Regulation of μ and δ opioid receptor functions: involvement of cyclin-dependent kinase 5

Abstract

Background and purpose: Phosphorylation of δ opioid receptors (DOP receptors) by cyclin-dependent kinase 5 (CDK5) was shown to regulate the trafficking of this receptor. Therefore, we aimed to determine the role of CDK5 in regulating DOP receptors in rats treated with morphine or with complete Freund's adjuvant (CFA). As μ (MOP) and DOP receptors are known to be co-regulated, we also sought to determine if CDK5-mediated regulation of DOP receptors also affects MOP receptor functions.

Experimental approach: The role of CDK5 in regulating opioid receptors in CFA- and morphine-treated rats was studied using roscovitine as a CDK inhibitor and a cell-penetrant peptide mimicking the second intracellular loop of DOP receptors (C11-DOPri2). Opioid receptor functions were assessed in vivo in a series of behavioural experiments and correlated by measuring ERK1/2 activity in dorsal root ganglia homogenates.

Key results: Chronic roscovitine treatment reduced the antinociceptive and antihyperalgesic effects of deltorphin II (Dlt II) in morphine- and CFA-treated rats respectively. Repeated administrations of C11-DOPri2 also robustly decreased Dlt II-induced analgesia. Interestingly, DAMGO-induced analgesia was significantly increased by roscovitine and C11-DOPri2. Concomitantly, in roscovitine-treated rats the Dlt II-induced ERK1/2 activation was decreased, whereas the DAMGO-induced ERK1/2 activation was increased. An acute roscovitine treatment had no effect on Dlt II- or DAMGO-induced analgesia.

Conclusions and implications: Together, our results demonstrate that CDK5 is a key player in the regulation of DOP receptors in morphine- and CFA-treated rats and that the regulation of DOP receptors by CDK5 is sufficient to modulate MOP receptor functions through an indirect process.

Figure 1

Determination of efficient roscovitine dose in morphine-treated rats. Sprague-Dawley rats were injected s.c. once every 12 h with escalating doses of morphine (5, 8, 10 and 15 mg·kg⁻¹). Twelve hours after the last morphine injection, tail flick latencies (in s) were measured every 10 min (from 0 to 60 min) after Dlt II i.t. injection (10 μg) using the tail immersion test. Data at the 20 min time point, representing the peak effect of Delt II, were used for the calculation of the %MPE. (A) i.t. roscovitine (1, 3, 10, 30 μg) administered 30 min before each morphine injection produced a dose-dependent decrease in Dlt II-induced antinociception. *P < 0.05 (n= 6 animals per group). (B) The antinociceptive effect of increasing doses of i.t. Dlt II expressed as the %MPE (percentage of the MPE) is shown for animals pretreated with morphine over 48 h and with vehicle or roscovitine 30 μg. **P < 0.01 (n= 4–6 animals per group).

Figure 2

Effect of roscovitine on DOP receptor-mediated antihyperalgesia and antinociception. (A) Sprague-Dawley rats were injected with CFA in the plantar surface of the hindpaw. Thirty minutes before the CFA injection and every 12 h thereafter, rats were injected i.t. with roscovitine (30 μg) or vehicle (30 μL). Seventy-two hours after CFA injection, paw withdrawal latencies (in s) to noxious heat (plantar test) were recorded every 15 min for a period of 60 min following Dlt II administration (10 μg, i.t.). I.t.-administered roscovitine (30 μg) induced a significant decrease in DOP receptor-mediated antihyperalgesia. *P < 0.05 and ****P < 0.0001. (B) Sprague-Dawley rats injected s.c. once every 12 h with escalating doses of morphine (5, 8, 10 and 15 mg·kg⁻¹) received roscovitine (30 μg) or vehicle (30 μL) 30 min before each morphine injection. Twelve hours after the last morphine injection, tail flick latencies (in s) were measured every 10 min (from 0 to 60 min) after Dlt II injection (10 μg, i.t.) using the tail immersion test. Roscovitine injection induced a significant decrease in DOP receptor-mediated antinociception. **P < 0.01 and ***P < 0.001. (C) Results presented in (A) are expressed as the AUC obtained between 0 and 60 min after Dlt II injection (the Y-axis baseline was set for each animal according to their latency to paw withdrawal after inflammation). **P < 0.01. (D) Results presented in (B) are expressed as the AUC obtained between 0 and 60 min after Dlt II injection (Y-axis baseline was set for each animal according to their latency to tail withdrawal at 0 min). *P < 0.05. Numbers given in parentheses represent the number of animals per group.

Figure 3

Effect of C11-DOPri2 on DOP receptor-mediated antihyperalgesia and antinociception. (A) Sprague-Dawley rats were injected with CFA in the plantar surface of the hindpaw. Thirty minutes before the CFA injection and every 12 h thereafter, rats were injected i.t. with C11-DOPri2 (6 and 15 μg) or C11-DOPrscrambled peptides (6 and 15 μg). Seventy-two hours after the CFA injection, paw withdrawal latencies (in s) to noxious heat (plantar test) were recorded every 15 min (from 0 to 60 min) following Dlt II administration (10 μg, i.t.). I.t.-administered C11-DOPri2 (6 and 15 μg) induced a significant decrease in DOP receptor-mediated antihyperalgesia. ****P < 0.0001. (B) Sprague-Dawley rats injected s.c. once every 12 h with escalating doses of morphine (5, 8, 10 and 15 mg·kg⁻¹) received i.t. injections of C11-DOPri2 (6 and 15 μg) or C11-DOPrscrambled (6 and 15 μg) 30 min before each morphine injection. Twelve hours after the last morphine injection, tail flick latencies (in s) were measured every 10 min (from 0 to 60 min) after Dlt II injection (10 μg, i.t.) using the tail immersion test. C11-DOPri2 induced a significant decrease in DOP receptor-mediated antinociception. **P < 0.01. (C) Results presented in (A) are expressed as the AUC obtained between 0 and 60 min after Dlt II injection (the Y-axis baseline was set for each animal according to their latency to paw withdrawal after inflammation). *P < 0.05. (D) Results presented in (B) are expressed as the AUC obtained between 0 and 60 min after Dlt II injection (the Y-axis baseline was set for each animal according to their latency to tail withdrawal at 0 min). **P < 0.01. Numbers given in parentheses represent the number of animals per group.

Figure 4

Effect of roscovitine on MOP receptor-mediated antihyperalgesia and antinociception. (A) Sprague-Dawley rats were injected with CFA in the plantar surface of the hindpaw. Thirty minutes before the CFA injection and every 12 h thereafter, rats were injected i.t. with roscovitine (30 μg) or vehicle (30 μL). Seventy-two hours after the CFA injection, paw withdrawal latencies (in s) to noxious heat (plantar test) were recorded every 15 min (from 0 to 60 min) following DAMGO administration (100 ng, i.t.). I.t.-administered roscovitine (30 μg) induced a significant increase in MOP receptor-mediated antihyperalgesia. *P < 0.05, **P < 0.01 and ***P < 0.001. (B) Sprague-Dawley rats injected s.c. once every 12 h with escalating doses of morphine (5, 8, 10 and 15 mg·kg⁻¹) received a roscovitine (30 μg) or a vehicle (30 μL) i.t. injection 30 min before each morphine injection. Twelve hours after the last morphine injection, tail flick latencies (in s) were measured every 10 min (from 0 to 60 min) after DAMGO injection (30 ng, i.t.) using the tail immersion test. Roscovitine induced a significant increase in MOP receptor-mediated antinociception. **P < 0.01. (C) Results presented in (A) are expressed as the AUC obtained between 0 and 60 min after DAMGO injection (the Y-axis baseline was set for each animal according to their latency to paw withdrawal after inflammation). **P < 0.01. (D) Results presented in (B) are expressed as the AUC obtained between 0 and 60 min after DAMGO II injection (the Y-axis baseline was set for each animal according to their latency to tail withdrawal at 0 min). *P < 0.05. Numbers given in parentheses represent the number of animals per group.

Figure 5

Effect of C11-DOPri2 on MOP receptor-mediated antihyperalgesia and antinociception. (A) Sprague-Dawley rats were injected with CFA in the plantar surface of the hindpaw. Thirty minutes before the CFA injection and every 12 h thereafter, rats were injected i.t. with C11-DOPri2 (6 and 15 μg) or C11-DOPrscrambled peptides (6 and 15 μg). Seventy-two hours after the CFA injection, paw withdrawal latencies (in s) to noxious heat (plantar test) were recorded every 15 min (from 0 to 60 min) following DAMGO administration (30 ng, i.t.). I.t.-administered C11-DOPri2 induced a significant increase in MOP receptor-mediated antihyperalgesia. ****P < 0.0001. (B) Sprague-Dawley rats injected s.c. once every 12 h with escalating doses of morphine (5, 8, 10 and 15 mg·kg⁻¹) received i.t. injections of C11-DOPri2 (6 and 15 μg) or C11-DOPrscrambled peptides (6 and 15 μg) 30 min before each morphine injection. Twelve hours after the last morphine injection, tail flick latencies (in s) were measured every 10 min (from 0 to 60 min) after DAMGO injection (30 ng, i.t.) using the tail immersion test. C11-DOPri2 induced a significant increase in MOP receptor-mediated antinociception. *P < 0.05. (C) Results presented in (A) are expressed as the AUC obtained between 0 and 60 min after DAMGO injection (the Y-axis baseline was set for each animal according to their latency to paw withdrawal after inflammation). **P < 0.01. (D) Results presented in (B) are expressed as the AUC obtained between 0 and 60 min after DAMGO II injection (the Y-axis baseline was set for each animal according to their latency to tail withdrawal at 0 min). *P < 0.05. Numbers given in parentheses represent the number of animals per group.

Figure 6

Effect of acute roscovitine on DOP and MOP receptor-mediated analgesia. (A) Sprague-Dawley rats were injected with CFA in the plantar surface of the hindpaw. Seventy-two hours after the CFA injection, rats received an i.t. injection of roscovitine (30 μg) or vehicle (30 μL). Thirty minutes after the injection of roscovitine or vehicle, the paw withdrawal latencies (in s) to noxious heat (plantar test) were recorded every 15 min for 60 min following Dlt II administration (10 μg, i.t.). I.t.-administered roscovitine (30 μg) did not modify DOP receptor-mediated antihyperalgesia. (B) Sprague-Dawley rats were injected s.c. once every 12 h with escalating doses of morphine (5, 8, 10 and 15 mg·kg⁻¹). Twelve hours after the last morphine injection they received an i.t. injection of roscovitine (30 μg) or vehicle (30 μL). 30 min thereafter, tail flick latencies (in s) were measured every 10 min (from 0 to 60 min) after Dlt II injection (10 μg, i.t.) using the tail immersion test. Roscovitine injection did not modify DOP receptor-mediated antinociception. (C) Rats were injected with CFA in the plantar surface of the hindpaw. Seventy-two hours after CFA injection, they received an i.t. injection of roscovitine (30 μg) or vehicle (30 μL). Thirty minutes after the injection, paw withdrawal latencies (in s) to noxious heat (plantar test) were recorded every 15 min for 60 min following DAMGO administration (30 ng, i.t.). I.t.-administered roscovitine (30 μg) did not modify MOP receptor-mediated antihyperalgesia. (D) Sprague-Dawley rats were injected s.c. once every 12 h with escalating doses of morphine (5, 8, 10 and 15 mg·kg⁻¹). Twelve hours after the last morphine injection they received an i.t. injection of roscovitine (30 μg) or vehicle (30 μL). Thirty minutes after the injection, tail flick latencies (in s) were measured every 10 min (from 0 to 60 min) after DAMGO injection (30 ng, i.t.) using the tail immersion test. Roscovitine injection did not modify MOP receptor-mediated antinociception. Numbers given in parentheses represent the number of animals per group.

Figure 7

Roscovitine modifies the activity of DOP and MOP receptors ex vivo. Sprague-Dawley rats were injected with CFA in the plantar surface of the hindpaw. Thirty minutes before the CFA injection and every 12 h thereafter, rats were injected i.t. with vehicle or roscovitine (30 μg, i.t.). Seventy two hours after the CFA injection, rats received a single i.t. injection of Dlt II (10 μg) or DAMGO (30 ng) for 20 min, afterwards the DRGs were quickly collected and frozen in dry ice. Western blot analysis of phosphorylated ERK1/2 (pERK1 and pERK2) was performed as described in the Methods section. (A) Representative autoradiogram of ERK1/2 activation by Dlt II or DAMGO. The lower panel shows total ERK1/2. (B) Densitometric analysis of p42/p44MAPK activation. In CFA-inflamed rats, Dlt II and DAMGO increased ERK1/2 phosphorylation in vehicle-treated rats. In roscovitine-treated rats, Dlt II-induced ERK1/2 phosphorylation was reduced whereas DAMGO-induced ERK1/2 phosphorylation was increased. *P < 0.05, **P < 0.01 and ***P < 0.001. (n = 3–5 animals per group).