A pH-Sensitive Fluorescent Chemosensor Turn-On Based in a Salen Iron (III) Complex: Synthesis, Photophysical Properties, and Live-Cell Imaging Application.

pH regulation is essential to allow normal cell function, and their imbalance is associated with different pathologic situations, including cancer. In this study, we present the synthesis of 2-(((2-aminoethyl)imino)methyl)phenol (HL1) and the iron (III) complex (Fe(L1)2Br, (C1)), confirmed by X-ray diffraction analysis. The absorption and emission properties of complex C1 were assessed in the presence and absence of different physiologically relevant analytes, finding a fluorescent turn-on when OH- was added. So, we determined the limit of detection (LOD = 3.97 × 10-9 M), stoichiometry (1:1), and association constant (Kas = 5.86 × 103 M-1). Using DFT calculations, we proposed a spontaneous decomposition mechanism for C1. After characterization, complex C1 was evaluated as an intracellular pH chemosensor on the human primary gastric adenocarcinoma (AGS) and non-tumoral gastric epithelia (GES-1) cell lines, finding fluorescent signal activation in the latter when compared to AGS cells due to the lower intracellular pH of AGS cells caused by the increased metabolic rate. However, when complex C1 was used on metastatic cancer cell lines (MKN-45 and MKN-74), a fluorescent turn-on was observed in both cell lines because the intracellular lactate amount increased. Our results could provide insights about the application of complex C1 as a metabolic probe to be used in cancer cell imaging.

Understanding the Role of ATP Release through Connexins Hemichannels during Neurulation.

Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD+; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis. Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation.

Activation of Intra-nodose Ganglion P2X7 Receptors Elicit Increases in Neuronal Activity.

Vagus nerve innervates several organs including the heart, stomach, and pancreas among others. Somas of sensory neurons that project through the vagal nerve are located in the nodose ganglion. The presence of purinergic receptors has been reported in neurons and satellite glial cells in several sensory ganglia. In the nodose ganglion, calcium depletion-induced increases in neuron activity can be partly reversed by P2X7 blockers applied directly into the ganglion. The later suggest a possible role of P2X7 receptors in the modulation of neuronal activity within this sensory ganglion. We aimed to characterize the response to P2X7 activation in nodose ganglion neurons under physiological conditions. Using an ex vivo preparation for electrophysiological recordings of the neural discharges of nodose ganglion neurons, we found that treatments with ATP induce transient neuronal activity increases. Also, we found a concentration-dependent increase in neural activity in response to Bz-ATP (ED50 = 0.62 mM, a selective P2X7 receptor agonist), with a clear desensitization pattern when applied every ~ 30 s. Electrophysiological recordings from isolated nodose ganglion neurons reveal no differences in the responses to Bz-ATP and ATP. Finally, we showed that the P2X7 receptor was expressed in the rat nodose ganglion, both in neurons and satellite glial cells. Additionally, a P2X7 receptor negative allosteric modulator decreased the duration of Bz-ATP-induced maximal responses without affecting their amplitude. Our results show the presence of functional P2X7 receptors under physiological conditions within the nodose ganglion of the rat, and suggest that ATP modulation of nodose ganglion activity may be in part mediated by the activation of P2X7 receptors.

Resveratrol-Schiff Base Hybrid Compounds with Selective Antibacterial Activity: Synthesis, Biological Activity, and Computational Study.

Nowadays, antimicrobial resistance is a serious concern associated with the reduced efficacy of traditional antibiotics and an increased health burden worldwide. In response to this challenge, the scientific community is developing a new generation of antibacterial molecules. Contributing to this effort, and inspired by the resveratrol structure, five new resveratrol-dimers (9a−9e) and one resveratrol-monomer (10a) were synthetized using 2,5-dibromo-1,4-diaminobenzene (8) as the core compound for Schiff base bridge conformation. These compounds were evaluated in vitro against pathogenic clinical isolates of Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus sp., and Listeria monocytogenes. Antibacterial activity measurements of resveratrol-Schiff base derivatives (9a−9e) and their precursors (4−8) showed high selectivity against Listeria monocytogenes, being 2.5 and 13.7 times more potent than chloramphenicol, while resveratrol showed an EC50 > 320 µg/mL on the same model. Moreover, a prospective mechanism of action for these compounds against L. monocytogenes strains was proposed using molecular docking analysis, finding a plausible inhibition of internalin C (InlC), a surface protein relevant in bacteria−host interaction. These results would allow for the future development of new molecules for listeriosis treatment based on compound 8.

Roles of the Unsaturated Fatty Acid Docosahexaenoic Acid in the Central Nervous System: Molecular and Cellular Insights.

Fatty acids (FAs) are essential components of the central nervous system (CNS), where they exert multiple roles in health and disease. Among the FAs, docosahexaenoic acid (DHA) has been widely recognized as a key molecule for neuronal function and cell signaling. Despite its relevance, the molecular pathways underlying the beneficial effects of DHA on the cells of the CNS are still unclear. Here, we summarize and discuss the molecular mechanisms underlying the actions of DHA in neural cells with a special focus on processes of survival, morphological development, and synaptic maturation. In addition, we examine the evidence supporting a potential therapeutic role of DHA against CNS tumor diseases and tumorigenesis. The current results suggest that DHA exerts its actions on neural cells mainly through the modulation of signaling cascades involving the activation of diverse types of receptors. In addition, we found evidence connecting brain DHA and ω-3 PUFA levels with CNS diseases, such as depression, autism spectrum disorders, obesity, and neurodegenerative diseases. In the context of cancer, the existing data have shown that DHA exerts positive actions as a coadjuvant in antitumoral therapy. Although many questions in the field remain only partially resolved, we hope that future research may soon define specific pathways and receptor systems involved in the beneficial effects of DHA in cells of the CNS, opening new avenues for innovative therapeutic strategies for CNS diseases.

QSAR-driven synthesis of antiproliferative chalcones against SH-SY5Y cancer cells: Design, biological evaluation, and redesign.

Neuroblastoma is one of the most frequent types of cancer found in infants, and traditional chemotherapy has limited efficacy against this pathology. Thus, the development of new compounds with higher activity and selectivity than traditional drugs is a current challenge in medicinal chemistry research. In this study, we report the synthesis of 21 chalcones with antiproliferative activity and selectivity against the neuroblastoma cell line SH-SY5Y. Then, we developed three-dimensional quantitative structure-activity relationship models (comparative molecular field analysis and comparative molecular similarity index analysis) with high-quality statistical values (q2 > 0.7; r2 > 0.8; r2 pred > 0.7), using IC50 and selectivity index (SI) data as dependent variables. With the information derived from these theoretical models, we designed and synthesized 16 new molecules to prove their consistency, finding good antiproliferative activity against SH-SY5Y cells on these derivatives, with three of them showing higher SI than the referential drugs 5-fluorouracil and cisplatin, displaying also a proapoptotic effect comparable to these drugs, as proven by measuring their effects on executor caspases 3/7 activity induction, Bcl-2/Bax messenger RNA levels alteration, and DNA fragmentation promotion.

Inhibition of Caco-2 and MCF-7 cancer cells using chalcones: synthesis, biological evaluation and computational study.

Cancer is the second death cause worldwide, with breast and colon cancer among the most prevalent types. Traditional treatment strategies have several side effects that inspire the development of novel anticancer agents derived from natural sources, like chalcone derivatives. For this investigation, twenty-three chalcones (4a-w) were synthesized and evaluated as antiproliferative agents against MCF-7 and Caco-2 cells, finding three and two compounds with similar or higher antiproliferative activity than daunorubicin, while only two chalcones showed better selectivity indexes than daunorubicin on MCF-7. From these results, we developed good-performance QSAR models (r > 0.850, q2>0.650), finding several structural features that could modify chalcone activity and selectivity. According to these models, chalcones 4w and 4t have high potency and selectivity against Caco-2 and MCF-7, respectively, which make them attractive candidates for hit-to-lead development of ROS-independent pro apoptotic agents.

Purinergic P2Y2 and P2X4 Receptors Are Involved in the Epithelial-Mesenchymal Transition and Metastatic Potential of Gastric Cancer Derived Cell Lines.

Gastric cancer (GC) is a major health concern worldwide, presenting a complex pathophysiology that has hindered many therapeutic efforts so far. In this context, purinergic signaling emerges as a promising pathway for intervention due to its known role in cancer cell proliferation and migration. In this work, we explored in more detail the role of purinergic signaling in GC with several experimental approaches. First, we measured extracellular ATP concentrations on GC-derived cell lines (AGS, MKN-45, and MKN-74), finding higher levels of extracellular ATP than those obtained for the non-tumoral gastric cell line GES-1. Next, we established the P2Y2 and P2X4 receptors (P2Y2R and P2X4R) expression profile on these cells and evaluated their role on cell proliferation and migration after applying overexpression and knockdown strategies. In general, a P2Y2R overexpression and P2X4R downregulation pattern were observed on GC cell lines, and when these patterns were modified, concomitant changes in cell viability were observed. These modifications on gene expression also modified transepithelial electrical resistance (TEER), showing that higher P2Y2R levels decreased TEER, and high P2X4R expression had the opposite effect, suggesting that P2Y2R and P2X4R activation could promote and suppress epithelial-mesenchymal transition (EMT), respectively. These effects were confirmed after treating AGS cells with UTP, a P2Y2R-agonist that modified the expression patterns towards mesenchymal markers. To further characterize the effects of P2Y2R activation on EMT, we used cDNA microarrays and observed that UTP induced important transcriptional changes on several cell processes like cell proliferation induction, apoptosis inhibition, cell differentiation induction, and cell adhesion reduction. These results suggest that purinergic signaling plays a complex role in GC pathophysiology, and changes in purinergic balance can trigger tumorigenesis in non-tumoral gastric cells.

The Ω-3 fatty acid docosahexaenoic acid selectively induces apoptosis in tumor-derived cells and suppress tumor growth in gastric cancer.

Despite current achievements and innovations in cancer treatment, conventional chemotherapy has several limitations, such as unsatisfactory long-term survival, cancer drug resistance and toxicity against non-tumoral cells. In the search for safer therapeutic alternatives, docosahexaenoic acid (DHA) has shown promising effects inhibiting tumor growth without significant side effects in several types of cancer, but in gastric cancer (GC) its effects have not been completely described. In this study, we characterized the effects of DHA in GC using in vivo and in vitro models. Among all of the evaluated Ω-3 and Ω-6 fatty acids, DHA showed the highest antiproliferative potency and selectivity against the GC-derived cell line AGS. 10-100 µM DHA decreased AGS cell viability in a concentration-dependent manner but had no effect on non-tumoral GES-1 cells. To evaluate if the effects of DHA were due to apoptosis induction, cells were stained with Annexin V-PI, observing that 75 and 100 µM DHA increased apoptosis in AGS, but not in GES-1 cells. Additionally, levels of several proapoptotic and antiapoptotic regulators were assessed by qPCR, western blot and activity assays, showing similar results. In order to evaluate DHA efficacy in vivo, xenografts in an immunodeficient mouse model (BALB/cNOD-SCID) were used. In these experiments, DHA treatment for six weeks consistently reduced subcutaneous tumor size, ascitic fluid volume and liver metastasis. In summary, we found that DHA has a selective antiproliferative effect on GC, being this effect driven by apoptosis induction. Our investigation provides promising features for DHA as potential therapeutic agent in GC.

Altered Glutaminase 1 Activity During Neurulation and Its Potential Implications in Neural Tube Defects.

The neurulation process is regulated by a large amount of genetic and environmental factors that determine the establishment, folding, and fusion of the neural plate to form the neural tube, which develops into the main structure of the central nervous system. A recently described factor involved in this process is glutamate. Through NMDA ionotropic receptor, glutamate modifies intracellular Ca2+ dynamics allowing the oriented cell migration and proliferation, essentials processes in neurulation. Glutamate synthesis depends on the mitochondrial enzyme known as glutaminase 1 (GLS1) that is widely expressed in brain and kidney. The participation of GLS 1 in prenatal neurogenic processes and in the adult brain has been experimentally established, however, its participation in early stages of embryonic development has not been described. The present investigation describes for the first time the presence and functionality of GLS1 in Xenopus laevis embryos during neurulation. Although protein expression levels remains constant, the catalytic activity of GLS1 increases significantly (~66%) between early (stage 12) and middle to late (stages 14-19) neurulation process. Additionally, the use of 6-diazo-5-oxo-L-norleucine (L-DON, competitive inhibitor of glutamine-depend enzymes), reduced significantly the GLS1 specific activity during neurulation (~36%) and induce the occurrence of neural tube defects involving its possible participation in the neural tube closure in Xenopus laevis embryos.

Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors.

Gastric cancer (GC) is the one of the most prevalent cancers and one of the leading causes of cancer-induced deaths. Previously, we found that the expression of purinergic P2Y2 receptor (P2Y2R) is increased in GC samples as compared to adjacent healthy mucosa taken from GC-diagnosed patients. In this work, we studied in detail purinergic signaling in the gastric adenocarcinoma-derived cell lines: AGS, MKN-45, and MKN-74, and compared them to a nontumoral epithelial cell line: GES-1. In GC-derived cells, we detected the expression of several purinergic receptors, and found important differences as compared to GES-1 cells. Functional studies revealed a strong contribution of P2Y2Rs in intracellular calcium increases, elicited by adenosine-triphosphate (ATP), uridine-triphosphate (UTP), and the P2Y2R agonist MRS2768. Responses were preserved in the absence of extracellular calcium and inhibited by P2Y2R antagonists. In GES-1 cells, ATP and UTP induced similar responses and the combination of P2X and P2Y receptor antagonists was able to block them. Proliferation studies showed that ATP regulates AGS and MKN-74 cells in a biphasic manner, increasing cell proliferation at 10-100 µM, but inhibiting at 300 µM ATP. On the other hand, 1-300 µM UTP, a P2Y2R agonist, increased concentration-dependent cell proliferation. The effects of UTP and ATP were prevented by both wide-range and specific purinergic antagonists. In contrast, in GES-1 cells ATP only decreased cell proliferation in a concentration-dependent manner, and UTP had no effect. Notably, the isolated application of purinergic antagonists was sufficient to change the basal proliferation of AGS cells, indicating that nucleotides released by the cells can act as paracrine/autocrine signals. Finally, in tumor-derived biopsies, we found an increase of P2Y2R and a decrease in P2X4R expression; however, we found high variability between seven different biopsies and their respective adjacent healthy gastric mucosa. Even so, we found a correlation between the expression levels of P2Y2R and P2X4R and survival rates of GC patients. Taken together, these results demonstrate the involvement of different purinergic receptors and signaling in GC, and the pattern of expression changes in tumoral cells, and this change likely directs ATP and nucleotide signaling from antiproliferative effects in healthy tissues to proliferative effects in cancer.

Characterization of the antagonist actions of 5-BDBD at the rat P2X4 receptor.

P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, we studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. We found that 5-BDBD is a potent P2X4R antagonist, with an IC50 of 0.75 µM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 µM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 µM concentration but not 0.75 µM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue.

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel.

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

Divalent metal modulation of Japanese flounder (Paralichthys olivaceus) purinergic P2X7 receptor.

Paralichthys olivaceus P2X7 receptor (poP2X7R) is a recently identified as a P2X7 purinergic receptor involved in innate immunity of the Japanese flounder Paralichthys olivaceus. Divalent metals are allosteric modulators of mammalian P2XRs, but there is no information for fish P2XRs. Here, we characterized the effects of divalent metals on poP2X7R channel activity by electrophysiology and molecular biology techniques. Copper, zinc and mercury inhibited poP2X7R-mediated currents with different maximal inhibition potency, while cadmium had no effect on poP2X7R activity. Mercury-induced inhibition was irreversible, but the inhibitory effects of copper and zinc were reversed after washout. Cooper and zinc also reduced poP2X7R-mediated interleukin-1 mRNA production. These findings suggest that divalent metals have potential effects on the Japanese flounder innate immune response through modulation of poP2X7R activity.

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372.

The purinergic P2X2 receptor (P2X2R) is an adenosine triphosphate-gated ion channel widely expressed in the nervous system. Here, we identified a putative cyclin-dependent kinase 5 (Cdk5) phosphorylation site in the full-size variant P2X2aR (TPKH), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5 activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells and P2X2/3R mediated increases of intracellular Ca in trigeminal neurons were Cdk5 dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.

Tonotopic action potential tuning of maturing auditory neurons through endogenous ATP.

KEY POINTS: Following the genetically controlled formation of neuronal circuits, early firing activity guides the development of sensory maps in the auditory, visual and somatosensory system. However, it is not clear whether the activity of central auditory neurons is specifically regulated depending on the position within the sensory map. In the ventral cochlear nucleus, the first central station along the auditory pathway, we describe a mechanism through which paracrine ATP signalling enhances firing in a cell-specific and tonotopically-determined manner. Developmental down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, suggesting a high-to-low frequency maturation pattern. Facilitated action potential (AP) generation, measured as higher firing rate, shorter EPSP-AP delay in vivo and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. The long lasting change in intrinsic neuronal excitability is mediated by the heteromeric P2X2/3 receptors. ABSTRACT: Synaptic refinement and strengthening are activity-dependent processes that establish orderly arranged cochleotopic maps throughout the central auditory system. The maturation of auditory brainstem circuits is guided by action potentials (APs) arising from the inner hair cells in the developing cochlea. The AP firing of developing central auditory neurons can be modulated by paracrine ATP signalling, as shown for the cochlear nucleus bushy cells and principal neurons in the medial nucleus of the trapezoid body. However, it is not clear whether neuronal activity may be specifically regulated with respect to the nuclear tonotopic position (i.e. sound frequency selectivity). Using slice recordings before hearing onset and in vivo recordings with iontophoretic drug applications after hearing onset, we show that cell-specific purinergic modulation follows a precise tonotopic pattern in the ventral cochlear nucleus of developing gerbils. In high-frequency regions, ATP responsiveness diminished before hearing onset. In low-to-mid frequency regions, ATP modulation persisted after hearing onset in a subset of low-frequency bushy cells (characteristic frequency< 10 kHz). Down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, thus suggesting a high-to-low frequency maturation pattern. Facilitated AP generation, measured as higher firing frequency, shorter EPSP-AP delay in vivo, and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. Finally, by combining recordings and pharmacology in vivo, in slices, and in human embryonic kidney 293 cells, it was shown that the long lasting change in intrinsic neuronal excitability is mediated by the P2X2/3R.

Role of domain calcium in purinergic P2X2 receptor channel desensitization.

Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using rat, mouse, and human receptors, we show here that two processes contribute to receptor desensitization: bath calcium-independent desensitization and calcium-dependent desensitization. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of the receptor but is pronounced in cells expressing shorter versions of receptors, indicating a role of the COOH terminus in control of receptor desensitization. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in bath ATP and calcium concentration-dependent manners. Experiments with substitution of bath Na(+) with N-methyl-d-glucamine (NMDG(+)), a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential from -60 to +120 mV further indicates that calcium influx through the channel pores at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and/or intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that a decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording shows development of calcium-dependent desensitization without an increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings.

Molecular characterization and expression analysis of ATP-gated P2X7 receptor involved in Japanese flounder (Paralichthys olivaceus) innate immune response.

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60-88% sequence similarity and 45-78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1ß and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.

Regulation of ATP-gated P2X channels: from redox signaling to interactions with other proteins.

SIGNIFICANCE: The family of purinergic P2X receptors (P2XRs) is a part of ligand-gated superfamily of channels activated by extracellular adenosine-5'-triphosphate. P2XRs are present in virtually all mammalian tissues as well as in tissues of other vertebrate and nonvertebrate species and mediate a large variety of functions, including fast transmission at central synapses, contraction of smooth muscle cells, platelet aggregation, and macrophage activation to proliferation and cell death. RECENT ADVANCES: The recent solving of crystal structure of the zebrafish P2X4.1R is a major advance in the understanding of structural correlates of channel activation and regulation. Combined with growing information obtained in the post-structure era and the reinterpretation of previous work within the context of the tridimensional structure, these data provide a better understanding of how the channel operates at the molecular levels. CRITICAL ISSUES: This review focuses on the relationship between redox signaling and P2XR function. We also discuss other allosteric modulation of P2XR gating in the physiological/pathophysiological context. This includes the summary of extracellular actions of trace metals, which can be released to the synaptic cleft, pH decrease that happens during ischemia and inflammation, and calcium, an extracellular and intracellular messenger. FUTURE DIRECTIONS: Our evolving understanding of activation and regulation of P2XRs is helpful in clarifying the mechanism by which these channels trigger and modulate cellular functions. Further research is required to identify the signaling pathways contributing to the regulation of the receptor activity and to develop novel and receptor-specific allosteric modulators, which could be used in vivo with therapeutic potential.