Aequorin Luminescence Research Articles

Mammalian type opsin 5 preferentially activates G14 in Gq-type G proteins triggering intracellular calcium response

Mammalian type opsin 5 (Opn5m), a UV-sensitive G protein-coupled receptor opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and Gα-KO cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by cotransfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially activated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-β5 and αG-α4 loops, αG and α4 helices, and the extreme C terminus, in the preferential activation of Gα14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types.

Abstract 1124: The identification and characterization of a novel A3R agonist with potent anti-cancer properties

Abstract Introduction: G protein-coupled receptors (GPCRs) are among the most druggable targets. Adenosine receptors (ARs) belong to the class A GPCRs. The role of adenosine and ARs in tumorigenesis is increasingly appreciated. Moreover, novel studies also point out pro-tumor and anti-tumor effects of modulating ARs. Besides A2AR and A2BR in immune-oncology, A3R was shown to be overexpressed in tumor tissue and play an important role in cancer. Especially A3R agonists decrease proliferation, induce cell cycle arrest and cytotoxicity selectively in cancer cells. However, most compounds fail in clinical trials due to their insufficient efficacy or high occurrence of adverse effects. Here we introduce a novel A3R agonist with plausible anti-cancer properties. Material and methods: First, we developed a screening pipeline of AR agonists and antagonists, where the hits from the primary screen (aequorin luminescence assay) were evaluated in the counter-screen (calcium mobilization assay) and confirmed in concentration-response experiments. In parallel, the identified modulators were subjected to cytotoxicity screening against a panel of tumor and non-tumor cell lines. The selected lead compound with potent anti-cancer properties was biologically and pharmacologically compared to a reference A3R agonist, 2-Cl-IB-MECA, recently evaluated in a clinical trial against hepatocellular carcinoma. To assess the relevance of the in vitro assays, the lead compound was administered to mice and its in vivo efficacy was evaluated. Results: Our screening pipeline shows high reproducibility and robustness. We identified several potential hits among the newly synthesized nucleosides and selected an A3R agonist PNH173 with significant cytotoxic effects against several tumor cell types compared to the reference A3R agonist. PNH173 also demonstrates good pharmacological properties in non-clinical ADME tests, reduces tumor growth, and increases overall survival in vivo experiments. Conclusions: We set out to identify novel small molecule A3R agonists with strong anti-tumor effects using a screening campaign and subsequent series of biological assays. Our data indicate desired anti-cancer properties of a novel A3R agonist PNH173 in in vitro and in vivo experiments, with higher efficacy compared to the reference compound. In addition, PNH173 demonstrates biased signaling which could be further utilized against human malignancies. Funding: The European Regional Development Fund (Project ENOCH No. CZ.02.1.01/0.0/0.0/16_019/0000868), the Czech Ministry of Education, Youth and Sports (EATRIS-CZ, LM2018133, and CZ-OPENSCREEN, LM2018130), the Czech Science Foundation (GACR 19-08124S), the Technology Agency of the Czech Republic: Czech National Centres of Competence, project “PerMed” Personalized Medicine - Diagnostics and Therapy (TN01000013), and the Grant agency of Palacky University (IGA_LF_2021_036). Citation Format: Jana Kotulova, Sona Gurska, Barbora Liskova, Miroslav Popper, Katerina Jecmenova, Petr Dzubak, Marian Hajduch. The identification and characterization of a novel A3R agonist with potent anti-cancer properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1124.

Cardioluminescence in Transgenic Zebrafish Larvae: A Calcium Imaging Tool to Study Drug Effects and Pathological Modeling.

Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetyl h-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The β-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.

Capsaicin Is a Negative Allosteric Modulator of the 5-HT3 Receptor.

In this study, effects of capsaicin, an active ingredient of the capsicum plant, were investigated on human 5-hydroxytryptamine type 3 (5-HT3) receptors. Capsaicin reversibly inhibited serotonin (5-HT)-induced currents recorded by two-electrode voltage clamp method in Xenopus oocytes. The inhibition was time- and concentration-dependent with an IC50 = 62 μM. The effect of capsaicin was not altered in the presence of capsazepine, and by intracellular BAPTA injections or trans-membrane potential changes. In radio-ligand binding studies, capsaicin did not change the specific binding of the 5-HT3 antagonist [3H]GR65630, indicating that it is a noncompetitive inhibitor of 5-HT3 receptor. In HEK-293 cells, capsaicin inhibited 5-HT3 receptor induced aequorin luminescence with an IC50 of 54 µM and inhibition was not reversed by increasing concentrations of 5-HT. In conclusion, the results indicate that capsaicin acts as a negative allosteric modulator of human 5-HT3 receptors.

OR24-02 Identification of Membrane Proteins That Enhance the Responsiveness of the Ghrelin Receptor

Ghrelin, a 28-amino acid peptide gut hormone, occurs in acylated (AG) and unacylated (UAG) variants. AG is a GH secretagogue as well as being orexigenic and diabetogenic, acting via the growth hormone secretagogue receptor (GHSR1a) in the hypothalamus and pituitary. UAG counteracts these metabolic effects through unknown mechanisms. While screening for potential UAG receptor(s) we discovered previously uncharacterised interactions of AG and UAG with five cell membrane proteins (MPs), three of which are known to modulate metabolism. Here, we studied if two of these MPs (MP1 & 2) could modulate GHSR1a signalling by expressing their transgenes in HEK293 cells. As GHSR1a is coupled with calcium signalling via Gq proteins, aequorin luminescence was used to evaluate Ca2+ influx into the cells. Transfected cells were treated with either AG, UAG, or soluble parts of the MPs, or combinations thereof.MP2 markedly enhanced the efficacy (~5.5-fold), but not the potency, of AG-induced Ca2+ influx, whereas MP1 had no effect on Ca2+ influx. Neither MP1 nor MP2 overexpression altered cellular GHSR1a levels. In the absence of GHSR1a, MP2 was unable to stimulate an AG-induced Ca2+ influx. UAG treatment (100nM) had no effect on GHSR1a-mediated Ca2+ influx in the presence or absence of MP2. MP2 is post-translationally modified and we suspected this to be important for its activity. However, removal or blockade of these modifications had no effect on the ability of MP2 to enhance GHSR1a signalling. Moreover, incubating the cells with soluble ectodomain of MP2 did not alter its effect on GHSR1a signalling. Nevertheless, induction of ectodomain shedding with PMA (0.4-1µM) dose-dependently reduced the AG-induced Ca2+ response to 0.5-0.2 of control levels (DMSO) in MP2-GHSR1a co-transfected cells. Unlike MP2, which has a transmembrane and intracellular domain, MP1 is attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor and lacks an intracellular domain. Since MP1 is otherwise structurally similar to MP2, we suspected that the intracellular domain of MP2 may be important for its function. Therefore, we expressed chimeras of MP2 and MP1 in which the GPI linkage site and the transmembrane/intracellular domains were exchanged. The MP2 ectodomain with a GPI-anchor had similar stimulatory effects on GHSR1a signalling as full-length MP2, whereas the MP1 ectodomain with MP2 transmembrane and intracellular domain only enhanced GHSR1a signalling by approximately 3-fold.In conclusion, we have identified a membrane protein as a novel component of the ghrelin signalling pathway that markedly enhances the response of the ghrelin receptor to AG. Our current data suggest its ectodomain is important in mediating this effect. Studies are ongoing to fully delineate the mode of interaction and to determine the role of MP2 in ghrelin signalling in vivo.

Functional Identification and Characterization of the Diuretic Hormone 31 (DH31) Signaling System in the Green Shore Crab, Carcinus maenas.

The functional characterization of crustacean neuropeptides and their cognate receptors has not kept pace with the recent advances in sequence determination and, therefore, our understanding of the physiological roles of neuropeptides in this important arthropod sub-phylum is rather limited. We identified a candidate receptor-ligand pairing for diuretic hormone 31 (DH31) in a neural transcriptome of the crab, Carcinus maenas. In insects, DH31 plays species -specific but central roles in many facets of physiology, including fluid secretion, myoactivity, and gut peristalsis but little is known concerning its functions in crustaceans. The C. maenas DH31 transcript codes for a 147 amino acid prepropeptide, and a single receptor transcript translates to a secretin-like (Class B1) G protein-coupled receptor (GPCR). We used an in vitro aequorin luminescence Ca2+ mobilization assay to demonstrate that this candidate DH31R is activated byCarcinus and insect DH31s in a dose-dependent manner (EC50 15–30 nM). Whole mount immunohistochemical and in situ hybridization localization revealed extensive DH31 expressing neurons throughout the central nervous system, most notably in the abdominal ganglion where large, unpaired cells give rise to medial nerves, which terminate in extensive DH31 immunopositive dendritic fields intimately associated with oesophageal musculature. This system constitutes a large and hitherto undescribed neurohemal area adjacent to key muscle groups associated with the gastric system. DH31 expressing neurons were also seen in the cardiac, commissural, oesophageal, and stomatogastric ganglia and intense labeling was seen in dendrites innervating fore- and hindgut musculature but not with limb muscles. These labeling patterns, together with measurement of DH31R mRNA in the heart and hindgut, prompted us test the effects of DH31 on semi-isolated heart preparations. Cardiac superfusion with peptide evoked increased heart rates (10–100 nM). The neuroanatomical distribution of DH31 and its receptor transcripts, particularly that associated with gastric and cardiac musculature, coupled with the cardio- acceleratory effects of the peptide implicate this peptide in key myoactive roles, likely related to rhythmic coordination.

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, Carcinus maenas.

Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.

Roles of the N- and C-Termini in the Function of the Yvc1p Saccharomyces Cerevisiae TRP Channel

In the budding yeast Saccharomyces cerevisiae, cytoplasic calcium levels increase in response to hyperosmotic stress. The Yvc1p TRP channel is responsible for for this response: stress activates Yvc1p, and the channel releases Ca2+ from the vacuolar storage compartment into the cytoplasm. Changes in cytoplasmic Ca2+ concentrations in Saccharomyces are easily observed by monitoring the luminescence of cytoplasmic aequorin (Batiza et al, 1996, JBC). We have explored the utility of this assay for examining Yvc1p function, and have used the assay to study the consequence of truncation mutations of Yvc1p.From the N-terminus, the deletion constructs sequentially remove residues from the start up to the first putative transmembrane domain. We find that the first 30 amino acids are not essential for basic Yvc1p function and response to hyperosmotic shock. At the C-terminus, the panel of deletion constructs spans the segment after TM6 through the last residue. Here we find a more complicated pattern, but Yvc1p function, as judged by aequorin luminescence, is clearly adversely affected by deletions of more than the last 12 residues.

Aequorin as Intracellular Ca2+ Indicator Incorporated in Follicular Lymphoma Cells by Hypoosmotic Shock Treatment

Natural proteins can be used in measuring intracellular Ca2+ concentration. As one of the Ca2+- regulated photoproteins, aequorin has several advantages in comparison to widely used Ca2+ fluorescence indicators (e.g., fura-2, indo-1 and fluo-3), including high dynamic range and resistance to motion artefacts. However, incorporation of aequorin into cells remains a challenge. Hypoosmotic shock treatment was optimized and used as a method for loading aequorin into the cytoplasm of follicular lymphoma cells. Measurement of aequorin luminescence in the cells was performed using a luminometer with a sensitive photomultiplier tube and the luminescence intensity was recalculated into intracellular [Ca2+]. The value of (0.85 ± 0.52)·10-6 M was found. We show that the optimized method of incorporation was effective for loading aequorin into follicular lymphoma cells in vitro. The cell viability remains high immediately after the procedure. This method can also be used for measuring intracellular Ca2+ concentration in other types of non-adherent cells.

Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca2+ concentrations in the mutant strain Aspergillus awamori 66A, containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity ofsporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 microM) stimulated spore germination: the number of germinating spores was 2-3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 microM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca2+ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 microM) were shown to cause an instantaneous increase in Ca2+ concentrations, compared to the control and lower amphotericin concentration (2.5 microM). Ca2+ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.

Measuring circadian oscillations of cytosolic-free calcium in Arabidopsis thaliana.

Circadian oscillations of cytosolic-free calcium can be measured in plants by observing luminescence of the bioluminescent calcium binding protein aequorin. Here we describe the use of intensified photon-counting CCD cameras to measure circadian oscillations in aequorin bioluminescence from Arabidopsis thaliana.

Mitogen-activated protein kinase 6 controls root growth in Arabidopsis by modulating Ca2+-based Na+ flux in root cell under salt stress

Little is known about the role of mitogen-activated protein kinase 6 (MPK6) in Na+ toxicity and inhibition of root growth in Arabidopsis under NaCl stress. In this study, we found that root elongation in seedlings of the loss-of-function mutants mpk6-2 and mpk6-3 was less sensitive to NaCl or Na-glutamate, but not to KCl or mannitol, as compared with that of wild-type (WT) seedlings. The less sensitive characteristic was eliminated by adding the Ca2+ chelator EGTA or the Ca2+ channel inhibitor LaCl3, but not the Ca2+ ionophore A23187. This suggested that the tolerance of mpk6 to Na+ toxicity was Ca2+-dependent. We measured plasma membrane (PM) Na+-conducted currents (NCCs) in root cells. Increased concentrations of NaCl increased the inward NCCs while decreased the outward NCCs in WT root cells, attended by a positive shift in membrane potential. In mpk6 root cells, NaCl significantly increased outward but not inward NCCs, accompanied by a negative shift in membrane potential. That is, mpk6 decreased NaCl-induced the Na+ accumulation by modifying PM Na+ flux in root cells. Observations of aequorin luminescence revealed a NaCl-induced increase of cytosolic Ca2+ in mpk6 root cells, resulting from PM Ca2+ influx. An increase of cytosolic Ca2+ was required to alleviate the NaCl-increased Na+ content and Na+/K+ ratio in mpk6 roots. Together, these results show that mpk6 accumulated less Na+ in response to NaCl because of the increased cytosolic Ca2+ level in root cells; thus, its root elongation was less inhibited than that of WT by NaCl.

The effect of micro-environment on luminescence of aequorin: The role of amino acids and explicit water molecules on spectroscopic properties of coelenteramide

Despite the fact that the luminescence reaction mechanism of aequorin has been intensively investigated, details in luminescence such as the effect of important amino acids residues and explicit water molecules on spectroscopic properties of coelenteramide remain unclear. In this work, the effect of amino acids residues His16, Tyr82, Trp86, Phe113, Trp129, Tyr132, explicit water molecules Wat505 and Wat405 on the spectral properties of CLM− has been studied by CAM-B3LYP, TD M06L and TD CAM-B3LYP methods in hydrophobic environment and aqueous solution. In hydrophobic environment, the amino acids or water molecules have no significant effect on the absorption. Tyr82 and Trp86 move close to CLM− changes the hydrogen bond network, and thus, the spectral properties is significantly affected by the hydrogen bonds between His16H++Tyr82+Trp86 and CLM−. Tyr82, Trp86 hydrogen bonding to CLM− upshifts the excited energy and helps emission spectra shift to blue region. Therefore, it is concluded that His16H++Tyr82+Trp86 modify the emission spectra. The molecular electrostatic potential indicated that the greater electron density is located at the oxygen atom of 6-p-hydroxyphenyl group of CLM−, and it facilitates the formation of hydrogen bond with His16H++Tyr82+Trp86. It is a critical condition for the modification of emission spectra. It is expected to help to understand the interactions between emitter and amino acids in the micro environment.

Role of C-Terminus in the Function of the Yvc1p Saccharomyces Cerevisiae TRP Channel

Yvc1p is a TRP channel found on the vacuolar membrane of Saccharomyces cerevisiae. Yvc1p plays a key role in Ca2+ homeostasis and can be activated by hyperosmotic shock1. The transmembrane topology of TRP channels is very similar to voltage-dependent K+ channels: there are six transmembrane domains and the N- and C-termini are generally depicted as being cytoplasmic. Yvc1p is unusual in that its N- and C-termini are much lengthier than most related TRP channels. In this study, we have focused on understanding the contribution of the C-terminus to the activity of Yvc1p.The C- terminus contains ∼210 amino acid residues. within this segment, there is only one putative domain: a DDDD motif that is thought to bind calcium2. We are using two different functional assays to examine a panel of C-terminal deletion constructs. We used an in vivo luminescence assay to monitor channel activity induced by hyperosmotic shock. In this assay, luminescence is generated when Ca2+, released from the vacuole by Yvc1p, binds to cytoplasmic aequorin. We find that aequorin luminescence is adversely affected by deletions of more than the last 12 residues. The second functional assay examines a Yvc1p-dependent phenotype observed during growth on agar plates: Yvc1p overexpression causes diminished growth in high [Ca2+]2. With this assay, constructs truncated in the region of 578 and 633 affect growth in high [Ca2+], but also in the control conditions of low salt and high [Mg2+] as well, suggesting perturbations of this region may have non-specific consequences for channel function. Ongoing experiments examine how the level and localization of Yvc1p is affected by truncations of the C-terminus.1Denis V, Cyert MS (2002) J Cell Biol 156: 29-34.2Su Z. et al., (2009) J Membr Biol 227:141-150.

The Effect of Indole and Related Compounds on Aequorin Luminescence and Hyperosmotic Shock Response in Saccharomyces cerevisiae

In eukaryotic cells, Transient Receptor Potential (TRP) channels act in response to various stimuli to produce responses. Multiple stimuli may affect TRP channels, although the mechanism for this convergence has been incompletely characterized to date. There is a single TRP channel gene in the Saccharomyces cerevisiae genome coding for a 676 amino acid vacuolar protein, Yvc1p. Ca2+ ions travel from the vacuole through Yvc1p to reach the cytoplasm; this process is activated directly by both hyperosmotic shock (Zhou XL et al (2003) Proc Natl Acad Sci USA 100: 7105-7110) and cytoplasmic Ca2+ (Bertl A, Slayman CL (1990) Proc Natl Acad Sci USA 87: 7824-7828). John Haynes, et al (Haynes et al FEBS Letters 582 (2008) 1514 – 1518) demonstrated direct activation of Yvc1p by Indole and related compounds (Tryptamine, Indoleacetic acid, Tyramine, Quinoline, and Parabens). They additionally showed that the hyperosmotic shock effect could be enhanced by the same compounds.In this study, we use an in vivo luminescence assay to detail the effects of Indole-related compounds on cytoplasmic aequorin expressed in Saccharomyces cerevisiae. We observe a variable response to the aromatic compounds Indole and Tryptamine. In some cases, the compounds appear to act selectively on Yvc1p: they enhance luminescence above that observed in wild-type strains, but have no effect on ΔYVC1 control strains. At other times, ΔYVC1 control strains show a response to the compounds, indicating the compounds either activate aequorin directly, or work through a non-Yvc1p dependent mechanism. We are currently working to understand the basis of this variability.

A High-Throughput Assay for Connexin 43 (Cx43,GJA1) Gap Junctions Using Codon-Optimized Aequorin

Gap junctions (GJs) are intercellular channels which are composed of the connexin family of proteins that allow electrical and chemical communications and synchronization in tissue ensembles. Evidence suggests that pharmaceutical modulators of these channels may have therapeutic potential or carry undesired liability. In this report, we exogenously expressed human connexin 43 (Cx43, GJA1) and demonstrated functionality in a 96-well flow cytometry assay detecting intercellular transfer of the calcein dye. We have designed a 384-well high-throughput method for detecting the transfer of calcium between HeLa cells expressing Cx43. In this assay, donor cells coexpress Cx43 and the α1A adrenergic Gα-coupled receptor, while recipient cells coexpress Cx43 and the cytoplasmic version of the calcium-sensitive luminescent protein aequorin enhanced by codon optimization (cytoAeq). The two cell populations were mixed, dispensed to 384-well plates, and incubated for 3 h to allow the formation of GJs. Activation of α1A by epinephrine in donor cells led to dose-dependent calcium increases in recipient cells, which were detected by measuring the intensity of aequorin luminescence. The response was dependent on the expression of Cx43 and inhibited by the GJ blocker 18α-glycyrrhetinic acid, suggesting Cx43 GJ-mediated activity. In a parallel experiment with capsaicin and the TrpV1 ion channel in place of phenylephrine and α1A, a similar magnitude of difference in the maximal calcium response was detected in both donor and recipient cells, suggesting that calcium is likely the permeant ion through the GJ. This assay may pave the way for high-throughput screening of GJ modulators for drug discovery.

Yvc1p and the Hyperosmotic Shock Response in Saccharomyces Cerevisiae

In addition to their roles as environmental sensors on cytoplasmic membranes, TRP channels are also involved in signaling from intracellular organelles. Yvc1p channel is a TRP channel located in the vacuolar membrane in the yeast Saccharomyces cerevisiae. The vacuole is responsible for the storage of Ca2+ in the yeast cell. Yvc1p controls the release of Ca2+ into the cell. We are using several different approaches to examine the role of Yvc1p in the shock response. First, we are monitoring the release of Ca2+ from the vacuole in response to hyperosmotic shock solution. The Ca2+ release is monitored through the aequorin luminescence assay. Ca2+ binds to aequorin to release luminescence. The release of Ca2+ is affected by pH, solute species and concentration of the hyperosmotic shock solution. We are currently using fluorescence microscopy to examine whether osmotic shock-induced Yvc1p activation effects any morphological changes, and growth and complementation assays to assess Yvc1p function under prolonged conditions of shock.

Pharmacological characterization of receptor guanylyl cyclase reporter cell lines

Results In our reporter cell lines, receptor guanylyl cyclase activity can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different natural as well as synthetic receptor ligands of the natriuretic and guanylin peptide families, we could show that our reporter assay monitors guanylyl cyclase activity with unrivalled high sensitivity. Receptor antagonists were also tested and, unexpectedly, were characterized as partial agonists.

Prevention of Copper-Induced Calcium Influx and Cell Death by Prion-Derived Peptide in Suspension-Cultured Tobacco Cells

Impact of copper on the oxidative and calcium signal transductions leading to cell death in plant cells and the effects of the copper-binding peptide derived from the human prion protein (PrP) as a novel plant-protecting agent were assessed using a cell suspension culture of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species (ROS), such as hydroxyl radicals, and stimulation of calcium channel opening, allowing a transient increase in cytosolic calcium concentrations. The former was proven by the action of specific ROS scavengers blocking the calcium responses and the latter was proven by an increase in aequorin luminescence and its inhibition by specific channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 h in a dose-dependent manner. Addition of a synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper, effectively blocked both calcium influx and cell death induced by copper. Lastly, a possible mechanism of peptide action and future applications of this peptide in the protection of plant roots from metal toxicity or in favour of phytoremediation processes are discussed.

A Novel PDE2A Reporter Cell Line: Characterization of the Cellular Activity of PDE Inhibitors

We report here the generation and pharmacological characterization of a phosphodiesterase 2A (PDE2A) reporter cell line. Human PDE2A was stably transfected in a parental cell line expressing the atrial natriuretic peptide (ANP) receptor and the cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the biosensor for intracellular cGMP. In this reporter cell line, cGMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different PDE inhibitors, we could show that our PDE2A reporter assay specifically monitors PDE2A inhibition with high sensitivity. In the absence of ANP stimulation, the PDE2A selective inhibitors EHNA, BAY 60-7550 and PDP did not increase basal luminescence levels in this experimental setting. However, in combination with ANP, these inhibitors stimulated luminescence signals and induced leftward shifts of ANP concentration-response curves. Similar results were obtained when using IBMX, trequinsin and dipyridamole, which inhibit PDE2A nonselectively with lower potency. PDP, the most potent PDE2A inhibitor known to date, was found to exhibit much lower cellular activity as anticipated from its biochemical PDE2A inhibitory activity. By cellular uptake and transport studies we could show that PDP's cell permeability is low and that the compound is a substrate for an efflux transporter. Other PDE inhibitors including vinpocetine, milrinone, rolipram, sildenafil, zaprinast, BRL 50481 and BAY 73-6691 did not stimulate luminescence signals on our PDE2A reporter cell line. The results imply that this novel PDE2A reporter assay provides an efficient, high throughput means for the identification and characterization of PDE2A inhibitors.