R7BP modulates opiate analgesia and tolerance but not withdrawal

Abstract

The adaptor protein R7 family binding protein (R7BP) modulates G protein coupled receptor (GPCR) signaling and desensitization by controlling the function of regulator of G protein signaling (RGS) proteins. R7BP is expressed throughout the brain and appears to modulate the membrane localization and stability of three proteins that belong to R7 RGS family: RGS6, RGS7, and RGS9-2. RGS9-2 is a potent negative modulator of opiate and psychostimulant addiction and promotes the development of analgesic tolerance to morphine, whereas the role of RGS6 and RGS7 in addiction remains unknown. Recent studies revealed that functional deletion of R7BP reduces R7 protein activity by preventing their anchoring to the cell membrane and enhances GPCR responsiveness in the basal ganglia. Here, we take advantage of R7BP knockout mice in order to examine the way interventions in R7 proteins function throughout the brain affect opiate actions. Our results suggest that R7BP is a negative modulator of the analgesic and locomotor activating actions of morphine. We also report that R7BP contributes to the development of morphine tolerance. Finally, our data suggest that although prevention of R7BP actions enhances the analgesic responses to morphine, it does not affect the severity of somatic withdrawal signs. Our data suggest that interventions in R7BP actions enhance the analgesic effect of morphine and prevent tolerance, without affecting withdrawal, pointing to R7BP complexes as potential new targets for analgesic drugs.

Figure 1

Expression of R7 family members and interacting proteins in the mouse brain. (a) The expression of R7BP, Gβ5, RGS9-2, and RGS6/7 and in different brain regions of the mouse. Western blot analysis was also used to determine possible regulation of R7BP levels in the mouse striatum by acute (30 min or 2 h, n=4–5 per group) or repeated morphine application (once a day for 4 days, n=4 per group). As shown in (b), acute or repeated morphine treatments do not affect R7BP levels. Co-immunoprecipitation assays in the striatum demonstrate an increase in RGS9-2/R7BP complexes following acute fentanyl or morphine administration (c; n=3 per group). Striata from C57/Bl/6 mice treated with saline (sal) fentanyl (fent) or morphine (mor) for 30 min were immunoprecipitated with anti-RGS9-2 antibody and assayed with anti-R7BP antibody. Total lysates (TL) were assayed for R7BP levels. Data are expressed as mean±SEM, n=3 per group ^*p<0.05, one-way ANOVA followed by Dunnett's post hoc test.

Figure 2

A role of R7BP in the locomotor activating actions of morphine. Knockout of R7BP increases sensitivity to the locomotor activating actions of morphine. In particular, R7BPKO mice show a significant locomotor activation at a dose of 5 mg/kg (a). Wild-type animals do not show activation at this dose (p<0.001 two-way ANOVA followed by Bonferroni test). At higher doses of morphine (10 and 20 mg/kg), locomotor activation starts much earlier in R7BPKO mice compared with their wild-type controls (b, c, p<0.001, followed by Bonferroni post hoc test). Data are expressed as mean±SEM, n=5–9 per group.

Figure 3

A role of R7BP in opiate analgesia. Knockout of R7BP does not affect paw withdrawal latencies in the hot plate test (a). R7BPKO mice show greater responses to morphine (10 and 15 mg/kg, n=10–11 per group) than their wild-type controls (b). R7BPKO mice show greater response than their wild-type controls to the analgesic actions of fentanyl (0.125 mg/kg, n=9–10 per group) in the hot plate assay (c). ^*p<0.05, t-test. Data are expressed as mean±SEM.

Figure 4

Blockade of R7BP actions prevents the development of morphine tolerance. Mice received 20 mg/kg morphine once a day for 5 consecutive days and 52 °C hot plate responses were monitored before and 30 min after morphine administration. R7BPKO mice show the same analgesic response throughout the study, whereas wild-type animals develop tolerance by the third day of morphine administration. Data are expressed as mean±SEM, ^*p<0.001, two-way ANOVA, followed by the Bonferroni post hoc test (n=8–11 per group).

Figure 5

Knockout of R7BP delays tolerance to the antiallodynic effects of morphine in neuropathic pain-suffering animals. We examined the influence of R7BP in the mechanical allodynia in the SNI model of neuropathic pain. (a) The von Frey responses of R7BPWT and R7BPKO mice following 1–10 days of spared nerve injury. Both wild-type and mutant animals develop the same degree of mechanical allodynia. Morphine (3 mg/kg s.c.) produces an antiallodynic effect on both genotypes, but responses of R7BPKOs are greater than those of their WT controls (n=7–11 per group). A delay in the development of morphine tolerance is also observed in R7BP mutants (b). Data are expressed as mean±SEM, ^*p<0.001 two-way ANOVA, followed by the Bonferroni post hoc test.

Figure 6

R7BP does not affect morphine withdrawal. Mice received increasing morphine doses as described in Methods and withdrawal was precipitated using naloxone hydrochloride (1 mg/kg). Several withdrawal signs (jumps, wet dog shakes, tremor, diarrhea, and weight loss) were monitored for 30 min after naloxone administration. No significant difference was observed between genotypes, although there was a trend for decreased diarrhea and weigh loss in mutant mice. Data are expressed as mean±SEM, n=12–14 per group.