Agonist-Specific Regulation of G Protein-Coupled Receptors after Chronic Opioid Treatment

Abstract

Chronic treatment of animals with morphine results in a long lasting cellular tolerance in the locus coeruleus and alters the kinase dependent desensitization of opioid and nonopioid G protein-coupled receptors (GPCRs). This study examined the development of tolerance and altered regulation of kinase activity after chronic treatment of animals with clinically relevant opioids that differ in efficacy at the µ-opioid receptors (MOR). In slices from oxycodone treated animals, no tolerance to opioids was observed when measuring the MOR induced increase in potassium conductance, but the G protein receptor kinase 2/3 blocker, compound 101, no longer inhibited desensitization of somatostatin (SST) receptors. Chronic fentanyl treatment induced a rightward shift in the concentration response to [Met⁵]enkephalin, but there was no change in the kinase regulation of desensitization of the SST receptor. When total phosphorylation deficient MORs that block desensitization, internalization, and tolerance were virally expressed, chronic treatment with fentanyl resulted in the altered kinase regulation of SST receptors. The results suggest that sustained opioid receptor signaling initiates the process that results in altered kinase regulation of not only opioid receptors, but also other GPCRs. This study highlights two very distinct downstream adaptive processes that are specifically regulated by an agonist dependent mechanism. SIGNIFICANCE STATEMENT: Persistent signaling of MORs results in altered kinase regulation of nonopioid GPCRs after chronic treatment with morphine and oxycodone. Profound tolerance develops after chronic treatment with fentanyl without affecting kinase regulation. The homeostatic change in the kinase regulation of nonopioid GPCRs could account for the systems level in vivo development of tolerance that is seen with opioid agonists, such as morphine and oxycodone, that develop more rapidly than the tolerance induced by efficacious agonists, such as fentanyl and etorphine.

Fig. 1.

Decreased sensitivity to ME after chronic fentanyl but not oxycodone treatment. (A) Representative trace showing outward currents induced by ME in a slice from an untreated animal. (B) Representative trace from a fentanyl treated animal. (C) Concentration response curve of ME normalized to UK14304 (3 µM) in slices from untreated (black circle), oxycodone treated (gray circle), and fentanyl treated animals (blue circle). In slices from untreated animals the EC₅₀ was 166 nM (95% CI = 152–297 nM, N = 3, 5 cells/concentration). There was a 2-fold rightward shift in the concentration response curve in slices from fentanyl treated (EC₅₀ fentanyl treated animals: 518 nM, 95% CI = 496–644 nM, N = 3, 5 cells per concentration). The concentration response in slices from oxycodone treated animals was not changed relative to untreated animals (179 nM, 95% CI = 117–280 nM, N = 3, 5 cells/concentration).

Fig. 2.

Oxycodone concentration response curves. (A) Representative trace showing G protein–coupled inwardly rectifying potassium channel channel currents induced by oxycodone (1 µM) and reversal by naloxone. (1 µM), followed by UK14304 (3 µM) and reversal by idaxozan (1 µM). (B) Representative trace showing oxycodone induced current in a slice from oxycodone treated and oxycodone maintained animal. Application of naloxone (1 µM) revealed the oxycodone current, followed by UK14304 (3 µM) and reversal by idaxozan (1 µM). (C) Oxycodone concentration response curves normalized to UK14304 (3 µM) current in slices from untreated animals (black circle) and oxycodone treated animals (gray circle). There were no differences in oxycodone induced currents between untreated and oxycodone treated animals, (EC₅₀ untreated: 2.5 µM, 95% CI = 1.1–5.5 µM, N = 3, 5 cells/concentration; EC₅₀ oxycodone treated animals: 4.5 µM; 95% CI = 1.4–16.1 µM, N = 3, 4 cells/concentration). Additionally, summary of oxycodone induced current in slices maintained in oxycodone treatment normalized to UK14303 (3 µM; gray square). The current induced by oxycodone (1 µM) was not different between slices from untreated, oxycodone treated, and oxycodone treated maintained in oxycodone (untreated, 35.8 ± 7.4% of UK14304; oxycodone treated, 34.9 ± 6.1% OF UK14304; oxycodone treated maintained in oxycodone, 34.5 ± 11.3% of UK14304).

Fig. 3.

Morphine induced current is decreased in slices from fentanyl but not oxycodone treated animals. (A) Representative trace showing the current induced by morphine (1 µM), reversed by naloxone (1 µM), followed by UK14303 (3 µM), reversed by idaxozan (1 µM) in a slice from an untreated animal. (B) Representative trace showing the morphine induced current in a slice from an oxycodone treated animal. (C) Representative trace showing the current induced by morphine in a slice from a fentanyl treated animal. (D) Summary data of morphine induced currents normalized to UK14303 induced currents from untreated animals (black circle, N = 11 cells from 3 male and 3 female), oxycodone treated animals (gray circle, N = 7 cells from 3 male and 2 female), and fentanyl treated animals (blue circles, N = 10 cells from 4 male and 3 female). Chronic fentanyl treatment, but not chronic oxycodone treatment, significantly reduced morphine induced current, P < .05, one-way ANOVA followed by a Tukey test).

Fig. 4.

Increased MOR desensitization by ME after chronic fentanyl but not oxycodone. (A) Representative trace showing the current induced by ME (10 µM; 2 minutes) followed by morphine (10 µM) and reversed by naloxone (1 µM). Morphine induced current is normalized to current induced by UK14303 (3 µM). (B) Representative trace showing the morphine induced current after ME (10 µM; 2 minutes) in a slice from a fentanyl treated animal. (C) Summary showing oxycodone induced current after ME (10 µM; 2 minutes) in slices from untreated animals (black circle, N = 6) and oxycodone treated animals (gray circles, N = 5) and fentanyl treated animals (blue circles, N = 6). There was no change in desensitization MORs between untreated and oxycodone treated animals (P > 0.05, unpaired t test), but the morphine induced current was decreased in slices from fentanyl treatment animals compared with untreated controls (P < 0.05, unpaired t test).

Fig. 5.

Compound 101 blocks desensitization induced by SST in slices from buprenorphine and fentanyl treated animals but not after treatment with morphine or oxycodone. (A) Representative trace of the current induced by SST (10 µM; 10 minutes) in a slice from an oxycodone treated animal in control (left) and after treatment with compound 101 (right). (B) Representative trace of the SST induced current in a slice from a fentanyl treated animal in control (left) and after treatment with compound 101 (right). (C) Summary of the % decline. From the peak current induced by SST (10 µM; 10 minutes) in slices from (from left to right) untreated, morphine treated, oxycodone treated, buprenorphine treated, and fentanyl treated animals with and without compound 101. Compound 101 was effective in reducing the decline in current in slices from untreated, buprenorphine, and fentanyl treated animals but not in morphine or oxycodone treated animals.

Fig. 6.

Compound 101 is effective after morphine treatment in MOR knockout animals. (A) Representative trace the current induced by SST (10 µM; 10 minutes) in a slice from an untreated animal in control (left) and after treatment with compound 101 (right) in a MOR knockout animal. (B) Representative trace of the current induced by SST (10 µM; 10 minutes) in a slice from a morphine treated animal in control (left) and after treatment with compound 101 (right). (C) Summary of % decline from the peak current induced by SST (10 µM; 10 minutes) in slices from an untreated (left) and morphine treated animals. In MOR knockout animals compound 101 reduced SST induced desensitization in both untreated animals (open circles, N = 7) and morphine related animals (open circles, N = 6).

Fig. 7.

Compound 101 no longer blocks the decline in SST current in animals that express the TPD-MOR. (A) Representative trace the current induced by SST (10 µM; 10 minutes) in a slice from an untreated animal in control (left) and after treatment with compound 101 (right) in a MOR knockout animal expressing TPD-MORs. (B) Representative trace of the current induced by SST (10 µM; 10 minutes) in a slice from a fentanyl treated animal in control (left) and after treatment with compound 101 (right). (C) Summary of % decline from the peak current induced by SST (10 µM; 10 minutes) in slices from an untreated (left) and fentanyl treated animals. In animals expressing the TPD-MOR, compound 101 reduced SST induced desensitization in untreated animals (open circles, N = 5) and compound 101 was ineffective at blocking SST induced desensitization in slices from fentanyl treated animals (open circles, N = 7).