New Insights in Purinergic Therapy: Novel Antagonists for UTP-Activated P2Y Receptors from Natural Products

P2Y2 and P2Y4 receptors are physiologically activated by UTP and are widely expressed in many cell types in humans. They promote an increase in intracellular calcium via PLCβ/ IP3 and act on ion flux and water secretion. P2Y2 plays an important role in inflammation and proliferation of tumor cells, which could be attenuated with the use of antagonists. However, little is known about the physiological functions related to P2Y4 due to the lack of selective ligands for these receptors, which can be solved through the search of novel compounds with antagonistic activity. In the present study, we have applied a methodology of calcium measurement to identify new antagonist candidates for these receptors. Firstly, we established optimal conditions for calcium assay using J774.G8, a murine macrophage cell line, which expresses functional P2Y2 and P2Y4 receptors. J774.G8 cells were loaded with 2 μM of Fluo-4 to test its sensitivity in responding calcium stimuli. ATP and ionomycin, known as inductors of intracellular calcium rise, were used to stimulate cells. The EC50 obtained were 11 μM and 103 nM, respectively. Subsequently, investigation of P2Y2 and P2Y4 expression was performed. These cells responded with EC50 of 1.021 μM to the UTP stimulation. Screening assays were performed and a total of 100 extracts from Brazilian natural products were tested. JA2, RA3, and RB3 extracts stood out for their ability to inhibit UTP-induced responses without causing cytotoxicity and presented IC50 of 32.32 μg/mL, 14.99 μg/mL, and 12.98 μg/mL, respectively. Collectively, our results point to the discovery of potential antagonists candidates from natural products for UTP-activated receptors.

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Pregnancy-enhanced Ca2+ responses to ATP in uterine artery endothelial cells is due to greater capacitative Ca2+ entry rather than altered receptor coupling

Journal of Endocrinology ◽

10.1677/joe.1.06635 ◽

2006 ◽

Vol 190 (2) ◽

pp. 373-384 ◽

Cited By ~ 18

Author(s):

Shannon M Gifford ◽

Fu-Xian Yi ◽

Ian M Bird

Keyword(s):

Endothelial Cells ◽

Uterine Artery ◽

Purinergic Receptor ◽

Cell Types ◽

P2y Receptors ◽

Receptor Expression ◽

Receptor Subtype ◽

Initial Transient ◽

Receptor Coupling ◽

Specific Variation

Uterine artery endothelial cells (UAEC) derived from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes retain pregnancy-specific differences in cell signaling as well as vasodilator production through passage 4. In particular, when P- and NP-UAEC are stimulated with ATP over a 2.5 min recording period, they exhibit similar initial transient peaks in the intracellular free Ca2+ concentration ([Ca2+]i), but the P-UAEC show a heightened sustained phase. In order to establish whether thiswas due to an altered subclass of purinergic receptor (P2), both the dose dependencyof [Ca2+]i responses to ADP and UTP and the profile of purinergic receptor expression are determined in NP- and P-UAEC. Our findings indicate that while several isoforms of P2X and P2Y receptors are present, it is P2Y2 that is responsible for the ATP-induced initial transient peak in both cell types. We also characterized several key components of the ATP-induced Ca2+ signaling cascade, including the inositol 1,4,5-trisphosphate receptor and G-proteins, but could not confirm any pregnancy-specific variation in the protein expression that correlated with pregnancy-specific differences in prolonged Ca2+ signaling. We thus investigated whether such a difference may be inherent to the cell itself rather than specific to the purinergic receptor-signaling pathway. Using thapsigargin (Tg), we were able to demonstrate that the initial Tg-sensitive intracellular pool of Ca2+is nearly identical with the capacity in both cell types, but the P-UAEC is nonetheless capable of greater capacitative Ca2+ entry (CCE) than NP-UAEC. Furthermore, CCE induced by Tg could be dramatically inhibited by 2-aminoethoxydiphenyl borate, suggesting a role for store-operated channels in the ATP-induced [Ca2+]i response. We conclude that changes at the level of capacitative entry mechanisms rather than switching of receptor subtype or coupling to phospholipase C underlies pregnancy adaptation of UAEC at the level of Ca2+signaling.

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Control of secretion in anterior pituitary cells--linking ion channels, messengers and exocytosis

Journal of Experimental Biology ◽

10.1242/jeb.139.1.287 ◽

1988 ◽

Vol 139 (1) ◽

pp. 287-316

Author(s):

W. T. Mason ◽

S. R. Rawlings ◽

P. Cobbett ◽

S. K. Sikdar ◽

R. Zorec ◽

...

Keyword(s):

Ion Channels ◽

Intracellular Calcium ◽

Anterior Pituitary ◽

Hormone Secretion ◽

Cell Types ◽

Pituitary Cell ◽

Pituitary Cells ◽

Calcium Activity ◽

Anterior Pituitary Cells ◽

Voltage Dependent

Normal anterior pituitary cells, in their diversity and heterogeneity, provide a rich source of models for secretory function. However, until recently they have largely been neglected in favour of neoplastic, clonal tumour cell lines of pituitary origin, which have enabled a number of studies on supposedly homogeneous cell types. Because many of these lines appear to lack key peptide and neurotransmitter receptors, as well as being degranulated with accompanying abnormal levels of secretion, we have developed a range of normal primary anterior pituitary cell cultures using dispersion and enrichment techniques. By studying lactotrophs, somatotrophs and gonadotrophs we have revealed a number of possible transduction mechanisms by which receptors for hypothalamic peptides and neurotransmitters may control secretion. In particular, the transduction events controlling secretion from pituitary cells may differ fundamentally from those found in other cell types. Patch-clamp recordings in these various pituitary cell preparations have revealed substantial populations of voltage-dependent Na+, Ca2+ and K+ channels which may support action potentials in these cells. Although activation of these channels may gate Ca2+ entry to the cells under some conditions, our evidence taken with that of other laboratories suggests that peptide-receptor interactions leading to hormone secretion occur independently of significant membrane depolarization. Rather, secretion of hormone and rises in intracellular calcium measured with new probes for intracellular calcium activity, can occur in response to hypothalamic peptide activation in the absence of substantial changes in membrane potential. These changes in intracellular calcium activity almost certainly depend on both intracellular and extracellular calcium sources. In addition, strong evidence of a role for multiple intracellular receptors and modulators in the secretory event suggests we should consider the plasma membrane channels important for regulation of hormone secretion to be predominantly agonist-activated, rather than of the more conventional voltage-dependent type. Likewise, evidence from new methods for recording single ion channels suggests the existence of intracellular sites for channel modulation, implying they too may play an important role in secretory regulation. We shall consider new data and new technology which we hope will provide key answers to the many intriguing questions surrounding the control of pituitary hormone secretion. We shall highlight our work with recordings of single ion channels activated by peptides, and recent experiments using imaging of intracellular ionized free calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

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Study Cellular Hypoxic Response under Cyclic Oxygen Gradients Generated in Microfluidic Devices Using Real-Time Fluorescence Imaging

10.21203/rs.3.rs-382697/v1 ◽

2021 ◽

Author(s):

Dao-Ming Chang ◽

Yi-Chung Tung

Keyword(s):

Cell Culture ◽

Intracellular Calcium ◽

Real Time ◽

Oxygen Tension ◽

Calcium Concentration ◽

A549 Cells ◽

Cell Types ◽

Hypoxic Response ◽

Oxygen Gradient ◽

Oxygen Gradients

Abstract In this paper, an integrated approach to study cellular hypoxic response under cyclic oxygen gradients is developed. In the experiments, a cell culture system based on a microfluidic device is constructed to generate cyclic oxygen gradients with fast response. The oxygen gradient is generated by alternatively introducing gases with specific compositions by a computer-controlled gas supply system into the microfluidic channels next to the cell culture one separated by thin channel walls. Observation of cellular hypoxic responses is performed using real-time fluorescence imaging of dyes sensitive to extra-and intra-cellular oxygen tensions as well as intracellular calcium concentrations. The capability of consistent cyclic oxygen gradient generation is confirmed by characterizing spatiotemporal oxygen tension profiles within the device. In the cell experiments, cellular hypoxic responses of human aortic smooth muscle cells (AoSMCs) and lung carcinoma epithelium (A549) cells including intracellular oxygen and calcium levels are measured. The relationships between the oxygen probe intensity and extracellular oxygen tension for both cell types are highly linear, and difference between the intensity measured from the two cell types is observed between 4 to 8% O2 culture environment. In addition, both cell types show detectable intracellular calcium concentration variation when the environmental O2 varies between normoxia and lower than 4%. The AoSMCs and A549 cells show decrease and increase of the intracellular calcium concentration during hypoxic response when facing change from normoxia to hypoxia environments, respectively. With the capability of real-time cellular responses monitoring under cyclic oxygen gradients, the developed approach provides a useful scheme to investigate cellular hypoxic response in vitro under microenvironments mimicking various in vivo physiological and pathological conditions.

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Degradation of extracellular ATP by the retinal pigment epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.00542.2004 ◽

2005 ◽

Vol 289 (3) ◽

pp. C617-C624 ◽

Cited By ~ 32

Author(s):

David Reigada ◽

Wennan Lu ◽

Xiulan Zhang ◽

Constantin Friedman ◽

Klara Pendrak ◽

...

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Adenosine Diphosphate ◽

Cell Types ◽

P2y Receptors ◽

Extracellular Atp ◽

Pcr Analysis ◽

Subretinal Space ◽

Rpe Cells ◽

Mrs 2179

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and βγ-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca2+. Both responses were inhibited by thapsigargin and P2Y1 receptor inhibitor MRS 2179. Message for both P2Y1 and P2Y12 receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca2+ signaling in the RPE.

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Intracellular calcium measurement in isolated rat arterioles during phasic and tonic contractions

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)60028-4 ◽

1992 ◽

Vol 58 ◽

pp. 367

Author(s):

Akihiko Karibe ◽

Mitsumasa Keitoku ◽

Jun Watanabe ◽

Tamotsu Takishima

Keyword(s):

Intracellular Calcium ◽

Calcium Measurement ◽

Isolated Rat

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Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate-mediated intracellular calcium homeostasis

Genome ◽

10.1139/gen-2019-0113 ◽

2020 ◽

Vol 63 (2) ◽

pp. 61-90

Author(s):

Vidhya Balasubramanian ◽

Bharath Srinivasan

Keyword(s):

Intracellular Calcium ◽

Calcium Binding ◽

Model Organism ◽

Cell Types ◽

Single Copy ◽

Structural Determinants ◽

Α Subunit ◽

Behavioral Abnormalities ◽

Mutant Phenotypes ◽

Trisphosphate Receptor

Nucleobindin-1 is an EF-hand calcium-binding protein with a distinctive profile, predominantly localized to the Golgi in insect and wide-ranging vertebrate cell types, alike. Its putative involvements in intracellular calcium (Ca2+) homeostasis have never been phenotypically characterized in any model organism. We have analyzed an adult-viable mutant that completely disrupts the G protein α-subunit binding and activating (GBA) motif of Drosophila Nucleobindin-1 (dmNUCB1). Such disruption does not manifest any obvious fitness-related, morphological/developmental, or behavioral abnormalities. A single copy of this mutation or the knockdown of dmnucb1 in restricted sets of cells variously rescues pleiotropic mutant phenotypes arising from impaired inositol 1,4,5-trisphosphate receptor (IP3R) activity (in turn depleting cytoplasmic Ca2+ levels across diverse tissue types). Additionally, altered dmNUCB1 expression or function considerably reverses lifespan and mobility improvements effected by IP3R mutants, in a Drosophila model of amyotrophic lateral sclerosis. Homology modeling-based analyses further predict a high degree of conformational conservation in Drosophila, of biochemically validated structural determinants in the GBA motif that specify in vertebrates, the unconventional Ca2+-regulated interaction of NUCB1 with Gαi subunits. The broad implications of our findings are hypothetically discussed, regarding potential roles for NUCB1 in GBA-mediated, Golgi-associated Ca2+ signaling, in health and disease.

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Activation of P2Y but not P2X4 nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.6.c1531 ◽

2001 ◽

Vol 280 (6) ◽

pp. C1531-C1539 ◽

Cited By ~ 27

Author(s):

A. Frederik Weidema ◽

S. Jeffrey Dixon ◽

Stephen M. Sims

Keyword(s):

Endoplasmic Reticulum ◽

Patch Clamp ◽

Cyclopiazonic Acid ◽

Cell Types ◽

P2y Receptors ◽

P2x Receptors ◽

Extracellular Nucleotides ◽

Nucleotide Receptors ◽

Inward Current ◽

Store Depletion

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of [Ca2+]ipersisted on removal of extracellular Ca2+(Ca[Formula: see text]), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca2+-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of [Ca2+]i. After store depletion in the absence of Ca[Formula: see text], addition of Ca[Formula: see text] led to a rise of [Ca2+]i consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La3+. In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X4 nucleotide receptors, but with no rise of [Ca2+]i. We conclude that nucleotide-induced elevation of [Ca2+]i in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.

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Immunocytochemical localization of the nuclear 3,5,3'-triiodothyronine receptor in the adult rat: liver, kidney, heart, lung and spleen

Acta Endocrinologica ◽

10.1530/acta.0.1200451 ◽

1989 ◽

Vol 120 (4) ◽

pp. 451-458 ◽

Cited By ~ 11

Author(s):

M. Luo ◽

R. Faure ◽

Y. A. Tong ◽

J. H. Dussault

Keyword(s):

Ascitic Fluid ◽

Cell Types ◽

Type Ii ◽

Myocardial Cells ◽

Cell Nuclei ◽

Macrophage Cell ◽

Specific Staining ◽

Adult Rat ◽

Type Ii Pneumocytes ◽

Mature Lymphocyte

Abstract. A monoclonal antibody was used for the localization of the nuclear T3 receptor in different tissues of the adult rat: the liver, kidney, heart, lung, spleen, testis, and pituitary. In the liver, the immunoreactivity was found uniformly distributed in the nuclei of hepatocytes. Sections incubated with a control ascitic fluid or with the same ascitic fluid pre-adsorbed with purified receptor showed no specific staining. In the kidney, the immunoreactivity was higher in the epithelial cell of the proximal convoluted tubes and juxtaglomerular cells. In the heart, only the myocardial cells were stained. In the lung, the immunoreactivity was confined to type II pneumocytes and alveolar macrophages. In the spleen, only a few mature lymphocyte and macrophage cell nuclei were stained. These results show that: 1) the abundance of the nuclear T3 correlates with previous studies using hormone binding techniques; 2) the nuclear T3 receptor is selectively located in certain cell types, which possess a precise local function.

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Modulation of Pineal Melatonin Synthesis by Glutamate Involves Paracrine Interactions between Pinealocytes and Astrocytes through NF-κB Activation

BioMed Research International ◽

10.1155/2013/618432 ◽

2013 ◽

Vol 2013 ◽

pp. 1-14 ◽

Cited By ~ 12

Author(s):

Darine Villela ◽

Victoria Fairbanks Atherino ◽

Larissa de Sá Lima ◽

Anderson Augusto Moutinho ◽

Fernanda Gaspar do Amaral ◽

...

Keyword(s):

Intracellular Calcium ◽

Glutamate Receptors ◽

Metabotropic Glutamate Receptors ◽

Calcium Content ◽

Cell Types ◽

Inhibitory Effect ◽

Metabotropic Glutamate ◽

Melatonin Synthesis ◽

Group I ◽

Glutamatergic Signaling

The glutamatergic modulation of melatonin synthesis is well known, along with the importance of astrocytes in mediating glutamatergic signaling in the central nervous system. Pinealocytes and astrocytes are the main cell types in the pineal gland. The objective of this work was to investigate the interactions between astrocytes and pinealocytes as a part of the glutamate inhibitory effect on melatonin synthesis. Rat pinealocytes isolated or in coculture with astrocytes were incubated with glutamate in the presence of norepinephrine, and the melatonin content, was quantified. The expression of glutamate receptors, the intracellular calcium content and the NF-κB activation were analyzed in astrocytes and pinealocytes. TNF-α's possible mediation of the effect of glutamate was also investigated. The results showed that glutamate's inhibitory effect on melatonin synthesis involves interactions between astrocytes and pinealocytes, possibly through the release of TNF-α. Moreover, the activation of the astrocytic NF-κB seems to be a necessary step. In astrocytes and pinealocytes, AMPA, NMDA, and group I metabotropic glutamate receptors were observed, as well as the intracellular calcium elevation. In conclusion, there is evidence that the modulation of melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through the activation of the astrocytic NF-κB transcription factor and possibly by subsequent TNF-αrelease.

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Ryanodine receptor Ca2+ release channels: does diversity in form equal diversity in function?

Physiological Reviews ◽

10.1152/physrev.1996.76.4.1027 ◽

1996 ◽

Vol 76 (4) ◽

pp. 1027-1071 ◽

Cited By ~ 282

Author(s):

J. L. Sutko ◽

J. A. Airey

Keyword(s):

Intracellular Calcium ◽

Ryanodine Receptor ◽

Skeletal Muscles ◽

Calcium Release ◽

Cell Types ◽

Calcium Transients ◽

Eukaryotic Cells ◽

Calcium Release Channels ◽

Primary Focus ◽

Fast Twitch

Complexities in calcium signaling in eukaryotic cells require diversity in the proteins involved in generating these signals. In this review, we consider the ryanodine receptor (RyR) family of intracellular calcium release channels. This includes species, tissue, and cellular distributions of the RyRs and mechanisms of activation, deactivation, and inactivation of RyR calcium release events. In addition, as first observed in nonmammalian vertebrate skeletal muscles, it is now clear that more than one RyR isoform is frequently coexpressed within many cell types. How multiple ryanodine receptor release channels are used to generate intracellular calcium transients is unknown. Therefore, a primary focus of this review is why more than one RyR is required for this purpose, particularly in a tissue, such as vertebrate fast-twitch skeletal muscles, where a relatively simple and straightforward change in calcium would appear to be required to elicit contraction. Finally, the roles of the RyR isoforms and the calcium release events they mediate in the development of embryonic skeletal muscle are considered.

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