Cloning, expression, and functional analysis of rhesus monkey trace amine-associated receptor 6: evidence for lack of monoaminergic association.

Several recent studies report an association between trace amine-associated receptor 6 (TAAR6) and susceptibility to schizophrenia and bipolar affective disorder in humans. However, endogenous TAAR6 agonists and the receptor signaling profile and brain distribution remain unclear. Here, we clone TAAR6 from the rhesus monkey and use transfected cells to investigate whether this receptor interacts with brain monoamines and a psychostimulant drug to trigger cAMP signaling or extracellular signal-regulated kinase (ERK) phosphorylation, while investigating its expression profile in the rhesus monkey brain. Unlike TAAR1, rhesus monkey TAAR6 did not alter cAMP levels in response to 10 microM of monoamines (dopamine, norepinephrine, serotonin, beta-phenylethylamine (beta-PEA), octopamine, tryptamine, and tyramine) or methamphetamine in stably transfected cells in vitro. Real-time cell electronic sensing analysis indicated that the receptor did not alter cell impedance or change the effect of forskolin on cell impedance at exposure to 20 microM of each monoamine, suggesting a lack of either Gs or Gi-linked signaling. Whereas kappa opioid receptor activation led to ERK phosphorylation at exposure to 1 microM U69593, rhesus monkey TAAR6 had no such effect at exposure to 10 microM of monoamines or methamphetamine. Membrane and cell surface localization of TAAR6 was confirmed by immunocytochemistry, biotinylation, and Western blot testing with a TAAR6 antibody in the transfected cells. Real-time reverse transcriptase-polymerase chain reaction amplification showed that TAAR6 mRNA was undetectable in selected rhesus monkey brain regions. Together, the data reveal that TAAR6 is unresponsive to brain monoamines and is not expressed in rhesus monkey brain monoaminergic nuclei, suggesting TAAR6 lacks direct association with brain monoaminergic neuronal function.

The application of cell-based label-free technology in drug discovery.

Cell-based assays are an important part of the drug discovery process allowing for interrogation of targets and pathways in a more physiological setting compared to biochemical assays. One of the main hurdles in the cell-based assay field is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the pathway or target being investigated. Conventional label and reporter-based cell assays may be more prone to artifacts due to considerable manipulation of the cell either by the label or over-expression of targets or reporter proteins. Cell-based label-free technologies preclude the need for cellular labeling or over-expression of reporter proteins, utilizing the inherent morphological and adhesive characteristics of the cell as a physiologically relevant and quantitative readout for various cellular assays. Furthermore, these technologies utilize non-invasive measurements allowing for time resolution and kinetics in the assay. In this article, we have reviewed the various label-free technologies that are being used in drug discovery settings and have focused our discussion on impedance-based label-free technologies and its main applications in drug discovery.

Live cell quality control and utility of real-time cell electronic sensing for assay development.

In this paper we have explored the utility of the real-time cell electronic sensing (RTCES, ACEA Biosciences Inc., San Diego, CA) system for monitoring the quality of live cells in cell-based assays as well as for assay development. We have demonstrated that each cell type displays unique growth kinetic profiles that provide a quantitative account of cell behavior and can be used as a diagnostic tool for cellular quality control. The utility of the specific signature patterns was shown by demonstrating the significant differences in primary cell behavior depending on the supplier. In addition, the RT-CES system was able to differentiate cell behavior depending on the passage stage of the cells. The utility of the RT-CES system as an assay development tool was demonstrated in cytotoxicity assays. The RT-CES system not only provides information regarding the potency of cytotoxic compounds, but in addition relates potency to the rate of the response for each concentration of the compound tested, which is important for understanding the mechanism of compound action. Moreover, real-time display of cytotoxicity data by the RT-CES system allows for calculation of real-time 50% inhibitory concentration (IC50) values or determination of optimal IC(50) value. In summary, the RT-CES system provides high content and information-rich data that are beyond the scope of single-point assays.

Dynamic and label-free cell-based assays using the real-time cell electronic sensing system.

Cell-based assays have become an integral part of the preclinical drug development process. Recently, noninvasive label-free cell-based assay technologies have taken center stage, offering important and distinct advantages over and in addition to traditional label-based endpoint assays. Dynamic monitoring of live cells, the preclusion of label, and kinetics are some of the fundamental features of cell-based label-free technologies. In this article we will discuss the real-time cell electronic sensing (RT-CES, ACEA Biosciences Inc., San Diego, CA) system and some of its key applications for cell-based assays such as cell proliferation and cytotoxicity, functional assays for receptor-ligand analysis, cell adhesion and spreading assays, dynamic monitoring of endothelial barrier function, and dynamic monitoring of cell migration and invasion. Also, where appropriate we will briefly discuss other label-free technologies in an application-specific manner.

Label-free and real-time cell-based kinase assay for screening selective and potent receptor tyrosine kinase inhibitors using microelectronic sensor array.

Kinases are the 2nd largest group of therapeutic targets in the human genome. In this article, a label-free and real-time cell-based receptor tyrosine kinase (RTK) assay that addresses limitation of existing kinase assays and can be used for high-throughput screening and lead optimization studies was validated and characterized. Using impedance, growth factor-induced morphological changes were quantitatively assessed in real time and used as a measure of RTK activity. COS7 cells treated with epidermal growth factor (EGF) and insulin results in a rapid increase in cell impedance. Assessment of these growth factor-induced morphological changes and levels of receptor autophosphorylation using fluorescent microscopy and enzyme-linked immunosorbent assay, respectively, demonstrates that these changes correlate with changes in impedance. This assay was used to screen, identify, and characterize a potent EGF receptor inhibitor from a compound library. This report describes an assay that is simple in that it does not require intensive optimization or special reagents such as peptides, antibodies, or probes. More important, because the assay is cell based, the studies are done in a physiologically relevant environment, allowing for concurrent assessment of a compound's solubility, stability, membrane permeability, cytotoxicity, and off-target interaction effects.

Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute important targets for drug discovery against a wide range of ailments including cancer, inflammatory, and cardiovascular diseases. Efforts are underway to screen selective modulators of GPCRs and also to deorphanize GPCRs with unidentified natural ligands. Most GPCR-based cellular screens depend on labeling or recombinant expression of receptor or reporter proteins, which may not capture the true physiology or pharmacology of the GPCRs. In this paper, we describe a noninvasive and label-free assay for GPCRs that can be used with both engineered and nonengineered cell lines. The assay is based on using cell-electrode impedance to measure minute changes in cellular morphology as a result of ligand-dependent GPCR activation. We have used this technology to assay the functional activation of GPCRs coupled to different signaling pathways and have compared it to standard assays. We have used pharmacological modulators of GPCR signaling pathways to demonstrate the specificity of impedance-based measurements. Our data indicate that cell-electrode impedance measurements offer a convenient, sensitive, and quantitative method for assessing GPCR function. Moreover, the noninvasive nature of the readout offers the added advantage of performing multiple treatments in the same well to study events such as desensitization and receptor cross-talk.

Sphingosine 1-phosphate receptor agonists attenuate relapsing-remitting experimental autoimmune encephalitis in SJL mice.

FTY720 is a prodrug for FTY-phosphate, an agonist at four of the five known receptors for sphingosine-1-phosphate (S1P). We show that administration of either FTY720 or FTY-P to SJL mice with established relapsing-remitting experimental autoimmune encephalitis (EAE) results in a rapid and sustained improvement in their clinical status, and a reversal of changes in expression of mRNAs encoding some myelin proteins and inflammatory mediators. EAE produced by adoptively transferring lymph node cells from immunized mice to naïve hosts is similarly ameliorated by FTY-P. Treatment with FTY-P is accompanied by a dose-responsive peripheral lymphopoenia.

Growth factor pre-treatment differentially regulates phosphoinositide turnover downstream of lysophospholipid receptor and metabotropic glutamate receptors in cultured rat cerebrocortical astrocytes.

Reactive gliosis is an aspect of neural plasticity and growth factor (GF) stimulation of astrocytes in vitro is widely regarded as a model system to study astrocyte plasticity. Astrocytes express receptors for several ligands including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), agonists for the G-protein-coupled lysophospholipid receptors (lpRs). Activation of lpRs by LPA or S1P leads to multiple pharmacological effects including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK), release of arachidonic acid, and induces mitogenesis. Treatment of astrocytes in vitro with a growth factor cocktail (containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF] and insulin) led to a marked attenuation of lpR-induced PI hydrolysis. In contrast, under identical conditions, GF treatment led to marked potentiation of PI hydrolysis downstream of activation of another abundantly expressed G-protein coupled receptor, mGluR5. Quantitative gene expression analysis of GF-treated or control astrocytes by TaqMan RT-PCR indicated that GF treatment did not change gene expression of lpa1 and s1p1, but increased gene expression of s1p5 which is expressed at very low levels in basal conditions. These results suggest that GF differentially affected PLC activation downstream of mGluR5 versus lpR activation and that the changes in mRNA levels of lpRs do not account for marked attenuation of agonist-induced phosphoinositide turnover.

Characterization of lysophosphatidic acid and sphingosine-1-phosphate-mediated signal transduction in rat cortical oligodendrocytes.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been proposed to play a key role in oligodendrocyte maturation and myelinogenesis. In this study, we examined lysophospholipid receptor gene expression in differentiated rat oligodendrocyte cultures and signaling downstream of lysophospholipid receptor activation by LPA and S1P. Differentiated oligodendrocytes express mRNAs encoding lysophospholipid receptors with the relative abundance of lpa1>s1p5>s1p1=s1p2=lpa3>s1p3. LPA and S1P transiently increased phosphorylation of extracellular signal-regulated kinase (ERK) with EC50 values of 956 and 168 nM, respectively. LPA- and S1P-induced ERK phosphorylation was dependent on the activation of mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and protein kinase C (PKC), but was insensitive to pertussis toxin (PTX). LPA increased intracellular calcium levels in oligodendrocytes and these increases were partially blocked by a PLC inhibitor but not by PTX. In contrast, S1P was not found to induce measurable changes of intracellular calcium. These results taken together suggest that lysophospholipid receptor activation involves receptor coupling to heterotrimeric Gq subunits with consequent activation of PLC, PKC, and MAPK pathways leading to ERK phosphorylation.

Microglial activation state and lysophospholipid acid receptor expression.

We used a simple commercial magnetic immunobead method for the preparation of acutely isolated microglial cells from postnatal days 1-3 rat brain. With the exception of a 15 min enzyme incubation, all stages are carried out at 4 degrees C, minimizing the opportunity for changes in gene expression during the isolation to be reflected in changes in accumulated mRNA. The composition of the isolated cells was compared with that of microglial cultures prepared by conventional tissue culture methods, and the purity of microglia was comparable between the two preparations. RT-PCR analysis of several genes related to inflammatory products indicated that the acutely prepared cells were in a less activated condition than the conventionally tissue cultured cells. We examined the pattern of expression of receptors for lysophosphatidic acid (lpa) and sphingosine-1-phosphate (S1P) using quantitative real-time PCR (TaqMan PCR) techniques. mRNA for LPA1, S1P1, S1P2, S1P3 and S1P5 was detected in these preparations, but the levels of the different receptor mRNAs varied according to the state of activation of the cells. mRNA for LPA3 was only detected significantly in cultured cell after lipopolysaccharide (LPS) stimulation, being almost absent in cultured microglia and undetectable in the acutely isolated preparations. The levels of mRNA of LPA1 and S1P receptors was reduced by overnight exposure to S1P, while the same treatment significantly up-regulated the level of LPA3 mRNA.

Pharmacological characterization of lysophospholipid receptor signal transduction pathways in rat cerebrocortical astrocytes.

Lysophosphatidic acid (1-acyl-2-lyso-sn-glycero-3-phosphate; LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids which respectively act as agonists for the G-protein-coupled lpA receptors (LPA1, LPA2, and LPA3) and s1p receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), collectively referred to as lysophospholipid receptors (lpR). Since astrocytes are responsive to LPA and S1P, we examined mechanisms of lpR signaling in rat cortical secondary astrocytes. Rat cortical astrocyte mRNA expression by quantitative TaqMan polymerase chain reaction (PCR) analysis revealed the following order of relative expression of lpR mRNAs: s1p3>s1p1>lpa1>s1p2=lpa3>>s1p5. Activation of lpRs by LPA or S1P led to multiple pharmacological effects, including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK) and release of [3H]-arachidonic acid (AA). These signalling events downstream of lpR activation were inhibited to varying degrees by pertussis toxin (PTX) pretreatment or by the inhibition of sphingosine kinase (SK), a rate-limiting enzyme in the biosynthesis of S1P from sphingosine. These results suggest that astrocyte lpR signalling mechanisms likely involve both Gi- and Gq-coupled GPCRs and that receptor-mediated activation of SK leads to intracellular generation of S1P, which in turn amplifies the lpR signalling in a paracrine/autocrine manner.

Glutamate-dependent glutamine, aspartate and serine release from rat cortical glial cell cultures.

Glia play a pivotal role in glutaminergic excitatory neurotransmission in the central nervous system by regulating synaptic levels of glutamate and by providing glutamine as the sole precursor for the neurotransmitter pool glutamate to neurons through the glutamate-glutamine cycle. In the present investigation, we examined the influence of glutamate application on glutamine, serine and aspartate release from rat cortical glial cultures. The glial glutamate transporters rapidly cleared exogenously applied glutamate and this was accompanied by rapid increases in aspartate and glutamine, and a delayed increase in serine levels in the glial-conditioned medium. While glutamate-induced increases in glutamine and serine were sustained for up to 24 h, increases in aspartate lasted only for up to 6 h. The glutamate-induced increases in aspartate and glutamine were dependent both on the concentration and the duration of glutamate stimulus, but were largely insensitive to the inhibition of non-N-methyl-D-aspartate receptors or the metabotropic glutamate receptor 5. Inhibition of the glutamate transporter function by L-trans-pyrrolidine 2,4-dicarboxylate decreased the rate of glutamate uptake but not completely abrogated the uptake process, and this resulted in the attenuation of rate of glutamate induced glutamine synthesis. Dexamethasone treatment increased serine and glutamine levels in conditioned medium and increased glutamate induced glutamine release suggesting an upregulation of glutamine synthase activity. These results further substantiate coupling between glutamate and glutamine, and shed light on glutamate-dependent release of serine and aspartate, which may further contribute to excitatory neurotransmission.