Gi/o-coupled receptors compete for signaling to adenylyl cyclase in SH-SY5Y cells and reduce opioid-mediated cAMP overshoot

Abstract

Organization of G protein-coupled receptors and cognate signaling partners at the plasma membrane has been proposed to occur via multiple mechanisms, including membrane microdomains, receptor oligomerization, and protein scaffolding. Here, we investigate the organization of six types of Gi/o-coupled receptors endogenously expressed in SH-SY5Y cells. The most abundant receptor in these cells was the μ-opioid receptor (MOR), the activation of which occluded acute inhibition of adenylyl cyclase (AC) by agonists to δ-opioid (DOR), nociceptin/orphanin FQ peptide (NOPr), α2-adrenergic (α2AR), cannabinoid 1, and serotonin 1A receptors. We further demonstrate that all receptor pairs share a common pool of AC. The MOR agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) also occluded the ability of DOR agonist to stimulate G proteins. However, at lower agonist concentrations and at shorter incubation times when G proteins were not limiting, the relationship between MOR and DOR agonists was additive. The additive relationship was confirmed by isobolographic analysis. Long-term coadministration of MOR and DOR agonists caused cAMP overshoot that was not additive, suggesting that sensitization of AC mediated by these two receptors occurs by a common pathway. Furthermore, heterologous inhibition of AC by agonists to DOR, NOPr, and α2AR reduced the expression of cAMP overshoot in DAMGO-dependent cells. However, this cross-talk did not lead to heterologous tolerance. These results indicate that multiple receptors could be tethered into complexes with cognate signaling proteins and that access to shared AC by multiple receptor types may provide a means to prevent opioid withdrawal.

Fig. 1.

G_i/o-coupled receptors endogenously expressed in SH-SY5Y cells share a common pool of AC. A, short-term inhibition of 5 μM forskolin-stimulated AC by 1 μM concentration of the indicated agonist alone. Agonist (receptor): DAMGO (MOR), SNC80 (DOR), OFQ (NOPr), UK14,304 (α₂AR), clonidine (α₂AR), CP 55,940 (CB₁), and 8-OH-DPAT (5-HT_1A). ***, p < 0.001 compared with DAMGO by one-way ANOVA with Bonferroni's post-test. B, coincubation with 1 μM DAMGO occludes inhibition by 1 μM concentration of all indicated agonists. All bars are not statistically different from 1 μM DAMGO alone (p > 0.05 by one-way ANOVA with Bonferroni's post-test). C, lower efficacy agonists (1 μM) were also not additive when coadministered in the indicated pairs (p > 0.05 for all pairs compared with the most efficacious agonist of the pair by one-way ANOVA with Bonferroni's post test). Data are presented as mean ± S.E.M. (n = 4, in duplicate) of the percentage of cAMP inhibition, where stimulation by 5 μM forskolin alone is represented as 0%.

Fig. 2.

MOR and DOR share pertussis toxin-sensitive G proteins. A, stimulation of [³⁵S]GTPγS binding in membranes from SH-SY5Y cells after 60-min incubation with 1 μM DAMGO or SNC80 alone or in combination (DAMGO/SNC80). Incubation with DAMGO and SNC80 in combination (DAMGO/SNC80) did not significantly increase [³⁵S]GTPγS binding more than DAMGO alone (p > 0.05 by one-way ANOVA with Bonferroni's post test) and stimulated significantly less [³⁵S]GTPγS binding than the theoretical additive of the individual responses (R₁ + R₂) (**, p < 0.01 by one-way ANOVA with Bonferroni's post test). B, pretreatment of SH-SY5Y cells with PTX (100 ng/ml) for 24 h before membrane preparation blocked the stimulation of [³⁵S]GTPγS binding by 1 μM DAMGO, SNC80, or the combination (DAMGO/SNC80) (***, p < 0.001 by two-way ANOVA with Bonferroni's post test). Pertussis toxin treatment did not alter spontaneous [³⁵S]GTPγS binding in the absence of agonist (p > 0.05 by two-way ANOVA with Bonferroni's post test). Data are presented as mean ± S.E.M. (n = 3, in triplicate); n.s., not significant.

Fig. 3.

DAMGO and SNC80 activation of G protein is additive at concentrations or time points when G protein is not limiting. A, concentration-dependent stimulation of [³⁵S]GTPγS binding in SH-SY5Y membranes after 60-min incubation with various concentrations of DAMGO alone (■) or DAMGO with 30 nM SNC80 (□). The EC₅₀ of DAMGO is not significantly altered by the addition of 30 nM SNC80 (DAMGO alone, 121 ± 32 nM, DAMGO + 30 nM SNC80, 64 ± 12 nM; p = 0.14 by two-tailed Student's t test). Coincubation of DAMGO with 30 nM SNC80 produces additive [³⁵S]GTPγS binding similar to the theoretical additive curve (●), which diverges only when DAMGO becomes occlusive at maximal concentrations (*, p < 0.05 at 10 μM for DAMGO + 30 nM SNC80 compared with the “theoretical addition” by two-way ANOVA with Bonferroni's post test; n = 4, in duplicate). B, isobologram for agonists with a variable potency ratio calculated as described under Materials and Methods. Stimulation of [³⁵S]GTPγS binding by DAMGO was conducted in the presence of indicated concentrations of SNC80. Concentration combinations that produced 50% of the maximum effect of DAMGO alone are plotted from three separate experiments as mean ± S.E.M. Points on the line indicate additivity between DAMGO and SNC80. C, stimulation of [³⁵S]GTPγS binding in SH-SY5Y membranes after incubation with 1 μM DAMGO or SNC80 alone or in combination for 10 or 20 min, before the incubation reaches steady state. At both time points, coincubation with DAMGO and SNC80 (DAMGO/SNC80) is greater than DAMGO alone (*, p < 0.05 by one-way ANOVA with Bonferroni's post test) and similar to the theoretical additive (R₁ + R₂) (p > 0.05 by one-way ANOVA with Bonferroni's post test; n = 2, in triplicate); n.s., not significant.

Fig. 4.

MOR and DOR share AC during long-term agonist administration. SH-SY5Y cells were treated overnight with vehicle (□) or 10 μM DPDPE () in the presence or absence of the MOR agonist DAMGO (10 or 100 nM) to induce dependence. Withdrawal was precipitated with the opioid antagonist naloxone (100 μM) in the presence of 5 μM forskolin. Data are presented as mean ± S.E.M. (n = 4, in duplicate) of the percentage of forskolin-stimulated cAMP, where forskolin alone is 100% and is indicated by the dashed line. Overnight incubation with DPDPE produced overshoot on its own and enhanced the overshoot produced by 10 nM but not 100 nM DAMGO. ***, p < 0.001 compared with the vehicle with the same concentration of DAMGO by two-way ANOVA and Bonferroni's post test.

Fig. 5.

DAMGO-mediated cAMP overshoot is reduced by heterologous inhibition of shared AC by agonist to DOR, NOPr, or α₂AR. AC sensitization was developed by incubating SH-SY5Y cells overnight with 100 nM DAMGO. To precipitate withdrawal, DAMGO-containing media were replaced with media containing 5 μM forskolin, 1 mM IBMX, and 1 μM CTAP in the presence or absence of 1 μM concentration of a non-MOR agonist, as indicated, for 10 min. Data are presented as mean ± S.E.M. of the percentage of cAMP overshoot, where stimulation by forskolin alone is represented as 0%. Three of six experiments, in duplicate, were compiled that produced >100% DAMGO overshoot in the absence of a non-MOR agonist. **, p < 0.01; ***, p < 0.001 compared with DAMGO overshoot without non-MOR agonist by one-way ANOVA with Bonferroni's post test.

Fig. 6.

Inhibition of cAMP by MOR or DOR agonists is similar for sensitized or nonsensitized AC. A, SH-SY5Y cells were incubated with vehicle (■) or the MOR agonist DAMGO (100 nM, □) overnight to induce dependence. Withdrawal was precipitated with the MOR antagonist CTAP (1 μM) in the presence of 5 μM forskolin. Short-term cAMP production was inhibited by including various concentrations of the DOR agonist SNC80 in the precipitating media. The concentration-response of SNC80 to inhibit cAMP was similar in control and DAMGO-dependent cells (EC₅₀: vehicle-treated = 14.6 ± 7.8 nM, DAMGO-treated, 13.8 ± 7.2 nM; p > 0.05 by two-tailed student's t test). B, cells were incubated with vehicle (■) or the DOR agonist DPDPE (10 μM, □) overnight to induce dependence. Receptor-specific withdrawal was precipitated with the DOR antagonist ICI 174,864 (1 μM) in the presence of 30 μM forskolin. Various concentrations of the MOR agonist DAMGO were included in the precipitating media. The concentration-response of DAMGO to inhibit cAMP production was similar in control and DPDPE-withdrawn cells (EC₅₀: vehicle-treated, 32.2 ± 12.5 nM, DPDPE-treated, 29.0 ± 7.1 nM; p > 0.05 by two-tailed Student's t test). Data are presented as mean picomoles of cAMP per milligram of protein ± S.E.M. (n = 3 or 4, in duplicate). cAMP produced by forskolin alone is indicated by the dashed line.

Fig. 7.

Lack of heterologous tolerance between MOR and DOR. SH-SY5Y cells were incubated with 1 μM concentration of the MOR agonist DAMGO (A and B) or the DOR agonist SNC80 (C and D) for 24 h before membrane preparation. [³⁵S]GTPγS binding in membranes from treated cells was stimulated by incubation for 60 min with various concentrations of DAMGO (A and D) or SNC80 (B and C). Long-term treatment with 1 μM DAMGO reduced the maximal effect and potency of DAMGO (EC₅₀: vehicle-treated, 86 ± 16 nM; DAMGO-treated, 260 ± 62 nM, p = 0.04) but not SNC80 (EC₅₀: vehicle-treated, 29.8 ± 12.5 nM; DAMGO-treated EC₅₀, 34.6 ± 10.8 nM; p > 0.05). Likewise, long-term treatment with 1 μM SNC80 almost completely abolished SNC80-mediated [³⁵S]GTPγS binding but did not alter the potency of DAMGO-mediated [³⁵S]GTPγS binding (EC₅₀: vehicle-treated, 69 ± 8.5 nM; SNC80-treated, 79 ± 1.6 nM, p > 0.05). EC₅₀ statistical comparisons were made by two-tailed Student's t test. E, Gα_o and Gα_i2 were detected in membranes from cells treated overnight with vehicle (DMEM, V), 1 μM DAMGO (D), or 1 μM SNC80 (S). Immunoreactive density was quantified, normalized to tubulin loading control, and compared with vehicle-treated cells (n = 3). There was no difference in G protein levels after agonist treatment (p > 0.05 by one-way ANOVA with Bonferroni's post test).

Fig. 8.

Schematic depicting the accessibility of G_i/o-coupled receptors to portions of the total AC pool. The amount of the AC pool used by each receptor type is related to the agonist-mediated activity of each receptor. The most active and most highly expressed receptor, MOR, shares AC with all other receptor types. The other receptor types share AC in a manner predicted by receptor density and/or the ability of agonist to inhibit AC such that three receptor groups exist: one that contains MOR only; one that contains MOR, DOR, and NOPr; and one that contains MOR, DOR, NOPr, α₂AR, CB₁, and 5-HT_1A.