Expression of uroplakin II and GATA-3 in bladder cancer mimickers: caveats in the use of a limited panel to determine cell of origin in bladder lesions.

Antibodies targeting uroplakin II (UPII) are highly specific for urothelial cells and are frequently used to determine if a primary bladder lesion or a metastatic lesion originates from the urothelium. However, to date, no studies have tested the expression of UPII in histological mimickers of bladder cancer that are nonurothelial in origin. Given the potential risk of misdiagnosis, immunohistochemical markers are often used to better characterize these lesions. In the present study, we analyzed the immunohistochemical expression of UPII in a set of urothelial carcinoma mimickers that included conventional nephrogenic adenoma (n = 8), papillary nephrogenic adenoma (n = 6), endometriosis/endosalpingiosis (n = 5), inflammatory myofibroblastic tumor (n = 4), ectopic prostate tissue (n = 2), and malakoplakia (n = 2). We also examined the expression of GATA-3, another commonly used immunohistochemical marker in bladder cancer diagnosis, in the same lesions. Weak immunoreactivity for UPII was identified in 6 of 27 mimickers (22%), and GATA-3 was expressed in 16 of 27 mimickers (59%). Strong immunoreactivity for UPII appeared to be a specific marker for urothelial cell of origin, although weak staining was seen in a significant proportion of mimickers. GATA-3 immunostaining was present in a greater number and broader spectrum of mimickers; however, only one case of papillary nephrogenic adenoma showed dual positivity for UPII and GATA-3. These findings support the immunohistochemical panel-based approach in the diagnosis of bladder lesions, especially if nonurothelial bladder cancer mimickers are in the differential diagnosis. Additional larger studies would be of value to expand on these findings.

Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.

Application of MS Transport Assays to the Four Human γ-Aminobutyric Acid Transporters.

γ-Aminobutyric acid (GABA) transporters (GATs) are promising drug targets for various diseases associated with imbalances in GABAergic neurotransmission. For the development of new drugs or pharmacological tools addressing GATs, screening techniques to identify new inhibitors and to characterize their potency at each GAT subtype are indispensable. By now, the technique by far dominating is based on radiolabeled GABA. We recently described "MS Transport Assays" for hGAT-1 by employing ((2) H6 )GABA as the substrate. In the present study, we applied this approach to all four human GAT subtypes and determined the KM values for GAT-mediated transport of ((2) H6 )GABA at each subtype. Furthermore, a comprehensive set of GAT inhibitors reflecting the whole range of potency and subtype selectivity known so far was evaluated for their potency. The comparison of pIC50 values obtained in conventional [(3) H]GABA uptake assays with those obtained in MS Transport Assays indicated the reliability of the latter. The MS Transport Assays enable a throughput similar to that of conventional radiometric transport assays performed in a 96-well format but avoid the use of radiolabeled substrates.

A quantitative analysis of cellular and synaptic localization of GAT-1 and GAT-3 in rat neocortex.

High-affinity plasma membrane GABA transporters GAT-1 and GAT-3 contribute to the modulation of GABA-mediated inhibition in adult mammalian cerebral cortex. How GATs regulate inhibition in neocortical circuits remains however poorly understood for the lack of information on key localizational features. In this study, we used quantitative pre- and post-embedding electron microscopy to define the distribution of GAT-1 and GAT-3 in elements contributing to synapses and to unveil their ultrastructural organization at adult cortical GABAergic synapses. GAT-1 and GAT-3 were found in both neuronal and astrocytic processes: GAT-1 was prevalently segregated in neuronal elements and in profiles contributing to synapses, whereas GAT-3 was mostly expressed in astrocytes and did not exhibit a preferential distribution in elements contributing to synapses. Analysis of the ultrastructural distribution of GAT-1 and GAT-3 in the plasma membrane of axon terminals and perisynaptic astrocytic processes of symmetric synapses in relation to the active zone revealed that GAT-1 was more concentrated in restricted perisynaptic and extrasynaptic regions, whereas GAT-3 was prominent in extrasynaptic areas. These studies provide a basis for understanding the role GAT-1 and GAT-3 play in the modulation of GABA-mediated phasic and tonic inhibition in cerebral cortex.

Development and validation of an LC-ESI-MS/MS quantification method for a potential γ-aminobutyric acid transporter 3 (GAT3) marker and its application in preliminary MS binding assays.

Binding assays for the γ-aminobutyric acid (GABA) transporter GAT3 can be assumed to significantly facilitate screening for respective inhibitors. As appropriate labeled ligands for this promising drug target are not available so far, we started efforts to set up mass spectrometry-based binding assays (MS binding assays), for which labeled markers are not required. Therefore, we developed a sensitive and rapid LC-ESI-MS/MS quantification method for DDPM-1007 {(RS)-1-[4,4,4-Tris(4-methoxyphenyl)but-2-en-1-yl]piperidine-3-carboxylic acid}, one of the most potent GAT3 inhibitors yet known, as a potential GAT3 marker. Using a 50 × 2 mm C(8) column in combination with a mobile phase composed of 10 mM ammonium bicarbonate buffer pH 8.0 and acetonitrile (60:40, v/v) at a flow rate of 450 µL/min DDPM-1007 could be analyzed in the positive multiple reaction monitoring mode [(m/z) 502.5 → 265.4] within a chromatographic cycle time of 3 min. Deuterated DDPM-1007 [((2)H(9))DDPM-1007] was synthesized and employed as internal standard. This way DDPM-1007 could be quantified in a range from 100 pM to 10 nM in the matrix resulting from respective binding experiments without any sample preparation. The established quantification method met the requirements of the FDA guidance for bioanalytical method validation concerning linearity and intra- and inter-batch accuracy. Based on this LC-ESI-MS/MS quantification preliminary MS binding assays employing membrane preparations obtained from a stably GAT3 expressing HEK293 cell line and DDPM-1007 as nonlabeled GAT3 marker could be performed. In these experiments specific binding of DDPM-1007 at GAT3 could be unambiguously detected. Additionally, the established LC-MS method provides a suitable analytical tool for further pharmacokinetic characterization of DDPM-1007, as exemplified for its logD determination.

Somatostatin varicosities contain the vesicular GABA transporter and contact orexin neurons in the hypothalamus.

Somatostatin (SST) is a neuropeptide with known inhibitory actions in the hypothalamus, where it inhibits release of growth hormone-releasing hormone (GHRH), while also influencing the sleep-wake cycle. Here we investigated in the rat whether SST neurons might additionally release GABA (gamma-aminobutyric acid) or glutamate in different regions and whether they might contact orexin neurons that play an important role in the maintenance of wakefulness. In dual-immunostained sections viewed by epifluorescence microscopy, we examined if SST varicosities were immunopositive for the vesicular transporter for GABA (VGAT) or glutamate (VGLUT2) in the posterolateral hypothalamus and neighboring arcuate nucleus and median eminence. Of the SST varicosities in the posterolateral hypothalamus, 18% were immunopositive for VGAT, whereas ≤ 1% were immunopositive for VGLUT2. In the arcuate and median eminence, 26 and 64% were VGAT+ and < 3% VGLUT2 + , respectively. In triple-immunostained sections viewed by epifluorescence and confocal microscopy, SST varicosities were seen in contact with orexin somata, and of these varicosities, a significant proportion (23%) contained VGAT along with synaptophysin, the presynaptic marker for small synaptic vesicles, and a similar proportion (25%) abutted puncta that were immunostained for gephyrin, the postsynaptic marker for GABAergic synapses. Our results indicate that a significant proportion of SST varicosities in the hypothalamus have the capacity to release GABA, to form inhibitory synapses upon orexin neurons, and accordingly through their peptide and/or amino acid, to inhibit orexin neurons, as well as GHRH neurons. Thus while regulating GHRH release, SST neurons could serve to attenuate arousal and permit progression through the sleep cycle.

Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity.

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.

Expanding the scope of MS binding assays to low-affinity markers as exemplified for mGAT1.

Following a recently developed concept of MS binding assays based on the quantification of a native marker by LC-MS a procedure to study binding of a low-affinity marker in kinetic, saturation, and competition experiments was established. Separation of bound and unbound marker-the most crucial step of the assay-could be effectively achieved by filtration in a 96-well-format. MS binding assays according to this procedure allowed the reliable characterization of NO 711 binding to mGAT1 in presence of physiological NaCl concentrations. Comparing the results obtained in the present study with those from experiments using 1 mol L(-1) NaCl in the incubation milieu reveals remarkable differences with respect to the marker's affinity and kinetics and to the investigated test compound's potency. [figure: see text]

The Caenorhabditis elegans snf-11 gene encodes a sodium-dependent GABA transporter required for clearance of synaptic GABA.

Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na+ and Cl- dependent, has an EC50 value of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.

Voltage-gated ion channels in the axon initial segment of human cortical pyramidal cells and their relationship with chandelier cells.

The axon initial segment (AIS) of pyramidal cells is a critical region for the generation of action potentials and for the control of pyramidal cell activity. Here we show that Na+ and K+ voltage-gated channels, together with other molecules involved in the localization of ion channels, are distributed asymmetrically in the AIS of pyramidal cells situated in the human temporal neocortex. There is a high density of Na+ channels distributed along the length of the AIS together with the associated proteins spectrin betaIV and ankyrin G. In contrast, Kv1.2 channels are associated with the adhesion molecule Caspr2, and they are mostly localized to the distal region of the AIS. In general, the distal region of the AIS is targeted by the GABAergic axon terminals of chandelier cells, whereas the proximal region is innervated, mostly by other types of GABAergic interneurons. We suggest that this molecular segregation and the consequent regional specialization of the GABAergic input to the AIS of pyramidal cells may have important functional implications for the control of pyramidal cell activity.

A homogeneous assay to assess GABA transporter activity.

This unit describes a convenient functional uptake assay for GABA transport into cell lines transiently transfected with GABA transporter-1 (GAT-1) and other GAT isoforms. This facile, homogeneous assay allows for the determination of K(m), V(max), and K(i) values. The assay utilizes commercially available microtiter plates that contain scintillant embedded in the bottom of the wells. Whereas a signal is generated as the cell accumulates the labeled neurotransmitter, label in the medium is undetected. While GABA uptake is observed in several cell lines transfected with GAT-1, K(m) values for GABA uptake may vary with the cell line. This indicates that the choice of cell line is an important consideration when conducting uptake assays.