G protein-coupled receptor kinase 2 mediates µ-opioid receptor desensitization in GABAergic neurons of the nucleus raphe magnus

Authors


Address correspondence and reprint requests to H.-L. Wang, Department of Physiology, Chang Gung University School of Medicine, Kwei-San, Tao-Yuan, Taiwan, R.O.C. E-mail: hlwns@mail.cgu.edu.tw

Abstract

Nucleus raphe magnus (NRM) sends the projection to spinal dorsal horn and inhibits nociceptive transmission. Analgesic effect produced by µ-opioid receptor agonists including morphine partially results from activating the NRM-spinal cord pathway. It is generally believed that µ-opioid receptor agonists disinhibit spinally projecting neurons of the NRM and produce analgesia by hyperpolarizing GABAergic interneurons. In the present study, whole-cell patch-clamp recordings combined with single-cell RT-PCR analysis were used to test the hypothesis that DAMGO ([D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin), a specific µ-opioid receptor agonist, selectively hyperpolarizes NRM neurons expressing mRNA of glutamate decarboxylase (GAD67). Homologous desensitization of µ-opioid receptors in NRM neurons could result in the development of morphine-induced tolerance. G protein-coupled receptor kinase (GRK) is believed to mediate µ-opioid receptor desensitization in vivo. Therefore, we also investigated the involvement of GRK in mediating homologous desensitization of DAMΑΜGO-induced electrophysiological effects on NRM neurons by using two experimental strategies. First, single-cell RT-PCR assay was used to study the expression of GRK2 and GRK3 mRNAs in individual DAMGO-responsive NRM neurons. Whole-cell recording was also performed with an internal solution containing the synthetic peptide, which corresponds to Gβγ-binding domain of GRK and inhibits Gβγ activation of GRK. Our results suggest that DAMGO selectively hyperpolarizes NRM GABAergic neurons by opening inwardly rectifying K+ channels and that GRK2 mediates short-term homologous desensitization of µ-opioid receptors in NRM GABAergic neurons.

Abbreviations used
DAMGO

[D-Ala2, N-methyl-Phe4,Gly-ol5]enkephalin

G protein

guanine nucleotide binding protein

GRK

G protein-coupled receptor kinase

NRM

nucleus raphe magnus.

Nucleus raphe magnus (NRM), a serotonergic nucleus, plays a major role in supraspinal pain regulation by projecting to the dorsal horn of spinal cord and inhibiting nociceptive transmission (Basbum and Fields 1984; Fields et al. 1991). The physiological importance of this descending pathway in pain modulation is indicated by previous studies showing that endogenous opioid peptides and morphine, through activating µ-opioid receptors, produce the antinociceptive effect by activating NRM-spinal dorsal horn pathway (Fields et al. 1991). Enkephalin-containing nerve terminals are present in the NRM (Menetrey and Basbaum 1987). Autoradiographic studies showed that a high density of µ-opioid receptors is expressed in the NRM (Bowker and Dilts 1988). When microinjected to NRM, morphine or opioid peptide produces a potent antinociceptive effect by inhibiting noxious stimulus-evoked activity of nociception-specific neurons of spinal dorsal horn (Azami et al. 1982; Fields et al. 1991). Furthermore, opioid-induced analgesia in the NRM is greatly reduced following the destruction of NRM-spinal cord projection (Basbaum and Fields 1984). Opioid peptides and morphine exert a direct inhibitory effect on CNS neurons by enhancing the inwardly rectifying K+ conductance via pertussis toxin-sensitive Gi/o proteins (Grudt and Williams 1994; Fiorillo and Williams 1996; Pan et al. 1997; Brunton and Charpak 1998). Thus, it has been hypothesized that µ-opioid receptor agonists disinhibit spinally projecting neurons of the NRM and produce an analgesic effect by hyperpolarizing GABAergic interneurons, which tonically inhibit descending output neurons (Fields et al. 1991; Pan et al. 1997). However, a direct hyperpolarization of NRM GABAergic neurons by µ-opioid receptor agonist has not been reported.

Continuous administration of morphine-like drugs leads to a development of tolerance and dependence (Nestler 1996). In the face of sustained or repeated exposure to agonist, µ-opioid receptors are also rapidly inactivated by a process referred to as the desensitization (Law and Loh 1999). Therefore, homologous desensitization of µ-opioid receptors in vivo is likely to be involved in the development of morphine-induced tolerance (Nestler 1996). Molecular cloning studies indicated that µ-opioid receptor is a member of G protein-coupled receptor family (Reisine and Bell 1993; Law and Loh 1999). Recently, multiple lines of evidence propose that agonist-induced desensitization of G protein-linked receptor results from the receptor phosphorylation by G protein-coupled receptor kinase (GRK) (Krupnick and Benovic 1998; Pitcher et al. 1998; Bunemann and Hosey 1999). According to this proposal, agonist-bound G protein-coupled receptor causes the dissociation of G protein, which is consisted of α (Gα) and βγ subunits (Gβγ). In addition to the subsequent Gα-or Gβγ-mediated activation of effector systems, Gβγ subunits also bind to GRK and translocate GRK from the cytoplasm to the cell membrane. After being phosphorylated by GRK, the receptors bind to inhibitory proteins, β-arrestins, and are uncoupled from G proteins, which results in homologous desensitization (Haga et al. 1994; Krupnick and Benovic 1998).

Up to now, six subtypes of GRKs have been cloned and characterized (Krupnick and Benovic 1998; Pitcher et al. 1998). Among these GRKs, GRK2 and GRK3 are widely distributed in the nervous system and likely to mediate homologous desensitization of µ-opioid receptors in the brain (Arriza et al. 1992). Consistent with this hypothesis, it has been reported that when expressed in CHO or HEK 293 cells, short-term desensitization of µ-opioid receptors was accompanied by the phosphorylation of receptor (Zhang et al. 1996; Yu et al. 1997). Overexpression of GRK2 or GRK3 in cell lines expressing µ-opioid receptors has been shown to promote the agonist-induced receptor phosphorylation (Zhang et al. 1998). Our recent investigation demonstrated that homologous desensitization of µ-opioid receptors expressed in HEK 293 cells was greatly impaired following the transfection of cDNA fragment encoding GRK2(495–689) polypeptide, which acts as a specific Gβγ antagonist and blocks various Gβγ-mediated transduction events including the activation of GRK2 (Wang 2000). It has also been shown that GRK3 accelerates homologous desensitization of µ-opioid receptors expressed in Xenopus oocytes (Kovoor et al. 1998). These findings observed from heterologous expression systems suggest that agonist-induced desensitization of µ-opioid receptors results from GRK2- or GRK3-mediated phosphorylation. However, it is still unknown whether GRK mediates homologous desensitization of µ-opioid receptors in the brain.

In the present study, whole-cell patch-clamp recording was combined with single-cell reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis and used to test the hypothesis that DAMGO ([D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin), a specific µ-opioid receptor agonist, selectively hyperpolarizes NRM neurons expressing mRNA of glutamate decarboxylase (GAD67), the synthesizing enzyme of GABA. Two experimental strategies were also used to investigate the involvement of GRK2 and GRK3 in mediating homologous desensitization of DAMGO-induced electrophysiological effects on NRM neurons. First, single-cell RT-PCR assay was performed to study the expression of GRK2 and GRK3 mRNAs in individual DAMGO-responsive NRM neurons. Synthetic peptide corresponding to Gβγ-binding domain of GRK2 or GRK3 has been shown to inhibit Gβγ activation of GRK2 or GRK3 (Koch et al. 1993; Boekhoff et al. 1994; Diverse-Pierluissi et al. 1996). Thus, we also performed whole-cell patch-clamp recordings with an internal solution containing the synthetic peptide that interferes with the translocation and activation of GRK. Our results suggest that DAMGO selectively hyperpolarizes NRM GABAergic neurons by opening inwardly rectifying K+ channels and that GRK2 mediates short-term homologous desensitization of µ-opioid receptors in NRM GABAergic neurons.

Materials and methods

Acute isolation of nucleus raphe magnus neurons

Neurons of the rat NRM were acutely dissociated according to the procedures described previously (Wu et al. 1995; Wu and Wang 1996). Briefly, 14- to 16-day-old Sprague–Dawley rats were terminally anesthetized with sodium pentobarbital and decapitated. The whole brain was quickly removed, and 300 µm-thick brain stem slices containing the NRM were prepared by using a Vibratome slicer in the ice-cold PIPES-buffered Ringer solution containing: NaCl 120 m m, KCl 5 m m, NaHCO3 20 m m, MgSO4 2 m m, CaCl2 2 m m, KH2PO4 1 m m, glucose 10 m m and PIPES 15 m m (pH = 7.4). Segments containing the NRM were excised, and incubated for 20 min at 32°C in an oxygenated PIPES saline solution (NaCl 125 m m, KCl 5 m m, CaCl2 2 m m, MgSO4 2 m m, glucose 10 m m, PIPES 15 m m, pH = 7.4) containing pronase E (0.5 mg/mL, Sigma). Subsequently, tissue segments were triturated with a Pasteur pipette, and dissociated neurons were plated onto polylysine-coated coverslips. Dissociated neurons were kept at a 100% O2 atmosphere for 30 min and then used for whole-cell patch-clamp recordings.

Whole-cell voltage-and current-clamp recordings

Acutely dissociated NRM neurons were voltage- and current-clamped by using the conventional whole-cell version of patch-clamp techniques (Hamil et al. 1981). Patch pipettes with a resistance of 3–4 Mohms were fabricated from hard borosillicate glasses using a pipet puller (P-87, Sutter). Holding potentials, data acquisition and analysis were controlled by an on-line personal computer programmed with AxoTape 2.0 and pCLAMP 6.0 (Axon Instruments). Current and voltage signals obtained by a patch-clamp amplifier (Axopatch-200 A, Axon Instruments) were filtered at 2 KHz, digitized (Digidata 1200 A interface, Axon Instruments) and stored for a later analysis. The external solution had the following composition: NaCl 145 m m, KCl 3 m m, CaCl2 2 m m, MgCl2 1 m m, glucose 15 m m and HEPES 10 m m (pH 7.3 with NaOH). The patch pipette was filled with: KCl 65 m m, KF 70 m m, MgCl2 1 m m, CaCl2 0.1 m m, EGTA 1.1 m m, ATP 2 m m, GTP 0.3 m m and HEPES 5 m m (pH 7.3 with KOH). Series resistance was usually < 10 MΩ, and the compensation circuitry of the amplifier was used to minimize the series resistance error. Liquid junction potentials were corrected as described previously (Barry and Lynch 1991). DAMGO (Peninsula) was dissolved in the external solution and applied to neurons using pressure ejections (Picospritzer, General Valve) from blunt micropipettes (diameter = 20–30 µm). For desensitization experiments, DAMGO (10 µm) was applied to NRM neuron for 10–12 s, and neuron was washed. Following an interval of 5 min, DAMGO (10 µm) was applied to NRM neuron again. P rism program (GraphPad Software) was used to analyze the dose–response curve. Experiments were performed at 22–25°C.

Single-cell RT-PCR assay

Harvesting cellular RNA of single NRM neurons and subsequent reverse transcription were performed by using the procedures as described previously (Wang and Wu 1996; Wu and Wang 1996). Briefly, patch electrodes were heated at 250°C for 5 h, and then filled with 8 µL of the autoclaved internal solution for whole-cell recordings. After the electrophysiological investigation, the cellular content was aspirated into the tip of patch electrode by applying a gentle suction. In order to prevent the contamination of genomic DNA, cellular contents were treated with 0.5 U of RNase-free DNase (Promega). Then, the first-strand cDNA was synthesized in a reaction volume of 30 µL containing 3 m m MgCl2, 50 m m Tris–HCl (pH = 8.3), 77 m m KCl, 10 m m dithiothreitol, 8 ng/µL hexamers, 1 m m of each deoxynucleotide 5′-trisphosphate, 20 U of ribonuclease inhibitor, and 150 U of Molony murine leukemia virus reverse transcriptase (Promega) for 1 h at 42°C. Then, the reaction mixture was heated at 90°C for 5 min, chilled on the ice, and used as the DNA template for PCR amplification.

PCR was carried out in a programmable thermal controller (Minicycler, NJ Research Inc.) with following oligonucleotide primers:

(a) Forward primer for GAD67 was 5′-GGCTACCTCTTCCAGCCAGACAAG-3′ and corresponded to nucleotides 1422–1445 of rat GAD67 cDNA (Julien et al. 1990).

(b) Reverse primer was 5′-GGAGATGACCATCCGGAAGAAGTT-3′ and corresponded to nucleotides 1815–1838 of rat GAD67. The expected size of cDNA fragment encoding GAD67 is 417 bp.

(c) Sense primer for GRK2 was 5′-GATGAGGAGGACACAAAAGGAATC-3′ and corresponded to nucleotides 1465–1488 of rat GRK2 cDNA (Arriza et al. 1992).

(d) Antisense primer was 5′-TCAGAGGCCGTTGGCACTGCCACGCTG-3′ and corresponded to nucleotides 2044–2070 of rat GRK2 cDNA.

(e) Forward primer for GRK3 was 5′-AATTGAGGCCAGGAAGAAGGCTA-3′ and corresponded to nucleotides 1605–1627 of rat GRK3 cDNA (Arriza et al. 1992).

(f) Reverse primer was 5′-TCAGAGGCCGCTGCTATTTCTGTGACA-3′ and corresponded to nucleotides 2041–2067 of rat GRK3 cDNA.

The expected sizes of cDNA fragments coding GRK2 and GRK3 are 606 bp and 463 bp, respectively.

PCR amplification was performed in a final volume of 100 µL containing the one-third of reverse transcription product (10 µL), 1.5 m m MgCl2, 50 m m KCl, 10 m m Tris–HCl, 0.1% Triton X-100, 0.2 m m of each deoxynucleotide 5′-trisphosphate, 0.2 µm of each primer and 5 U of Taq DNA polymerase (Takara). Aliquots of PCR products were separated and visualized in an ethidium bromide-stained agarose gel (1.5%) by the electrophoresis. PCR DNA fragment encoding GAD67, GRK2 or GRK3 was also gel-purified and used for the dideoxy chain-termination DNA sequencing (Thermo Sequenase cycle sequencing kit, Amersham).

Synthesis of GRK2 peptides

Peptide corresponding to Gβγ-binding site of rat GRK2 (W643-S670, WKKEL RDAVREAQQLVQRVPKMKNKPRS) or control GRK2 peptide (A531-Y553, AETDRLEARKKAKNKQLGHEEDY) (Arriza et al. 1992; Koch et al. 1993) was custom synthesized as the N-terminal-acylated and C-terminal-amidated form using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by using reverse HPLC (Research Genetics). Synthetic peptides were dissolved in the internal solution and dialyzed into NRM neurons during whole-cell patch-clamp recordings.

Statistics

All results are expressed as the mean ± SEM value of n experiments. Mann–Whitney test (two-tailed) was used to determine whether the difference was statistically significant (p < 0.01).

Results

DAMGO hyperpolarizes NRM GABAergic neurons by enhancing the inwardly rectifier K+ conductance

Consistent with previous studies using the slice preparation of rat NRM (Pan et al. 1990, 1997), two subtypes of neurons, primary and secondary cells, were observed from acutely dissociated NRM neurons. Primary cells were large oval-shaped serotonergic neurons (diameter = 30–45 µm) with two to three primary dendrites and did not fire action potentials spontaneously. Primary serotonergic neurons of NRM had the resting membrane potential of − 61 ± 5 mV and membrane input resistance of 350 ± 23 mΩ (n = 12). In agreement with previous studies (Pan et al. 1990, 1997), Current- and voltage-clamp recordings indicated that DAMGO (5 µm), a selective µ-opioid receptor agonist, failed to affect the electrical property of primary serotonergic neurons (n = 12).

Secondary neurons were small oval or multipolar cells (diameter = 15–25 µm) and generally spontaneously active. The spontaneous firing frequency was 10 ± 2 Hz (n = 12), and membrane input resistance was 610 ± 47 MΩ (n = 12). During whole-cell current-clamp recordings, DAMGO (5 µm) hyperpolarized NRM secondary cells (n = 8 out of 12 cells) and inhibited spontaneous action potentials (Fig. 1a). During the voltage-clamp recording at a holding potential (VH) of −40 mV, DAMGO (5 µm) evoked an outward current reversibly (Fig. 1b; the mean amplitude = 19 ± 2 pA, n = 15) and with a concentration-dependent manner (EC50 = 230 ± 27 n m, n = 6). Morphine (10 µm) also induced the outward current (the mean magnitude = 14 ± 2 pA, n = 5, VH = −40 mV). DAMGO (5 µm)-induced outward currents were reversibly inhibited by 5 µm naloxone (n = 6; data not shown). To elucidate the ionic mechanism by which DAMGO evokes the outward current, current (I)–voltage (V) curve was constructed by measuring DAMGO (5 µm)-induced currents at various holding potentials (− 140 mV to − 40 mV). DAMGO-evoked currents reversed the direction at about −100 ± 5 mV (n = 10), a reversal potential expected for potassium channels, and exhibited the inward rectification (Fig. 1c). DAMGO-evoked potassium current was blocked by 2 m m Cs+ or Ba2+, blocker of inward rectifier potassium channels (data not shown). These results indicate that DAMGO hyperpolarizes NRM secondary neurons by enhancing the inwardly rectifying K+ conductance.

Figure 1.

DAMGO, a selective µ-opioid receptor agonist, hyperpolarizes and inhibits NRM secondary neurons by enhancing the inwardly rectifying K+ conductance. (a) During the whole-cell current-clamp recording, DAMGO (5 µm) reversibly hyperpolarized a NRM secondary neuron and inhibited spontaneous action potentials. (b) Under the whole-cell voltage-clamp recording, DAMGO (5 µm) evoked an outward current from the same neuron with a reversible manner. Holding potential (VH) = – 40 mV (c) DAMGO-evoked currents were obtained at various holding potentials (− 140 to − 40 mV), and current (I)–voltage (V) curve was constructed. Note that DAMGO-induced currents reversed the polarity at about − 100 mV and exhibited the inward rectification. Each point shows the mean ± SEM value from 10 neurons.

Most of NRM secondary cells are believed to be GABAergic interneurons (Pan et al. 1990, 1997). To test the hypothesis that NRM secondary cells hyperpolarized by DAMGO were indeed GABAergic neurons, cellular RNA of the DAMGO-responsive secondary neurons was harvested following the electrophysiological investigation and used as the template for the subsequent single-cell RT-PCR analysis using primers specific for glutamate decarboxylase (GAD67), the synthesizing enzyme of GABA (Julien et al. 1990). Expected size for the partial cDNA fragment encoding GAD67 is 417 bp. GAD67 cDNA fragment with the expected size was amplified from all 14 NRM secondary neurons, which responded to DAMGO with a membrane hyperpolarization and the induction of K+ outward current (Figs 2a and b, lane 1). DNA sequencing using PCR DNA product indicated that 417-bp DNA fragment corresponded to rodent GAD67 cDNA. Amplified PCR DNA band was not observed when reverse transcriptase was omitted (data not shown, n = 4 neurons), indicating that PCR DNA product derived from GAD67 mRNA rather than contaminating genomic DNA. These findings suggest that DAMGO selectively hyperpolarizes GABAergic secondary neurons of the NRM.

Figure 2.

(a) Agonist-induced short-term desensitization of µ-opioid receptors in the NRM secondary neurons. DAMGO (10 µm) evoked outward K+ current from a NRM secondary neuron (upper trace). Following an interval of 5 min, the amplitude of potassium current elicited by the second application of DAMGO was greatly reduced (lower trace). VH = − 40 mV (b) GAD67 and GRK2 mRNA are expressed in DAMGO-responsive NRM secondary neurons. After finishing the electrophysiological investigation, the cellular content of NRM neuron shown in (a) was aspirated to the tip of patch electrode and used for the subsequent reverse transcription. One-third of reverse transcription product was utilized as DNA template for PCR amplification of GAD67, GRK2 or GRK3 cDNA fragment. Amplified cDNA fragments encoding GAD67 (lane 1) and GRK2 (lane 2) were obtained from this neuron. The same finding was also observed from 13 other NRM secondary neurons that responded to DAMGO. (c) As control experiments, cDNA fragments encoding rat GAD67 (lane 1), GRK2 (lane 2) and GRK3 (lane 3) were amplified using rat brainstem cDNA as DNA template. The locations of DNA size markers (φX174 DNA digested with Hinf I) are shown on the right side.

GRK2 mediates short-term homologous desensitization of µ-opioid receptors in NRM GABAergic neurons

Previous studies of locus coeruleus neurons have shown that µ-opioid receptors, which open inward rectifier K+ channels when activated, are rapidly desensitized following a short-term exposure to high concentrations of agonists (Osborne and Williams 1995; Fiorillo and Williams 1996). Consistent with this finding, µ-opioid receptor enhancement of the potassium conductance in NRM secondary neurons exhibits the phenomenon of acute homologous desensitization. The first application of DAMGO (10 µm), a supramaximally effective concentration, to NRM secondary neurons resulted in a induction of outward potassium current with a mean amplitude of 20 ± 3 pA (n = 14; VH = − 40 mV; upper trace of Fig. 2a). The magnitude of inwardly rectifying K+ current declined during the sustained application of DAMGO (Fig. 2a). After an interval of 5 min, the amplitude of outward potassium current evoked by the second application of 10 µm DAMGO was greatly reduced (lower trace of Fig. 2a; the mean magnitude = 8 ± 2 pA, n = 14). The dose–response curve for DAMGO enhancement of inwardly rectifying K+ conductance significantly shifted to the right, with the EC50 value increasing from 230 ± 27 n m to 950 ± 85 n m (n = 4). Morphine-evoked K+ current also developed short-term homologous desensitization. The amplitude of inwardly rectifying K+ current evoked by the first application of morphine (10 µm) was 14 ± 2 pA (n = 5, VH = −40 mV). Following an interval of 5 min, the magnitude of outward K+ current induced by the second application of 10 µm morphine was significantly reduced (the mean value = 6 ± 1 pA, n = 5). These results suggest that approximately 60% of µ-opioid receptors in NRM GABAergic neurons develops short-term homologous desensitization following an exposure to the saturating dose of DAMGO or morphine. With a longer interval (15–20 min) between DAMGO applications, the induction of homologous desensitization of DAMGO-evoked K+ currents was impaired (data not shown), indicating that acute µ-opioid receptor desensitization in NRM secondary neurons is a reversible process.

To test the hypothesis that GRK2 or GRK3 mediates short-term µ-opioid receptor desensitization, we first investigated the expression of mRNAs encoding GRK2 and GRK3 in individual NRM secondary neurons with the aid of single-cell RT-PCR assay. Expected sizes for GRK2 and GRK3 cDNA fragments are 606 bp and 463 bp, respectively. All 14 GAD67 mRNA-positive NRM neurons, which exhibited the phenomenon of acute desensitization of DAMGO-evoked K+ currents (Fig. 2a), contained GRK2 mRNA (Fig. 2b, lane 2) without expressing GRK3 mRNA (Fig. 2b, lane 3). DNA sequencing using PCR DNA product indicated that 606-bp DNA fragment corresponded to rodent GRK2 cDNA. To validate our single-cell RT-PCR method, cDNA was also synthesized using mRNA purified from rat brainstem. When brainstem cDNA was used as the template, partial GAD67, GRK2 and GRK3 cDNA fragments with the expected size were obtained (Fig. 2c). These results suggest that GRK2 is expressed in DAMGO-responsive NRM GABAergic neurons and likely to mediate µ-opioid receptor desensitization.

Following the agonist activation of G protein-coupled receptors, Gβγ mediates translocation of cytosolic GRK2 to the cell membrane where it phosphorylates activated receptors and initiates the process of desensitization (Haga et al. 1994; Krupnick and Benovic 1998; Pitcher et al. 1998). A previous study by Lefkowitz and co-workers demonstrated that C-terminal 28 amino acids (W643 to S670) of GRK2 or GRK3 function as Gβγ binding site and that synthetic (W643-S670) peptide blocks Gβγ activation of GRK2 or GRK3 in vitro (Koch et al. 1993). When internally perfused into cells, synthetic (W643-S670) peptide corresponding to Gβγ-binding domain of GRK3 has been successfully used to inhibit Gβγ activation of GRK3 and GRK3-meidated homologous desensitization of odorant receptors and α2-adrenergic receptors (Boekhoff et al. 1994; Diverse-Pierluissi et al. 1996).

In the present study, the functional role of GRK2 in mediating µ-opioid receptor desensitization was further investigated by dialyzing NRM GABAergic neurons with synthetic 28-mer peptide corresponding to Gβγ-binding site of GRK2. According to a previous study (Diverse-Pierluissi et al. 1996), NRM secondary neurons were internally perfused with 0.2 m m GRK2(W643-S670) peptide for 10 min. Subsequently, whole-cell voltage-clamp recording was performed as described above to determine whether µ-opioid receptors still undergo agonist-induced desensitization. Internal perfusion of GRK2(W643-S670) peptide failed to affect resting electrical properties of NRM GABAergic neurons and the amplitude of DAMGO-evoked potassium current, indicating that GRK2(W643-S670) peptide did not interfere with µ-opioid receptor activation of inwardly rectifying K+ conductance via Gi/o proteins. In the presence of GRK2(W643-S670) peptide, the initial magnitude of DAMGO (10 µm)-induced K+ current was 20 ± 2 pA (n = 14; control value = 20 ± 3 pA). Following an interval of 5 min, potassium current evoked by the second application of DAMGO (Fig. 3a, lower trace; the mean amplitude = 18 ± 2 pA; n = 14) was not significantly decreased compared to K+ current induced by the first application of DAMGO (Fig. 3a, upper trace). As a result, percentage of µ-opioid receptor desensitization was reduced to 10 ± 1% (control value = 61 ± 11%) following the intracellular administration of GRK2(W643-S670) peptide (Fig. 3c). The specificity of GRK2(W643-S670)–mediated inhibitory effect on µ-opioid receptor desensitization was tested by using GRK2(A531-Y553) peptide, which derived from a sequence located outside of Gβγ-binding domain (Koch et al. 1993). Intracellular administration of GRK2(A531-Y553) peptide for 10 min did not affect the magnitude of DAMGO-evoked potassium current (with GRK2(A531-Y553) peptide, the mean value = 24 ± 1 pA, n = 14; control DAMGO K+ current = 20 ± 3 (pA). Dialyzing NRM GABAergic neurons with 0.4 m m GRK2(A531-Y553) peptide also failed to affect acute desensitization of DAMGO-evoked outward K+ currents (Fig. 3b,c; control percentage of desensitization = 61 ± 11%; with GRK2(A531-Y553) peptide,% of desensitization = 63 ± 7%; n = (14). These results suggest that GRK2 mediates short-term homologous desensitization of µ-opioid receptors in NRM GABAergic neurons.

Figure 3.

GRK2 mediates short-term µ-opioid receptor desensitization in NRM GABAergic neurons. (a) DAMGO (10 µm) evoked the potassium current from a NRM GABAergic neuron dialyzed with 0.2 m m GRK2(W643-S670) peptide for 10 min (upper trace). Following the interval of 5 min, DAMGO still induced an outward K+ current without exhibiting homologous desensitization (lower trace). VH = − 40 mV. (b) After internally perfusing a NRM secondary neuron with 0.4 m m control peptide, GRK2(A531-Y553), for 10 min, the amplitude of potassium current evoked by the second application of 10 µm DAMGO (lower trace; interval = 5 min) was significantly reduced compared to that of K+ current induced by the first application of DAMGO (upper trace). VH = − 40 mV. (c) Intracellular administration of GRK2(W643-S670), a synthetic peptide corresponding to Gβγ-binding site of GRK2, impairs µ-opioid receptor desensitization in NRM GABAergic neurons. Percentage of desensitization was calculated as: [1-(potassium current evoked by the second application of DAMGO/K+ current induced by the first application of DAMGO)] × 100. The interval between DAMGO applications was 5 min. Each bar shows the mean ± SEM value of 14 neurons. *p < 0.01 compared to control.

In CHO cells stably expressing µ-opioid receptors, mitogen-activated protein kinase (MAPK) pathway has been proposed to mediate µ-opioid receptor desensitization that is accompanied by a decrease in receptor density (Polakiewicz et al. 1998). In the present study, the involvement of MAPK pathway in mediating short-term µ-opioid receptor desensitization was tested by using PD98059 (20 µm), which blocks MAPK transduction pathway by inhibiting the activity of mitogen-activated protein kinase kinase (Alessi et al. 1995; Polakiewicz et al. 1998). After internally perfusing NRM secondary neurons with 20 µm PD98059 for 10 min, DAMGO (10 µm) enhancement of the inward rectifier K+ conductance still developed short-term homologous desensitization (control percentage of desensitization = 61 ± 11%; with PD98059,% of desensitization = 58 ± 3%; n = 8). Internal perfusion of PD98059 failed to affect the initial amplitude of DAMGO (10 µm)-induced potassium current (control DAMGO K+ current = 20 ± 3 pA; with PD98059, the mean value = 22 ± 2 pA, n = 8). This finding suggests that mitogen-activated protein kinase is not involved in initiating short-term µ-opioid receptor desensitization in NRM secondary neurons.

Discussion

Nucleus raphe magnus is an essential component of brainstem descending antinociceptive pathway and plays an important role in opioid-induced analgesia in the brain (Fields et al. 1991). To gain insight into the cellular mechanism by which µ-opioid receptor agonist produces an analgesic effect in the NRM, we investigated the electrophysiological effect of DAMGO on acutely dissociated NRM neurons. Consistent with previous studies using brain slices (Pan et al. 1990, 1997), the present study identified two subtypes of acutely isolated NRM neurons, primary and secondary cells. Our results demonstrate that DAMGO selectively hyperpolarizes NRM GABAergic secondary neurons by opening inwardly rectifying K+ channels. Although previous studies showed that µ-opioid receptor agonists including Met-enkephalin and DAMGO hyperpolarize a subpopulation of NRM secondary cells (Pan et al. 1990, 1997), the present study, for the first time, provides the direct evidence that DAMGO disinhibits NRM primary neurons and produces an analgesic effect in the NRM by hyperpolarizing secondary GABAergic interneurons, which tonically inhibit spinally projecting primary serotonergic neurons (Fields et al. 1991; Pan et al. 1997). Consistent with this hypothesis, it has been shown that DAMGO reduces GABA-mediated postsynaptic potentials in NRM primary neurons without affecting the excitatory glutamatergic neurotransmission (Pan et al. 1990).

In addition to opioid peptides, neurotensin and nicotinic receptor agonists also produce an analgesic effect by stimulating NRM-spinal dorsal horn antinociceptive neuronal circuitry (Behbehani 1992; Bitner et al. 1998). In contrast to selective inhibition of NRM GABAergic neurons by DAMGO, neurotensin and nicotinic receptor agonists, which exert an excitatory effect on CNS neurons, have been shown to selectively depolarize and excite primary serotonergic neurons of the NRM, which project to the spinal cord and inhibit nociceptive transmission (Bitner et al. 1998; Li and Wang, unpublished results). Thus, excitatory and inhibitory neurotransmitters in the NRM produce antinociceptive effects by affecting the electrical activity of different populations of NRM neurons.

In agreement with previous studies (Osborne and Williams 1995; Fiorillo and Williams 1996), the present investigation indicates that µ-opioid receptor-mediated enhancement of inward rectifier K+ conductance in NRM GABAergic neurons develops short-term homologous desensitization. Recently, several lines of evidence have implicated GRK-mediated phosphorylation in initiating homologous desensitization of G protein-coupled receptors (Krupnick and Benovic 1998; Pitcher et al. 1998; Bunemann and Hosey 1999). Among six members of the GRK family, GRK2 and GRK3 are highly expressed in the brain and likely to mediate µ-opioid receptor desensitization in vivo (Arriza et al. 1992). With the aid of cell lines or Xenopus oocytes expressing µ-opioid receptors and GRK, previous studies have shown that both GRK2 and GRK3 promote agonist-induced phosphorylation of µ-opioid receptors and attenuate µ-opioid receptor-induced cellular responses, which include the inhibition of adenylate cyclase and opening of inwardly rectifier K+ channels (Kovoor et al. 1998; Whistler and von Zastrow 1998; Zhang et al. 1998). To determine the functional significance of GRK2 or GRK3 in regulating the activity of µ-opioid receptors in the NRM, we first investigated the expression of GRK2 and GRK3 in individual NRM neurons that respond to DAMGO with outward K+ current. In accordance with a previous investigation showing that mRNA and protein levels of GRK2 in the rat brain are more abundant that those of GRK3 (Arriza et al. 1992), our single-cell RT-PCR analysis demonstrates that DAMGO-responsive NRM GABAergic neurons contain GRK2 mRNA without expressing GRK3 mRNA, suggesting the physiological importance of GRK2 in mediating µ-opioid receptor desensitization. GRK2 and GRK3 proteins share a high structural similarity, with over 80% amino acid identity (Arriza et al. 1992). Both isozymes have been shown to equally phosphorylate several G protein-coupled receptors, which include δ-, κ- and µ-opioid receptors (Pitcher et al. 1998; Bunemann and Hosey 1999). For G protein-coupled receptors that are substrates for both GRK2 and GRK3, the specificity of GRK–receptor interaction is likely to be determined by differential cellular expression of GRK2 and GRK3. This hypothesis is supported by the finding that GRK3 is selectively expressed in olfactory receptor neurons and dorsal root ganglion (DRG) neurons. As a result, GRK3 has been reported to specifically mediate homologous desensitization of odorant receptors in olfactory receptor cells and α2-adrenergic receptors in DRG neurons (Boekhoff et al. 1994; Diverse-Pierluissi et al. 1996).

Homologous desensitization of opioid receptor-mediated cellular responses could result from a decrease in the agonist affinity or the downregulation of opioid receptors (Law and Loh 1999). Short-term opioid receptor desensitization occurs within minutes of agonist exposure and is believed to result from the uncoupling of µ-opioid receptor and Gi/o proteins, which leads to a reduction of agonist affinity (Puttfarcken et al. 1988; Raynor et al. 1994; Law and Loh 1999). In contrast to short-term pretreatment, a prolonged exposure to agonist induces the functional desensitization of opioid receptors by decreasing the density of opioid receptors in the cell membrane (Cvejic et al. 1996; Burd et al. 1998; Law and Loh 1999). The present study indicates that 5 min after an exposure to the saturating dose of DAMGO, µ-opioid receptor enhancement of inwardly rectifying K+ conductance develops homologous desensitization. Therefore, short-term µ-opioid receptor desensitization in NRM secondary neurons is likely to result from a reduction in the agonist affinity. Consistent with single-cell RT-PCR assay showing that DAMGO-responsive NRM GABAergic neurons express GRK2 mRNA, our recent study demonstrated that GRK2 mediates short-term homologous desensitization of µ-opioid receptors expressed in HEK 293 cells by causing receptor-Gi/o protein uncoupling and decreasing the agonist affinity (Wang 2000).

Gβγ subunits mediate the translocation and activation of GRK2 by binding to the carboxyl terminus of GRK2 (Pitcher et al. 1998). It has been shown that C-terminal 28 amino acids (Trp643 to Ser670) of GRK2 act as the principal Gβγ binding site (Koch et al. 1993). When internally perfused into cells, 28-mer GRK2(W643-S670) peptide is expected to inhibit GRK2 activation by blocking the association between Gβγ and endogenous GRK2. In the absence of specific pharmacological inhibitors of GRK2, we further investigated the functional role of GRK2 in initiating short-term µ-opioid receptor desensitization by dialyzing NRM GABAergic neurons with synthetic GRK2(W643-S670) peptide. The present study demonstrates that short-term homologous desensitization of µ-opioid receptors in NRM GABAergic neurons is greatly impaired following the dialysis of GRK2(W643-S670) peptide. In addition to GRK2, Gβγ also activate other effectors including adenylate cyclase II, phospholipase C-β2 ioform and mitogen-activated protein kinase (MAPK) (Koch et al. 1994; Hawes et al. 1996). It has been reported that neither cAMP-dependent protein kinase nor protein kinase C is involved in initiating short-term homologous desensitization of µ-opioid receptors (Zhang et al. 1996; Chakrabarti et al. 1998). The present study also indicates that internal perfusion of PD98059, which inhibits MAPK transduction pathway, fails to block short-term desensitization of µ-opioid receptors in NRM GABAergic neurons. Instead of short-term homologous desensitization, mitogen-activated protein kinase and protein kinase C have been reported to play an important role in mediating agonist-induced down-regulation of µ-opioid receptors (Polakiewicz et al. 1998; Kramer and Simon 2000; Schmidt et al. 2000). Therefore, it is very likely that intracellular dialysis of GRK2(W643-S670) peptide blocks short-term µ-opioid receptor desensitization in NRM GABAergic neurons by inhibiting Gβγ-mediated activation of GRK2.

The results presented here suggest that short-term desensitization of µ-opioid receptors in the brain results from Gβγ-mediated translocation of GRK2 to the cellular membrane and subsequent GRK2-induced µ-opioid receptor phosphorylation. GRK2 is believed to mediate µ-opioid receptor desensitization by phosphorylating serine or threonine residues located in the third intracellular loop or cytoplasmic carboxyl tail (Haga et al. 1994; Pitcher et al. 1998). In accordance with this hypothesis, our recent study using various C-terminal deletion mutants showed that a cluster of Ser/Thr residues (T354S355S356T357) at the carboxyl tail is required for GRK2-mediated homologous desensitization of µ-opioid receptors stably expressed in HEK 293 cells (Wang 2000). Further studies using point mutant µ-opioid receptors suggest that two C-terminal amino acids, Ser355 and Thr357, play a critical role in short-term homologous desensitization of µ-opioid receptors (Wang et al. unpublished results).

It has been reported that GRK2 phosphorylates δ-opioid receptors expressed in mammalian cell lines and mediates short-term desensitization of δ-opioid receptors (Pei et al. 1995). Pretreating hippocampal slices with U50,488H, a specific κ-opioid receptor agonist, results in the phosphorylation of κ-opioid receptor protein (Appleyard et al. 1997). GRK2 and GRK3 have been shown to mediate agonist-induced desensitization of κ-opioid receptors expressed in mammalian cell lines or Xenopus oocytes (Raynor et al. 1994; Appleyard et al. 1999). However, it remains unknown that whether GRK mediates homologous desensitization of δ-and κ-opioid receptors in vivo. The experimental strategy described in the present study could also be used to investigate the involvement of GRK2 or GRK3 in mediating homologous desensitization of δ- and κ-opioid receptors in the brain.

In conclusion, the present study provides the evidence that GRK2 mediates short-term homologous desensitization of µ-opioid receptors in NRM GABAergic neurons. Together with previous studies showing that the expression of GRK2 is upregulated in the brains of chronic opiate-treated rats and human opiate addicts (Nestler 1996; Ozaita et al. 1998), the results reported here further support the hypothesis that GRK2 is an important regulator of µ-opioid receptor activity in vivo.

Acknowledgements

This work was supported by the Chang Gung Research Foundation (CMRP 555) and National Science Council (NSC89-2320-B182-057).

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