Phorbol ester receptors and protein kinase C in primary neuronal cultures: development and stimulation of endogenous phosphorylation.

Embryonic rat neurons cultured in defined medium, essentially in the absence of glia, were highly enriched in phorbol ester receptors. The neurons displayed a single class of phorbol 12,13-dibutyrate binding sites with a maximum binding capacity, after 10 d in culture, of 18.6 pmol/mg protein and an apparent dissociation constant of 7.1 nM. Phorbol ester binding sites were associated with protein kinase C, which represented a major protein kinase activity in primary neuronal cultures. Ca2+-phosphatidylserine-sensitive phosphorylation of endogenous substrates was more marked than that observed in the presence of cyclic AMP or Ca2+ and calmodulin. Phorbol ester receptors and protein kinase C levels were critically dependent on the culture age. Thus, about a 20-fold increase in binding sites occurred during the first week in culture and was accompanied by a corresponding increase in Ca2+-phosphatidylserine-sensitive protein phosphorylation in soluble neuronal extracts. These changes largely paralleled a similar rise in phorbol ester binding during fetal development in vivo. The apparent induction of phorbol ester receptors was specific relative to other cellular proteins and could be inhibited by cycloheximide or Actinomycin D. Phosphorylation of endogenous substrates in intact cultured neurons paralleled the age-dependent increase in protein kinase C. Furthermore, 32P incorporation into several major phosphoproteins was markedly augmented by treating the neuronal cultures with phorbol esters. Such phosphorylation events may provide a clue to the significance of protein kinase C in developing neurons.

Bile acids: natural ligands for an orphan nuclear receptor.

Bile acids regulate the transcription of genes that control cholesterol homeostasis through molecular mechanisms that are poorly understood. Physiological concentrations of free and conjugated chenodeoxycholic acid, lithocholic acid, and deoxycholic acid activated the farnesoid X receptor (FXR; NR1H4), an orphan nuclear receptor. As ligands, these bile acids and their conjugates modulated interaction of FXR with a peptide derived from steroid receptor coactivator 1. These results provide evidence for a nuclear bile acid signaling pathway that may regulate cholesterol homeostasis.

A novel N-aryl tyrosine activator of peroxisome proliferator-activated receptor-gamma reverses the diabetic phenotype of the Zucker diabetic fatty rat.

The discovery that peroxisome proliferator-activated receptor (PPAR)-gamma was the molecular target of the thiazolidinedione class of antidiabetic agents suggested a key role for PPAR-gamma in the regulation of carbohydrate and lipid metabolism. Through the use of high-throughput biochemical assays, GW1929, a novel N-aryl tyrosine activator of human PPAR-gamma, was identified. Chronic oral administration of GW1929 or troglitazone to Zucker diabetic fatty (ZDF) rats resulted in dose-dependent decreases in daily glucose, free fatty acid, and triglyceride exposure compared with pretreatment values, as well as significant decreases in glycosylated hemoglobin. Whole body insulin sensitivity, as determined by the euglycemic-hyperinsulinemic clamp technique, was significantly increased in treated animals. Comparison of the magnitude of glucose lowering as a function of serum drug concentrations showed that GW1929 was 2 orders of magnitude more potent than troglitazone in vivo. These data were consistent with the relative in vitro potencies of GW1929 and troglitazone. Isolated perfused pancreas studies performed at the end of the study confirmed that pancreata from vehicle-treated rats showed no increase in insulin secretion in response to a step change in glucose from 3 to 10 mmol/l. In contrast, pancreata from animals treated with GW1929 showed a first- and second-phase insulin secretion pattern. Consistent with the functional data from the perfusion experiments, animals treated with the PPAR-gamma agonist had more normal islet architecture with preserved insulin staining compared with vehicle-treated ZDF rats. This is the first demonstration of in vivo efficacy of a novel nonthiazolidinedione identified as a high-affinity ligand for human PPAR-gamma. The increased potency of GW1929 compared with troglitazone both in vitro and in vivo may translate into improved clinical efficacy when used as monotherapy in type 2 diabetic patients. In addition, the significant improvement in daily meal tolerance may impact cardiovascular risk factor management in these patients.

Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library.

BACKGROUND: The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown. RESULTS: A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS: The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.

A synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), is a ligand for the peroxisome proliferator-activated receptor gamma.

A novel synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), previously reported to have potent differentiating, antiproliferative, and antiinflammatory activities, has been identified as a ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma). CDDO induces adipocytic differentiation in 3T3-L1 cells, although it is not as potent as the full agonist of PPARgamma, rosiglitazone. Binding studies of CDDO to PPARgamma using a scintillation proximity assay give a Ki between 10(-8) to 10(-7) M. In transactivation assays, CDDO is a partial agonist for PPARgamma. The methyl ester of CDDO, CDDO-Me, binds to PPARgamma with similar affinity, but is an antagonist. Like other PPARgamma ligands, CDDO synergizes with a retinoid X receptor (RXR)-specific ligand to induce 3T3-L1 differentiation, while CDDO-Me is an antagonist in this assay. The partial agonism of CDDO and the antagonism of CDDO-Me reflect the differences in their capacity to recruit or displace cofactors of transcriptional regulation; CDDO and rosiglitazone both release the nuclear receptor corepressor, NCoR, from PPARgamma, while CDDO-Me does not. The differences between CDDO and rosiglitazone as either partial or full agonists, respectively, are seen in the weaker ability of CDDO to recruit the coactivator CREB-binding protein, CBP, to PPARgamma. Our results establish the triterpenoid CDDO as a member of a new class of PPARgamma ligands.

Substrate specificity in short-chain phospholipid analogs at the active site of human synovial phospholipase A2.

The substrate specificity at the active site of recombinant human synovial fluid phospholipase A2 (hs-PLA2) was investigated by the preparation of a series of short-chain phospholipid analogs and measurement of their enzymatic hydrolysis at concentrations well below the critical micelle concentration. Substrates used in the study included 1,2-dihexanoylglycerophospholipids, 1,2-bis(alkanoylthio)glycerophospholipids, and 1-O-alkyl-2-(alkanoylthio)phospholipids. Turnover was observed for only a few of the 1,2-dihexanoylglycerophospholipids, and the rate of hydrolysis was very low, near the limit of detection of the assay. In contrast, selected 2-(alkanoylthio)-glycerophospholipids were hydrolyzed by hs-PLA2 at much higher rates at concentrations well below their critical micelle concentration (cmc). Thus, the 1,2-bis(hexanoylthio)glycerophosphatidylmethanol exhibits a k(cat)/K(M) = 1800 L mol-1 s-1. Over the calculated log P (cLogP) range of 3-9, cLogP and log(k(cat)/K(M) were linearly related for compounds with straight-chain sn-1 and sn-2 substituents. At comparable cLogP's, the sn-1 ethers and thioesters were hydrolyzed at comparable rates. A negative charge in the phosphate head group was required for enzyme activity. Unsaturation, aromaticity, and branching in the sn-2 substituent reduce turnover dramatically. The same structural modifications in the sn-1 substituent have less effect on turnover. Certain of these substrates, e.g., 1,2-bis(hexanoylthio)glycerophosphatidylmethanol, may be useful in assaying for active site inhibitors of PLA2. The structure--activity relationships established here for substrates should serve as a reference for the structure--activity relationships of substrate-based inhibitors.

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 2. Structure-activity relationship and optimization of the phenyl alkyl ether moiety.

We previously reported the identification of (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (2) (PPARgamma pKi = 8.94, PPARgamma pEC50 = 9.47) as a potent and selective PPARgamma agonist. We now report the expanded structure-activity relationship around the phenyl alkyl ether moiety by pursuing both a classical medicinal chemistry approach and a solid-phase chemistry approach for analogue synthesis. The solution-phase strategy focused on evaluating the effects of oxazole and phenyl ring replacements of the 2-(5-methyl-2-phenyloxazol-4-yl)ethyl side chain of 2 with several replacements providing potent and selective PPARgamma agonists with improved aqueous solubility. Specifically, replacement of the phenyl ring of the phenyloxazole moiety with a 4-pyridyl group to give 2(S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-pyridin-4-yloxazol+ ++- 4-yl)ethoxy]phenyl¿propionic acid (16) (PPARgamma pKi = 8.85, PPARgamma pEC50 = 8.74) or a 4-methylpiperazine to give 2(S)-((2-benzoylphenyl)amino)-3-(4-¿2-[5-methyl-2-(4-methylpiperazin+ ++- 1-yl)thiazol-4-yl]ethoxy¿phenyl)propionic acid (24) (PPARgamma pKi = 8.66, PPARgamma pEC50 = 8.89) provided two potent and selective PPARgamma agonists with increased solubility in pH 7.4 phosphate buffer and simulated gastric fluid as compared to 2. The second strategy took advantage of the speed and ease of parallel solid-phase analogue synthesis to generate a more diverse set of phenyl alkyl ethers which led to the identification of a number of novel, high-affinity PPARgamma ligands (PPARgamma pKi's 6.98-8.03). The combined structure-activity data derived from the two strategies provide valuable insight on the requirements for PPARgamma binding, functional activity, selectivity, and aqueous solubility.

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 3. Structure-activity relationship and optimization of the N-aryl substituent.

3-¿4-[2-(Benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propionic acid (1) and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propionic acid (2) are peroxisome proliferator-activated receptor gamma (PPARgamma) agonists and have antidiabetic activity in rodent models of type 2 diabetes. As part of an effort to develop the SAR of the N-2-benzoylphenyl moiety of 1 and 2, a series of novel carboxylic acid analogues, 23-66, modified only in the N-2-benzoylphenyl moiety were synthesized from L-tyrosine and evaluated as PPARgamma agonists. In general, only modest changes in the N-2-benzoylphenyl moiety of 1 and 2 are tolerated. More specifically, the best changes involve bioisosteric replacement of one of the two phenyl rings of this moiety. Addition of substituents to this moiety generally produced compounds that are less active in the cell-based functional assays of PPARgamma activity although binding affinity to PPARgamma may be maintained. A particularly promising set of analogues is the anthranilic acid esters 63-66 in which the phenyl ring in the 2-benzoyl group of 1 and 2 has been replaced by an alkoxy group. In particular, (S)-2-(1-carboxy-2-¿4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phen yl¿ ethylamino)benzoic acid methyl ester (63) has a pKi of 8.43 in the binding assay using human PPARgamma ligand binding domain and a pEC50 of 9.21 in the in vitro murine lipogenesis functional assay of PPARgamma activity. Finally, 63 was found to normalize glycemia when dosed at 3 mg/kg bid po in the Zucker diabetic fatty rat model of type 2 diabetes.

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 1. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents.

We have identified a novel series of antidiabetic N-(2-benzoylphenyl)-L-tyrosine derivatives which are potent, selective PPARgamma agonists. Through the use of in vitro PPARgamma binding and functional assays (2S)-3-(4-(benzyloxy)phenyl)-2-((1-methyl-3-oxo-3-phenylpropenyl)+ ++amin o)propionic acid (2) was identified as a structurally novel PPARgamma agonist. Structure-activity relationships identified the 2-aminobenzophenone moiety as a suitable isostere for the chemically labile enaminone moiety in compound 2, affording 2-((2-benzoylphenyl)amino)-3-(4-(benzyloxy)phenyl)propionic acid (9). Replacement of the benzyl group in 9 with substituents known to confer in vivo potency in the thiazolidinedione (TZD) class of antidiabetic agents provided a dramatic increase in the in vitro functional potency and affinity at PPARgamma, affording a series of potent and selective PPARgamma agonists exemplified by (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(methylpyridin-2-ylamino+ ++)ethoxy ]phenyl¿propionic acid (18), 3-¿4-[2-(benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propanoic acid (19), and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (20). Compounds 18 and 20 show potent antihyperglycemic and antihyperlipidemic activity when given orally in two rodent models of type 2 diabetes. In addition, these analogues are readily prepared in chiral nonracemic fashion from L-tyrosine and do not show a propensity to undergo racemization in vitro. The increased potency of these PPARgamma agonists relative to troglitazone may translate into superior clinical efficacy for the treatment of type 2 diabetes.

HIV protease inhibitors block adipogenesis and increase lipolysis in vitro.

AIDS therapies employing HIV protease inhibitors (PIs) are associated with changes in fat metabolism. However, the cellular mechanisms affected by PIs are not clear. Thus, the affects of PIs on adipocyte differentiation were examined in vitro using C3H10T1/2 stem cells. In these cells the PIs, nelfinavir, saquinavir, and ritonavir, reduced triglyceride accumulation, lipogenesis, and expression of the adipose markers, aP2 and LPL. Histological analysis revealed nelfinavir, saquinavir and ritonavir treatment decreased oil red O-staining of cytoplasmic fat droplets. Inhibition occurred in the presence of the RXR agonist LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Moreover, these three PIs increased acute lipolysis in adipocytes. In contrast, two HIV PIs, amprenavir and indinavir, had little effect on lipolysis, lipogenesis, or expression of aP2 and LPL. Although, saquinavir, inhibited ligand-binding to PPARgamma with an IC(50) of 12.7+/-3.2 microM, none of the other PIs bound to the nuclear receptors RXRalpha or PPARgamma, (IC(50)s>20 microM), suggesting that inhibition of adipogenesis is not due to antagonism of ligand binding to RXRalpha or PPARgamma. Taken together, the results suggest that some, but not all, PIs block adipogenesis and stimulate fat catabolism in vitro and this may contribute to the effects of PIs on metabolism in the clinic.

Studies on the mechanism of enkephalin receptor down regulation.

The association of [3H] [D-Ala2, D-Leu5] enkephalin ([3H]DADLE]) with mouse neuroblastoma cells (N4TG1) was investigated. Under identical conditions the time course, dose response curve and temperature dependence for ligand uptake were similar to those for ligand-induced receptor loss (down regulation). Uptake of [3H]DADLE was inhibited by opiate ligands as well as by the metabolic inhibitors sodium azide and 2,4 dinitrophenol. Comparison of the effects of these inhibitors on receptor binding, ligand uptake and receptor loss indicated that these cells accumulate [3H]DADLE in excess of their surface receptor number. The data suggest that receptor recycling occurs and that ligand is internalized via receptor mediated endocytosis.

125I aqueous waste volume reduction at a pharmaceutical research laboratory.

A wide variety of radioactive waste is generated by pharmaceutical research. One particularly high volume low specific activity waste stream produced by a research and development facility is aqueous 125I waste. This waste is generally held for decay and released to the sanitary sewer when the activity is low enough to ensure that concentrations are well below the appropriate regulatory limits. However, the large volume of this particular waste stream rapidly exhausted available space to store it for decay. This study investigated safe, inexpensive, and efficient methods for removing 125I from the aqueous waste, with the goal of implementing a practical process for concentrating the activity and thus reducing the stored volume. The implemented treatment (volume reduction) process used commercially available low cost activated carbon/particulate filters and inexpensive resin beds manufactured in-house. The much smaller volumes of spent filters and resin beds are then held for decay and eventual incineration. This technique has proven an effective means for managing this high volume waste stream.

Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog.

Procaine amide azide, a derivative of the local anesthetic procaine amide, was prepared and its interactions with acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax were studied. Procaine amide azide was radiolabeled by a method that may be of general use for the preparation of other radioactive tertiary amines. When low concentrations of 3H-labeled procaine amide azide were photolyzed in the presence of receptor-rich membrane preparations, a simple pattern of incorporated radioactivity was seen after electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. Only two major labeled bands were seen, corresponding to apparent Mr of about 43,000 and 90,000. When the length of the gels was increased, the labeled band of lower Mr was resolved into two labeled proteins, one major and one minor, with apparent Mr of 43,000 and 40,000, respectively. The radioactivity incorporated into the protein of Mr 40,000 could be attributed to interaction of [3H]procaine amide azide with cholinergic ligand-binding sites, whereas labeling of the polypeptide of Mr 43,000 appears to represent interaction of the photolabile derivative with another class of sites. The labeling component of 90,000 Mr could be removed by preparation of membrane fragments in iodoacetamide-containing buffer and therefore appeared unrelated to the acetylcholine receptor.

Kinetics of carbamylcholine binding to membrane-bound acetylcholine receptor monitored by fluorescence changes of a covalently bound probe.

The fluorescent probe 5-(iodoacetamido)salicylic acid has been used to alkylate acetylcholine receptor enriched membrane fragments from Torpedo californica following their reduction with low concentrations of dithiothreitol. This modification did not affect the equilibrium binding of carbamylcholine to the receptor. The fluorescence of bound 5-(iodoacetamido)salicyclic acid was enhanced when the labeled membrane fragments were mixed with carbamylcholine. This increase in fluorescence was abolished by preincubation of the membrane fragments with excess alpha-bungarotoxin and was therefore specific for the acetylcholine receptor. Estimates of dissociation constants obtained from centrifugation experiments with radioactive ligand and from fluorescence titration data were in good agreement, showing that the observed fluorescence enhancement was an accurate reflection of receptor-carbamylcholine complex formation. The kinetics of carbamylcholine binding to labeled membrane fragments have been investigated over a wide range of ligand concentrations by using stopped-flow fluorescence techniques. The kinetic signal was complicated, and four distinct exponential phases were observed. A kinetic mechanism has been proposed to account for this behavior.

Effects of local anesthetics and histrionicotoxin on the binding of carbamoylcholine to membrane-bound acetylcholine receptor.

The effects of local anesthetics and perhydrohistrionicotoxin on the kinetic mechanism of carbamoylcholine binding to the membrane-bound acetylcholine receptor have been studied by stopped-flow methods. Receptor-enriched membrane fragments from Torpedo californica were reduced and then alkylated by 5-(iodoacetamido)salicylic acid, and the agonist binding kinetics were monitored by the fluorescence changes of this bound probe. The alkylation procedures did not alter the ability of the receptor to mediate agonist-induced cation flux. Preincubation of such modified receptor preparations with saturating concentrations of lidocaine, prilocaine, or dimethisoquin did not significantly affect the equilibrium dissociation constant for carbamoylcholine binding. The multiphasic kinetic signal which accompanies the binding of the agonist was, however, much simplified in the presence of local anesthetics, and the observed kinetics could be described by a mechanism in which a single conformational change follows the formation of the initial complex. Perhydrohistrionicotoxin did not act in the same way as the local anesthetics examined since saturating concentrations did not significantly perturb the agonist binding kinetics.

Effects of reduction and alkylation on ligand binding and cation transport by Torpedo californica acetylcholine receptor.

The effects of sulfhydryl group modification on ligand binding and functional properties of the membrane-bound acetylcholine receptor from Torpedo californica have been investigated. Agonist binding kinetics were monitored by changes in fluorescence of the probe 5-(iodoacetamido)-salicylic acid which was covalently bound to the receptor after reduction of a reactive disulfide bond(s) by low concentrations of dithiothreitol. These labeling procedures did not affect either the equilibrium binding constant for [3H]acetylcholine or the number of high-affinity binding sites measured in centrifugation experiments. Further reduction of these labeled receptor preparations by higher concentrations of dithiothreitol and subsequent alkylation by excess iodoacetamide resulted in a more than 10-fold decrease in the affinity of the receptor for [3H]acetylcholine. This reduction and alkylation did not, however, radically alter the observed kinetics of acetylcholine binding. The fluorescence signal change on binding consisted of at least three phases similar to those observed for the control preparations, and the ligand concentration dependencies of the measured rate constants could be described by the same kinetic mechanism involving sequential binding of two ligand molecules and three conformational changes. Variation in the values of some of the kinetic parameters describing the formation of the monoliganded complex adequately accounted for the measured decrease in affinity for [3H]acetylcholine. Stopped-flow fluorescence experiments showed that extensive reduction and alkylation resulted in an apparent loss of the ability of the acetylcholine receptor to mediate agonist-induced cation flux. These results show that reduction of disulfide bonds by high concentrations of dithiothreitol followed by alkylation with iodoacetamide seriously perturbs receptor function although the receptor can still undergo its characteristic conformational changes on the binding of acetylcholine but with altered concentration dependence accounting for the reduced affinity for agonist.

A fluorescence-based assay for human type II phospholipase A2.

A fluorescence assay for quantitation of human Type II Phospholipase A2 activity is described. Hydrolysis of 1-Acyl-2-(N-4-nitrobenzo-2-oxo-1,3-diazole)aminododecanoyl Phosphatidylethanolamine is accompanied by an increase in fluorescence intensity that is linearly proportional to enzyme activity. Substrate is prepared in the absence of detergents as a sonicated dispersion in aqueous buffer. Hydrolysis of the corresponding phosphatidylcholine derivative is more than an order of magnitude slower under identical assay conditions. A plot of initial rate versus substrate concentration could be fit to a simple Michaelis-Menten relationship with Km = 13 microM. In contrast to commonly used radiochemical assays for this enzyme, the method described here is continuous and allows estimation of enzyme activity without separation of substrate from product. Thus, the method is suitable for both kinetic analysis and large-scale screening using automated readers for 96-well tissue culture plates. The fluorescence-based assay displays advantages over other continuous assays for human Type II Phospholipase A2 based on (a) high sensitivity and (b) the use of a commercially available substrate.

Benzomorphan sites are ligand recognition sites of putative epsilon-receptors.

The binding characteristics of benzomorphan sites of rat brain membranes are compared with those of kappa-sites of human placenta and guinea pig brain membranes. Enkephalins and the stable analog [D-Ala2,D-Leu5]enkephalin, which are virtually inactive at kappa-sites, possess moderate binding affinity at benzomorphan sites. In contrast, a dynorphin analog, PL017-dynorphin A(6-17), binds well to kappa-sites but poorly to benzomorphan sites. Among all opioid peptides tested, beta h-endorphin, which is essentially inactive at the kappa-receptor sites, is the most potent ligand at benzomorphan sites. The potencies of beta h-endorphin and its fragments at epsilon-receptors of the rat vas deferens correlate well with their binding affinities of benzomorphan sites but not of mu- and delta-sites. These data, as well as the data which show the distinct distribution of benzomorphan sites in rat brain as compared with the distribution of mu- and delta-sites of rat brain and of kappa-sites of guinea pig brain, suggest that benzomorphan sites of rat brain are the ligand-binding sites of epsilon-receptors.