Plasticity of Signaling by Spinal Estrogen Receptor α, κ-Opioid Receptor, and Metabotropic Glutamate Receptors over the Rat Reproductive Cycle Regulates Spinal Endomorphin 2 Antinociception: Relevance of Endogenous-Biased Agonism

Co-IP and coexpression of mGluR₁ and mGluR_2/3 in proestrus versus diestrus. A, solubilized spinal cord membrane fractions were immunoprecipitated using an antibody selective for mGluR₁. Immunoprecipitates were Western blotted using an antibody selective for mGluR_2/3 (+). The co-IP of mGluR_2/3 with mGluR₁ is markedly elevated in proestrus (P) versus diestrus (D). Specificity of anti-mGluR_2/3 antibody was confirmed by the absence of the Western blot signal when the sample was incubated with preadsorbed (−) anti-mGluR_2/3 antibody. Bar graph in A shows quantification of signal intensity after normalizing with the corresponding mGluR₁ that was directly immunoprecipitated. Specificity of the anti-mGluR₁ antibodies was demonstrated by Liu et al. (2017). *p < 0.05, D versus P, n = 6 for each group. B, left, Coexpression of mGluR₁ (red; arrows) and mGluR_2/3 (green; arrowheads) in a MOR-expressing neuron. Right, 2× higher-magnification images of bottom-middle part of that neuron, showing expression in that region. MOR, Blue; mGluR₁, red; mGluR_2/3, green. These data provide a cellular basis for the cooperative modulation of intrathecal EM2 antinociception by mGluR₁ and mGluR_2/3. Data in bar graph are expressed as mean + SEM.

Basal spinal dynorphin release depends on stage of estrous cycle. Magnitude of basal spinal dynorphin (Dyn) release is greater in proestrus versus diestrus (*p < 0.05; n = 18 and 21, respectively). This suggests a positive correlation between the magnitude of basal spinal Dyn release and spinal EM2 antinociception. Data are expressed as mean + SEM.

Activity of endogenous spinal dynorphin/KOR is a prerequisite for the spinal EM2 analgesia unveiled by blocking mERα or mGluR₁ in diestrus. A–C, Analgesic responsiveness to intrathecal EM2 (45 nmol) was determined in diestrus following intrathecal pretreatment with either MPP (ERα blocker; A), YM298198 (mGluR₁ blocker; B, YM), or dynorphin (C, Dyn). MPP (10 nmol) or YM was administered alone or in combination with anti-dynorphin antibody (DyAb, 300 ng) or norBNI (BNI, 26 nmol). MPP, YM, or DyAb was administered 30 min preceding intrathecal EM2. BNI was administered 18 h before intrathecal EM2. TFL was determined at the indicated times. Either intrathecal DyAb or norBNI abolished the spinal EM2 antinociception unveiled by blocking either spinal mERα or mGluR₁. In contrast, intrathecal Dyn unmasked intrathecal EM2 antinociception during diestrus. Pretreatment with the vehicle (water) for DyAb/BNI did not alter the ability of mERα or mGluR₁ antagonists to unmask intrathecal EM2 antinociception. Furthermore, neither of the indicated treatments, in the absence of intrathecal EM2, but in the presence of its vehicle (3% DMSO), altered TFL. n = 4–7. Since BNI was added 18 h before intrathecal EM2, its indicated TFL at zero time represents that obtained 17.5 h after the spinal application of BNI. Veh1, DMSO (vehicle for MPP); Veh2, water (vehicle for DyAb/BNI/YM/Dyn); Veh3, 3% DMSO (vehicle for EM2). These data indicate that suppression of endogenous spinal dynorphin/KOR signaling is causally associated with the absence of spinal EM2 analgesic responsiveness during diestrus. Data are expressed as mean ± SEM.

Activity of mERα and mGluR₁ is necessary for EM2 inhibition of spinal dynorphin (Dyn) release in diestrus but not proestrus. Intrathecal EM2 (45 nmol) inhibits spinal Dyn release similarly in proestrus and diestrus, but regulation of this modulation by spinal mERα or mGluRs depends on stage of estrous cycle. In proestrus, blocking mGluR₁ (via YM298198, 25 nmol, 30 min), mGluR_2/3 (via LY341495, 25 nmol, 30 min), or mERα (via MPP, 10 nmol, 30 min) fails to alter the EM2 inhibition of spinal Dyn release. In contrast, YM298198 and MPP profoundly attenuate EM2 inhibition of Dyn release during diestrus (n = 6–8). The effect on EM2 inhibition of Dyn release of blocking mGluR_2/3 during diestrus and mERα during proestrus was not determined since these treatments did not alter spinal EM2 antinociception. Although either YM298198 or MPP attenuates EM2 inhibition of Dyn release, these blockers failed to increase Dyn release in the absence of EM2 (but in the presence of its vehicle; data not shown). The ability of mGluR₁ or ERα blockade to reduce EM2 inhibition of Dyn release during diestrus suggests that spinal Dyn tone is a critical determinant of spinal EM2 analgesic responsiveness. *p < 0.05 for YM/MP+EM2 versus EM2 in diestrus. YM, YM298198; LY, LY341495; MP, MPP; Veh, vehicle (DMSO). Data are expressed as mean + SEM.

Glutamate is required for the manifestation of spinal EM2 analgesia during proestrus. Analgesic responsiveness to intrathecal EM2 (45 nmol) was determined using TFL following intrathecal pretreatment with riluzole (Rilu; glutamate release inhibitor, 43 nmol, 1 h) or its water vehicle (Veh1). Attenuation of spinal glutamatergic activity (via Rilu) eliminated spinal EM2 antinociception. Intrathecal Rilu, in the absence of intrathecal EM2, but in the presence of its vehicle, did not alter TFL. n = 4–6. Veh2, 3% DMSO. Data are expressed as mean + SEM.

A single spinal dynorphinergic neuron coexpresses ERα and mGluR₁ and is apposed by glutamatergic terminals. The two images are from adjacent 5 μm sections through the same neuron. Left, mGluR₁ (green; arrowheads), ERα (blue), and dynorphin (Dyn; red) are coexpressed in or near the plasma membrane in a single neuron in the spinal superficial dorsal horn of segment L6 during proestrus. Right, Glutamatergic terminals (green; arrow, stained for VGLUTs) appose the neuron expressing ERα and Dyn.

Spinal KOR and mGluR_2/3 are present in an oligomer containing MOR, mGluR₁, ERα, and aromatase. Solubilized spinal cord membranes were electrophoresed using blue native (BN) gels. A, E, A ≈360 kDa band identified with Coomassie Blue (A) was eluted and subjected to reducing SDS PAGE Western analysis using anti-KOR and anti-mGluR_2/3 antibodies (E). Since mGluR_2/3 that coimmunoprecipitated with mGluR₁ appeared at the predicted molecular mass (≈110 kDa; Fig. 2), its detected Western blot signal (≈200 kDa) derived from the oligomer likely represents its dimerized form. B, To validate the idea that the visualized KOR and mGluR_2/3 originated from the same oligomer as that previously shown to contain aromatase (Aro), ERα, mGluR₁, and MOR, solubilized spinal membranes were subjected to sequential IP using antibodies (in order) against Aro, ERα, mGluR₁, KOR, and mGluR_2/3. C, D, MOR Western blotting of the final immunoprecipitate following BN gel electrophoresis revealed a ≈360 kDa band (D), as was obtained when using the same procedure but absent IP with anti-KOR and anti-mGluR_2/3 antibodies (C). A–C [previously reported (Liu et al., 2017), reproduced with permission from Pain) are included to integrate current findings with the previously defined oligomer. The discrepancy between the apparent molecular mass of the oligomer and the sum of its monomeric components (≈360 vs ≈450 kDa) likely results from the oligomer being resolved using nonreducing and nondenaturing conditions (BN PAGE), maintaining structure and charge density (shape, hydrodynamic diameter, and charge influence apparent molecular mass). In contrast, oligomer components were resolved using SDS-PAGE, eliminating three-dimensional structure, making electrophoretic mobility dependent predominantly on size. Additionally, notwithstanding inaccuracies of molecular mass estimation using BN gels, summing apparent molecular mass of oligomer components resolved on SDS PAGE exacerbates the commonly accepted 10–15% variability of using this method for molecular mass estimation (Goetz et al., 2004).