Enhanced Morphine Analgesia in Mice Lacking β-Arrestin 2
Tóm tắt
The ability of morphine to alleviate pain is mediated through a heterotrimeric guanine nucleotide binding protein (G protein)–coupled heptahelical receptor (GPCR), the μ opioid receptor (μOR). The efficiency of GPCR signaling is tightly regulated and ultimately limited by the coordinated phosphorylation of the receptors by specific GPCR kinases and the subsequent interaction of the phosphorylated receptors with β-arrestin 1 and β-arrestin 2. Functional deletion of the β-arrestin 2 gene in mice resulted in remarkable potentiation and prolongation of the analgesic effect of morphine, suggesting that μOR desensitization was impaired. These results provide evidence in vivo for the physiological importance of β-arrestin 2 in regulating the function of a specific GPCR, the μOR. Moreover, they suggest that inhibition of β-arrestin 2 function might lead to enhanced analgesic effectiveness of morphine and provide potential new avenues for the study and treatment of pain, narcotic tolerance, and dependence.
Từ khóa
Tài liệu tham khảo
A bacteriophage λ library of mouse 129SvJ genomic DNA (Stratagene) was screened with the rat βarr2 cDNA (24). Positive phages were identified and analyzed by restriction endonuclease digestion. A 12-kb βarr2 fragment was digested with Bam HI subcloned into pBluescript KS(-) and sequenced. The targeting vector was assembled by blunt-end ligation of a pHSV-TK cassette (from plasmid pIC19R/MCI-TK) a 2.8-kb Nco I–Bam HI βarr2 fragment a pGK-neo cassette (from plasmid pD383) that replaced the 0.8-kb Bam HI–Hind III fragment of βarr2 and a 4.5-kb Hind III βarr2 fragment into pBluescript KS(-). This targeting vector was linearized with Not I and was electroporated into mouse embryonic stem cells. Genomic DNA from transfectants resistant to G418 and gancyclovir were isolated and screened by Southern blot analysis using a 0.2-kb 5′ external βarr2 probe and a 0.3-kb 3′ external βarr2 probe. Supplemental information regarding the targeting construct is available at Science Online (www.sciencemag.org/feature/data/1045481.shl). Chimeric animals were generated by microinjecting these embryonic stem cells into C57BL/6 blastocysts. Five chimeric male pups were obtained and mated with C57BL/6 females. Germ line transmission was confirmed by Southern blotting. Heterozygous offspring were intercrossed to obtain homozygous mice. Wild-type and mutant mice used in this study were age-matched 3- to 5-month-old male siblings. For protein immunoblot analysis whole-cell lysates were prepared by polytron homogenization in lysing buffer [10 mM tris (pH 7.4) 5 mM EDTA one protease inhibitor tablet per 10 ml (Roche Molecular Biochemicals Indianapolis IN) and 1% Nonidet-40]. Polyacrylamide gels were loaded with 25 μg of protein per lane and equivalent protein loading was confirmed by Ponceau S staining of the gels. After transfer to polyvinyldifluoride membranes proteins were blotted with polyclonal antibodies to β-arrestin 2 or β-arrestin 1 (24). Bands were visualized with secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence detection system (Amersham). All experiments were conducted in accordance with the NIH guidelines for the care and use of animals and with an approved animal protocol from the Duke University Animal Care and Use Committee.
F.-T. Lin unpublished data.
Porreca F., Mosberg H. I., Hurst R., Hruby V. J., Burks T. F., J. Pharmacol. Exp. Ther. 230, 341 (1984);
L. M. Bohn unpublished data.
Mice were injected with morphine (10 mg/kg sc). After 30 min or 2 hours wild-type mice were killed and blood was collected in vials containing sodium fluoride and potassium oxalate. Morphine concentrations in blood samples pooled from three mice per sample were 1500 ng/ml after 30 min and 83 ng/ml after 2 hours as measured by mass spectroscopy analysis (Occupational Testing Division LabCorp Inc. Research Triangle Park NC). In similar experiments βarr2-KO mice had a concentration of 93 ng/ml in the blood after 2 hours.
Naloxone (2.5 mg/kg sc) which immediately reverses the effects of opiates was given 30 min after morphine (10 mg/kg). Naltrindole [P. S. Portoghese M. Sultana A. E. Takemori. J. Med. Chem. 88 1547 (1990)] was given 20 min before morphine and nor-binaltorphimine [
Takemori A. E., Ho B. Y., Naeseth J. S., Portoghese P. S., J. Pharmacol. Exp. Ther. 246, 255 (1988);
] was given 1 hour before morphine [
. Rectal body temperatures were determined with a digital thermometer (TH8; Physitemp Clifton NJ) [
]. The probe was inserted into the rectum and maintained until the temperature reading stabilized.
Membranes were prepared for binding assays as follows. Brain regions were dissected and immediately frozen in liquid nitrogen and were stored at −80°C for less than 1 week before use. Samples were placed on ice and homogenized by polytron in membrane preparation buffer [50 mM tris (pH 7.4) 1 mM EDTA 3 mM MgCl 2 ] and crude membranes were prepared by centrifugation at 20 000 g for 15 min at 4°C. Membranes were resuspended in either 50 mM tris-HCl (pH 7.4) for radioligand binding assays or in assay buffer [50 mM tris-HCl (pH 7.4) 100 mM NaCl 3 mM MgCl 2 and 0.2 mM EDTA] containing 10 μM guanosine diphosphate for [ 35 S]GTP- γ -S binding assays. For both binding assays reactions were terminated by rapid filtration over GF/B filters (Brandel Inc. Gaithersburg MD) using a Brandel cell harvester. Filters were washed three times with ice-cold 10 mM tris-HCl (pH 7.4) and then counted in a liquid scintillation counter.
P. S. Portoghese in Handbook of Experimental Pharmacology: Opioids I A. Herz Ed. (Springer-Verlag New York 1993) pp. 279-293
A. D. Corbett S. J. Paterson H. W. Kosterlitz in Handbook of Experimental Pharmacology: Opioids I A. Herz Ed. (Springer-Verlag New York 1993) pp. 645-679.
R. R. Gainetdinov unpublished data.
We thank B. H. Koller for homologous recombinant mouse embryonic stem cells and chimeric mice M. R. Capecchi for plasmid pIC19R/MCI-TK and R. Hen for plasmid pD383. R.R.G. is a visiting scientist from the Institute of Pharmacology Russian Academy of Medical Sciences Moscow. Supported in part by NIH grants NS 19576 (M.G.C.) and HL16037 and by a cardiovascular unrestricted grant from Bristol-Myers Squibb (R.J.L.). R.J.L. and M.G.C. are Investigators of the Howard Hughes Medical Institute.