Src Kinase Inhibition Attenuates Morphine Tolerance without Affecting Reinforcement or Psychomotor Stimulation

Abstract

Background: Prolonged opioid administration leads to tolerance characterized by reduced analgesic potency. Pain management is additionally compromised by the hedonic effects of opioids, the cause of their misuse. The multifunctional protein β-arrestin2 regulates the hedonic effects of morphine and participates in tolerance. These actions might reflect µ opioid receptor up-regulation through reduced endocytosis. β-Arrestin2 also recruits kinases to µ receptors. We explored the role of Src kinase in morphine analgesic tolerance, locomotor stimulation, and reinforcement in C57BL/6 mice.

Methods: Analgesic (tail withdrawal latency; percentage of maximum possible effect, n = 8 to 16), locomotor (distance traveled, n = 7 to 8), and reinforcing (conditioned place preference, n = 7 to 8) effects of morphine were compared in wild-type, µ, µ, and β-arrestin2 mice. The influence of c-Src inhibitors dasatinib (n = 8) and PP2 (n = 12) was examined.

Results: Analgesia in morphine-treated wild-type mice exhibited tolerance, declining by day 10 to a median of 62% maximum possible effect (interquartile range, 29 to 92%). Tolerance was absent from mice receiving dasatinib. Tolerance was enhanced in µ mice (34% maximum possible effect; interquartile range, 5 to 52% on day 5); dasatinib attenuated tolerance (100% maximum possible effect; interquartile range, 68 to 100%), as did PP2 (91% maximum possible effect; interquartile range, 78 to 100%). By contrast, c-Src inhibition affected neither morphine-evoked locomotor stimulation nor reinforcement. Remarkably, dasatinib not only attenuated tolerance but also reversed established tolerance in µ mice.

Conclusions: The ability of c-Src inhibitors to inhibit tolerance, thereby restoring analgesia, without altering the hedonic effect of morphine, makes c-Src inhibitors promising candidates as adjuncts to opioid analgesics.

Fig. 1

Pathways implicated in tolerance that converge on Src. Neurons contain high levels of c-Src. Recent studies have identified several pathways that converge on Src, their inhibition reduces morphine tolerance,,– potentially implicating the non-receptor tyrosine kinase as a hub in this process. Red spots represent targets of Src-mediated phosphorylation.,, μ Receptors (grey), the chemokine receptor type 4 (CXCR4), the leptin receptor (LEPR), N-methyl D-aspartate receptors (NMDA-R) and platelet derived growth factor receptor β (PDGF-β) are depicted in grey, red, green, dark blue and light blue, respectively. β-Arrestin2 (β-arr2), Src kinase (Src), c-Jun N-terminal kinase (JNK), mechanistic target of rapamycin (mTOR) and N-type Ca²⁺ channels (Ca_V2.2) are depicted in green, pink, orange, yellow and light blue, respectively.

Fig. 2

Dasatinib attenuates morphine tolerance. (A) The dose-dependence of morphine prolongation of latency for tail withdrawal from noxious heat in wild type (n = 29) and μ+/- mice (n = 15). The ED₅₀ was significantly greater in μ+/- mice (Table 1). Morphine (10 mg/Kg) had no effect on tail withdrawal latency when applied to μ-/- mice (n = 15). (B) The development of morphine (10 mg/Kg) analgesic tolerance in wild type (n = 16), μ+/- (n = 15) and β-arr2-/- (n = 15) mice. Data identified with asterisks were significantly different from equivalent wild type data (*p < 0.05, Kruskall-Wallis test, post hoc Dunn’s correction). (C) The dose-dependence of morphine prolongation of latency for tail withdrawal from noxious heat in μ+/- mice, in which tolerance was induced by 4 once daily injections of morphine (10 mg/Kg s.c.). The morphine dose-response relationship was examined on day 5 (n = 8). Compared to naïve MOP+/- mice, tolerance caused a reduction in the analgesic potency of morphine (Table 1). (D) The dose-response relationship for morphine in β-arr2-/- (n = 16) was similar to that of wild type mice (Table 1). Inset, tail withdrawal latency was longer for β-arr2-/- compared to WT mice (*p < 0.001, student’s t-test). (E) Dasatinib (5 mg/Kg i.p.), applied 30 mins before morphine (10 mg/Kg), reduced morphine tolerance in wild type mice (n = 8) compared to vehicle treated controls (n = 8). Inset, the schematic represents the dosing regimen. Data identified with asterisks were significantly different from equivalent vehicle data (*p < 0.01, Kruskall-Wallis test, post hoc Dunn’s correction). (F) Dasatinib neither affected the morphine dose-response relationship nor the time for tail withdrawal measured in the absence of morphine (inset bar graph). Data points in (B) and (E) represent median values and error bars are ± IQR. All other data are expressed as mean ± SD.

Fig. 3

Inhibition of c-Src attenuates morphine tolerance. (A) Dasatinib (n = 8) reduced tolerance in μ+/- mice compared to vehicle injections (n = 8). Data identified with asterisks were significantly different from equivalent vehicle data (*p < 0.05, Kruskall-Wallis test, post hoc Dunn’s correction). (B) Western blot showing total c-Src (left panel) and phosphorylated c-Src (right panel) extracted from SW620 colon cancer cells treated with either DMSO (vehicle), PP2 (10 μM), dasatinib (10 μM) or PP3 (10 μM). β-Actin was used as a loading control. PP2 and dasatinib reduced phosphorylated c-Src levels, while PP3 had no effect relative to vehicle. (C) The relatively selective c-Src inhibitor, PP2 (5 mg/Kg i.p.), also attenuated the development of morphine tolerance, while the inactive analogue, PP3 (5 mg/Kg i.p.) did not. Data identified with asterisks were significantly different from equivalent PP3 data (*p < 0.01, Kruskall-Wallis test, post hoc Dunn’s correction). Data are presented as median ± IQR.

Fig. 4

Either fewer μ receptors or the absence of β-arrestin2 diminishes psychomotor stimulation by morphine. (A) Morphine (10 mg/Kg s.c.) stimulated locomotor activity in WT mice averaged over the 3 days of conditioning (*p < 0.001, n = 8, paired t-test compared to saline). This effect was not seen in μ-/- mice in which morphine had no effect on the averaged locomotion (n = 7). By contrast, morphine stimulated locomotion in β-arr2-/- mice (*p < 0.01, paired t-test, n = 8), but the average distance travelled was less than that of WT mice (#p < 0.01, unpaired t-test). (B) Morphine (10 mg/Kg) was without effect on distance travelled by μ-/- mice (n = 7) on all days of conditioning. (C) Morphine (3 mg/Kg) administration to WT mice (n = 8) showed modest increase on distance travelled on days 2 and 3 of conditioning (*p < 0.05, two-way ANOVA, post hoc Bonferroni test). (D) At a higher dose morphine (10 mg/Kg s.c.) increased distance travelled on all 3 days and this effect exhibited sensitization (*p < 0.0001, two-way ANOVA, post hoc Bonferroni test; n = 8). (E) The locomotor effect of morphine (10 mg/Kg) was diminished in μ+/- mice (n = 8) and there was no sensitisation (*p < 0.01 on day 1, p < 0.0001 on day 2 and 3, two-way ANOVA, post hoc Bonferroni test). (F) A higher dose of morphine (30 mg/Kg) enhanced locomotion, but there was no effect of time over the 3 days of conditioning (*p < 0.0001, two-way ANOVA, post hoc Bonferroni test; n = 8). Morphine (3 mg/Kg) had no effect on distance travelled by β-arr2-/- mice on days 1-3 (n = 8). (G) At a higher dose morphine (10 mg/Kg) increased distance travelled by β-arr2-/- mice on all 3 days and this effect exhibited sensitization (*p < 0.01 on day 1, p < 0.0001 on day 2 and 3, two-way ANOVA, post hoc Bonferroni test; n = 8).

Fig. 5

Dasatinib does not affect psychomotor stimulation by morphine. (A) The graph of locomotion averaged over 3 days of conditioning with dasatinib and/or morphine. Dasatinib had no effect on locomotion when administered alone to wild type mice (n = 8). Dasatinib (n = 8) also had no effect compared to vehicle (n = 8) on the stimulation of locomotion by morphine, which was significant in both cases (*p < 0.001, paired t-test). (B) Dasatinib (5 mg/Kg) was without effect on distance travelled by mice (n = 8) on all 3 days of conditioning. (C) Morphine caused locomotor stimulation with sensitization over the 3 days of conditioning when mice were administered either vehicle (left panel) or dasatinib (right panel) i.p (*p < 0.0001, two-way ANOVA, post hoc Bonferroni test).

Fig. 6

Unlike the absence of β-arrestin2, which enhances reinforcement by morphine, dasatinib had no effect. (A) Morphine (3 and 10 mg/Kg s.c.) caused a dose-dependent preference of wild type mice (n = 8) for the paired environment (*p < 0.01, one-way ANOVA, post hoc Dunnett’s test). Morphine preference was lacking in μ-/- mice (n = 7). (B) Following conditioning, the duration of occupancy of the morphine-paired environment increased significantly compared to the saline-paired environment in wild type mice at all three 5 min intervals (*p < 0.0001, two-way ANOVA, post hoc Bonferroni test; n = 8). (C) μ-/- mice (n = 7) exhibited no morphine conditioned place preference at any stage during the 15 min test period. (D) μ+/- mice (n = 8) also exhibited no morphine (10 mg/Kg) conditioned place preference. (E) By contrast, μ+/- mice (n = 8) exhibited conditioned place preference to a higher dose of morphine (30 mg/Kg) (*p < 0.05 for 300-600 s, p < 0.001 for 600-900 s, two-way ANOVA, post hoc Bonferroni test). (F) Morphine (10 and 30 mg/Kg) caused a dose-dependent preference of μ+/- mice (n = 8) for the paired environment (*p < 0.05, one way ANOVA, post hoc Dunnett’s test). (G) Morphine preference occurred at a lower dose in β-arr2-/- mice (*p < 0.01, one way ANOVA, post hoc Dunnett’s test; n = 8). (H) By contrast, dasatinib had no effect morphine preference (*p < 0.01, one way ANOVA, post hoc Dunnett’s test; n = 8).

Fig. 7

Reversal of morphine tolerance by dasatinib. The diagram depicts the morphine, dasatinib/vehicle injection schedule on days 1 - 5. Data in the graph are average tail withdrawal latencies expressed as percentage of maximal possible effect (%MPE). μ+/- mice injected with vehicle (n = 8) 30 min prior to morphine on days 4 and 5 continued to develop tolerance. By contrast, μ+/- mice receiving dasatinib (n = 8) 30 min prior to morphine on days 4 and 5 exhibited reversal of analgesic tolerance. Data identified with asterisks were significantly different from equivalent vehicle data (*p < 0.05, Kruskall-Wallis test, post hoc Dunn’s correction). Data are presented as median ± IQR.