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p38 MAPK and β-Arrestin 2 Mediate Functional Interactions between Endogenous μ-Opioid and α2A-Adrenergic Receptors in Neurons

2009; Elsevier BV; Volume: 284; Issue: 10 Linguagem: Inglês

10.1074/jbc.m806742200

1083-351X

Miao Tan, Wendy Walwyn, Christopher J. Evans, Cui-Wei Xie,

Neuroscience and Neuropharmacology Research

Formation of receptor complexes between μ-opioid and α2A-adrenergic receptors has been demonstrated in transfected cells. The functional significance and underlying mechanisms of such receptor interactions remain to be determined in neuronal systems. We examined functional interactions between endogenous μ and α2A receptors in mouse dorsal root ganglion neurons. Acute application of the μ agonist [d-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) or the α2 agonist clonidine inhibited voltage-gated Ca2+ currents in these neurons. Prolonged treatment with either DAMGO or clonidine induced a mutual cross-desensitization between μ and α2A receptor-mediated current inhibition. The cross-desensitization was closely associated with simultaneous internalization of μ and α2A receptors. Morphine, a μ agonist triggering little μ receptor endocytosis, induced neither cross-desensitization nor internalization of α2A receptors. Furthermore, inhibition of p38 MAPK prevented the cross-desensitization as well as cointernalization of μ and α2A receptors. Changes in receptor trafficking profiles suggested that p38 MAPK activity was required for initiating μ receptor internalization and maintaining possible μ-α2A association during their cointernalization. Finally, the μ-α2A cross-desensitization was absent in dorsal root ganglion neurons lacking β-arrestin 2. These findings demonstrated p38 MAPK- and β-arrestin 2-dependent cross-regulation between neuronal μ and α2A receptors. By promoting receptor cross-desensitization and cointernalization, such functional interactions may serve as negative feedback mechanisms triggered by prolonged agonist exposure to modulate the signaling of functionally related G protein-coupled receptors. Formation of receptor complexes between μ-opioid and α2A-adrenergic receptors has been demonstrated in transfected cells. The functional significance and underlying mechanisms of such receptor interactions remain to be determined in neuronal systems. We examined functional interactions between endogenous μ and α2A receptors in mouse dorsal root ganglion neurons. Acute application of the μ agonist [d-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) or the α2 agonist clonidine inhibited voltage-gated Ca2+ currents in these neurons. Prolonged treatment with either DAMGO or clonidine induced a mutual cross-desensitization between μ and α2A receptor-mediated current inhibition. The cross-desensitization was closely associated with simultaneous internalization of μ and α2A receptors. Morphine, a μ agonist triggering little μ receptor endocytosis, induced neither cross-desensitization nor internalization of α2A receptors. Furthermore, inhibition of p38 MAPK prevented the cross-desensitization as well as cointernalization of μ and α2A receptors. Changes in receptor trafficking profiles suggested that p38 MAPK activity was required for initiating μ receptor internalization and maintaining possible μ-α2A association during their cointernalization. Finally, the μ-α2A cross-desensitization was absent in dorsal root ganglion neurons lacking β-arrestin 2. These findings demonstrated p38 MAPK- and β-arrestin 2-dependent cross-regulation between neuronal μ and α2A receptors. By promoting receptor cross-desensitization and cointernalization, such functional interactions may serve as negative feedback mechanisms triggered by prolonged agonist exposure to modulate the signaling of functionally related G protein-coupled receptors. G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; DRG, dorsal root ganglion; DAMGO, [d-Ala2,N-MePhe4,Gly-ol5]enkephalin; Gβγ, G protein βγ subunits; MAPK, mitogen-activated protein kinase; PPF, prepulse facilitation; β-arr2, β-arrestin 2; PBS, phosphate-buffered saline; NE, norepinephrine; GTPγS, guanosine 5′-3-O-(thio)triphosphate; CTAP, Cys2, Tyr3, Arg5, Pen7-amide; ERK, extracellular signal-regulated kinase. interact with each other through formation of receptor complexes, including homo- or heterodimers and possibly higher order oligomers (1Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (584) Google Scholar, 2Marshall F.H. Curr. Opin. Pharmacol. 2001; 1: 40-44Crossref PubMed Scopus (46) Google Scholar, 3Devi L.A. Trends. Pharmacol. Sci. 2001; 22: 532-537Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 4Gurevich V.V. Gurevich E.V. Trends Neurosci. 2008; 31: 74-81Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Heterooligomerization of GPCRs has been shown to enable cross-regulation between different receptor systems, resulting in various changes in receptor binding, signaling, and trafficking. For example, dimerization of μ-opioid and NK1 (neurokinin type 1) receptors in HEK-293 cells promotes agonist-induced cross-phosphorylation and cointernalization of the two receptors, whereas receptor binding and functional coupling are relatively unaffected (5Pfeiffer M. Kirscht S. Stumm R. Koch T. Wu D. Laugsch M. Schroder H. Hollt V. Schulz S. J. Biol. Chem. 2003; 278: 51630-51637Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). A similar pattern has been observed in cells expressing heterodimers of δ-opioid and β2-adrenergic receptors (6Jordan B.A. Trapaidze N. Gomes I. Nivarthi R. Devi L.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 343-348PubMed Google Scholar). Formation of μ- and δ-opioid receptor complexes alters receptor properties, leading to synergistic enhancement of receptor binding and signaling by μ and δ ligands (7Gomes I. Jordan B.A. Gupta A. Trapaidze N. Nagy V. Devi L.A. J. Neurosci. 2000; 20 (1-5): RC110Crossref PubMed Google Scholar). The μ-δ heterodimer may form as early as in the endoplasmic reticulum during receptor processing and allows co-trafficking of the two receptors (8Hasbi A. Nguyen T. Fan T. Cheng R. Rashid A. Alijaniaram M. Rasenick M.M. O'Dowd B.F. George S.R. Biochemistry. 2007; 46: 12997-13009Crossref PubMed Scopus (75) Google Scholar). Controversial evidence exists, however, for agonist-induced, separate endocytosis of μ and δ receptors (9Law P.Y. Erickson-Herbrandson L.J. Zha Q.Q. Solberg J. Chu J. Sarre A. Loh H.H. J. Biol. Chem. 2005; 280: 11152-11164Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Although these studies and many others have underscored the dynamic nature and divergent roles of receptor heterodimerization in GPCR modulation, the molecular basis and regulatory mechanisms for such interactions remain to be elucidated. In particular, most studies addressing this issue have been conducted in heterologous cells or in systems where receptors are overexpressed, which may lead to interactions nonexistent with endogenously expressed receptors. Further studies are necessary to identify and characterize interactions between naturally existing GPCRs in primary neurons. We examined interactions between endogenous μ-opioid and α2A-adrenergic receptors in mouse dorsal root ganglion (DRG) neurons. Both μ and α2A receptors are coupled to Gi and Go proteins and induce similar cellular responses, such as inhibition of voltage-gated Ca2+ channels and activation of inwardly rectifying potassium channels. These cellular effects can lead to presynaptic inhibition of neurotransmitter release or hyperpolarization of postsynaptic neurons. Both are crucial mechanisms for opioid and adrenergic modulation of nociception. A functional synergy between the two systems has been demonstrated in vivo, evidenced by potentiation of morphine analgesia (10Ossipov M.H. Harris S. Lloyd P. Messineo E. J. Pharmacol. Exp. Ther. 1990; 255: 1107-1116PubMed Google Scholar, 11Fairbanks C.A. Wilcox G.L. J. Pharmacol. Exp. Ther. 1999; 288: 1107-1116PubMed Google Scholar) and alleviation of opiate withdrawal (12Maldonado R. Neurosci. Biobehav. Rev. 1997; 21: 91-104Crossref PubMed Scopus (199) Google Scholar) by the α2-adrenergic agonist clonidine. Studies in transgenic mice lacking functional α2A receptors further indicate that the α2A receptor is the principal subtype mediating α2 agonist-induced analgesia at the spinal level (13Lakhlani P.P. MacMillan L.B. Guo T.Z. McCool B.A. Lovinger D.M. Maze M. Limbird L.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9950-9955Crossref PubMed Scopus (304) Google Scholar) and responsible for the synergistic potentiation of morphine analgesia (14Stone L.S. MacMillan L.B. Kitto K.F. Limbird L.E. Wilcox G.L. J. Neurosci. 1997; 17: 7157-7165Crossref PubMed Google Scholar). The exact mechanisms for this adrenergic-opioid synergy, however, remain unclear. Recently, the μ-α2A receptor heterodimers have been detected in HEK-293 cells co-expressing both receptors (15Jordan B.A. Gomes I. Rios C. Filipovska J. Devi L.A. Mol. Pharmacol. 2003; 64: 1317-1324Crossref PubMed Scopus (165) Google Scholar, 16Zhang Y.Q. Limbird L.E. Biochem. Soc. Trans. 2004; 32: 856-860Crossref PubMed Scopus (28) Google Scholar). It is of great interest to further explore whether heterodimerization of μ and α2A receptors serves as a novel mechanism coordinating the function of both receptors in pain-processing pathways. Here we report that endogenous μ and α2A receptors interact in DRG sensory neurons via p38 MAPK- and β-arrestin 2-dependent mechanisms, which promote agonist-selective cross-regulation of receptor signaling and internalization. DRG Cultures-Primary DRG cultures were prepared as described previously (17Tan M. Groszer M. Tan A.M. Pandya A. Liu X. Xie C.W. J. Neurosci. 2003; 23: 10292-10301Crossref PubMed Google Scholar). Briefly, the ganglia were collected from postnatal day 0–3 pups of C57 BL/6 mice, enzymatically dissociated for 30 min with minimal essential medium containing 0.25% trypsin at 37 °C, and triturated with fire-polished Pasteur pipettes. Dissociated neurons were plated onto glass coverslips coated with poly-l-ornithine and laminin. The cultures were maintained at 37 °C in 5% CO2; fed with serum-free Neurobasal-A medium supplemented with B-27, l-glutamine, 2.5s nerve growth factor (0.1 μg/ml; Invitrogen), and 5-fluoxy-d-uridine (0.1 mg/ml; Sigma); and studied after 2–5 days in vitro. DRG cultures were also prepared using postnatal day 0–3 mice lacking α2A adrenergic receptors (α2A-/-) or β-arrestin 2 (β-arr2-/-) and their respective wild-type (+/+) controls. The α2A-/- (stock number 004367; Jackson Laboratory) (18Hein L. Limbird L.E. Eglen R.M. Kobilka B.K. Ann. N. Y. Acad. Sci. 1999; 881: 265-271Crossref PubMed Scopus (40) Google Scholar) and β-arr2-/- lines (19Bohn L.M. Lefkowitz R.J. Gainetdinov R.R. Peppel K. Caron M.G. Lin F.T. Science. 1999; 286: 2495-2498Crossref PubMed Scopus (811) Google Scholar) have both been fully back-crossed to the C57 BL/6 background (10 generations). The +/+ mice used in the same experiments with the knockouts were within two generations of heterozygous mating. Electrophysiological Recordings-The voltage-gated Ca2+ currents were recorded from DRG neurons with 15–30-μm diameters under whole-cell voltage clamp conditions, as described (17Tan M. Groszer M. Tan A.M. Pandya A. Liu X. Xie C.W. J. Neurosci. 2003; 23: 10292-10301Crossref PubMed Google Scholar). Cells were perfused with an external solution containing 10 mm CaCl2, 130 mm tetraethylammonium chloride, 5 mm HEPES, 25 mm d-glucose, and 0.25 μm tetrodotoxin at pH 7.35. The patch electrode was filled with an internal solution composed of 105 mm CsCl, 40 mm HEPES, 5 mm d-glucose, 2.5 mm MgCl2, 10 mm EGTA, 2 mm Mg-ATP, and 0.5 mm GTP at pH 7.2. Ca2+ currents were evoked every 10 s by 40-ms voltage steps from -80 to +10 mV using an Axopatch 200A patch clamp amplifier. Capacitance and series resistance were corrected with the compensation circuitry on the amplifier. Series resistance was compensated by 80–90%. Leak currents were subtracted using a P/6 protocol. Recorded signals were acquired and analyzed using Axon pCLAMP version 8.0 software (Axon Instruments). The amplitude of peak Ca2+ currents was determined using the peak detect feature of the software. Drug Application and Desensitization Protocols-The μ and α2 receptor ligands (Sigma) were prepared as stock solutions in water, diluted with external solution to the final concentration for acute bath application, or added into culture medium for pretreatment. Various kinase inhibitors (Sigma) were dissolved in DMSO and diluted with culture medium for pretreatment with a final DMSO concentration of 0.1%. Cells pretreated with the medium containing 0.1% DMSO served as vehicle controls in these experiments. In additional control experiments, DAMGO- or clonidine-induced current inhibition was compared between untreated cells and cells pretreated with 0.1% DMSO for 4 h. No significant differences were observed between the two treatments (data not shown). During recording, the external solution was continuously applied at 2 ml/min through a 0.5-ml recording chamber carrying the culture coverslip. After establishing a stable base line, the μ or α2 agonist was applied for up to 1 min to observe the maximal change in Ca2+ currents. Agonist-induced current inhibition was measured as the maximal reduction in the peak current amplitude during drug perfusion and expressed as percentage changes from the base-line level. The voltage dependence of agonist effect was assessed using a prepulse facilitation (PPF) protocol consisting of two normal test pulses (P1 and P2) and in between a strong depolarizing prepulse (-80 to +80 mV, 40 ms) delivered 10 ms before P2. The PPF was expressed by the amplitude ratio of currents activated by the two test pulses (P2/P1). To induce chronic desensitization, DRG cultures were pretreated with either μ or α2 agonist for 4 h. After extensive washing, whole-cell recording was performed in pretreated cells, and the acute inhibitory effect of μ or α2 agonist on Ca2+ currents was measured during a brief perfusion (0.5–1 min). The extent of desensitization was determined by the percentage reduction of the agonist effect in pretreated neurons relative to untreated neurons. Immunocytochemical Analysis and Fluorescence Confocal Microscopy-Cellular distribution of μ and α2A receptors in DRG cultures was determined by immunocytochemical double labeling. After drug treatment, the cultures were fixed with 4% paraformaldehyde for 10 min, washed in phosphate-buffered saline (PBS), permeabilized, and preblocked with PBS containing 10% normal donkey serum and 0.1% Triton X-100 for 2 h at room temperature. The cultures were then incubated overnight at 4 °C with the primary antibodies against the C-terminal sequence of the μ receptor (20Keith D.E. Anton B. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Monteillet-Agius G. Stewart P.L. Evans C.J. von Zastrow M. Mol. Pharmacol. 1998; 53: 377-384Crossref PubMed Scopus (293) Google Scholar) or of the α2A receptor (21Nasser Y. Ho W. Sharkey K.A. J. Comp. Neurol. 2006; 495: 529-553Crossref PubMed Scopus (64) Google Scholar) (sc-1478 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); ab45871 (Abcam)). After washing, the cells were further incubated for 2 h with Alexa Fluor 488-labeled donkey anti-rabbit IgG for visualization of μ receptors and with biotinylated anti-goat IgG (Calbiochem) and Cy3-conjugated streptavidin (Calbiochem) to detect α2A receptors. Control samples were prepared in the absence of the primary or secondary antibodies. The specificity of the anti-α2A antibody was further confirmed by the absence of α2A receptor labeling in neurons from the α2A-/- mice (see Fig. 4B). The images of neurons with receptor labeling were acquired using a Leica TCS-SP confocal laser-scanning microscope and processed either as a single scan or as maximum intensity projections of multiple scans taken at successive 1-μm depths. Flow Cytometric Measurement of Surface μ Receptors-Internalization of μ receptors was further accessed in living neurons by quantifying cell surface receptors with flow cytometry as described previously (22Walwyn W.M. Keith Jr., D.E. Wei W. Tan A.M. Xie C.W. Evans C.J. Kieffer B.L. Maidment N.T. Neuroscience. 2004; 123: 111-121Crossref PubMed Scopus (12) Google Scholar). DRG cultures were treated with clonidine, DAMGO, morphine, or control medium for 4 h at 37 °C. Monensin (10 μm) was added during the treatment to block recycling of internalized receptors in all experiments. DRG cells were then harvested in PBS containing 2 mm EDTA, spun at 300 × g for 5 min, and resuspended with PBS containing 1% normal goat serum and 0.1% NaN3. Cell surface μ receptors were labeled at 4 °C for 30 min with a polyclonal antibody against the third extracellular loop of the receptor (Chemicon International) (23Williams J.P. Thompson J.P. McDonald J. Barnes T.A. Cote T. Rowbotham D.J. Lambert D.G. Anesth. Analg. 2007; 105: 998-1005Crossref PubMed Scopus (45) Google Scholar) and visualized with Alexa Fluor 488-labeled donkey anti-rabbit IgG at 4 °C for another 30 min. Cell surface immunofluorescence was measured with a FACScan flow cytometer at 5,000–10,000 cells/sample. Data were acquired and analyzed using Cell Quest version 3.0.1 (BD Bioscience). The loss of cell surface μ receptors after agonist exposure was quantified by the reduction in the proportion of cells expressing detectable surface μ receptors (μ-positive cells) and in the density of surface receptors of μ-positive cells reflected by their mean fluorescence intensity. Nonspecific background fluorescence was determined in control samples processed without the primary antibody and was subtracted to obtain mean relative fluorescence intensity of experimental samples. Measurement of Phospho-p38 MAPK-The cellular level of phospho-p38 MAPK was measured with flow cytometry (24Perez O.D. Nolan G.P. Nat. Biotechnol. 2002; 20: 155-162Crossref PubMed Scopus (768) Google Scholar), using a polyclonal antibody recognizing p38 MAPK dually phosphorylated at Thr180 and Tyr182 (Cell Signaling Technology) (25Uddin S. Lekmine F. Sharma N. Majchrzak B. Mayer I. Young P.R. Bokoch G.M. Fish E.N. Platanias L.C. J. Biol. Chem. 2000; 275: 27634-27640Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). After drug treatment, DRG cells were harvested from culture plates with PBS buffer containing 4 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 0.1 mm calyculin A, 1 μg/ml leupeptin, and protease inhibitor mixture tablet (Sigma) (26Kummer J.L. Rao P.K. Heidenreich K.A. J. Biol. Chem. 1997; 272: 20490-20494Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). The cells were fixed in 2% formaldehyde for 10 min at room temperature, permeabilized in ice-cold 90% methanol for 30 min, incubated with the primary antibody for 60 min at room temperature, and labeled with an Alexa Fluor 488-conjugated second antibody for another 60 min. In between these steps, the cells were washed, centrifuged at 450 × g for 5 min, and resuspended with fresh PBS. After the final wash, the fluorescence signal of the cells was analyzed with a FACScan flow cytometer at 5,000 cells/sample, and the data were processed with CellQuest software. Statistical Analysis-All data are presented as means ± S.E. One-way analysis of variance was applied for overall statistical significance across multiple group means, followed by the Bonferroni post hoc test for pairwise comparisons. Statistical significance was defined as p < 0.05. α2-Adrenergic Agonist-induced Cross-desensitization to μ-Opioids-Brief bath application of norepinephrine (NE) at 10 μm induced acute inhibition of voltage-gated Ca2+ currents in DRG neurons (Fig. 1A). This effect was significantly attenuated by a selective α2 antagonist yohimbine, confirming involvement of α2 receptors in NE action (27.2 ± 5.0% current inhibition without yohimbine versus 9.7 ± 1.6% with 10 μm yohimbine, n = 12 and 8, p < 0.05). Acute application of a selective α2 agonist, clonidine (10 μm), induced similar reductions in Ca2+ currents (20.0 ± 4.2%, n = 12) reversible by co-application of yohimbine (5.7 ± 1.6%, n = 10, p < 0.01 compared with clonidine alone). The effect of NE or clonidine was substantially reduced in neurons pretreated with the same agonist for 4 h (6.4 ± 1.7%, n = 9 for NE; 7.6 ± 1.1%, n = 8 for clonidine, p < 0.01 compared with the effects in untreated neurons), indicating development of homologous desensitization to α2 receptor-mediated effect. As expected, this desensitization was blocked by yohimbine added during NE or clonidine pretreatment (Fig. 1A). We then determined the influence of prolonged α2 agonist exposure on μ-opioid-induced Ca2+ current inhibition. In DRG neurons pretreated with 10 μm clonidine for 0.5 or 4 h, the subsequent DAMGO application (1 μm, 1 min) reduced Ca2+ currents by 40.5 ± 6.7% (n = 4) and 37.4 ± 3.0% (n = 18), respectively. The latter was significantly smaller than the responses in untreated cells (0 h, 54.7 ± 2.5%, n = 62, p < 0.01), indicative of a cross-desensitization to DAMGO. Similarly, morphine application (1 μm, 1 min) reduced Ca2+ currents by 47.6 ± 6.2% in control cells (0 h, n = 10) but by only 27.2 ± 4.4% in cells pretreated with clonidine for 4 h (n = 9, p < 0.05). Thus, prolonged NE or clonidine exposure heterologously reduced the effect of subsequently applied μ-opioid agonists. This cross-desensitization was prevented by yohimbine co-treatment (Fig. 1B). Inhibition of neuronal Ca2+ channels by GPCRs is mediated by both voltage-dependent and voltage-independent mechanisms. The voltage-dependent inhibition requires direct binding of G-protein βγ subunits (Gβγ) to the Ca2+ channel, a process reversible by strong depolarization. To determine whether this direct Gβγ-channel interaction was affected by clonidine, we examined voltage dependence of clonidine action using the PPF protocol (Fig. 1, C and D). In cells acutely perfused with clonidine (10 μm, 1 min), the depolarizing prepulse produced little relief of inhibition, with a P2/P1 ratio similar to that under basal conditions (1.06 ± 0.04 versus 0.99 ± 0.02, n = 5 for each group, p > 0.05). Clonidine pretreatment for 4 h was also without effect on the P2/P1 ratio subsequently tested either in control perfusion medium (0.97 ± 0.02, n = 8) or during acute clonidine test (1.06 ± 0.03, n = 8). These results suggested that at the concentration we used, the effect of clonidine was primarily mediated by voltage-independent mechanisms. We then measured PPF induced by intracellular application of 0.1 mm GTPγS that can directly activate G proteins and release Gβγ subunits. GTPγS-induced PPF was not significantly different between untreated control cells and clonidine-pretreated cells (1.68 ± 0.23 versus 1.68 ± 0.18 at 8 min after the application, n = 12 for each group, p > 0.05) (Fig. 1, C and D). This confirmed that clonidine-induced desensitization was not associated with a diminished cell capacity for voltage-dependent Gβγ-Ca2+ channel interactions. DAMGO-induced Cross-desensitization to α2A Adrenergic Responses-As we previously reported (17Tan M. Groszer M. Tan A.M. Pandya A. Liu X. Xie C.W. J. Neurosci. 2003; 23: 10292-10301Crossref PubMed Google Scholar, 22Walwyn W.M. Keith Jr., D.E. Wei W. Tan A.M. Xie C.W. Evans C.J. Kieffer B.L. Maidment N.T. Neuroscience. 2004; 123: 111-121Crossref PubMed Scopus (12) Google Scholar), application of 1 μm DAMGO or morphine strongly inhibited Ca2+ currents in DRG neurons, but their effects were desensitized dramatically following a 4-h pretreatment (Fig. 2A). The inhibitory effect of 10 μm NE or clonidine on Ca2+ currents also significantly decreased in DAMGO-treated cells (11.4 ± 2.5%, n = 9 for NE; 5.3 ± 1.6%, n = 6 for clonidine, p < 0.05 for both as compared with untreated cells). This cross-desensitization was prevented by a selective μ receptor antagonist, Cys2, Tyr3, Arg5, Pen7-amide (CTAP), added during DAMGO pretreatment (Fig. 2B). Interestingly, the effect of NE or clonidine was not significantly reduced in morphine-pretreated cells (21.7 ± 6.3%, n = 7 for NE; 16.3 ± 3.3%, n = 11 for clonidine, p > 0.05 for both as compared with untreated cells), despite development of homologous morphine desensitization in these cells (Fig. 2A). Thus, the cross-desensitization to clonidine was induced by prolonged treatment with DAMGO but not morphine. Since clonidine is relatively nonselective to different subtypes of the α2 receptors, we examined the role of α2A receptors in the acute and chronic effects of clonidine. Acute application of 10 μm clonidine induced negligible current inhibition in DRG neurons derived from the α2A-/- mice (2.5 ± 1.1%, n = 6, p > 0.05). In contrast to the wild-type neurons, the α2A-/- neurons also displayed no cross-desensitization to DAMGO following clonidine pretreatment (Fig. 4A). These results suggested that in our preparation, the α2A receptor was the major subtype responsible for clonidine-induced Ca2+ current inhibition and its cross-desensitization to DAMGO. Agonist-induced Cointernalization of μ and α2A Receptors- Next we determined whether the mutual cross-desensitization between μ and α2A receptors was associated with changes in agonist-induced receptor internalization. Immunohistochemical double labeling and confocal microscopy showed that a substantial portion of μ and α2A receptors were colocalized on the cell membrane throughout the cell body and processes of cultured DRG neurons under basal conditions (Fig. 3A). Incubation with DAMGO for 30 min to 4 h induced simultaneous internalization of both receptors, which was blocked by co-incubation with the μ antagonist CTAP (Fig. 3C). Clonidine also elicited internalization of both μ and α2A receptors, reversible by the α2 antagonist yohimbine (Fig. 3D). In contrast, morphine treatment up to 4 h failed to internalize either μ or α2A receptors (Fig. 3B). Furthermore, clonidine did not induce μ receptor endocytosis in α2A-/- neurons (Fig. 4B). Thus, similar to the cross-desensitization, the cointernalization was agonist-selective and dependent upon the presence of functional μ and α2A receptors. Fluorescence flow cytometry was then conducted in living DRG neurons to quantify agonist-induced μ receptor internalization. Surface μ receptors were detected in 77.7 ± 3% of control cells. DAMGO treatment for 4 h reduced the portion of cells expressing detectable levels of surface μ receptors (μ-positive cells) by 26.3 ± 5.8% (p < 0.05) relative to the controls (Fig. 5B). Furthermore, the intensity of surface μ receptor labeling in the μ-positive cells decreased by 43.1 ± 7.8% (p < 0.05) following DAMGO treatment (Fig. 5A). These changes confirmed loss of surface μ receptors due to DAMGO-induced receptor internalization. Consistent with previous reports in heterologous cells (20Keith D.E. Anton B. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Monteillet-Agius G. Stewart P.L. Evans C.J. von Zastrow M. Mol. Pharmacol. 1998; 53: 377-384Crossref PubMed Scopus (293) Google Scholar, 27Whistler J.L. von Zastrow M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9914-9919Crossref PubMed Scopus (318) Google Scholar), morphine was much less effective in triggering μ receptor internalization, causing little change in surface μ receptor expression after a 4-h treatment. Importantly, clonidine pretreatment reduced the portion of μ-positive cells by 16.7 ± 5.7% and the level of surface μ receptors by 30.8 ± 10.9% (p < 0.05 for both compared with the controls), and both effects were blocked by yohimbine (Fig. 5). Blockade of μ-α2A Cross-desensitization and Cointernalization by p38 MAPK Inhibitors-To explore the signaling mechanisms underlying μ-α2A interactions, we examined potential involvement of several opioid-activated protein kinases in the cross-desensitization. DRG neurons were pretreated with clonidine (10 μm, 4 h) in the presence of a selective kinase inhibitor, washed, and tested for acute DAMGO responses. As shown in Fig. 6A, co-treatment with a selective p38 MAPK inhibitor PD169316 (26Kummer J.L. Rao P.K. Heidenreich K.A. J. Biol. Chem. 1997; 272: 20490-20494Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar) attenuated clonidine-induced cross desensitization. Acute DAMGO responses were significantly greater in cells co-treated with 5 or 20 μm PD169316 (49.0 ± 4.8% (n = 8) or 54.9 ± 8.1% (n = 10)) compared with cells treated with clonidine alone (37.4 ± 3.0%, n = 18, p < 0.05 for both comparisons). Another selective p38 MAPK inhibitor, SB239063 (28Underwood D.C. Osborn R.R. Bochnowicz S. Webb E.F. Rieman D.J. Lee J.C. Romanic A.M. Adams J.L. Hay D.W. Griswold D.E. Am. J. Physiol. 2000; 279: L895-L902Crossref PubMed Google Scholar), also significantly reduced the cross-desensitization when co-applied with clonidine. In contrast, inhibitors of several other protein kinases failed to prevent clonidine-induced cross-desensitization, which included the phosphoinositide 3-kinase inhibitor LY294002 (10 μm), the extracellular signal-regulated kinase (ERK) inhibitor PD98059 (10 μm), the protein kinase A inhibitor rpCAMP (1 mm), and the protein kinase C inhibitor Go6987 (0.1 μm). Acute DAMGO responses in cells co-treated with these inhibitors was 41.7 ± 2.7% (n = 14), 38.5 ± 6.3% (n = 7), 31.5 ± 4.5% (n = 7), and 36.8 ± 6.6% (n = 6), respectively, not significantly different from that in cells treated with clonidine alone. In a separate set of experiments, DRG neurons were pretreated with DAMGO (1 μm, 4 h) in the presence of one of the kinase inhibitors and tested for acute responses to clonidine. Blocking p38 MAPK activity with PD169316 (5 and 20 μm) during pretreatment attenuated DAMGO-induced cross-desensitization in a dose-dependent manner (Fig. 6B). The responses to subsequent clonidine test were significantly greater in cells co-treated with 20 μm PD169316 compared with those treated with DAMGO alone (13.9 ± 2.4% versus 5.7 ± 0.8%, n = 17 and 18, p < 0.01). Co-treatment with another specific p38 inhibitor SB203580 (20 μm) (29Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F.