Differential expression of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension.

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nomega-nitro-L-arginine methyl ester (L-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from L-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-alpha-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

Epidermal Growth Factor Receptor Activity Determines Response of Colorectal Cancer Cells to Gefitinib Alone and in Combination with Chemotherapy.

Purpose: Up to now, there have been no established predictive markers for response to epidermal growth factor receptor (EGFR/HER1/erbB1) inhibitors alone and in combination with chemotherapy in colorectal cancer. To identify markers that predict response to EGFR-based chemotherapy regimens, we analyzed the response of human colorectal cancer cell lines to the EGFR-tyrosine kinase inhibitor, gefitinib (Iressa, AstraZeneca, Wilmington, DE), as a single agent and in combination with oxaliplatin and 5-fluorouracil (5-FU). Experimental Design: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays and analyzed by ANOVA. Apoptosis was measured by flow cytometry, poly(ADP-ribose) polymerase, and caspase 3 cleavage. EGFR protein phosphorylation was detected by Western blotting. Results: Cell lines displaying high constitutive EGFR phosphorylation (a surrogate marker for EGFR activity) were more sensitive to gefitinib. Furthermore, in cell lines exhibiting low constitutive EGFR phosphorylation, an antagonistic interaction between gefitinib and oxaliplatin was observed, whereas in cell lines with high basal EGFR phosphorylation, the interaction was synergistic. In addition, oxaliplatin treatment increased EGFR phosphorylation in those cell lines in which oxaliplatin and gefitinib were synergistic but down-regulated EGFR phosphorylation in those lines in which oxaliplatin and gefitinib were antagonistic. In contrast to oxaliplatin, 5-FU treatment increased EGFR phosphorylation in all cell lines and this correlated with synergistic decreases in cell viability when 5-FU was combined with gefitinib. Conclusions: These results suggest that phospho-EGFR levels determine the sensitivity of colorectal cancer cells to gefitinib alone and that chemotherapy-mediated changes in phospho-EGFR levels determine the nature of interaction between gefitinib and chemotherapy.

Phase II study of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C alpha, in patients with previously treated low-grade non-Hodgkin's lymphoma

Background: The purpose of this study was to assess the efficacy and safety of ISIS 3521, an antisense phosphorothioate oligonucleotide to protein kinase C in patients with relapsed low-grade non-Hodgkin's lymphoma (NHL). Patients and methods: Twenty-six patients received ISIS 3521 (2 mg/kg/day) as a continuous infusion over 21 days of each 28-day cycle. Results: The median age of the patients was 53 years (range 37–77). Histological subtypes were low-grade follicular lymphoma (n=22) and B-cell small lymphocytic lymphoma (n=4). Twenty-one (81%) had stage III/IV disease. The median number of previous lines of chemotherapy was two (range one to six). A total of 87 cycles of ISIS 3521 were administered. Twenty-three patients were assessable for response. Three patients achieved a partial response. No complete responses were observed. Ten patients had stable disease. Grade 3–4 toxicity was as follows: neutropenia (3.8%) and thrombocytopenia (26.9%). Conclusions: ISIS 3521 has demonstrated anti-tumour activity in patients with relapsed low-grade NHL. There may be a potential role for this agent in combination with conventional chemotherapy for advanced low-grade lymphoma, and further trials are warranted.

Limitations in using peptide drugs to characterize calcitonin gene-related peptide receptors

Calcitonin gene-related peptide (CGRP) is an endogenous vasodilator peptide that produces its effects by activation of CGRP(1) and CGRP(2) receptor subtypes, These receptor subtypes are characterized in functional studies using the agonist Cys(Acm)(2,7)-human-alpha-calcitonin gene-related peptide (Cys(ACM)(2,7)-h-alpha-CGRP), which activates CGRP(2) receptors, and the antagonist h-alpha CGRP(8-37) which has a high affinity for CGRP, receptors and a low affinity for CGRP(2) receptors. Our aim was to identify factors that may limit the use of these drugs to characterize CGRP receptor subtypes. We studied CGRP receptors using isolated ring segments of pig coronary and basilar arteries studied in vitro. The affinity of the antagonist h-alpha CGRP(8-37) for inhibiting h-alpha CGRP-induced relaxation of coronary arteries (log(10) of the antagonist equilibrium dissociation constant = -5.33) was determined from Schild plots that had steep slopes. Therefore, we used capsaicin to investigate the role of endogenous CGRP in confounding affinity measurements for h-alpha CGRP(8-37). After capsaicin treatment, the slopes of the Schild plots were not different from one, and a higher affinity of h-CGRP(8-37) in blocking relaxation was obtained (log(10) of the antagonist equilibrium dissociation constant = -6.01). We also investigated the agonist activity of the putative CGRP, receptor selective agonist Cys(Acm)(2,7)-h-alpha-CGRP. We found that maximal relaxation of coronary arteries caused by Cys(Acm)(2,7)-h-alpha CGRP was dependent upon the level of contractile tone induced by KCI. We also determined the K-A for Cys(Acm)(2,7)-h-alpha CGRP and found that the K-A (817 nM) was not significantly different from the EC50 (503 nM) for this drug in causing relaxation, indicating that Cys(Acm)(2,7)-h-alpha CGRP is a partial agonist. Because experimental conditions affect the actions of h-CGRP(8-37) and Cys(Acm)(2,7)-h-alpha CGRP, the conditions must be carefully controlled to reliably identify CGRP receptor subtypes.

Role of conformational constraints of position 7 of the disulphide bridge of h-alpha-CGRP derivatives in their agonist versus antagonist properties

Previous structure-activity studies have shown that the disulphide bridge of calcitonin gene-related peptide (CGRP) is important for the highly potent, CGRP receptor-mediated effects of this peptide. In this study penicillamine (Pen) was substituted for one or both of the cysteinyl residues to determine conformational and topographical properties of the disulphide bridge favourable for binding to CGRP receptors and/or receptor activation. Pen constrains the conformational flexibility of disulphide bridges in other peptides. Binding affinities were measured using a radioligand binding assay with membranes prepared from pig coronary arteries and I-125-h-alpha-CGRP. Functional effects were characterized using a previously reported pig coronary artery relaxation bioassay. The binding affinity of [Pen(2)]h-alpha-CGRP was not significantly different from that of h-alpha-CGRP. All other analogues showed reduced affinity for CGRP receptors. [Pen(2)]h-alpha-CGRP also caused relaxation of coronary arteries. The remaining analogues either caused relaxation with significantly reduced potency or failed to relax the arteries at concentrations up to 1 x 10(-5) M. All analogues that did not relax coronary arteries contained a D-Pen in position 7 and inhibited CGRP-induced relaxation. [D-Pen(2,7)]h-alpha- CGRP was the most potent antagonist with a K-B value of 630 nM. This affinity is similar to that of the classical CGRP receptor antagonist, h-alpha-CGRP(8-37), on these arteries (K-B, 212 nM). These studies show that modifying the topography of the disulphide bridge can cause large and variable effects on ligand binding and activation of CGRP receptors. The contribution of position 7 to the conformation and topography of the disulphide bridge of h-alpha-CGRP is crucial to the future design of agonists of CGRP receptors. Furthermore, position 7 is important for the development of new CGRP receptor antagonists with structures based on the whole sequence of h-alpha-CGRP.

Structure-activity studies on position 14 of human alpha-calcitonin gene-related peptide

A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala(14)]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, [Asn(14)]-h-alpha-CGRP, and [Pro(14)]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala(1)4]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, and [Asn(14)]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly(14) of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro(14)]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro(14)]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.

Influence of atenolol and nifedipine on nitric oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)). RESULTS: In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3. CONCLUSIONS: normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

Immunocolloidal Targeting of the Endocytotic Siglec-7 Receptor Using Peripheral Attachment of Siglec-7 Antibodies to Poly(Lactide-co-Glycolide) Nanoparticles

Purpose: To prepare a nanoparticulate formulation expressing variable peripheral carboxyl density using non-endcapped and endcapped poly(lactide-co-glycolide), conjugated to antibodies recognising the siglec-7 receptor, which is expressed on most acute myeloid leukaemias. The aim is to exploit this receptor as a therapeutic target by constructing an internalising drug-loaded nanoparticle able to
translocate into cytoplasm by siglec receptor-mediated internalisation.

Materials and Methods: Antibodies to the siglec-7 (CD33-like) receptor were conjugated to dye-loaded nanoparticles using carbodiimide chemistry, giving 32.6 mg protein per mg of nanoparticles using 100% of the non-endcapped PLGA. Binding studies using cognate antigen were used to verify preservation of antibody function following conjugation.

Results: Mouse embryonic fibroblasts expressing recombinant siglec-7 receptor and exposed to NileRed-loaded nanoparticles conjugated to antibody accumulated intracellular fluorescence, which was not observed if either antibody or siglec-7 receptor was absent. Confocal microscopy revealed internalised perinuclear cytoplasmic staining, with an Acridine Orange-based analysis showing red staining in localised foci, indicating localisation within acidic endocytic compartments.

Conclusions: Results show antibody-NP constructs are internalised via siglec-7 receptor-mediated internalisation. If loaded with a therapeutic agent, antibody-NP constructs can cross into cytoplasmic
space and delivery drugs intracellularly to cells expressing CD33-like receptors, such as natural killer cells and monocytes.

Transient receptor potential melastatin 8 (TRPM8) channel involvement in the regulation of vascular tone

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue- and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 microM). However, both menthol and another agonist icilin (50 microM) caused relaxation of vessels precontracted with KCl (60 mM) or the alpha-adrenoceptor agonist phenylephrine (2 microM) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of Ca(2+)-independent phospholipase A(2) activation in TRPM8-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 microM). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of l-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 microA). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms.

Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER)(1). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes(2). Controversy exists, however, concerning whether ER has a role outside the nucleus(3), particularly in mediating the cardiovascular protective effects of oestrogen(4). Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85 alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ERa are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85 alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ERa with PI(3)K.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFa and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NF?B was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Vitamin D Receptor Modulates the Neoplastic Phenotype Through Antagonistic Growth Regulatory Signals

Vitamin D receptor (VDR) can modulate functionally antagonistic growth regulatory pathways, involving beta-catenin/E-cadherin on one hand and osteopontin (OPN) on the other. This study investigates effects of VDR ligand treatment on the balance of these discordant signals and on associated cell behavior. Treatment of Rama 37 or SW480 cells by 1 alpha,25-(OH)(2) D-3 or analogs suppressed beta-catenin/Lef-1/Tcf signaling and upregulated E-cadherin, consistent with a cancer-inhibitory action. Conversely, treatment also increased transcription of OPN that may be implicated in tumor progression. Molecular crosstalk was observed between the antagonistic VDR-dependent signals, in that beta-catenin/Lef-1/Tcf molecules modulated VDR activation of OPN. Treatment effects on cell growth were related to a constitutive balance of OPN and E-cadherin expression. No growth effects were observed in Rama 37 cells that have low OPN and high E-cadherin expression. Conversely, treatment of Rama 37 stably transfected subclones that had high OPN and/or low level E-cadherin induced small but significant increases of cell attachment to fibronectin, anchorage-independent growth or invasion. This study shows that relative expression levels of key VDR downstream genes may influence growth regulation by 1 alpha,25-(OH)(2) D-3 or analogs. These findings may be relevant to the cell- or tissue-specificity of vitamin D growth regulation. (C) 2009 Wiley-Liss, Inc.

Evidence for a Direct and Functional Interaction between the Regulators of G Protein Signaling-2 and Phosphorylated C Terminus of Cholecystokinin-2 Receptor

Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active G alpha subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be G(q-)dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.

Discovery of substituted maleimides as liver X receptor agonists and determination of a ligand-bound crystal structure

Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 (4) as an LXR ligand. 4 recruits the steroid receptor coactivator-1 to human LXR alpha and LXRP with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]iodophenyl]2-ethoxy propanoic acid], which is specific for the ? isoform of the peroxisomal proliferator activated receptor (PPAR?), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPAR?1 and to a GST (glutathione S-transferase) fusion protein contg. the ligand binding domain of human PPAR?1 (KD = 70 nM). Using this ligand, the authors characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition expts., rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, resp.). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPAR?1 were comparable with those detd. in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an ?-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPAR?1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPAR? and that the pharmacol. of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.