Membrane Homogenates Research Articles

Fatty acid composition in matrix vesicles and in microvilli from femurs of chicken embryos revealed selective recruitment of fatty acids

Hypertrophic chondrocytes participate in matrix mineralization by releasing matrix vesicles (MVs). These MVs, by accumulating Ca2+ and phosphate initiate the formation of hydroxyapatite. To determine the types of lipids essential for mineralization, we analyzed fatty acids (FAs) in MVs, microvilli and in membrane fractions of chondrocytes isolated from femurs of chicken embryos. The FA composition in the MVs was almost identical to that in microvilli, indicating that the MVs originated from microvilli. These fractions contained more monounsaturated FAs especially oleic acid than in membrane homogenates of chondrocytes. They were enriched in 5,8,11-eicosatrienoic acid (20:3n−9), in eicosadienoic acid (20:2n−6), and in arachidonic acid (20:4n−6). In contrast, membrane homogenates from chondrocytes were enriched in 20:1n−9, 18:3n−3, 22:5n−3 and 22:5n−6. Due to their relatively high content in MVs and to their selective recruitment within microvilli from where MV originate, we concluded that 20:2n−6 and 20:3n−9 (pooled values), 18:1n−9 and 20:4n−6 are essential for the biogenesis of MVs and for bone mineralization.

Influence of cosurfactant on the behavior of structured emulsions under simulated intestinal lipolysis conditions

Structured emulsion-based delivery systems, fabricated from natural lipids and polymers, are finding increasing use to control the biological fate of ingested lipids within the gastrointestinal tract. The purpose of this study was to elucidate the impact of cosurfactants on the digestion behavior of structured emulsions using a simulated intestinal lipolysis model. Lipid droplets were first prepared by membrane homogenization using a globular protein (β-lactoglobulin) as the primary surfactant, and a non-ionic surfactant (Tween 20) as the cosurfactant. The structured emulsion was fabricated by mixing lipid droplets (+), alginate solution (−), and subsequently chitosan solution (+). With an outer chitosan coating, the electrical charge on the structured emulsion droplets changed from positive to negative when the pH was varied from 2 to 7.5 both in the absence and presence of cosurfactant. However, the particle size was much smaller in the presence of the cosurfactant. The rate and extent of lipid digestion under simulated intestinal lipolysis conditions were influenced by cosurfactant, interfacial structure, and digestion conditions (fasted versus fed). Under high calcium fed conditions (20 mM Ca2+), lipid digestion was highly suppressed and delayed in delivery systems containing cosurfactant and chitosan, which was similar to the system containing alginate but no cosurfactant. This reduction in lipid digestion could be largely overcome by including cosurfactant in the delivery systems. The information obtained in this study may prove useful in designing oral delivery systems that control the digestion and release of lipids in the gastrointestinal tract.

Nanocomposite membranes based on sulfonated polysulfone and sulfated nano-titania/NMPA for proton exchange membrane fuel cells

Abstract Proton conducting nano-composite membranes have been prepared via ternary mixtures comprising sulfated nanotitania (TS), sulfonated polysulfone (SPSU) and nitrilotri(methyl triphosphonic acid) (NMPA). The surface morphology and homogeneity of the composite membranes were searched by scanning electron microscopy (SEM). The existence of ionic interaction between sulfonic acid units in SPSU/TS and phosphonic acid units in NMPA was illustrated in the FT-IR results. The spectroscopic measurements and water uptake studies confirmed the complexation between SPSU/TS and NMPA that inhibited the exclusion of NMPA up on swelling in excess water. The TGA results verified that the membranes are thermally stable up to 270 °C. According to DSC results, the Tg of the materials shifts to lower temperatures with increasing nitrilotri(methyl triphosphonic acid) (NMPA) content. From the methanol permeability experiments it was found that the permeability of the composite membranes is lower than that of commercial Nafion 112. The maximum proton conductivity of SPSU–TS–NMPA is 0.002 S cm− 1 at 150 °C. From the conductivity and spectroscopic results as well as literature data the possible proton conductivity mechanism of the membrane has been modeled.

Characterization of FlipIDAM as a SERT-selective SPECT imaging agent

Biological evaluation of ([(125)I]4), a new single-photon emission computed tomography (SPECT) radioligand for imaging the serotonin transporter (SERT) which displayed improved in vivo kinetics for mapping SERT binding sites in the brain. In vitro binding studies of [(125)I]4 were performed with membrane homogenates of LLC-PK1 cells stably transfected and overexpressing one of the monoamine transporter (SERT, DAT or NET) and rat cortical homogenates. Biodistribution and ex vivo autoradiography studies were carried out in rats. In vivo competition experiments were evaluated to determine the SERT selectivity of [(125)I]4 vs. ([(125)I]1). In vitro binding studies of 4 showed excellent binding affinity (Ki,SERT=0.90 ± 0.05 nM) and excellent selectivity over the other monoamine transporters (100 fold and >4000 fold for NET and DAT respectively). Scatchard analysis of saturation binding of [(125)I]4 to rat cortical homogenates gave a Kd value of 0.5 ± 0.09 nM and a Bmax value of 801.4 ± 58.08 fmol/mg protein. The biodistribution study showed rapid high brain uptake (3.09 ± 0.11% dose/organ at 2 min) and a good target to non-target ratio (hypothalamus to cerebellum) at 30 min (2.62) compared to [(125)I]1 (2.19). Ex vivo autoradiography showed that FlipIDAM localizes in accordance with SERT distribution patterns in the brain. In vivo and ex vivo competition experiments with specific and non-specific SERT compounds also showed that [(125)I]4 binds specifically to SERT rich regions. The biological evaluation of [(125)I]4 demonstrates that [(123)I]4 would be a good candidate for SPECT imaging of SERT.

Modulating lipid droplet intestinal lipolysis by electrostatic complexation with anionic polysaccharides: Influence of cosurfactants

The food, supplement, and pharmaceutical industries are interested in developing delivery systems that can control the biological fate of ingested lipids within the gastrointestinal tract. In this study, a simulated intestinal lipolysis model was used to elucidate the impact of cosurfactants and anionic polysaccharides on the digestion of emulsified fats. Lipid droplets were prepared by membrane homogenization using a globular protein (β-lactoglobulin) as the primary surfactant, and a non-ionic surfactant (Tween 20) as the cosurfactant. Electrostatic complexes were formed by mixing the resulting cationic lipid droplets with anionic alginate molecules. In the absence of cosurfactant, multilayer emulsions were formed consisting of lipid droplets coated by a layer of alginate. In the presence of cosurfactant, microclusters were formed that contained aggregates of alginate-coated lipid droplets linked together. The electrical charge on the complexes remained negative from pH 2 to 7.5, with the complexes formed in the presence of cosurfactant having a lower charge magnitude. The rate and extent of lipid digestion under simulated intestinal lipolysis conditions depended on cosurfactant, alginate, and digestion conditions (fasted versus fed). Under high calcium fed conditions (20 mM Ca2+), lipid digestion was highly suppressed in delivery systems containing alginate but no cosurfactant, which was attributed to the formation of a calcium alginate gel that restricted access of lipase to the lipid droplets. This reduction in lipid digestion could be largely overcome by including cosurfactant in the delivery systems. The information obtained in this study may prove useful for designing oral delivery systems that control the digestion and release of lipids in the gastrointestinal tract.

DIDS and the Janus-faced Na+-K+-activated ATPase. Focus on “DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism”

the maintenance of mammalian cell volume fundamentally rests on net cation extrusion through Na+-K+-activated ATPase, the Na+ pump. The publication by Shahidullah et al. ([11][1]) persuasively identifies the series of events by which a widely used inhibitor of anion transport, DIDS, can reduce Na+

Influence of non-ionic surfactant on electrostatic complexation of protein-coated oil droplets and ionic biopolymers (alginate and chitosan)

The purpose of this study was to examine the influence of a non-ionic cosurfactant (Tween 20) on the formation and properties of electrostatic complexes consisting of charged oil droplets and charged biopolymers. The mean droplet diameters in oil-in-water emulsions prepared using a membrane homogenizer were considerably larger when β-lactoglobulin (BLG) was used alone (≈8 μm), than when it was used in combination with Tween 20 (≈2 μm). The cationic oil droplets formed by membrane homogenization (4.0 μm pore size) were mixed with either alginate (anionic) solution (1% oil: 0–0.5% alginate: pH 3.5) or with alginate (anionic) and then chitosan (cationic) solutions (0.4% oil: 0.1% alginate; 0–0.2% chitosan: pH 4.5). The electrical characteristics, microstructure, and physical stability of the electrostatic complexes formed were determined. Under certain conditions multilayer emulsions consisting of oil droplets coated by alginate or alginate/chitosan layers were formed, whereas under other conditions microclusters consisting of aggregated oil droplets embedded within alginate or alginate/chitosan complexes were formed. The presence of the cosurfactant had a major impact on the electrical charge and dimensions of the electrostatic complexes formed. This study shows that various kinds of electrostatic complexes can be formed from charged oil droplets and charged biopolymers, and that their functional characteristics can be controlled using non-ionic cosurfactants.

In vitro sealing of iatrogenic fetal membrane defects by a collagen plug imbued with fibrinogen and plasma

We aimed to demonstrate local thrombin generation by fetal membranes, as well as its ability to generate fibrin from fibrinogen concentrate. Furthermore, we aimed to investigate the efficacy of collagen plugs, soaked with plasma and fibrinogen, to seal iatrogenic fetal membrane defects. Thrombin generation by homogenized fetal membranes was measured by calibrated automated thrombography. To identify the coagulation caused by an iatrogenic membrane defect, we analyzed fibrin formation by optical densitometry, upon various concentrations of fibrinogen. The ability of a collagen plug soaked with fibrinogen and plasma was tested in an ex vivo model for its ability to seal an iatrogenic fetal membrane defect. Fetal membrane homogenates potently induced thrombin generation in amniotic fluid and diluted plasma. Upon the addition of fibrinogen concentrate, potent fibrin formation was triggered. Measured by densiometry, fibrin formation was optimal at 1250 µg/mL fibrinogen in combination with 4% plasma. A collagen plug soaked with fibrinogen and plasma sealed an iatrogenic membrane defect about 35% better than collagen plugs without these additives (P = 0.037). These in vitro experiments suggest that the addition of fibrinogen and plasma may enhance the sealing efficacy of collagen plugs in closing iatrogenic fetal membrane defects.

Novel proton conductive hybrid membranes based on sulfonated polysulfone and benzotriazole

Abstract

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

Design, synthesis, and preliminary in vitro and in vivo pharmacological evaluation of 4-{4-[2-(4-(2-substitutedquinoxalin-3-yl)piperazin-1-yl)ethyl]phenyl}thiazoles as atypical antipsychotic agents

A series of 4-{4-[2-(4-(2-substitutedquinoxalin-3-yl)piperazin-1-yl)ethyl] phenyl} thiazoles were synthesized in an effort to prepare novel atypical antipsychotic agents. The compounds were designed, synthesized, and characterized by spectral data (IR, 1H NMR, and MS) and the purity was ascertained by microanalysis. The D2 and 5-HT2A affinity of the synthesized compounds was screened in vitro by radioligand displacement assays on membrane homogenates isolated from rat striatum and rat cortex, respectively. Furthermore, all the synthesized final compounds (10a–g; 11a–g; 12a–g) were screened for their in vivo pharmacological activity in Swiss albino mice. D2 antagonism studies were performed using climbing mouse assay model and 5-HT2A antagonism studies were performed using quipazine-induced head twitches in mice. It was observed that none of the new chemical entities exhibited catalepsy and 12d, 11f, and 10a were found to be the most active compounds with 5-HT2A/D2 ratio of 1.23077, 1.14286, and 1.12857, respectively, while the standard drug risperidone exhibited 5-HT2A/D2 ratio of 1.0989. Among the twenty one new chemical entities, three compounds (12d, 11f, and 10a) were found to exhibit better atypical antipsychotic activity as they were found to have higher Meltzer index than the standard drug risperidone.

Epithelium of human fresh amniotic membrane has antimicrobial effects in vitro

This study was carried out to investigate the antimicrobial effects of human fresh amniotic membrane and human amniotic homogenate supernatant (HAHS) in vitro. Fresh human amniotic membranes were harvested and used to make a cellular amniotic membranes (fresh amniotic membranes without the epithelium) and HAHS. Then, the antimicrobial effects of both the fresh human amniotic membrane with the epithelium and fresh amniotic membranes without the epithelium were observed. The antimicrobial effects of fresh HAHS were examined with Kirby-Bauer method. The antimicrobial capacity was compared with ten antibiotics with the Broth Micro-dilution Method, and the impacts of temperatures and pH values and preserved time of HAHS on the antimicrobial capacity were investigated. Finally, transmission electron microscopy (TEM) was used to explore the possible mechanism of the antibiosis of HAHS. Human fresh amniotic membrane with the epithelium had antimicrobial effects, while fresh amniotic membrane without the epithelium did not have any antimicrobial activities. HAHS showed antimicrobial activities for the three standard strains:Staphylococcus aureus ATCC, Escherichia coli, Pseudomonas aeruginosa; four clinically isolated sensitive bacterial strains: Streptococcus pneumonia, Staphylococcus epidermidis,Staphylococcus haemolyticus, and Proteus mirabilis; three clinically isolated fungal strains:Blastomyces albicans, Fusarium solani, Aspergillus fumigatus; and one clinically isolated multi-drug-resistant strain: Enterococcus faecalis. The minimum inhibitory concentration (MIC) of HAHS for Staphylococcus aureus was less than that of Chloramphenicol, equal to that of Clindamycin, greater than that of Penicillin, Rifampicin, and Levofloxacin. Fresh amniotic membrane with epithelium and HAHS had antimicrobial activities, and fresh amniotic membrane without the epithelium did not, indicating that antimicrobial activities are related with the epithelium. HAHS had broad antimicrobial activities and stable characteristics. The mechanism of antimicrobial effect of HAHS was probably interacting with the plasma membrane of the bacteria. Key words: Human amniotic membrane, antimicrobial effects, amniotic homogenate supernatant, minimum inhibitory concentration, minimal bactericidal concentration.

Studying homogeneity and zeta potential of membranes using electroacoustics

Abstract The goal of this paper is to show that electroacoustic spectroscopy can be used as a viable means for characterizing membranes. A novel set-up was designed for performing these experiments with a standard electroacoustic zeta potential probe typically used for measuring ζ-potential in concentrated dispersions. Calculation of zeta (ζ) potential from the measured signal was performed with a newly derived electroacoustic theory that takes into account overlap of double layers in the membrane pores (nano-filtration membrane NF 270 manufactured by Dow Chemicals). Several titrations were performed: two salt titrations with potassium chloride and copper sulfate, and pH titration in several solutions of KCl. In all these experiments, changes in ionic environment caused changes in the ζ-potential within the pores of the membrane, as was measured by changes in electroacoustic current. In all cases the results were consistent with theoretical expectations based on prior research and knowledge of ion effects on ζ-potential. Lastly, differences in electroacoustic signal from one area of the membrane to the next (all of the same material) shed some light into the homogeneity of the membrane itself.

Regulation of the Distribution and Function of [125I]Epibatidine Binding Sites by Chronic Nicotine in Mouse Embryonic Neuronal Cultures

Chronic nicotine produces up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) (* denotes that an additional subunit may be part of the receptor). However, the extent of up-regulation to persistent ligand exposure varies across brain regions. The aim of this work was to study the cellular distribution and function of nAChRs after chronic nicotine treatment in primary cultures of mouse brain neurons. Initially, high-affinity [(125)I]epibatidine binding to cell membrane homogenates from primary neuronal cultures obtained from diencephalon and hippocampus of C57BL/6J mouse embryos (embryonic days 16-18) was measured. An increase in α4β2*-nAChR binding sites was observed in hippocampus, but not in diencephalon, after 24 h of treatment with 1 μM nicotine. However, a nicotine dose-dependent up-regulation of approximately 3.5- and 0.4-fold in hippocampus and diencephalon, respectively, was found after 96 h of nicotine treatment. A significant fraction of total [(125)I]epibatidine binding sites in both hippocampus (45%) and diencephalon (65%) was located on the cell surface. Chronic nicotine (96 h) up-regulated both intracellular and surface binding in both brain regions without changing the proportion of those binding sites compared with control neurons. The increase in surface binding was not accompanied by an increase in nicotine-stimulated Ca(2+) influx, suggesting persistent desensitization or inactivation of receptors at the plasma membrane occurred. Given the differences observed between hippocampus and diencephalon neurons exposed to nicotine, multiple mechanisms may play a role in the regulation of nAChR expression and function.

Manufacture and Application of Water-in-Oil Emulsions to Induce the Aggregation of Sucrose Crystals in Oil: A Model for Melt-resistant Chocolate

The goal of this work was to determine if water-in-oil emulsions produced via a lab-scale cross flow membrane emulsification system are suitable for use in the production of melt resistant chocolate. Water-in-oil (w/o) emulsions (30 wt%) were prepared with either polyglycerol ricinoleate (PGPR, 1–6%) or lecithin (1–6 wt%) using a cross-flow membrane homogenizer. PGPR produced stable emulsions with smaller average diameters compared to lecithin. Lecithin produced larger particles that quickly phase separated. Aliquots (2 g) of a stable (2% PGPR-stabilized) and unstable (2% lecithin-stabilized) w/o emulsion were added to a sugar-in-molten confectionery coating fat (CCF) dispersion (50:50 sucrose:lipid, 120 g total) and cooled to produce a solid sample. After washing out the solid fat with hexane, the samples containing added water were found to have formed a self-supporting sucrose skeleton compared to control prepared without water which collapsed. Microscopy revealed the primary mechanism of water-induced sugar crystal adhesion was via capillary forces. The meltability of the solid samples was measured as change in shape following heating at 50 °C (i.e., above the melting point of the CCF). The degree of spread was less for the samples containing water and was affected by the mode of water addition. This work has relevance to the manufacture of melt resistant chocolate where a sugar skeleton is used to maintain a solid product even after the fat has melted.

Coupling microwave-assisted and classical heating methods for scaling-up MFI zeolite membrane synthesis

Silicalite-1 (S-1) nano-seeds obtained by microwave-assisted (MW) heating have been used to coat industrial supports and to up-scale the synthesis of MFI zeolite membranes by secondary growth applying a classical heating (CH) method. The MW-assisted method was adapted/optimized for fast synthesis and high yield of uniform and non-aggregated S-1 nano-seeds suspensions which were directly used for seeding macroporous industrial α-Al2O3- based supports by dip-coating. The CH secondary growth yielded uniform MFI membranes, by applying optimal reaction conditions while minimizing the reaction time and consumption of chemicals. The protocol coupling MW-assisted and classical heating in zeolite membrane synthesis revealed high reproducibility and has been validated on various industrial ceramic supports: single tubes (Pall-Exekia, Atech, Inocermic, CTI) and also single capillaries and capillary bundles, from Hyflux, providing a higher surface/volume ratio (typically S/V > 100 m2/m3). Membrane homogeneity has been validated by both SEM and single gas permeation measurements with N2 and SF6. The N2/SF6 ideal selectivity was used to predict the ethanol/water separation factor for the prepared MFI membranes.

On alloying and low-temperature stability of thin, supported PdAg membranes

Electroless plating is the technically most facile and most frequently studied method for preparation of PdAg/ceramic composite membranes. Limited high-temperature stability of such membranes requires alloying of sequentially deposited Pd and Ag layers far below their melting points, however. Here it is demonstrated that 600–800 h are needed for forming 2–4 μm thick, homogeneous alloy layers from Pd–Ag bilayers at 823 K under atmospheric H2 pressure. This is also the time scale on which the activation energy for H2 permeation becomes stable so that this characteristic can be employed for non-destructive, in-process monitoring of the alloying progress. High-temperature H2 permeation rates are shown to be less well suited for this purpose because they are not sufficiently sensitive to the homogeneity of PdAg membranes. The activation energies for the well-alloyed membranes indicate that diffusion through the bulk of the PdAg layer limits H2 permeation through these composite membranes. It is further shown that a fully alloyed Pd75Ag25 membrane tolerates temperature cycling under H2 well down to 373 K while H2/N2 exchanges at that temperature trigger a rapid growth of the N2 leak rate of that membrane. The defect formation is attributed to mechanical stress caused by the substantial expansion and shrinking of the alloy lattice during hydriding and dehydriding at low temperatures.

Synthesis and beta 1-, beta 2-adrenergic receptor binding studies of 4-acylamino-substituted phenoxypropanolamine and 5-acylamino-substituted naphthyloxypropanolamine derivatives.

The object of this study was to investigate the beta-adrenergic receptor binding affinity of 4-acylaminophenoxypropanolamine (10-15) and 5-acylaminonaphthyloxypropanolamine (21-24) derivatives, which were prepared from 4-aminophenol (5) and 5-amino-1-naphthol (16), respectively. The in vitro beta 1- and beta 2-adrenergic receptor binding affinities of the newly synthesized compounds were assessed in turkey erythrocyte membrane (beta 1) and lung homogenates of rats (beta 2). The binding affinities were compared with that of propranolol (3) (propranolol hydrochloride, CAS 318-98-9). The compound N-[5-(3-tert-butylamino-2-hydroxy-propoxy)-naphthalen-1-yl]-acetamide (22) has beta-adrenergic receptor affinity comparable with that of propranolol and shows selectivity to beta 1-adrenergic receptors.

Endogenously Expressed Muscarinic Receptors in HEK293 Cells Augment Up-regulation of Stably Expressed α4β2 Nicotinic Receptors

Nicotine-induced up-regulation of neuronal nicotinic receptors (nAChRs) has been known and studied for more than 25 years. Other nAChR ligands can also up-regulate nAChRs, but it is not known if these ligands induce up-regulation by mechanisms similar to that of nicotine. In this study, we compared up-regulation by three different nicotinic agonists and a competitive antagonist of several different nAChR subtypes expressed in HEK293 cells. Nicotine markedly increased α4β2 nAChR binding site density and β2 subunit protein. Carbachol, a known nAChR and muscarinic receptor agonist, up-regulated both α4β2 nAChR binding sites and subunit protein 2-fold more than did nicotine. This increased up-regulation was shown pharmacologically to involve endogenously expressed muscarinic receptors, and stimulation of these muscarinic receptors also correlated with a 2-fold increase in α4 and β2 mRNA. Muscarinic receptor activation in these cells appears to affect CMV promoter activity only minimally (∼1.2 fold), suggesting that the increase in α4 and β2 nAChR mRNA may not be dependent on enhanced transcription. Instead, other mechanisms may contribute to the increase in mRNA and a consequent increase in receptor subunits and binding site density. These studies demonstrate the possibility of augmenting nAChR expression in a cell model through mechanisms and targets other than the nAChR receptor itself.

Sigma receptor binding of tetrabenazine series tracers targeting VMAT2 in rat pancreas

The vesicular monoamine transporter type II (VMAT2) is highly expressed in pancreatic β-cells and thus has been proposed to be a potential target for measuring β-cell mass (BCM) by molecular imaging. Several tracers based on the TBZ backbone, including 9-fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]AV-133), have shown some promising results as potential biomarkers for BCM despite a relatively high background signal in the pancreas. In the present study, we explore the background binding characteristics of [(18)F]AV-133 in rat pancreas. Pancreatic exocrine cells and islet cells were isolated and purified from Sprague-Dawley rats. Membrane homogenates, prepared from both pancreatic exocrine and islet cells as well as from brain striatum regions, were used for in vitro binding studies of [(18)F]AV-133 under a selective masking condition. 1,3-Di-o-tolylguanidine (DTG), displaying high and roughly equal affinity for both sigma-1 and sigma-2 receptors, was chosen at 5 μM concentration for the masking/blocking studies. [(18)F]AV-133 binding to rat striatum homogenates was not significantly altered by the presence of DTG. In contrast, [(18)F]AV-133 showed significant competition with DTG for binding sites in rat pancreatic exocrine homogenates as well as in rat islet cell homogenates. Importantly, in the presence of DTG, [(18)F]AV-133 showed a single high-affinity binding site on islet cell homogenates with a K(d) value of 3.8 nM which is consistent with the affinity reported previously for VMAT2 sites in rat pancreas. [(18)F]AV-133, in addition to a high-affinity VMAT2 binding site, binds with low affinity (but high capacity) to sigma components that are present in the rat pancreas. Identification of the cause of background binding of [(18)F]AV-133 to rat pancreatic tissue may lead to improved methods for quantification.