Introduction

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Opioid receptors play important roles in many physiological functions and mediate pharmacological effects of opiate and opioid compounds. The presence of multiple types of opioid receptors (mu, delta, kappa and epsilon) in the peripheral and central nervous system has been established by pharmacological and binding studies as well as differential anatomical localization (for a review, Chang, 1984). Activation of opioid receptors couples to various effectors via pertussis toxin-sensitive G protein, including adenylate cyclase and K⁺ and Ca⁺⁺ channels (for reviews, Loh and Smith, 1990; Childers, 1991).

Activation of kappa opioid receptors produces many effects including analgesia (Von Voigtlander et al., 1983; Dykstra et al., 1987), dysphoria (Pfeiffer et al., 1986; Dykstra et al., 1987), water diuresis (VonVoigtlander et al., 1983; Dykstra et al., 1987) and hypothermia (Adler and Geller, 1993). After the cloning of the mouse delta opioid receptor (Kieffer et al., 1992; Evans et al., 1992), we (Zhu et al., 1995), Mansson et al. (1994) and Simonin et al. (1995) reported cloning of the human kappa opioid receptor. Deduced amino acid sequences of these clones display the motif of seven transmembrane helices, characteristics of G protein-coupled receptors.

Many G protein-coupled receptors show attenuated responsiveness to agonists after prolonged or repeated activation. Three processes are involved in response to agonists occurring across a time scale ranging from seconds to days: desensitization (seconds to hours), sequestration (minutes to hours) and down-regulation (hours to days). These processes were best studied in the beta-2 adrenergic receptor (for a review, Hausdorff et al., 1990). Desensitization is thought to be mediated partly through the uncoupling of the high-affinity receptor-G protein complex, which explains the loss of receptor responsiveness to agonists. Down-regulation involves a reduction in the number of receptors. Compared with mu and delta opioid receptors, relatively few studies have been conducted on desensitization and down-regulation of the kappa opioid receptor and reports are conflicting (Attali et al., 1989; Attali and Vogel, 1990; Joseph and Bidlack, 1995; Raynor et al., 1994; Blake et al., 1997; Jin et al., 1997). Attali et al. (1989) showed that pretreatment of rat spinal cord-dorsal root ganglion co-cultures with etorphine or U50,488H led to heterologous desensitization of kappa opioid, muscarinic and alpha-1 adrenergic receptors. After 72 h exposure to 1 µM etorphine, a slight down-regulation of the kappa opioid receptor was observed (Attali and Vogel, 1990). Incubation of R1.1 cells with 0.1 µM U50,488H for 24 h or 48 h resulted in 50% reduction of B_max of [³H]U69,593 and [³H]naloxone (Joseph and Bidlack, 1995). However, there was no change in the inhibition of forskolin-stimulated adenylate cyclase activity by U50,488H in terms of potency and maximal response (Joseph and Bidlack, 1995). Incubation of COS-7 cells transiently expressing the cloned mouse kappa opioid receptor with 1 µM U50,488H for 4 h diminished kappa opioid agonist-induced inhibition of forskolin-stimulated adenylate cyclase, with no change in the total receptor number (Raynor et al., 1994). Treatment of CHO cells stably expressing the rat kappa opioid receptor with U69,593 up to 10 µM for 4 h did not lead to reduction of the capacity of the agonist to inhibit forskolin-stimulated adenylate cyclase (Avidor-Reiss et al., 1995). Pretreatment of HEK-293 cells stably transfected with the human kappa opioid receptor with 1 µM U50,488H for 3 h led to a 6-fold increase in the EC₅₀ value, with no change in the maximal response, of U50,488H in inhibition of forskolin-stimulated adenylate cyclase activity (Blake et al., 1997). Concomitantly, the B_max value of [³H]diprenorphine binding was reduced by 31% with no change in its K_d value (Blake et al., 1997). U69,593 elicited a large K⁺ current in Xenopus oocytes injected with mRNA of the kappa opioid receptor and a G protein-linked inwardly rectifying potassium channel, and this effect was quickly desensitized with a T_1/2 of 10.5 min (Henry et al., 1995). Chronic in vivo administration of U50,488H shifted the dose-response curve of U69,593-induced electrophysiological responses to the right by 3-fold and reduced the maximal effect in guinea pig hippocampal slices in vitro (Jin et al., 1997).

We recently established a cell line of CHO-hkor cells (Zhu et al., 1995). Hkor expressed in CHO cells exhibited binding affinity and specificity for opioid ligands expected of the kappa opioid receptor (Zhu et al., 1997). In addition, we demonstrated that activation of hkor by kappa opioid agonists enhanced [³⁵S]GTPS binding. Pretreatment with pertussis toxin abolished agonist-induced increase in [³⁵S]GTPS binding, which indicates the coupling of the kappa opioid receptor to G_i and/or G_o proteins. This assay provides a sensitive functional measure for interaction between kappa opioid receptors and pertussis toxin-sensitive G proteins (Zhu et al., 1997). In this study, we examined whether desensitization and down-regulation of the human kappa opioid receptors expressed in the CHO cell line occurred after exposure to the kappa opioid agonist ()U50,488H, by determining [³⁵S]GTPS binding, adenylate cyclase and receptor binding activities.

	Materials and Methods

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Materials. [³H]Diprenorphine (35 Ci/mmol) was obtained from Amersham Corp (Arlington Heights, IL). [³⁵S]GTPS (1,000-1,200 Ci/mmol), [³H]adenine (30 Ci/mmol) and [¹⁴C]cAMP (40 Ci/mmol) were purchased from NEN Life Sciences (Boston, MA). Naloxone was a gift from DuPont/Merck Co. (Wilmington, DE). ()U50,488H and (+)U50,488H were provided by Upjohn Co. (Kalamazoo, MI). GDP, GTPS and Dulbecco's modified Eagle's medium were purchased from Sigma (St. Louis, MO). Geneticin was purchased from Mediatech Co. (Herndon, VA); fetal calf serum from Hyclone Co. (Logan, UT) and penicillin and streptomycin from GIBCO-BRL Co. (Gaithersburg, MD).

Stable expression of the human kappa opioid receptor in CHO cells. CHO cell lines stably expressing the human kappa opioid receptor (CHO-hkor) were established as described previously (Zhu et al., 1997).

Pretreatment of CHO-hkor cells with the kappa opioid agonist ()U50,488H. CHO-hkor cells were cultured in 100-mm culture dishes in Dulbecco's modified Eagle's medium F12 HAM supplemented with 10% fetal calf serum, 0.2 mg/ml G418, 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere consisting of 5% CO₂ and 95% air at 37°C. At ~90% confluence, the cells were washed once with 100 mM PBS and treated with the kappa opioid agonist ()U50,488H in the medium mentioned above for a time at a concentration as indicated. Cells were harvested and membranes prepared by a procedure similar to that described previously (Zhu et al., 1997). Cells were washed twice with 100 mM PBS, harvested in Versene solution, centrifuged at 500 × g for 3 min and washed once with PBS. The cell pellet was resuspended in 50 mM Tris-HCl buffer containing 1 mM ethylene glycol-bis(-aminoethyl ether) N,N,N',N'-tetraacetic acid, 5 µM leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM NaF and 10 mM Na pyrophosphate, sonicated and centrifuged at 46,000 × g for 30 min. The pellet was resuspended in 50 mM Tris, pH 7.0, and centrifuged again. The membrane pellet was resuspended in 50 mM Tris, 0.32 M sucrose, pH 7.0, aliquoted at ~100 µg protein/ml, frozen in dry ice/ethanol and stored in 70°C until use. All procedures were performed at 4°C.

Kappa opioid receptor binding assay. Receptor binding was conducted with [³H]diprenorphine in 50 mM Tris-HCl buffer containing 1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 5 µM leupeptin (pH 7.4) as described previously (Zhu et al., 1995). ()Naloxone (10 µM) was used to define nonspecific binding. Saturation experiments were performed with various concentrations of [³H]diprenorphine (ranging from 0.02 nM to 2 nM). Competitive inhibition of [³H]diprenorphine binding by ()U50,488Hwas performed with [³H]diprenorphine at a concentration close to its K_d (~0.2 nM) and various concentrations of ()U50,488H. Binding was conducted at 25°C for 60 min in duplicate in a volume of 1 ml with 30 to 40 µg protein. Bound and free ligands were separated by rapid filtration under reduced pressure over GF/B filters presoaked with 0.2% polyethyleneimine and 0.01% bovine serum albumin in 50 mM Tris-HCl (pH 7.4) for 1 h. Binding data were analyzed with EBDA and LIGAND programs (McPherson, 1983).

[³⁵S]GTPS binding assay. [³⁵S]GTPS binding assay was performed as we described previously (Zhu et al., 1997). Before assay, membranes were thawed at 37°C, chilled on ice, passed through a 22-gauge needle and diluted with 50 mM HEPES, 100 mM NaCl, 5 mM MgCl₂ and 1 mM ethylenediaminetetraacetic acid with 1 mM dithiothreitol and 0.1% bovine serum albumin freshly added (pH 7.4). Membranes (~10 µg protein) were incubated in the buffer described above containing [³⁵S]GTPS (100,000-150,000 dpm, ~80 pM), GDP (3 µM) and varying concentrations of the kappa opioid agonist ()U50,488H (10¹¹ to 10⁵ M) in a total volume of 0.5 ml for 60 min at 30°C. Nonspecific binding was defined by incubation in the presence of 10 µM GTPS. Bound and free [³⁵S]GTPS were separated by filtration with GF/B filters under reduced pressure. Radioactivity on filters was determined by liquid scintillation counting. EC₅₀ and maximal response values were calculated by use of the equation y = [E_max/[1 + (x/EC₅₀)^s]] + background, in which y is the response at the dose x, E_max is the maximal response, s is a slope factor.

Determination of adenylate cyclase activities. Adherent CHO-hkor cells grown in 6-well plates were incubated with [³H]adenine at 1 to 2 µCi/ml in serum-free medium to label adenine nucleotides in the cytoplasmic pool for at least 2 h. For 1-h pretreatment, cells were treated with or without 0.1 µM ()U50,488H at 37°C for the last 60 min. For 24-h pretreatment, cells were treated with ()U50,488H for 24 h at 37°C and labeled with [³H]adenine for the last 2 h. After removal of the medium, cells were washed three times with PBS, and serum-free medium containing 1 mM 3-isobutyl-1-methylxanthine was added. Cells were then treated with vehicle, forskolin (10 µM), forskolin (10 µM)/()U50,488H at 37°C for 10 min. The reaction was stopped with the addition of 0.1 N HCl. [¹⁴C]cAMP (~3,000 dpm) was added as the recovery standard. Radiolabeled cAMP was separated from other labeled nucleotides by the dual-column method of Salomon (1979). The radioactivities of eluted [³H]cAMP and [¹⁴C]cAMP were determined by two-channel scintillation counting. [³H]cAMP formed was calculated as percent of total adenine nucleotides taking into account the cross-over and recovery.

Protein assay. Protein contents of membranes were determined by the bicinchoninic acid method of Smith et al. (1985) with bovine serum albumin as the standard.

Statistical analysis. For comparison of multiple groups, data were analyzed with analysis of variance to determine whether there were significant differences among groups. If so, Sheffe F-test was performed to determine whether there was significant difference between the control and each treatment group. For comparison of two groups, Student's t test was performed. P < .05 was used as the level of significance.

	Results

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Effect of ()U50,488H pretreatment time on ()U50,488H-induced increase in [³⁵S]GTPS binding and the number of kappa opioid receptors. CHO-hkor cells were pretreated with 1 µM ()U50,488H for various periods of time and examined for their responses to the subsequent application of ()U50,488H. When membranes were prepared without 10 mM NaF and 10 mM Na pyrophosphate, desensitization was not observed consistently (data not shown). The levels of ()U50,488H-induced [³⁵S]GTPS binding were similar in untreated membranes prepared in the presence and absence of 10 mM NaF and 10 mM Na pyrophosphate. Hence, membranes for desensitization experiments were prepared in the presence of 10 mM NaF and 10 mM Na pyrophosphate.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effect of pretreatment time with 1 µM ()U50,488H on (A) [35S]GTPS binding induced by()U50,488H and (B) [3H]diprenorphine binding. CHO-hkor cells were treated at 37°C with or without 1 µM ()U50,488H for different periods of time, washed extensively and membranes prepared. [35S]GTPS binding in response to ()U50,488H and saturation [3H]diprenorphine binding were performed as described under "Materials and Methods." (A) Each point represents the mean ± S.E.M. of three to six experiments. Basal [35S]GTPS binding ranged from 83 to 88 fmol/mg protein and did not differ among the control and the treatment groups. (B) Scatchard analysis of [3H]diprenorphine binding after each treatment was performed. Each curve represents one of the three to six experiments performed. EC50 and maximal level values of [35S]GTPS binding and Kd and Bmax of [3H]diprenorphine binding are presented in table 1.

View this table: [in this window] [in a new window]   TABLE 1 Effect of pretreatment time with 1 µM ()U50,488H on U50,488H-stimulated [35S]GTPS binding and [3H]diprenorphine binding to the  receptor CHO-hkor cells were treated with 1 µM ()U50,488H for various times and washed, and the ()U50,488H-induced increases in [35S]GTPS binding and [3H]diprenorphine binding to the receptor were performed as described under "Materials and Methods." Each datum represents mean ± S.E.M. of three to six experiments. Data were analyzed by analysis of variance followed by Sheffe F-test.

Effect of ()U50,488H pretreatment concentration on ()U50,488H-induced increase in [³⁵S]GTPS binding and the number of kappa opioid receptors. Pretreatment for 1 h with 10 nM, 100 nM or 1 µM ()U50,488H, but not 1 nM, reduced [³⁵S]GTPS binding elicited by ()U50,488H (fig. 2A and table 2). Pretreatment with 10 nM and 100 nM ()U50,488H reduced maximal [³⁵S]GTPS binding to 82% and 64% of the control level, respectively, without affecting the EC₅₀ value. A 1 µM pretreatment increased the EC₅₀ value to 2.4-fold of the control and reduced the maximal response to 62% of the control level. One hour exposure to 100 nM or 1 µM ()U50,488H did not affect K_d or B_max value of [³H]diprenorphine binding (fig. 2B and table 2). Thus, 1 h pretreatment with 1 µM ()U50,488H led to desensitization, but not down-regulation, of the kappa opioid receptor.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of pretreatment concentration with ()U50,488H for 1 h on (A) [35S]GTPS binding induced by ()U50,488H and (B) [3H]diprenorphine binding. CHO-hkor cells were treated at 37°C for 1 h with different concentrations of ()U50,488H, washed extensively and membranes prepared. [35S]GTPS binding in response to ()U50,488H and saturation [3H]diprenorphine binding were performed as described under "Materials and Methods." (A) Each point represents the mean ± S.E.M. of three to six experiments. Basal [35S]GTPS binding ranged from 80 to 87 fmol/mg protein and did not differ among the control and the treatment groups. (B) Scatchard analysis of [3H]diprenorphine binding after each treatment was performed. Each curve represents one of the three to six experiments performed. EC50 and maximal level values of [35S]GTPS binding and Kd and Bmax of [3H]diprenorphine binding are presented in table 2.

View this table: [in this window] [in a new window]   TABLE 2 Effect of pretreatment concentration with ()U50,488H for 1 h on () U50,488H-stimulated [35S]GTPS binding and [3H]diprenorphine binding to the  receptor CHO-hkor cells were treated for 1 h with various concentrations of ()U50,488H and washed; the U50,488H-induced increases in [35S]GTPS binding and [3H]diprenorphine binding to the receptor were carried out as described under "Materials and Methods." Each datum represents mean ± S.E.M. of three to six experiments. Data were analyzed by analysis of variance followed by Sheffe F-test.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of pretreatment concentration with ()U50,488H-induced for 4 h on (A) [35S]GTPS binding induced by ()U50,488H and (B) [3H]diprenorphine binding. CHO-hkor cells were treated at 37°C for 4 h with different concentrations of ()U50,488H for 4 h, washed extensively and membranes prepared. [35S]GTPS binding in response to ()U50,488H and saturation [3H]diprenorphine binding were performed as described under "Materials and Methods." (A) Each point represents the mean ± S.E.M. of three to six experiments. Basal [35S]GTPS binding ranged from 77 to 87 fmol/mg protein and did not differ among the control and the treatment groups. (B) Scatchard analysis of [3H]diprenorphine binding after each treatment was performed. Each curve represents one of the three to six experiments performed. EC50 and maximal level values of [35S]GTPS binding and Kd and Bmax of [3H]diprenorphine binding are presented in table 3.

View this table: [in this window] [in a new window]   TABLE 3 Effect of 4-h pretreatment with different ()U50,488H concentrations on ()U50,488H-stimulated [35S]GTPS binding and [3H]diprenorphine binding to the  receptor CHO-hkor cells were treated for 4 h with various concentrations of ()U50,488H and washed; the ()U50,488H-induced increases in [35S]GTPS binding and [3H]diprenorphine binding to the receptor were carried out as described under "Materials and Methods." Each datum represents mean ± S.E.M. of three to six experiments. Data were analyzed by analysis of variance followed by Sheffe F-test.

Role of the kappa opioid receptor in ()U50,488H-induced desensitization and down-regulation. Pretreatment with 1 µM (+)U50,488H, an inactive isomer of ()U50,488H, for 4 h did not have any effect on ()U50,488H-induced [³⁵S]GTPS binding or the K_d and B_max values of [³H]diprenorphine (fig. 4 and table 4). Although naloxone (10 µM) alone did not affect responsiveness or number of the kappa opioid receptor, naloxone (10 µM) blocked 0.1 µM ()U50,488H (4 h)-induced desensitization of [³⁵S]GTPS response and reduction in B_max of [³H]diprenorphine binding (fig. 4 and table 4). These results indicate that both processes are mediated by receptor activation.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of preincubation with ()U50,488H, naloxone, ()U50,488H + naloxone or (+)U50,488H on (A) ()U50,488H-induced [35S]GTPS binding and (B) [3H]diprenorphine binding. CHO-hkor cells were treated at 37°C with 0.1 µM ()U50,488H, 10 µM naloxone, 0.1 µM ()U50,488H + 10 µM naloxone or 1 µM (+)U50,488H for 4 h, washed extensively and membranes prepared. Vehicle-treated cells served as the control. [35S]GTPS binding in response to ()U50,488H and saturation [3H]diprenorphine binding were performed as described under "Materials and Methods." (A) Each point represents the mean ± S.E.M. of three to six experiments. Basal [35S]GTPS binding ranged from 79 to 88 fmol/mg protein and did not differ among the control and the treatment groups. (B) Scatchard analysis of [3H]diprenorphine binding after each treatment was performed. Each curve represents one of the three to six experiments performed. EC50 and maximal level values of [35S]GTPS binding and Kd and Bmax of [3H]diprenorphine binding are presented in table 4.

View this table: [in this window] [in a new window]   TABLE 4 Effect on ()U50,488H-stimulated [35S]GTPS binding and [3H]diprenorphine binding to the  receptor of 4-h pretreatment with 0.1 µM ()U50,488H, 1 µM (+)U50,488H, 1 µM naloxone and 0.1 µM ()U50,488H + 1 µM naloxone, and 4 h of 0.1 µM ()U50,488H followed by 24-h recovery CHO-hkor cells were subjected to various treatments as indicated and washed; the ()U50,488H-induced increases in [35S]GTPS binding and [3H]diprenorphine binding to the receptor were conducted as described under "Materials and Methods." Each datum represents mean ± S.E.M. of three to six experiments. Data were analyzed by analysis of variance followed by the Sheffe F-test.

Reversibility of ()U50,488H-induced desensitization and down-regulation. CHO-hkor cells were treated with 0.1 µM ()U50,488H for 4 h to induce desensitization and down-regulation. Twenty-four hours after removal of ()U50,488H, both [³⁵S]GTPS binding induced by ()U50,488H and [³H]diprenorphine binding returned to the control levels (fig. 5 and table 4).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Reversibility of 0.1 µM ()U50,488H-induced kappa receptor desensitization and down-regulation. CHO-hkor cells were cultured at 37°C for 4 h in medium with or without 0.1 µM ()U50,488H, washed and then grown in medium without ()U50,488H for 24 h. Membranes were prepared and [35S]GTPS binding in response to ()U50,488H and saturation [3H]diprenorphine binding were performed as described under "Materials and Methods." (A) Each point represents the mean ± S.E.M. of three to six experiments. Basal [35S]GTPS binding ranged from 78 to 88 fmol/mg protein and did not differ among the control and the treatment groups. (B) Scatchard analysis of [3H]diprenorphine binding after each treatment was performed. Each curve represents one of the three to six experiments performed. EC50 and maximal level values of [35S]GTPS binding and Kd and Bmax of [3H]diprenorphine binding are presented in table 4.

Effect of ()U50,488H pretreatment on ()U50,488H-induced inhibition of forskolin-stimulated adenylate cyclase activity. Exposure of CHO-hkor cells to 0.1 µM ()U50,488H for 1 h shifted the dose-response curve to the right of ()U50,488H-elicited inhibition of forskolin-stimulated adenylate cyclase activity, compared with the control treatment (fig. 6A). The EC₅₀ value of ()U50,488H of the pretreated cells was about 2-fold of that of the control (control, 9.7 ± 1.1 nM; 1 h pretreatment, 21.0 ± 3.4 nM, n = 3, P < .05 by Student's t test). Maximal inhibition did not differ significantly (control, 16.6 ± 2.6%; 1 h pretreatment, 24.1 ± 2.6%, n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of ()U50,488H preincubation on inhibition of forskolin-stimulated adenylate cyclase by ()U50,488H. CHO-hkor cells were incubated without or with 100 nM ()U50,488H for 1 h (A) or 24 h (B) and washed extensively with PBS buffer. ()U50,488H-induced inhibition of forskolin-stimulated adenylate cyclase was determined as described under "Materials and Methods." In both control and ()U50,488H-treated cells, after forskolin stimulation, [3H]cAMP represented approximately 2% of total [3H]adenine nucleotides. Data are expressed as percent of forskolin-stimulated adenylate cyclase activity. Each point represents the mean ± S.E.M. of three determinations in triplicate.

Affinity and GTPS sensitivity of ()U50,488H binding to desensitized kappa opioid receptors. Affinity of ()U50,488H binding and effect of 100 µM GTPS on ()U50,488H binding affinity to control and desensitized receptors were compared. Cells were treated with or without 1 µM ()U50,488H for 1 h, which causes desensitization, but not down-regulation. Competitive inhibition by ()U50,488H of [³H]diprenorphine binding to membranes of control and treated cells was conducted in the presence or absence of 100 µM GTPS. For membranes of cells pretreated with 1 µM ()U50,488H for 1 h, the K_i value of ()U50,488H in inhibiting [³H]diprenorphine binding was 2.5 ± 0.5 nM (n = 3), which was significantly higher than that of the control cells (0.7 ± 0.1 nM, n = 3) (P < .05, analysis of variance followed by Sheffe F-test). In the presence of GTPS, K_i values of ()U50,488H binding were determined to be 2.9 ± 0.6 nM and 3.9 ± 0.7 nM for the pretreated and control receptors, respectively, and there was no significant difference. Thus, ()U50,488H pretreatment lowered the affinity of ()U50,488H for the receptor and abolished the effect of GTPS on the agonist affinity, which indicates that desensitized receptors are uncoupled from G proteins.

	Discussion

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In this study, we demonstrated that exposure to ()U50,488H led to a reduction in responsiveness to the agonist (desensitization) and a decrease in the receptor number (down-regulation) of the kappa opioid receptor. During desensitization, maximal response of ()U50,488-induced [³⁵S]GTPS binding was reduced with or without an increase in the EC₅₀ value. In addition, agonist affinity was decreased and GTPS effect on agonist affinity was abolished, yet affinity and receptor number of the antagonist [³H]diprenorphine binding were unchanged. With a longer incubation time and/or a higher agonist concentration, both desensitization and down-regulation occurred. During this phase, the EC₅₀ value of ()U50,488H in enhancing [³⁵S]GTPS binding was increased and the maximal response was reduced. In addition, the receptor number of [³H]diprenorphine binding was decreased, with no change in affinity. Both desensitization and down-regulation were mediated by receptor activation and were reversible after removal of the agonist.

In the present study, we have clearly shown that, temporally, desensitization precedes down-regulation. Initially the kappa opioid receptor undergoes desensitization without down-regulation, and later down-regulation concomitant with desensitization occurs. After 1 h incubation with 1 µM ()U50,488H or 4 h incubation with 1 nM ()U50,488H, desensitization, but not down-regulation, occurred. There were two different types of desensitization: one with a reduction in maximal response with no change in the EC₅₀ value of ()U50,488H and the other with a reduction in maximal response and an increase in the EC₅₀ value of ()U50,488H.

To the best of our knowledge, this study is the first one to use agonist-induced increase in [³⁵S]GTPS binding as the functional measure of the kappa opioid receptor responsiveness after agonist exposure. [³⁵S]GTPS binding is a direct measure of enhanced receptor-G protein coupling by receptor agonists. Basal levels of [³⁵S]GTPS binding in CHO-hkor membranes were about 85 fmol/mg protein and did not differ among the control and various treatment groups. The data shown in figures 1 to 5 and tables 1 to 4 are net stimulated increases with basal levels already subtracted. Thus, the maximal responses elicited by U50,488H represented about a 2-fold increase over the basal level, which were sufficient to allow examination of desensitization. Agonist-induced increase in [³⁵S]GTPS binding has been used to examine desensitization of mu and delta opioid receptors (Breivogel et al., 1997). Our results that pretreatment with 0.1 µM ()U50,488H for 1 h led to similar desensitization of both agonist-induced increase in [³⁵S]GTPS binding and inhibition of forskolin-stimulated adenylate cyclase indicate that [³⁵S]GTPS binding is a valid functional endpoint for examination of receptor desensitization.

The observation that in these CHO-hkor cells, agonist-induced increase in [³⁵S]GTPS binding was desensitized readily and the maximal response was lowered on desensitization suggests that there are few, if any, spare kappa opioid receptors for this response. A similar conclusion was reached in our previous study (Zhu et al., 1997). In this system, [³⁵S]GTPS binding assay allowed classification of highaffinity kappa opioid ligands into full agonists, partial agonists and antagonists, depending on the maximal response produced.

Although 0.1 µM ()U50,488H pretreatment for 1 h reduced maximal [³⁵S]GTPS binding by ()U50,488H, it did not affect maximal inhibition of forskolin-stimulated adenylate cyclase activities by ()U50,488H. These results suggest that, although there are few or no spare kappa opioid receptors for G proteins, there are spare inhibitory G proteins for adenylate cyclase.

The affinity and number of receptors determined by [³H]diprenorphine binding was not changed by several different ()U50,488H pretreatment paradigms (1 µM for 15 min, 1 nM for 1 h, 10 nM for 1 h, 100 nM for 1 h, 1 µM for 1 h) followed by extensive washing. These results indicate complete removal of ()U50,488H with our washing procedure.

Our finding that ()U50,488H treatment led to desensitization of the kappa opioid receptor is contrary to the reports of Joseph and Bidlack (1995) and Avidor-Reiss et al. (1995). We demonstrated that desensitization of ()U50,488H-induced enhancement of [³⁵S]GTPS binding occurred as early as 15 min after 1 µM ()U50,488H incubation. In addition, pretreatment with 0.1 µM ()U50,488H for 1 h or 24 h led to a desensitization in ()U50,488H-induced inhibition of forskolin-stimulated adenylate cyclase. Joseph and Bidlack (1995) observed no desensitization of U50,488H-elicited inhibition of forskolin-stimulated adenylate cyclase in R1.1 cells after 24 h or 48 h treatment with 0.1 µM U50,488H. In addition, culturing of R1.1 cells with 10 nM bremazocine for 24 h did not affect inhibition of adenylate cyclase by U50,488H (Joseph and Bidlack, 1995). This apparent discrepancy may be the result of the different systems used. Murine thymoma R1.1 cells were used by Joseph and Bidlack (1995), whereas we used CHO cells stably transfected with the human kappa opioid receptor. Different in vitro systems may vary in their responses to exposure to the kappa opioid agonist, because of the different levels of key proteins involved in desensitization. Joseph and Bidlack (1995) speculated that the lack of kappa opioid receptor desensitization in R1.1 cells might be caused by low levels of components involved in desensitization, such as beta adrenergic receptor kinase. Our finding is also different from that of Avidor-Reiss et al. (1995), who found that incubation of CHO cells stably transfected with the rat kappa opioid receptor with U69,593 up to 10 µM for 4 h inhibited forskolin-stimulated adenylate cyclase to the same extent as a 10-min incubation. The reason for this difference is not apparent. Their treatment paradigms were different from ours. Avidor-Reiss et al. (1995) did a 4-h incubation with U69,593 and measured inhibition of forskolin-stimulated adenylate cyclase activity, whereas we preincubated with ()U50,488H followed by removal of ()U50,488H with washes and determined inhibition of forskolin-stimulated adenylate cyclase activity by the subsequent application of U50,488H. In addition, this discrepancy might reflect species difference in the kappa opioid receptor properties, i.e., rat versus human. Comparison of the C-terminal domain sequences of the rat and human kappa opioid receptors shows Ser358 in the human receptor instead of Asn358 in the rat. Serine residues can be phosphorylated, whereas Asn can not. Whether this amino acid difference contributes to differential desensitization will be investigated.

Our results on kappa opioid receptor desensitization, on the other hand, are consistent with those of Raynor et al. (1994) and Blake et al. (1997). They demonstrated that chronic exposure to U50,488H induced kappa opioid receptor desensitization in Cos-7 cells transiently expressing the mouse kappa opioid receptor and HEK-293 cells stably expressing the human kappa opioid receptor, respectively. Inhibition of forskolin-stimulated adenylate cyclase activity was used as the functional endpoint. However, there are some differences. Raynor et al. (1994 ) observed that pretreatment with 1 µM U50,488H for 4 h did not reduce the number of sites of [³H]naloxone binding, whereas we did find receptor down-regulation after the same treatment.

Prolonged exposure to ()U50,488H reduced the B_max value of [³H]diprenorphine binding with no change in the affinity. The extent of decrease in receptor number after a 4-h exposure to 10 nM ()U50,488H was ~30%. Incubation for 4 h with a higher concentration of ()U50,488H up to 1 µM or for 24 h with 1 µM did not lead to further reduction in the receptor number. A similar degree of down-regulation was reported by Blake et al. (1997) after a 3-h pretreatment with 1 µM U50,488H. However, this down-regulation was less than the 50% reduction observed by Joseph and Bidlack (1995) after incubation with 0.1 µM U50,488H for 24 h in R1.1 thymoma cells. This difference may be a reflection of difference in the levels of molecules required for the down-regulation process between the two systems.

GTP or its analog uncouples G proteins from the receptor and thus lowers the affinity of the receptor for agonists. The observations that desensitized kappa opioid receptors exhibited lower affinity for U50,488H and that the ability of GTPS to lower kappa opioid agonist affinity was abolished indicate that uncoupling of the kappa opioid receptor from G proteins occurs after incubation with U50,488H. These findings agree with those of Raynor et al. (1994). Similar observations have been reported for mu and delta opioid receptors (Puttfarcken et al., 1988; Werling et al., 1989; Law et al., 1983).

Both sodium fluoride and sodium pyrophosphate are phosphatase inhibitors. They have been used extensively in receptor phosphorylation studies to prevent de-phosphorylation (for example, Liggett et al., 1992; Pei et al., 1995; Zhang et al., 1996; Arden et al., 1995). The G protein-coupled receptor phosphatase was reported recently, and this activity was a latent form of protein phosphatase type 2A (Pitcher et al., 1995). The finding that the presence of sodium fluoride and sodium pyrophosphate during membrane preparation was necessary for ()U50,488H-induced desensitization of [³⁵S]GTPS binding to be observed suggests that ()U50,488H-induced kappa opioid receptor desensitization is associated with phosphorylation of certain proteins involved in signal transduction, possibly of the kappa opioid receptor. Whether the kappa opioid receptor is phosphorylated after prolonged exposure to an agonist is currently under investigation. Mu and delta opioid receptors have undergone phosphorylation under conditions that cause desensitization (Pei et al., 1995; Zhang et al., 1996; Arden et al., 1995).

Pretreatment with 0.1 µM ()U50,488H for 1 h led to desensitization of ()U50,488H-induced inhibition of forskolin-stimulated adenylate cyclase without the presence of phosphatase inhibitors. The difference may be caused by a lengthy process of membrane preparation that had to be performed before [³⁵S]GTPS binding assay, during which many enzymes may be liberated. In contrast, adenylate cyclase assay was carried out in whole-cell preparations immediately after pretreatment with ()U50,488H and three washes of the cells.

In conclusion, we have demonstrated that the human kappa opioid receptor undergoes desensitization and down-regulation after prolonged exposure to the opioid agonist ()U50,488H. Temporally, desensitization occurs first, and if agonist exposure persists, down-regulation along with desensitization ensues. Both processes are mediated by receptor activation and are reversible after removal of the agonist. Because kappa opioid agonist-induced enhancement of [³⁵S]GTPS binding involved only receptor and G proteins, biochemical changes during desensitization must occur at the level of receptor and/or G proteins. With the availability of cloned kappa opioid receptors, we can start to delineate biochemical mechanisms of desensitization and down-regulation.