Coexpressed δ-, μ-, and κ-Opioid Receptors Modulate Voltage-Gated Ca2+ Channels in Gastric-Projecting Vagal Afferent Neurons

Abstract

Opioid analgesics are frequently associated with gastrointestinal side effects, including constipation, nausea, dysphagia, and reduced gastric motility. Though it has been shown that stimulation of opioid receptors expressed in enteric motor neurons contributes to opioid-induced constipation, it remains unclear whether activation of opioid receptors in gastric-projecting nodose ganglia neurons contributes to the reduction in gastric motility and emptying associated with opioid use. In the present study, whole-cell patch-clamp recordings were performed to determine the mechanism underlying opioid receptor-mediated modulation of Ca²⁺ currents in acutely isolated gastric vagal afferent neurons. Our results demonstrate that Ca_V2.2 channels provide the majority (71% ± 16%) of Ca²⁺ currents in gastric vagal afferent neurons. Furthermore, we found that application of oxycodone, U-50488, or deltorphin II on gastric nodose ganglia neurons inhibited Ca²⁺ currents through a voltage-dependent mechanism by coupling to the Gα _i/o family of heterotrimeric G-proteins. Because previous studies have demonstrated that the nodose ganglia expresses low levels of δ-opioid receptors, we also determined the deltorphin II concentration-response relationship and assessed deltorphin-mediated Ca²⁺ current inhibition following exposure to the δ-opioid receptor antagonist ICI 174,864 (0.3 µM). The peak mean Ca²⁺ current inhibition following deltorphin II application was 47% ± 24% (EC₅₀ = 302.6 nM), and exposure to ICI 174,864 blocked deltorphin II-mediated Ca²⁺ current inhibition (4% ± 4% versus 37% ± 20%). Together, our results suggest that analgesics targeting any opioid receptor subtype can modulate gastric vagal circuits. SIGNIFICANCE STATEMENT: This study demonstrated that in gastric nodose ganglia neurons, agonists targeting all three classical opioid receptor subtypes (μ, δ, and κ) inhibit voltage-gated Ca²⁺ channels in a voltage-dependent mechanism by coupling to Gα_i/o. These findings suggest that analgesics targeting any opioid receptor subtype would modulate gastric vagal circuits responsible for regulating gastric reflexes.

Graphical abstract

Fig. 1.

Ca_V2.2 are expressed in acutely dissociated gastric-projecting NG neurons. (A) Maximum intensity projection confocal images (40×) of isolated gastric-projecting NG neurons showing Ca_V2.2-immunopositive expression (480-nm excitation; left), DiI labeling (530-nm excitation; center), and merged Ca_V2.2 and DiI images with DIC to show cell outline (right). In total, 20 cells (n = 3 rats) were assessed for Ca_V2.2 expression. Of these 20 cells, 10 were DiI-positive, and 10 were DiI-negative. All analyzed neurons, including the non–DiI-labeled cells, showed Ca_V2.2 channel expression. Scale bar, 50 µm. (B) Representative traces of Ca²⁺ currents from a DiI-labeled gastric vagal afferent neuron generated using the described voltage protocol. (C) Mean I-V curve demonstrating Ca²⁺ currents activate around −20 mV and peak at +5 mV in gastric-projecting NG neurons. In total, 12 cells from three rats were used to generate the I-V curve. Data are presented as mean ± S.D. I-V, current-voltage.

Fig. 2.

Characterization of voltage-gated Ca²⁺ channel subtypes expressed in gastric-projecting NG neurons. (A) Time-course of peak Ca²⁺ current amplitude from a DiI-positive NG neuron during application of 50 nM ω-agatoxin IVA, 500 nM ω-conotoxin GVIA, and 10 µM nifedipine. (B) Representative Ca²⁺ current traces before (1) and during ω-agatoxin (2), ω-conotoxin (3), and nifedipine (4) application. (C) Mean ± S.D. peak Ca²⁺ current inhibition produced by application of ω-agatoxin (13% ± 12%, n = 13), ω-conotoxin (71% ± 16%, n = 13), and nifedipine (12% ± 12%, n = 11). Application of ω-conotoxin produced a significantly higher mean Ca²⁺ current inhibition when compared with both ω-agatoxin (P < 0.0001) and nifedipine (P < 0.0001) using a one-way ANOVA. (D) Plot of peak Ca²⁺ current percent inhibition following application of ω-conotoxin (red), nifedipine (gray), and ω-agatoxin (blue) versus cell capacitance of individual gastric NG neurons. Data are presented as mean ± S.D.

Fig. 3.

Deltorphin II concentration-response relationships in gastric-projecting NG neurons. (A) Deltorphin II concentration-response relationship in acutely isolated gastric NG neurons. Each point indicates the mean (± S.D.) peak Ca²⁺ current inhibition. The curve was generated by fitting the points to the Hill equation. The number of cells tested for each drug concentration is shown in parentheses. (B) Time-course of peak Ca²⁺ current amplitude resulting from the sequential application of deltorphin II (1 µM), ICI 174,864 (0.3 µM), and the simultaneous application of both drugs. (C) Representative Ca²⁺ current traces generated using the illustrated voltage protocol at baseline (1, 3), during initial exposure to deltorphin II (2), and during re-exposure to deltorphin II following application of ICI 174,864 (4). (D) Summary plot showing the mean ± S.D. Ca²⁺ current prepulse inhibition produced by deltorphin II exposure (37% ± 20%, n = 10) compared using a paired t test to the combined deltorphin II and antagonist application following the 3-minute period of ICI 174,864 application (4% ± 4% versus 37% ± 20%, P = 0.0012) and 3-minute period of external solution control (50% ± 25% versus 37% ± 20%, P = 0.34). Data are presented as mean ± S.D. **P < 0.01.

Fig. 4.

Characterization of opioid receptor subtypes expressed in gastric-projecting NG neurons. (A) Time-course of peak Ca²⁺ current amplitude from an acutely isolated gastric NG neuron during application of oxycodone (10 µM), deltorphin II (10 µM), and U-50488 (10 µM). (B) Representative Ca²⁺ current traces before (1) and during U-50488 (2), deltorphin II (3), and oxycodone (4) application. Note the evidence of kinetic slowing during each drug application. (C) Mean ± S.D. peak Ca²⁺ current inhibition produced by application of oxycodone (66% ± 11%, n = 7, P < 0.0001), deltorphin II (47% ± 18%, n = 8, P < 0.0001), and U-50488 (30% ± 17% n = 7, P = 0.0035) compared using a one-sample t test to zero. (D) Plot of peak Ca²⁺ current percentage of inhibition following application of U-50488 (red), deltorphin II (gray), and oxycodone (blue) versus cell capacitance of individual gastric NG neurons. Data are presented as mean ± S.D. **P < 0.01; ****P < 0.0001.

Fig. 5.

Opioid receptor agonists modulate Ca²⁺ currents in a VD manner by coupling to PTX-sensitive Gα_i/o proteins. (A) Representative Ca²⁺ current traces generated using the illustrated voltage protocol before (1, 2, 5, 6, 9, 10) and during oxycodone (3, 4), U-50488 (7, 8) and deltorphin II (11, 12) application. Ca²⁺ currents were evoked using the described triple-pulse voltage protocol. Note the presence of kinetic slowing during the prepulse portion of each drug application (traces 3, 7, 11). (B) Time-course of Ca²⁺ current inhibition resulting from the sequential application of each opioid receptor subtype agonist. The dark-gray circles represent peak prepulse Ca²⁺ current amplitude, and light-gray squares represent peak postpulse Ca²⁺ current amplitude. (C) Time-course of the facilitation ratio before and during drug application. (D) Mean ± S.D. peak Ca²⁺ current inhibition produced by application of subtype-specific agonists in control neurons (light gray) and neurons treated overnight with 0.5 μg/mL PTX (dark gray). The prepulse Ca²⁺ current inhibition during drug exposure were found to be 58% ± 14% for oxycodone (n = 7), 48% ± 24% for deltorphin II (n = 8), and 39% ± 17% for U-50488 (n = 9). For DiI-positive neurons that were incubated overnight with PTX, the effects of oxycodone (n = 5, P < 0.0001), deltorphin II (n = 5, P = 0.0027), and U-50488 (n = 5, P = 0.0017) on Ca²⁺ currents were reduced when compared with neurons not treated with PTX using an unpaired Student’s t test. Data are presented as mean ± S.D. **P < 0.01; ****P < 0.0001.