Receptor Dissociation Research Articles

Heterodimerization of glycosylated insulin-like growth factor-1 receptors and insulin receptors in cancer cells sensitive to anti-IGF1R antibody.

BackgroundIdentification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer.Methodology/Principal FindingsIn order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity.Conclusion and SignificanceThe data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells.

Systematic Evaluation of the Metabolic to Mitogenic Potency Ratio for B10-Substituted Insulin Analogues

BackgroundInsulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio.Methodology/Principal FindingsA panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed.Conclusions/SignificanceSeveral B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.

Azilsartan Medoxomil for Treating Hypertension—Clinical Implications of Recent Trials

Objective: To evaluate the efficacy, safety, and clinical role of azilsartan medoxomil, an angiotensin-II receptor blocker (ARB) that recently gained US Food and Drug Administration approval for lowering of blood pressure (BP) in patients with hypertension. Methods: A systematic review of the literature was performed through October 2011 using the keywords and medical subject headings azilsartan, azilsartan medoxomil, TAK-491, TAK-536, and Edarbi. Citations eligible for inclusion were in vitro or in vivo evaluations of azilsartan medoxomil with no restrictions on patient population or indication used. Data related to the patient populations and outcomes of interest were extracted from each citation. Results: Three trials are available in full publication form, with others available only as abstracts. Azilsartan medoxomil 40 mg and 80 mg daily significantly improves both systolic and diastolic BP from baseline compared with placebo, and the 80 mg dose has greater efficacy than other ARBs, including olmesartan 40 mg daily and valsartan 320 mg daily. Improvements in both 24-hour BP using ambulatory monitoring and clinic BPs, as well as a higher proportion of patients reaching goal, have been seen with azilsartan medoxomil. Additional information shows added BP lowering when azilsartan medoxomil is combined with chlorthalidone. Adverse events are similar with azilsartan medoxomil compared to other ARBs and include headache, dizziness, urinary tract infections, and fatigue. Conclusions: Azilsartan medoxomil is a safe and effective ARB with a unique pharmacologic profile compared with other agents, including slowed angiotensin-II type 1 (AT1) receptor dissociation rates and improved receptor specificity. Studies have shown azilsartan medoxomil 80 mg once daily to reduce BP to a greater extent than valsartan and olmesartan, with similar safety and tolerability.

Dissociation of the insulin receptor from caveolae during TNFα-induced insulin resistance and its recovery by d-PDMP

Previously, we demonstrated that an inhibitor of ganglioside biosynthesis, d-PDMP, could restore impaired insulin signaling in tumor necrosis factor α (TNFα)-treated adipocytes by blocking the increase of GM3 ganglioside. Here, we analyzed the interaction between insulin receptor (IR) and GM3 in the plasma membranes using immunoelectron microscopy. In normal adipocytes, most GM3 molecules localized at planar and non-caveolar regions. Approximately 19% of IR molecules were detected in caveolar regions. The relative ratio of IRs associated with caveolae in TNFα-treated adipocytes was decreased to one-fifth of that in normal adipocytes, but this decrease was restored by d-PDMP. Thus, we could obtain direct evidence that insulin resistance is a membrane microdomain disorder caused by aberrant expression of ganglioside.

Defective calcium signaling and disrupted CD20–B-cell receptor dissociation in patients with common variable immunodeficiency disorders

B cells of patients with common variable immunodeficiency (CVID) disorders display impairment in production of immunoglobulin class-switched antibodies, which is possibly contributed to by defects in early B-cell activation. On resting B cells, B-cell receptors (BCRs) are organized in oligomers that are signaling inactive. Their triggering by cognate antigen causes the lateral reorganization of BCRs and associated proteins into signalosomes, resulting in BCR-activated calcium entry. In resting cells the B-cell surface antigen CD20 is associated with the BCR but dissociates on signalosome formation. We sought to determine whether CD20 dissociation from the BCR during early B-cell activation might contribute to the development of CVID disorders. We evaluated BCR signalosome formation, internalization, and signaling in primary B cells of pediatric patients with CVID disorders and healthy control subjects. In many pediatric patients with CVID disorders, Bcells exhibit significant deficits in BCR triggering-mediated calcium entry in the cytosol, which correlates with impaired plasmablast differentiation invitro. These alterations did not originate from upregulation of CD22 or defects in calcium channels and did not involve gene mutations in phospholipase Cγ2 or Bruton tyrosine kinase. Instead, B cells from patients with CVID disorders exhibited reduced BCR dissociation from CD20. BCR or CD20 cross-linking induced less BCR internalization, and antibody-mediated CD20 triggering elicited less BCR downstream signaling, as measured based on secondary fluxes. We propose that CD20 dissociation from the BCR signalosome is pivotal to BCR-mediated calcium mobilization in the cytosol. Defects in CD20/BCR signalosome conformation might predispose to the spectrum of CVID disorders.

Azilsartan Medoxomil: A New Angiotensin II Receptor Antagonist for Treatment of Hypertension

To evaluate the efficacy, safety, and clinical role of azilsartan medoxomil, an angiotensin II receptor blocker (ARB) that recently gained Food and Drug Administration approval for lowering of blood pressure (BP) in patients with hypertension. A systematic review of the literature was performed through August 2011 using MEDLINE, Web of Science, and International Pharmaceutical Abstracts and the key words and MeSH terms azilsartan, azilsartan medoxomil, TAK-491, TAK-536, and Edarbi. Abstracts presented in the last 2 years from the annual meetings of appropriate medical societies were reviewed in addition to a search of clinicaltrials.gov. Studies eligible for inclusion were in vitro or in vivo evaluations of azilsartan medoxomil, with no restrictions on patient population or indication. Data related to the patient populations and outcomes of interest were extracted from each publication. Three trials are available in full publication form with others available only as abstracts. Azilsartan medoxomil 40 mg and 80 mg daily significantly improves both systolic and diastolic BP from baseline compared with placebo, and the 80-mg dose has greater efficacy than other ARBs, including olmesartan 40 mg daily and valsartan 320 mg daily. Improvements in both 24-hour BP using ambulatory monitoring and clinic monitoring have been seen with azilsartan medoxomil as well as a higher proportion of patients reaching the goal level. Additional information shows added BP lowering when azilsartan medoxomil is combined with chlorthalidone. Adverse events are similar with azilsartan medoxomil versus other ARBs and include headache, dizziness, urinary tract infections, and fatigue. Azilsartan medoxomil is a safe and effective ARB with a unique pharmacologic profile versus other agents, including slowed angiotensin II type 1 receptor dissociation rates and improved receptor specificity. Studies have shown azilsartan medoxomil 80 mg once daily to reduce BP to a greater extent than valsartan and olmesartan, with similar safety and tolerability.

Melatonin inhibits glucocorticoid-dependent GR–TIF2 interaction in newborn hamster kidney (BHK) cells

The antagonism exerted by melatonin on the glucocorticoid response has been well established, being strongly dependent on the cellular context. Previously, we found that melatonin inhibits glucocorticoid receptor (GR) dissociation from the chaperone hetero-complex and nuclear translocation on mouse thymocytes. Here, by performing confocal fluorescence microscopy and the Number and Brightness assay we show that in newborn hamster kidney cells (BHK21) melatonin neither affects GR nuclear translocation nor GR homodimerization. Instead, co-immunoprecipitation studies suggest that physiological concentrations of melatonin impair GR interaction with the transcriptional intermediary factor 2 (TIF2). This melatonin effect was not blocked by the MT1/MT2 receptor antagonist luzindole. Curiously, luzindole behaved as an antiglucocorticoid per se by impairing the glucocorticoid-dependent MMTV-driven gene expression affecting neither GR translocation nor GR–TIF2 interaction.

Glucoregulatory effects and prolonged duration of action of davalintide: a novel amylinomimetic peptide

Davalintide is a second-generation amylinomimetic peptide possessing enhanced pharmacological properties over rat amylin to reduce food intake in preclinical models. The current experiments in rats describe additional glucoregulatory actions of davalintide consistent with amylin agonism, and explore the duration of action of these effects. Subcutaneous (SC) injection of davalintide slowed gastric emptying with equal potency to amylin (ED₅₀'s = 2.3 and 4.1 µg/kg). This effect was maintained for 8 h with davalintide, but not amylin. Intraperitoneal injection of davalintide also reduced food intake with a potency similar to amylin (ED₅₀'s = 5.0 and 11.3 µg/kg). Consistent with amylin agonism, davalintide (10 µg/kg, SC) suppressed the plasma glucagon response over 90 min following an intravenous arginine bolus in anaesthetized rats. The elimination t(½) of davalintide (200 µg/kg, SC) was 26 min, similar to the t(½) of amylin, suggesting that pharmacokinetic-independent mechanisms contribute to davalintide's enhanced duration of action. Binding kinetic studies using ¹²⁵I davalintide revealed no appreciable dissociation from the amylin nucleus accumbens receptor after 7 h while ¹²⁵I rat amylin did dissociate from this receptor (K(off) = 0.013/min). Sustained SC infusion of davalintide (275 µg/kg/day) or amylin (300) decreased plasma glucose after an oral glucose challenge at 2 weeks (by 27 and 31%) and suppressed gastric emptying at 3 weeks (by 29 and 47%), demonstrating durable glucoregulatory actions of both peptides. These data show glucoregulatory properties of davalintide consistent with amylin agonism and suggest that slowed receptor dissociation plays a role in davalintide's prolonged pharmacodynamic actions.

Differential and Synergistic Effects of Epidermal Growth Factor Receptor Antibodies on Unliganded ErbB Dimers and Oligomers

Antibodies directed against the epidermal growth factor receptor (EGFR) offer a potentially powerful therapeutic approach against cancers driven by the EGFR pathway. EGFR antibodies are believed to halt cell surface activation by blocking ligand-induced receptor tyrosine kinase activation, i.e., ligand binding, a change in conformation, or the monomer-dimer transition. In this work, we demonstrate that wild-type EGFR and the truncated de2-7-EGFR (tumor-associated mutant) formed unliganded homo-oligomers and examined the effects of two clinically relevant antibodies on the conformation and quaternary state of these ligand-free EGFR oligomers on the surface of cells. The EGFR antibodies were mAb528, a ligand-blocking antibody that binds domain III, and mAb806, a conformationally sensitive antibody that binds near the dimer interface in domain II. We used a model cellular system, BaF/3 cells, with GFP-tagged receptors in the absence of interference from secreted ligands or other erbB receptor members. Different antibody-mediated effects (conformational transition, receptor cross-linking, or receptor dissociation) were distinguished by combining two complementary biophysical techniques: image correlation spectroscopy (submicrometer scale clustering) and homo-Forster resonance energy transfer (association and/or conformation on a 1-10 nm scale). mAb528 cross-linked EGFR into an inactive EGFR dimer of dimers but had no effect when added to de2-7-EGFR oligomers. mAb806 had a minor effect on EGFR dimers as expected from its poor binding to a conformationally shielded epitope on wtEGFR but bound de2-7-EGFR oligomers, causing a conformational change in the intracellular C-terminal GFP-tagged tail. The combination of the two antibodies had synergistic effects, increasing the level of cross-linking of de2-7-EGFR, but did not lead to enhanced cross-linking of EGFR. The results reveal new modes of receptor-antibody interactions for EGFR and de2-7-EGFR.

Assessment of receptor occupancy-over-time of two dopamine transporter inhibitors by [11C]CIT and target controlled infusion

Introduction. Occupancy-over-time was determined for two dopamine transporter (DAT) inhibitors through modeling of their ability to displace the PET ligand [11C]CIT. The tracer was held at a pseudo steady state in a reference tissue by target controlled infusion.Methods. Rhesus monkeys (n = 5) were given [11C]CIT and studied with a PET scanner. Tracer uptake in the reference tissue cerebellum was held at a pseudo steady state by use of target controlled infusion. The pharmacokinetics/pharmacodynamics(PK/PD) of [11C]CIT was assessed through the simplified reference tissue model (SRTM). Bupropion (n = 2) and GBR-12909 (n = 2) receptor occupancies were estimated through modeling of their effects on [11C]CIT displacement.Results. There was a high uptake of [11C]CIT in striatum, which contains a high DAT density. The reference tissue cerebellum had a comparatively low uptake. The modeling of [11C]CIT PK/PD properties in striatum showed high binding potential (BP = 5.34 ± 0.78). Both DAT inhibitors caused immediate displacement of [11C]CIT after administration. The occupancy-over-time was modeled as a mono-exponential function, describing initial maximal occupancy (Occ0) and rate of ligand–receptor dissociation (koff). GBR-12909 showed irreversible binding (koff = 0) after an initial occupancy of 76.1%. Bupropion had a higher initial occupancy (84.5%) followed by a release half-life of 33 minutes (koff = 0.021).Conclusions. The proposed model can be used for assessment of in-vivo occupancy-over-time of DAT ligands by use of target controlled infusion of [11C]CIT. The concept of assessing drug–receptor interactions by studying perturbations of a PET tracer from a pseudo steady state can be transferred to other CNS systems.

Importin β-type nuclear transport receptors have distinct binding affinities for Ran–GTP

Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin β family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran–GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran–GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran–GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of β-receptors and of other Ran-binding proteins was determined. We found that the number of β-receptors altogether about equals the amounts of yeast Ran, but Ran–GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.

Precision of Sensing Cell Length via Concentration Gradients

Unicellular organisms are typically found to have a characteristic cell size. To achieve a homeostatic distribution of cell sizes over many generations requires that cell length is actively sensed and regulated. However, the mechanisms by which cell size is controlled remain poorly understood. Recent experiments in fission yeast have shown that cell length is controlled in part by polar gradients of the protein Pom1 together with localized measurement of concentration at midcell. Dilution as the cell grows leads to a reduction in the midcell protein concentration, which lifts a block on mitosis. Here we analyze the precision of this mechanism for length sensing in the presence of inevitable intrinsic noise in the processes leading to formation and measurement of this gradient. We find that the use of concentration gradients allows for more robust length sensing than a comparable spatially uniform system, and allows for reliable length determination even if the average protein concentration throughout the cell remains constant as the cell grows. Optimal values for the gradient decay length and receptor dissociation constant emerge from maximizing sensitivity while minimizing the impact of density fluctuations.

Glycyrrhizic Acid and 18β-Glycyrrhetinic Acid Inhibit Inflammation via PI3K/Akt/GSK3β Signaling and Glucocorticoid Receptor Activation

Many lung-related diseases, such as asthma and chronic obstructive pulmonary disease, are initiated by airway inflammation, and several studies indicate that glycyrrhizic acid (GA) alleviates inflammatory lung disease. We previously showed that GA and 18beta-glycyrrhetinic acid (18betaGA), found in licorice, can act as neuroprotective agents by promoting downstream PI3K/Akt signaling. In this study, we investigate the effects of GA and 18betaGA on inflammation. We show that both GA and 18betaGA reduce inflammatory cytokine production and its resulting anti-inflammation. GA acts via PI3K/Akt/GSK3beta to reduce cytokine production, while 18betaGA leads to the dissociation of a glucocorticoid receptor (GR)-HSP90 complex to block inflammation. Our data suggest that GA and 18betaGA display anti-inflammatory activities but inhibit inflammation via different mechanisms. We propose that GA and 18betaGA may be valuable biological inhibitors of lung inflammation. Interestingly, these data may explain why licorice is frequently used to treat inflammatory disease and it might be a promising nutraceutical for remedying inflammation.

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [(3)H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([(3)H]SM-N(3)). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [(3)H]SM-N(3). However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Mechanism of LDL binding and release probed by structure-based mutagenesis of the LDL receptor

The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL. Several different molecular mechanisms have been proposed for the binding of LDL to LDL-R at neutral plasma pH and for its release at acidic endosomal pH. The crystal structure of LDL-R at acidic pH shows that the receptor folds back on itself in a closed form, obscuring parts of the ligand binding domain with the epidermal growth factor (EGF)-precursor homology domain. We have used a structure-based site-directed mutagenesis approach to examine 12 residues in the extracellular domain of LDL-R for their effect on LDL binding and release. Our studies show that the interface between the ligand binding domain and the EGF-precursor homology domain seen at acidic pH buries residues mediating both LDL binding and release. Our results are consistent with an alternative model of LDL-R whereby multiple modules of the extracellular domain interact with LDL at neutral pH, concurrently positioning key residues so that at acidic pH the LDL-R:LDL interactions become unfavorable, triggering release. After LDL release, the closed form of LDL-R may target its return to the cell surface.

The dopaminergic stabilizers pridopidine (ACR16) and (−)-OSU6162 display dopamine D 2 receptor antagonism and fast receptor dissociation properties

A new pharmacological class of CNS ligands with the unique ability to stimulate or suppress motor and behavioral symptoms depending on the prevailing dopaminergic tone has been suggested as “dopaminergic stabilizers”. The molecular mode-of-action of dopaminergic stabilizers is not yet fully understood, but they are assumed to act via normalization of dopaminergic signaling, through interactions with the dopamine D 2 receptor. Here we have evaluated the dopaminergic stabilizers pridopidine (ACR16) and (−)-OSU6162, as well as the new compound N-{[(2S)-5-chloro-7-(methylsulfonyl)-2,3-dihydro-1,4-benzodioxin-2-yl]methyl}ethanamine (NS30678) in a series of cellular in vitro dopamine D 2 receptor functional and binding assays. Neither ACR16, (−)-OSU6162, nor NS30678 displayed detectable dopamine D 2 receptor-mediated intrinsic activity, whereas they concentration-dependently antagonized dopamine-induced responses with IC 50 values of 12.9 μM, 5.8 μM, and 7.0 nM, respectively. In contrast to the high-affinity typical antipsychotics haloperidol and raclopride, the dopaminergic stabilizers ACR16 and (−)-OSU6162 both displayed fast dopamine D 2 receptor dissociation properties, a feature that has previously been suggested as a contributing factor to antipsychotic atypicality and attributed mainly to low receptor affinity. However, the finding that NS30678, which is equipotent to haloperidol and raclopride, also displays fast receptor dissociation, suggests that the agonist-like structural motif of the dopaminergic stabilizers tested is a critical dissociation rate determinant. The results demonstrate that dopaminergic stabilizers exhibit fast competitive dopamine D 2 receptor antagonism, possibly allowing for temporally variable and activity-dependent dopamine D 2 receptor occupancy that may partly account for their unique stabilization of dopamine dependent behaviors in vivo.

The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber

The Boyden chamber assay provides a convenient method of assessing cell migration and measuring cell motility coefficients at the population level. Previous models of this assay completely ignore cell sedimentation in the suspension, assuming that all cells have already settled on the filter surface before commencing migration within the filter. However, ignoring cell sedimentation could lead to poor data interpretation because the time required for cells to settle through the suspension is close to the incubation period of only a few hours. This study models the Boyden chamber assay by incorporating the cell settling process to account for the cells remaining in the upper well when other cells migrate in the filter. The simulations in this study elucidate the experiments in the literature that test the haptotactic and chemotactic responses of rabbit chondrocytes to type II collagen. This study determines the cell population random motility, as well as the haptotaxis and chemotaxis coefficients, by fitting the experimental data. Results show that the chemotactic motility coefficient is 100 times greater than the haptotactic coefficient, and the equilibrium collagen–receptor dissociation constant is about 10-fold the haptotactic counterpart. Diffusion causes the soluble collagen gradients in the chemotactic case to decline over time, while the coated collagen gradients in the haptotactic assay are likely to remain fixed. As a result, the chemotactic case exhibits a lower number of migrated cells than the haptotactic assay. This study also demonstrates the influences of the dimensionless parameters that control cell behavior in the Boyden assay, providing a reference for future experiment designs.

Preclinical Evaluation of Long-Acting Muscarinic Antagonists: Comparison of Tiotropium and Investigational Drugs

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation caused by persistent inflammatory processes in the airways. An increased cholinergic tone mediates different pathophysiological features of COPD, such as bronchoconstriction and mucus hypersecretion, mostly through activation of the human muscarinic M(3) receptor (hM(3)) subtype. Tiotropium bromide (Spiriva) is a well established muscarinic antagonist in the pharmacological management of COPD with a once-daily posology. The rationale behind the sustained bronchodilation obtained with tiotropium consists in its slow dissociation from hM(3) receptors. In this study, we performed a comprehensive preclinical comparison of tiotropium with other long-acting muscarinic antagonists (LAMAs) currently in clinical development, namely aclidinium bromide and glycopyrrolate. The different muscarinic antagonists were characterized for their 1) affinity toward the different human muscarinic receptor subtypes expressed in Chinese hamster ovary cells and kinetics of receptor dissociation, 2) potency in inhibiting the agonist-induced activation of muscarinic receptors through measurement of second messengers, and 3) efficacy and duration of bronchoprotection, as tested in a model of acetylcholine-induced bronchoconstriction in anesthetized dogs over a period of 24 h. All of the tested LAMAs showed high affinity and potency toward the hM(3) receptor (tiotropium, pA(2) = 10.4; aclidinium, pA(2) = 9.6; and glycopyrrolate, pA(2) = 9.7). However, dissociation half-lives of the LAMAs from the hM(3) receptor differed significantly (tiotropium, t((1/2)) = 27 h; aclidinium, t((1/2)) = 10.7 h; and glycopyrrolate, t((1/2)) = 6.1 h). In line with their kinetic properties at the hM(3), the tested LAMAs provided different levels of bronchoprotection in the in vivo setting 24 h after administration (tiotropium = 35%, aclidinium = 21%, and glycopyrrolate = 0% at 24 h) when applied at equieffective doses.

Atomic force and fluorescence microscopy imaging of cardiomyocyte plasma membrane, insulin receptor and caveolin‐3

Impaired insulin signaling is a component of diabetic cardiomyopathy. Our hypothesis is that myocardial insulin resistance involves a dissociation of insulin receptors (IR) from caveolae and its intracellular targets. Our aim was to develop techniques to image the spatial relationship between IR and caveolae using atomic force microscopy (AFM) and immunohistochemistry (IHC). We used an embryonic rat cardiac cell line (H9c2) to evaluate various fixation protocols and determined that 4% formaldehyde (rather than glutaldehyde, methanol or combinations), maintained the integrity of the plasma membrane. We tested a variety of AFM tips (standard, tungsten, carbon coated) in both contact and tapping modes on hydrated and dehydrated cells. We now can scan cells in solution, with a resolution of up to 10 nm. To validate our methods and reagents we conducted IHC and immunoblotting. IHC images included fixed and living cells. Future studies will use AFM techniques with gold conjugated antibodies for the IR and caveolin 3, in cells cultured in normal and high extracellular glucose. Ultimately, we intend to evaluate cell surface receptor distributions on adult rat ventricular myocytes. Resolving the localization of IR and caveolae in intact cells and comparing normal and diabetic‐like groups could further our understanding of the pathogenesis of insulin resistance in diabetic cardiomyopathy. APS Undergraduate Summer Research Fellowship (EGM)

Peroxisomal Targeting of PTS2 Pre‐Import Complexes in the Yeast Saccharomyces cerevisiae

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.