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Endocrinology Vol. 141, No. 7 2294-2300
Copyright © 2000 by The Endocrine Society

ARTICLES

A Subset of Kappa Opioid Ligands Bind to the Membrane Glucocorticoid Receptor in an Amphibian Brain

Simon J. Evans, Brian T. Searcy and Frank L. Moore

Zoology Department, Oregon State University, Corvallis, Oregon 97331

Address all correspondence and requests for reprints to: Frank L. Moore, Zoology Department, Oregon State University, Corvallis, Oregon 97331. E-mail: mooref{at}bcc.orst.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies demonstrated that a membrane receptor for glucocorticoids (mGR) exists in neuronal membranes from the roughskin newt (Taricha granulosa) and that this receptor appears to be a G protein-coupled receptor (GPCR). The present study investigated the question of whether this mGR recognizes nonsteroid ligands that bind to cognate receptors in the GPCR superfamily. To address this question, ligand-binding competition studies evaluated the potencies of various ligands to displace [3H]corticosterone (CORT) binding to neuronal membranes. Initial screening studies tested 21 different competitors and found that [3H]CORT binding was displaced only by dynorphin 1–13 amide (an endogenous {kappa}-selective opioid peptide), U50,488 (a synthetic {kappa}-specific agonist) and naloxone (a nonselective opioid antagonist). Follow-up studies revealed that the {kappa} agonists bremazocine (BRE) and ethylketocyclazocine (EKC) also displaced [3H]CORT binding to neuronal membranes, but that U69,593 (a {kappa} specific agonist) and nor-BNI (a {kappa} specific antagonist) were ineffective. The Ki values measured for the opioid competitors were in the subnanomolar to low micromolar range and had the following rank-order: dynorphin > U50,488 > naloxone > BRE > EKC. Because these ligands displaced, at most, only 70% of [3H]CORT specific binding, it appears that some [3H]CORT binding sites are opioid insensitive. Kinetic analysis of [3H]CORT off-rates in the presence of U50,488 and/or CORT revealed no differences in dissociation rate constants, suggesting that there is a direct, rather than allosteric, interaction with the [3H]CORT binding site. In summary, these results are consistent with the hypothesis that the high-affinity membrane binding site for [3H]CORT is located on a {kappa} opioid-like receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS increasing evidence that steroid hormones use multiple signal transduction pathways—the classic genomic pathway, and nongenomic pathways in which membrane receptors are coupled to second messenger systems [for a review see Wehling (1) or Moore and Evans (2)]. Nongenomic pathways are reportedly used by progestins (3, 4), androgens (5), estrogens (6, 7, 8) vitamin D (9), and corticosteroids (10).

An important, and largely unanswered, question concerns the molecular identity of the membrane receptor proteins used by steroid hormones. Recent research, mostly with estrogen and progestins, suggests that steroids use different types of receptors, including enzyme-linked receptors, G protein-coupled receptors, ligand-gated ion channels, or membrane-associated classical steroid receptors. First, one putative estrogen receptor protein is the epidermal growth factor (EGF)-like protein, ErbB2. This protein, when expressed in vitro, binds 17ß-estradiol with high affinity and shows estradiol-induced kinase activity (11). Similarly, a putative membrane receptor for progesterone is a protein that has structural similarities to enzyme-linked receptors (12) and is localized to endomembranes (13). This receptor also appears to have a pharmacological profile resembling the sigma receptor (14). Second, another set of examples indicate that estrogens and progestins can use ligand-gated ion channels. Specific progesterone metabolites (such as 3{alpha}-hydroxy-4-pregnene-20-one) have been found to rapidly modulate the GABAA receptor/chloride channel (15). Recent studies show that 17ß-estradiol can bind to the ß subunit of Maxi-K channels and thereby modulate calcium flux in vascular smooth muscles cells (16). Third, other studies indicate that intracellular estrogen receptors (ER{alpha}, ERß) might, in some cases, function as membrane-associated receptors (17). When ER{alpha} or ERß is expressed in cell lines that do not normally contain these receptor proteins, 17ß-estradiol can bind to the membranes and activate the MAP kinase pathways (18). Lastly, there also is evidence that progestins and estrogen use receptor proteins in the GPCR superfamily. For example, 17ß-estradiol modifies GRCR regulated K+ channels in guinea pig hypothalamic neurons (8). Other studies show that progesterone binds to and inhibits the activity of a known GPCR, namely the oxytocin receptor (19). Thus far, the evidence suggests that steroid hormones use multiple types of membrane-associated receptors and, relevant to the current research, that steroids can bind to and modulate known receptors used by other endogenous ligands.

There is considerably less information about the molecular identity of the membrane-associated receptors for corticosteroids, although there is good evidence that corticosteroids regulate various types of rapid responses in different vertebrates. In the cichlid fish Oreochromis mossambicus, cortisol administration to pituitary cells in vitro affects intracellular cAMP and Ca2+ concentrations in lactotrophs within seconds and inhibits PRL secretion within minutes (20). In rats and guinea pigs, neurophysiological studies show that corticosteroid administration decreases Ca2+ currents in specific neurons within a few seconds (21, 22). In an amphibian, the roughskin newt (Taricha granulosa), corticosterone administration decreases neuronal activity in medullary neurons within a few minutes (23, 24). Corticosteroids also can exert negative feedback effects on ACTH secretion within minutes (25). In in vitro studies with smooth muscle cells, aldosterone administration causes increases in DAG, IP3, and intracellular Ca2+ concentrations within a few seconds (26, 27). Corticosteroid administration also can cause rapid behavioral changes in birds (28), mammals (29), and amphibians (30).

Ligand-binding assays reveal that there are high-affinity binding sites for corticosteroids in membrane preparations from rodent brains, liver tissue, and pituitary glands (31, 32, 33, 34). The high-affinity binding site for [3H]CORT in neuronal membranes from T. granulosa has been studied in detail (35, 36, 37). Orchinik et al. (36) demonstrated that this high-affinity binding site for [3H]CORT meets all the criteria for being a functional membrane-associated corticosteroid receptor (mGR). Recent studies in T. granulosa solubilized and partially purified the mGR protein and found that it is an acidic glycoprotein with an apparent mass of 63 kDa (35, 38). This mGR appears to be in the GPCR superfamily because studies found that [3H]CORT specific binding was enhanced by Mg2+ and negatively modulated by guanyl nucleotides (37).

Considering the above evidence that the membrane corticosteroid receptor in T. granulosa is a GPCR and, also, that steroid hormones can bind to and modulate cognate receptors [see above (19)], a logical question is whether the high-affinity binding site for corticosterone is located on a known GPCR. If so, then the best candidates are GPCRs that mediate behavioral responses similar to those caused by corticosterone administration. In T. granulosa, corticosterone administration rapidly inhibits male sexual behaviors (30). Similarly, administration of CRH (CRH) (30) or {kappa}-selective opioid agonists (bremazocine, ethylketocyclazocine, and dynorphin) (39) can also suppress male sexual behaviors; whereas vasotocin (AVT) (40), GnRH (41), or corticotropin-like peptides (ACTH, {alpha}MSH) (42) enhance male sexual behaviors. Secondly, in T. granulosa, corticosterone administration rapidly suppresses the stress-induced increases in locomotor activity (43). Similar suppression of locomotor activity in newts is observed following injection of {kappa}-selective opioid agonists (44), whereas this behavior is enhanced by CRH administration (45, 46). Thus, results from these behavioral studies reveal that corticosterone and {kappa}-opioid receptor agonists cause similar responses.

The current study investigated the question of whether the high affinity binding site for corticosterone is located on a known GPCR. As a first step toward answering this question, ligand-binding competition studies were run to determine whether any of the peptides or opioid agonists that have been shown to affect newt behaviors (cited above) are recognized by the high affinity binding site for corticosterone in neuronal membranes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All buffers, salts, and drugs were from Sigma (St. Louis, MO) except diprenorphine, bremazocine (BRE), U50,488, and U69,593 were from RBI (Natick, MA); dynorphin (1–13 amide) was from Peninsula Laboratories, Inc. (Belmont, CA); ethylketocyclazocine (EKC) was originally from Sterling Winthrop (supplied by Dr. Thomas Murray, University of Georgia). [3H]-CORT (1,2,6,7- 3H corticosterone, 79 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). GF/C membranes were from Brandel (Gaithersburg, MD).

Adult male roughskin newts (T. granulosa) were collected from local ponds and maintained in tanks with flowthrough dechlorinated water. Animals were treated in compliance with IAUAC standards and guidelines as approved by the Animal Use and Care Committee at Oregon State University.

Preparation of neuronal membranes
Procedures for membrane preparation (P2 membrane pellets) followed the methods of Whittaker (47), as modified by Orchinik et al. (36). Brains were removed on wet ice and placed in 0.3 ml of chilled homogenization buffer [0.32 M sucrose, 5 mM HEPES, pH 7.45, 100 µM phenolmethylsulfonyl fluoride (PMSF)] per mg brain tissue (wet weight). Brain tissue was homogenized with a Teflon-on-glass tissue homogenizer. The homogenate was centrifuged at low speed (1,000 x g) for 10 min at 4 C, and the resultant supernatant (S1) was transferred to a clean centrifuge tube. The pellet (P1) was resuspended in homogenization buffer to the original concentration and centrifuged again as above. The pellet was discarded and the supernatant was pooled with S1 and centrifuged at higher speed (30,000 x g) for 40 min at 4 C. The pellet (P2) was quick frozen at -80 C, then thawed on ice and resuspended in 0.18 ml/mg (original tissue weight) of 25 mM HEPES, 10 mM EDTA, pH 7.45, 100 µM PMSF. The suspension of P2 was incubated at 4 C for 2 h to allow the dissociation of any endogenous ligand and centrifuged at 40,000 x g for 15 min. The resultant pellet was washed once by homogenization in 25 mM HEPES, pH 7.45 and recentrifuged at 40,000 x g for 15 min. This final pellet was quick frozen at -80 C. These well-washed P2 pellets were either stored at -80 C (for this study less than 3 weeks) or thawed on ice and used immediately. No loss of binding activity has been observed in stored pellets.

Radioligand binding assays
Ligand-binding assays used [3H]CORT and the methods described previously (36). Briefly, membrane preparations (P2 pellets) were suspended in 25 mM HEPES, pH 7.45, 10 mM MgCl2 at a concentration of 100 µg protein (as determined by the method of Lowry, et al. (48)) per 300 µl in each assay tube. Final concentrations were 0.5 nM for [3H]CORT, which approximates the Kd value (36), and 10 µM for the unlabeled corticosterone that was used to define nonspecific binding. Concentrations for the competitors were as described in Results. Assay tubes were incubated for 4–6 h at 30 C, conditions that allow binding to reach equilibrium as determined previously in kinetic studies at different temperatures (36, 38). Binding assays were terminated by rapid filtration over GF/C glass fiber filters equilibrated in cold assay buffer. Radioactivity in the filters was counted (cpm) in a Beckman Coulter, Inc. LS 6500 scintillation counter. Radioligand-binding data were analyzed with GraphPad Software, Inc. Prism software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Initial screen with ligands to G protein-coupled receptors
Ligand-binding competition studies used [3H]CORT with neuronal membranes and GPCR ligands as competitors. Because our objective was to screen as many GPCR ligands as possible for positive effects on displacing [3H]CORT specific binding without using too many animals, each ligand was tested at only one concentration of 10 µM. Figure 1Go shows that, of the 21 competitors tested, only three noticeably decreased [3H]CORT binding. Naloxone (a nonselective opioid antagonist) and U50,488 (a {kappa}-selective opioid agonist) inhibited [3H]CORT binding by >50%; whereas, dynorphin 1–13 amide (an endogenous peptide ligand for the {kappa} opioid receptor) inhibited [3H]CORT binding by approximately 25%.



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  		Figure 1. Multiple ligand screen. [3H]CORT competition binding assays were performed as described in the methods section. All competitors were added to assays to a final concentration of 10 µM. Values are reported as percent [3H]CORT specific binding in the presence of the various competitors. Zero percent specific binding (nonspecific) was determined in the presence of 10 µM cold corticosterone.

 
Equilibrium competition analysis with {kappa}-opioid receptor ligands
Based on the above results, ligand-binding competition assays investigated in detail the effects of specific opioid ligands on [3H]CORT binding. Each study was replicated at least once to confirm that the results are reproducible. Competition studies identified five opioid ligands with activity to displace [3H]-CORT binding and estimated their affinities in the subnanomolar to micromolar range (Fig. 2Go and Table 1Go). The rank-order potencies of these ligands to displace [3H]-CORT binding was dynorphin 1–13 amide > U50,488 > naloxone > BRE > EKC. Dynorphin only inhibited 25% of [3H]CORT specific binding, whereas, U50,488, naloxone, BRE, and EKC each inhibited 70%. The displacement curves for the nonpeptide ligands, as shown in Fig. 2AGo, conformed to a single-site competition model, according to curve-fitting analysis with r2 values of 0.98 (U50,48), 0.93 (naloxone), 0.96 (BRE), and 0.87 (EKC).



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  		Figure 2. Equilibrium competition analysis with {kappa} opioid receptor ligands. Binding assays were performed as described in the methods section. Competitors were present at the concentrations indicated on the graph. A, Competitors showing a maximum of 70% displacement of [3H]CORT binding. B, Dynorphin 1–13 amide competition curve showing a maximum of 25% displacement of [3H]CORT binding. Data were analyzed with GraphPad Software, Inc. Prism software using a single site competition model.

 

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  		Table 1. Summary of equilibrium competition binding with opioid competitors

 
Competition studies also revealed that the [3H]CORT binding site does not recognize four other ligands that bind to mammalian {kappa} opioid receptors. No inhibition of [3H]CORT specific binding was found in assays that included the competitors U69,593 ({kappa}-specific agonist), nor-BNI ({kappa}-specific antagonist), diprenorphine (nonselective opioid antagonist), and etorphine (nonselective opioid agonist).

Kinetic analysis for allosteric interactions
Kinetic experiments were performed to evaluate the possibility that the above results might reflect allosteric, rather than direct, interactions between opioid ligands and [3H]CORT binding sites (Fig. 3Go). In these experiments, neuronal membranes were incubated with [3H]CORT until equilibrium was reached followed by the induction of dissociation with excess unlabeled corticosterone and/or U50,488. Figure 3Go shows that the dissociation curves are similar for U50,488 and corticosterone competitors. Data analysis found that these dissociation curves fit a one-phase exponential decay model with r2 values of 0.94 for the U50,488 curve and 0.95 for the corticosterone curve. Dissociation rate constants for U50,488 and corticosterone were indistinguishable, with estimated k-1 values of 8.6 x 10-3 ± 0.0014 for U50,488 and 9.5 x 10-3 ± 0.0017 for CORT. The addition of both competitors (U50,488 plus corticosterone) produced a similar k-1 value 9.0 x 10-3 ± 0.0009 and a r2 value of 0.98. These data are best explained by a single site competition model.



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  		Figure 3. Kinetic analysis of dissociation rates. Binding assays were set up as described in the methods section and allowed to equilibrate for 6 h before adding competitors. Both U-50,488 and CORT competitors were added to achieve final concentrations of 10 µM. Dissociation time points were initiated in an inverse order and assays were terminated simultaneously by rapid filtration as described in Materials and Methods. Data were analyzed with GraphPad Software, Inc. Prism software using a one phase exponential decay to determine k-1 values. Data are normalized to maximum displacement of [3H]CORT by either U-50,488 or CORT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies in T. granulosa demonstrated that there is a high affinity binding site for [3H]CORT in neuronal membranes and that this binding site fits the criteria for being a functional receptor in the GPCR superfamily (36, 37). The present study revealed that this [3H]CORT binding site recognizes a subset of opioid ligands which include the {kappa}-selective agonists (dyn, BRE, U-50,488, and EKC) and the nonselective antagonist naloxone. It also found, however, that this [3H]CORT binding site does not recognize several other {kappa}-selective ligands. These findings are unique, showing for the first time in any species that specific opioid ligands can displace [3H]CORT binding to neuronal membranes, and as discussed below, support the working hypothesis that the [3H]CORT binding site is located on a {kappa} opioid-like receptor.

Although the current study is the first to find that specific opioid ligands are recognized by a corticosteroid binding site, previous research revealed that other steroids can interact with opioid receptors. Ligand-binding competition studies with neuronal membranes from rats and mice found that the synthetic steroid RU486 decreases specific binding of [3H]dihydromorphine (a µ-selective agonist) (49). Other studies with rat neuronal membranes found that 17ß-estradiol and other estrogens can inhibit specific binding of [3H]DAGO (a µ-selective agonist) and [3H]DADL (a {delta}-selective agonist) (50). In the current study in T. granulosa, competition studies found that a subset of {kappa}-selective ligands inhibit [3H]CORT specific binding. Furthermore, our kinetic analysis also provided evidence that this effect is probably due to direct competition between corticosterone and these {kappa}-selective ligands for the same binding pocket. In sum, these studies with mammals and an amphibian suggest that specific steroid hormones might interact with specific types of opioid receptors.

The current study found that the [3H]CORT binding site only recognizes a subset of {kappa} ligands. The pharmacological profile for the [3H]CORT binding site has similarities to, but is distinctly different from, the mammalian {kappa} opioid receptor; it recognizes dynorphin, U50,488, BRE, EKC, and naloxone but not U69,593, nor-BNI, diprenorphine, and etorphine. The mammalian {kappa} opioid receptor binds this entire set of ligands with high affinity. Unfortunately, it is not known whether {kappa} opioid receptors in T. granulosa have atypical ligand selectivity, because opioid receptors have not been characterized in this species or any other urodele amphibian. Considering studies in frogs, our prediction is that the {kappa} opioid receptor(s) in T. granulosa are pharmacologically similar to those of mammals. Research in Rana esculenta characterized ligand selectivity for the {kappa}-binding sites in the brain and found two subtypes of {kappa} opioid receptors with pharmacological profiles that resemble, but do not exactly match, the KOR1 and KOR2 subtypes in mammals (51, 52). In frogs and mammals, the defining feature of the KOR1 subtype of {kappa} opioid receptor is that it recognizes both U50,488 and U69,593, which is interesting because the [3H]CORT binding site in T. granulosa discriminates between these two {kappa}-specific agonists.

There is evidence that the opioid receptor system developed early in vertebrate evolution (53). A recent study found that complementary DNA sequences encoding each of the four types of opioid-like receptors [µ, {delta}, {kappa}, and the orphanin opioid receptor-like receptor (ORL)] occur in representative species of elasmobranchs to mammals (54). It therefore seems very likely that T. granulosa has these four types of opioid receptors as well. In mammals there are multiple subtypes of the {delta}, {kappa}, and ORL receptors, some of which are derived from stable splice variants with tissue-specific distribution (55, 56). None of these subtypes in mammals have been specifically characterized pharmacologically or linked to specific physiological functions. The presence of these multiple subtypes exposes the possibility that one of the subtypes could function as a membrane corticosteroid receptor.

Our earlier behavioral studies in T. granulosa are consistent with the working hypothesis that the [3H]CORT binding site is located on a {kappa} opioid-like receptor. Those studies found that male sexual behaviors are inhibited by exposure to acute stress (30) and that this stress-induced inhibition of sexual behaviors is linked to both corticosterone and the {kappa} opioid system. When male newts are exposed to acute stress, the incidence of amplectic clasping decreases in control animals, but not in males pretreated with metyrapone (the steroid synthesis inhibitor) (30) or naloxone (39). Furthermore, males injected with corticosterone or the {kappa}-selective agonist BRE exhibit a rapid and pronounced inhibition of male sexual behaviors (30, 39).

Stress-induced increase in locomotor activity is another behavioral response in T. granulosa that has been linked to both corticosterone and {kappa} opioid system. When male newts are exposed to acute stress, locomotor activity is enhanced in control animals, a response that is controlled by CRH acting centrally (45, 46). This stress-induced increase in locomotor activity can be rapidly suppressed by an injection of corticosterone (43) or the {kappa}-selective agonist BRE (44). Therefore, as with stress-induced inhibition of sexual behaviors, corticosteroids and {kappa}-selective agonists exert similar effects on locomotor activity.

There also are consistencies in the physiological functions of corticosteroids and the {kappa} opioid system, which adds indirect support for our working hypothesis. First, one important physiological function for both corticosteroids and {kappa} opioid receptors is to decrease ACTH activity by suppressing secretions of CRH and vasopression (VP) (57, 58, 59, 60, 61). Second, dynorphin, the endogenous {kappa}-selective neuropeptide, is colocalized in CRH- and VP-containing neurons (62, 63, 64, 65). Third, {kappa} opioid receptors have been found on presynaptic membranes of VP-containing neurons (66) and the activation of presynaptic {kappa} opioid receptors inhibits VP secretion (67). Likewise, corticosteroids have been shown to inhibit VP secretion (68). This convergence in the effects of corticosteroids and {kappa} opioid agonists on VP and CRH secretion may help to explain the behavioral responses in T. granulosa described above. In T. granulosa, male sexual behaviors are enhanced by AVT (69), and the stress-induced increase in locomotor activity is enhanced by CRH (46). Therefore, the inhibition of release of these two peptides (AVT and CRH) by corticosterone binding to and activating a specific {kappa} opioid-like receptor could provide a mechanism by which corticosteroids rapidly inhibit sexual behaviors and locomotor activity. Figure 4Go illustrates a model to summarize this hypothesis.



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  		Figure 4. Model for nongenomic actions of glucocorticoids through a {kappa} opioid-like receptor. Glucocorticoids are shown to signal through a {kappa}-opioid like receptor to inhibit the release of CRH and AVT and to inhibit Ca2+ currents and to effectively inhibit sex behavior and locomotor behavior. All of these have been shown to be activities of {kappa} opioid receptor agonists.

 
In summary, the main conclusion from the current study is that a subset of {kappa}-selective ligands inhibit [3H]CORT binding in neuronal membranes from T. granulosa. These findings, when considered in conjunction with information about the inhibitory effects of corticosteroids and {kappa} agonists on VP and CRH secretion and with the results from our behavioral studies in newts, are consistent with the hypothesis that the [3H]CORT binding site is located on a {kappa} opioid-like receptor. This hypothesis, if confirmed in future studies, would represent significant progress in understanding nongenomic actions of corticosteroids.

Received November 11, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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