Damgo-induced μ-opioid receptor desensitization in rat locus coeruleus neurones is mediated by G-protein-coupled receptor kinase 2

C. P. Bailey1, S. Oldfield1, C. J. Caunt2, A. G. Teschemacher3, S. Kasparov3, C. A. McArdle2, E. Kelly1, G. Henderson1. 1Department of Pharmacology 2Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology 3Department of Physiology, University of Bristol, Bristol, UK.

In rat locus coeruleus (LC) neurons, morphine causes significantly less rapid μ-opioid receptor (MOR) desensitization than full agonists such as DAMGO. However, desensitization is dramatically enhanced when protein kinase C (PKC) is activated (Bailey et al., 2004). In HEK293 cells, we have recently shown that DAMGO-induced MOR desensitization is mediated largely by G-protein-coupled receptor kinase 2 (GRK2), with no role for PKC, whereas morphine-induced MOR desensitiziation is largely PKC-dependent, with no significant role for GRK2 (Johnson et al., 2006). This suggests that there are two distinct agonist-specific mechanisms of MOR desensitization.

In this study, we examined the role of GRK2 in mediating MOR desensitization in adult mammalian neurones. Adenoviral vectors were constructed to express a kinase-deficient GRK2 mutant (GRK2-K220R) and enhanced green fluorescent protein (eGFP), under the control of the PRSx8 promoter. GRK2-K220R acts as a dominant-negative mutant, to inhibit the actions of endogenous GRK2 (Kong et al., 1994). 1 μl of viral suspension (1x1010 – 1x1011 pfu / ml) was injected, bilaterally, into the LC of anaesthetized rats (male Wistar, 130-180g). 2-3 days later, horizontal brain slices (200μm thick) were prepared and whole-cell patch-clamp recordings (Vh –60mV) were made from LC neurones. Activation of MORs results in opening of G-protein-coupled inwardly-rectifying K+ channels that provides a real-time measure of MOR activation. Virally-infected neurones could be identified by the presence of eGFP fluorescence. Data from individual neurones were normalized to each peak response and pooled. Data shown are means ± S.E.M., and analysed by Student’s t-test.

In non-virally infected neurones, DAMGO (10 μM) elicited a K+-current that rapidly desensitized to 45.4 ± 5.0% of peak after 7 minutes (n = 6). In virally-transfected neurones expressing the dominant-negative mutant GRK2-K220R, DAMGO-induced desensitization was significantly reduced (73.5 ± 3.0% of peak at 7 minutes; P < 0.05 vs. non-infected neurones; n = 3).

These data constitute the first evidence that DAMGO-induced MOR desensitization in adult mammalian neurones is mediated by GRK2. On-going experiments are being performed to examine whether morphine-induced MOR desensitization, which requires PKC activation in LC neurones, is also mediated by GRK2.

Bailey CP et al. (2004) Mol. Pharmacol 66:1592-1598
Johnson EA et al. (2006) Mol. Pharmacol 70:676-685
Kong G et al. (1994) J. Biol. Chem 269:13084-13087