentage of total cellular content (mean ± SEM). Both concentrations of gabapentin significantly reduced secretion compared to controls (n = 8; * p < 0.05; *** p < 0.001). (B) Data from all HPLC experiments were pooled and percent inhibition of evoked catecholamine secretion (mean ± SEM) plotted against Log ₁₀ of gabapentin concentration (10 μM, n = 4; 100 μM, n = 8; 1mM, n = 20; 3 mM, n = 4). A concentration response curve was generated by fitting the data to a Boltzmann function with a Hill slope = 1, and yielded an estimated EC₅₀ of 76 μM.

Figure 4. Gabapentin does not alter the density or kinetics of voltage-gated Ca²⁺ channel currents

(A) Whole cell patch-clamp electrophysiology was used to record I_Ca elicited by 20 ms step depolarizations (see inset), and the peak amplitude (mean ± SEM) plotted against time. Acute application of 1mM gabapentin (indicated by black bar) had no effect on I_Ca amplitude. (B) Cells were treated with vehicle (ctl) or 1mM gabapentin (GBP) for 18–24 h prior to recording. Peak I_Ca was normalized to cell size (membrane capacitance) to yield current density. There was no change in current density (mean ± SEM) in gabapentin treated cells (n = 17) compared to control cells (n = 21) (p = 0.92). (C) The current-voltage relationship for I_Ca was not shifted by treatment with 1mM gabapentin for 18 – 24 h. I_Ca amplitude (mean ± SEM) was normalized to the peak inward current in each cell to facilitate comparison of the voltage-dependence. (D) Inactivation of I_Ca during a 500 ms step depolarization in control cells and cells treated for 18– 24 h with 1mM gabapentin. Traces represent the data from 8 control cells and 10 gabapentin treated cells (mean ± SEM). For clarity, error bars are shown for only a few data points. (E) Inactivation of I_Ca during a 500 ms step was fit with a double exponential decay and the time constants from each cell were pooled. There was no difference in either the fast (τ fast) (p = 0.28) or slow (τ slow) (p = 0.31) inactivation time constants between control (n = 8) and gabapentin treated cells (n = 10) (mean ± SEM).

Figure 5. Gabapentin reduces the number of secretory vesicle fusion events but not the quantal size or kinetics of catecholamine release from each vesicle

Carbon fiber amperometry was used to quantify the number, quantal size, and kinetics of individual vesicular fusion events. (A) A representative amperometry recording from a control cell stimulated with 30 mM KCl. Each upward deflection (spike) on the current trace is produced by oxidation of the catecholamines released from a single vesicular fusion event. The inset shows a cartoon representation of the recoding configuration (above) and an expanded view of a few spikes is shown below. (B) The number of amperometric spikes was determined for each cell over a 2-min period. The box graph shows the 25^th percentile, median, and 75^th percentile distribution of vesicular fusion rate (spikes per minute) for control cells (n = 26 ) and cells treated for 18–24 hours with 1mM gabapentin (n = 25). The whiskers represent the smallest and largest non-outliers in each population of cells. Gabapentin produced a statistically significantly reduction in the rate of fusion events compared to matched controls (* p = 0.046, Mann-Whitney test). (C–F) Other parameters of the amperometric spikes (amplitude, charge, slope, and duration) were analyzed and a median value for each cell calculated. Pooled values (mean ± SEM) for each parameter are shown and were compared. No statistically significant differences were found between gabapentin treated cells (GBP) and control cells (ctl).