共查询到20条相似文献,搜索用时 31 毫秒
1.
Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of
many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships
(SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y1, P2Y2, and P2Y6 receptors and nucleotide antagonists selective for P2Y1 and P2Y12 receptors are now known. Selective nonnucleotide antagonists were reported for P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, and P2Y13 receptors. At the P2Y1 and P2Y12 receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering
the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues
with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y1 and P2Y6 receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y
receptor antagonists, such as competitive P2Y12 receptor antagonists with antithrombotic activity. Selective agonists for the P2Y4, P2Y11, and P2Y13 receptors and selective antagonists for P2Y4 and P2Y14 receptors have not yet been identified. The P2Y14 receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate
moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets. 相似文献
2.
Although extensive studies provided molecular and pharmacological characterization of metabotropic P2Y receptors for extracellular nucleotides, little is still known about their quaternary structure. By the use of transfected cellular systems and SDS-PAGE, in our previous work we established the propensity of P2Y4 receptor to form dimeric interactions. Here we focused on endogenously expressed P2Y4 and P2Y6 subtypes, comparing their oligomeric complexes under Blue Native (BN) gel electrophoresis. We provided evidence that P2Y4 and P2Y6 receptors form high order complexes in native neuronal phenotypes and that the oligomers can be disaggregated down to the dimeric P2Y4 or to the dimeric and monomeric P2Y6 receptor. Moreover, dimeric P2Y4 and monomeric P2Y6 proteins display selective microdomain partitioning in lipid rafts from specialized subcellular compartments such as synaptosomes. Ligand activation by UTP shifted the oligomerization of P2Y6 but not of P2Y4 receptor, as analysed by BN electrophoresis. Finally, whereas transfected P2Y4 and P2Y6 proteins homo-interact and posses the appropriate domains to associate with all P2Y1,2,4,6,11 subtypes, in naive PC12 cells the endogenous P2Y4 forms hetero-oligomers only with the P2Y6 subunit. In conclusion, our results indicate that quaternary structure distinguishing P2Y4 from P2Y6 receptors might be crucial for specific ligand activation, membrane partitioning and consequent functional regulation. 相似文献
3.
Flores RV Hernández-Pérez MG Aquino E Garrad RC Weisman GA Gonzalez FA 《Molecular and cellular biochemistry》2005,280(1-2):35-45
Purification of HA-tagged P2Y2 receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE
to be in the range of 57–76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The
protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized
state, suggesting a role for P2Y2 receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y2 nucleotide receptors from metabolically [32P]-labelled cells indicated a (3.8 ± 0.2)-fold increase in the [32P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated
control cells. Receptor sequestration studies indicated that ∼40% of the surface receptors were internalized after a 15-min
stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular
loop and C-terminal tail of the P2Y2 receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction
in the efficacy of UTP to desensitize P2Y2 receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation
of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the
level of P2Y2 receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases
in agonist-induced desensitization of the P2Y2 nucleotide receptor. (Mol Cell Biochem xxx: 35–45, 2005) 相似文献
4.
Ilya V. Fedorov 《生物化学与生物物理学报:生物膜》2007,1768(7):1727-1740
Here we elaborated an analytical approach for the simulation of dose-response curves mediated by cellular receptors coupled to PLC and Ca2+ mobilization. Based on a mathematical model of purinergic Ca2+ signaling in taste cells, the analysis of taste cells responsiveness to nucleotides was carried out. Consistently with the expression of P2Y2 and P2Y4 receptors in taste cells, saturating ATP and UTP equipotently mobilized intracellular Ca2+. Cellular responses versus concentration of BzATP, a P2Y2 agonist and a P2Y4 antagonist, implicated high and low affinity BzATP receptors. Suramin modified the BzATP dose-response curve in a manner that suggested the low affinity receptor to be weakly sensitive to this P2Y antagonist. Given that solely P2Y2 and P2Y11 are BzATP receptors, their high sensitivity to suramin is poorly consistent with the suramin effects on BzATP responses. We simulated a variety of dose-response curves for different P2Y receptor sets and found that the appropriate fit of the overall pharmacological data was achievable only with dimeric receptors modeled as P2Y2/P2Y4 homo- and heterodimers. Our computations and analytical analysis of experimental dose-response curves raise the possibility that ATP responsiveness of mouse taste cells is mediated by P2Y2 and P2Y4 receptors operative mostly in the dimeric form. 相似文献
5.
Our work aimed to provide a topographical analysis of all known ionotropic P2X1–7 and metabotropic P2Y1,2,4,6,11–14 receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices
from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the
exception of P2Y11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X5,6 and P2Y1,6,14 in striatum), medium (P2X3 in striatum and substantia nigra, P2X6,7 and P2Y1 in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament
light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic
(colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors
after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson’s
disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic
neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or
neuronal degeneration. Conversely, P2X1,3,4,6 on GABAergic neurons and P2Y4 on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory
reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal
circuit, and suggest their potential participation in the mechanisms of Parkinson’s disease. 相似文献
6.
The distribution of P2Y2 receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y2 with P2X2 and P2X3 receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining
methods. The results showed that P2Y2-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum,
ileum and colon. The typical morphology of P2Y2-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y2-ir neurons could be Dogiel type 1. About 40–60% P2X3-ir neurons were immunoreactive for P2Y2 in the myenteric plexus and all the P2X3-ir neurons expressed the P2Y2 receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive
for P2Y2, especially in the myenteric plexus of the small intestine; no P2Y2-ir neurons were immunoreactive for P2X2 receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated
depolarizations could be those neurons expressing both P2Y2-ir and P2X3-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut. 相似文献
7.
Lin-Chien Huang Peter R. Thorne Srdjan M. Vlajkovic Gary D. Housley 《Purinergic signalling》2010,6(2):231-248
Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic
P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides
by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation
or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been
well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used
immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in
the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the
driving force for sound transduction. In addition, early postnatal P2Y2 and P2Y4 protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation
and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular
compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral
ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca2+-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during
development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance
of extracellular nucleotide and nucleoside signaling to hearing. 相似文献
8.
Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic
nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated
kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway
involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate
(UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating
kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the
most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent
agonists, characteristic of the P2Y2 or P2Y4 receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The
receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially
inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2
phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor
GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is
mediated by a P2Y2 or P2Y4 receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR. 相似文献
9.
P2Y receptors are G protein coupled receptors that respond to extracellular nucleotides to promote a multitude of signaling events. Our laboratory has purified several P2Y receptors with the goal of providing molecular insight into their: (1) ligand binding properties, (2) G protein signaling selectivities, and (3) regulation by RGS proteins and other signaling cohorts. The human P2Y1 receptor and the human P2Y12 receptor, both of which are intimately involved in ADP-mediated platelet aggregation, were purified to near homogeneity and studied in detail. After high-level expression from recombinant baculovirus infection of Sf9 insect cells, approximately 50% of the receptors were successfully extracted with digitonin. Purification of nearly homogeneous epitope-tagged P2Y receptor was achieved using metal-affinity chromatography followed by other traditional chromatographic steps. Yields of purified P2Y receptors range from 10 to 100 g/l of infected cells. Once purified, the receptors were reconstituted in model lipid vesicles along with their cognate G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase assays demonstrated the functional activity of the reconstituted purified receptor. We have utilized this reconstitution system to assess the action of various nucleotide agonists and antagonists, the relative G protein selectivity, and the influence of other proteins, such as phospholipase C, on P2Y receptor-promoted signaling. Furthermore, we have identified the RGS expression profile of platelets and have begun to assess the action of these RGS proteins in a reconstituted P2Y receptor/G protein platelet model. 相似文献
10.
Xianmin Song Wei Guo Qiang Yu Xiaofeng Liu Zhenghua Xiang Cheng He Geoffrey Burnstock 《Purinergic signalling》2011,7(4):469-488
P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y1, 2, 4, 6, 11, 12, 13, 14), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y4 receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of
the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ
hybridization. Neurons expressing P2Y4 receptors were distributed widely in the rat CNS. Heavy P2Y4 receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine
nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus,
and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y4 receptors. P2Y4 receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y4 receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin
(OT) neurons and all the orexin A neurons were immunoreactive for P2Y4 receptors. All the neurons expressing P2Y4 receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release
of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y4 receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between
P2Y4 receptors and NR1. 相似文献
11.
In this study, the distribution patterns of P2Y1, P2Y2 P2Y4, P2Y6, P2Y12, and P2Y13 receptors in the anterior pituitary cells of rat were studied with double-labeling immunofluorescence and Western blot. The
results showed that P2Y receptors were widely expressed in the anterior pituitary. P2Y1 and P2Y4 receptors were found to be expressed in the majority of gonadotrophs and thyrotrophs, P2Y2 receptors were expressed in a small subpopulation of lactotrophs and almost all the folliculo-stellate cells, that were also
stained with S100 protein immunoreactivity. P2Y6 receptors were expressed in macrophages. P2Y13 receptors were expressed in a small subpopulation of cells in the rat anterior pituitary, the identity of which needs to
be clarified. P2Y1 and P2Y4 receptors are co-expressed in some gonadotrophs and thyrotrophs. Corticotrophs and somatotrophs were found not to express
P2Y receptors in this study. FSH and TSH were shown to coexist in the same endocrine cells in rat anterior pituitary. The
present data suggests that purines and/or pyrimidines could be involved in regulating the functions of gonadotrophs and thyrotrophs
via P2Y1 and P2Y4 receptors, some lactotrophs via P2Y2 receptors, and folliculo-stellate cells via P2Y2 receptors in the rat anterior pituitary. 相似文献
12.
Pintor J Sánchez-Nogueiro J Irazu M Mediero A Peláez T Peral A 《Purinergic signalling》2004,1(1):83-90
Nucleotides present an important role in ocular physiology which has been demonstrated by recent works that indicate their involvement in many ocular processes. P2Y are important among P2 receptors since they can control tear production, corneal wound healing, aqueous humour dynamics and retinal physiology. Commercial antibodies have allowed us to investigate the distribution of P2Y receptors in the cornea, anterior and posterior chamber of the eye and retina. The P2Y1 receptor was present mainly in cornea, ciliary processes, and trabecular meshwork. The P2Y2 receptors were present in cornea, ciliary processes and retinal pigmented epithelium. P2Y4 was present in cornea, ciliary processes, photoreceptors, outer plexiform layer and ganglion cell layer. The P2Y6 presented almost an identical distribution as the P2Y4 receptor. The P2Y11 was also detectable in the retinal pigmented epithelium. The detailed distribution of the receptors clearly supports the recent findings indicating the relevant role of nucleotides in the ocular function. 相似文献
13.
Schöneberg T Hermsdorf T Engemaier E Engel K Liebscher I Thor D Zierau K Römpler H Schulz A 《Purinergic signalling》2007,3(4):255-268
Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors
(GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates
or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and
species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y12, have been deorphanized. The P2Y12-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity
including nucleotides, their derivatives, and lipids. Besides the established function of P2Y12 in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies
implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and
response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some
aspects of the physiological relevance of P2Y12-like receptor members. 相似文献
14.
The distribution of P2Y6 and P2Y12 receptor-immunoreactive (ir) neurons and fibers and their coexistence with calbindin, calretinin and nitric oxide synthase
(NOS) has been investigated with single and double labeling immunostaining methods. The results showed that 30–36% of the
ganglion cells in the myenteric plexus are strongly P2Y6 receptor-ir neurons; they are distributed widely in the myenteric plexus of stomach, jejunum, ileum and colon, but not in
the submucosal plexus, with a typical morphology of multipolar neurons with a long axon-like process. About 42–46% of ganglion
cells in both the myenteric and submucosal plexuses show P2Y12 receptor-ir. About 28–35% of P2Y6 receptor-ir neurons were found to coexist with NOS and 41–47% of them coexist with calretinin, but there was no coexistence
of P2Y6 receptor-ir with calbindin. In contrast, all P2Y12 receptor-ir neurons were immunopositive for calbindin, although occasionally P2Y12 receptor-ir neurons without calbindin immunoreactivity were found, while none of the P2Y12 receptor-ir neurons were found to coexist with calretinin or NOS in the gastrointestinal system of guinea pig. The P2Y12 receptor-ir neurons coexpressing calbindin-ir in the small intestine are Dogiel type II/AH, intrinsic primary afferent neurons. 相似文献
15.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2020,1864(3):129501
The nucleotide receptors P2Y2 and P2Y4 are the most closely related G protein-coupled receptors (GPCRs) of the P2Y receptor (P2YR) family. Both subtypes couple to Gq proteins and are activated by the pyrimidine nucleotide UTP, but only P2Y2R is also activated by the purine nucleotide ATP. Agonists and antagonists of both receptor subtypes have potential as drugs e.g. for neurodegenerative and inflammatory diseases. So far, potent and selective, “drug-like” ligands for both receptors are scarce, but would be required for target validation and as lead structures for drug development. Structural information on the receptors is lacking since no X-ray structures or cryo-electron microscopy images are available. Thus, we performed receptor homology modeling and docking studies combined with mutagenesis experiments on both receptors to address the question how ligand binding selectivity for these closely related P2YR subtypes can be achieved. The orthosteric binding site of P2Y2R appeared to be more spacious than that of P2Y4R. Mutation of Y197 to alanine in P2Y4R resulted in a gain of ATP sensitivity. Anthraquinone-derived antagonists are likely to bind to the orthosteric or an allosteric site depending on their substitution pattern and the nature of the orthosteric binding site of the respective P2YR subtype. These insights into the architecture of P2Y2- and P2Y4Rs and their interactions with structurally diverse agonists and antagonist provide a solid basis for the future design of potent and selective ligands. 相似文献
16.
P2Y receptors and pain transmission 总被引:7,自引:0,他引:7
It is widely accepted that the most important ATP receptors involved in pain transmission belong to the P2X3 and P2X2/3 subtypes, selectively expressed in small diameter dorsal root ganglion (DRG) neurons. However, several types of the metabotropic ATP (P2Y) receptors have also been found in primary afferent neurons; P2Y1 and P2Y2 receptors are typically expressed in small, nociceptive cells. Here we review the results available on the involvement of P2Y receptors in the modulation of pain transmission. 相似文献
17.
Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon 总被引:4,自引:0,他引:4
Wolff SC Qi AD Harden TK Nicholas RA 《American journal of physiology. Cell physiology》2005,288(3):C624-C632
Eight human G protein-coupled P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current (Isc) or Ca2+ mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y1, P2Y11, P2Y12, and P2Y14 receptors reside at the basolateral membrane, P2Y2, P2Y4, and P2Y6 receptors are expressed at the apical membrane, and the P2Y13 receptor is unsorted. Biotinylation studies and Isc measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the Gq-coupled P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) in lung and colonic epithelial cells (16HBE14o and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells. Madin-Darby canine kidney; 16HBE14o; Caco-2; confocal microscopy; polarized targeting 相似文献
18.
Natalia Buzzi Paola Scodelaro BilbaoRicardo Boland Ana Russo de Boland 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009,1790(12):1651-1659
Background
ATP exerts diverse effects on various cell types via specific purinergic P2Y receptors. Intracellular signaling cascades are the main routes of communication between P2Y receptors and regulatory targets in the cell.Methods and results
We examined the role of ATP in the modulation of ERK1/2, JNK1/2, and p38 MAP kinases (MAPKs) in human colon cancer Caco-2 cells. Immunoblot analysis showed that ATP induces the phosphorylation of MAPKs in a time- and dose-dependent manner, peaking at 5 min at 10 µM ATP. Moreover, ATPγS, UTP, and UDP but not ADP or ADPβS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y2/P2Y4 and P2Y6 receptor subtypes. RT–PCR studies and PCR product sequencing supported the expression of P2Y2 and P2Y4 receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on Src family tyrosine kinases, calcium influx, and intracellular Ca2+ release and was partially dependent on the cAMP/PKA and PKC pathways and the EGFR.General significance
These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in colon adenocarcinoma Caco-2 cells. 相似文献19.
To study changes in the cytoplasmic Ca2+ concentration ([Ca2+]i) and the total amount of calcium in cells, we used, respectively, the fluorescent dye fura 2/AM and the metallochrome dye
arsenazo III. The total amount of calcium in acinar cells after their incubation in calcium-free ATP-containing extracellular
solution decreased. The action of ATP induced a dose-dependent increase in the [Ca2+]i; the EC50 was, on average, 130 ± ± 36 μM. Calcium transients induced by ATP demonstrated no desensitization. Against the background
of a blocker of ionotropic P2X receptors, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, we observed a decrease in
the ATP-induced calcium transients by 72%. In addition, these transients were reduced by 65% in the calcium-free milieu, while
after thapsigargin-induced exhaustion of the endoplasmic reticulum store they disappeared. This is indicative of the involvement
of metabotropic P2Y receptors in the formation of the above calcium transients. Therefore, P2X and P2Y receptors participate
in ATP-induced calcium signalling in acinar cells of the submandibular salivary gland; activation of these channels results
in a rise in the [Ca2+]i. The P2X receptors to a higher extent contribute to the formation of calcium signals; the P2Y-determined increase in the
[Ca2+]i is smaller (equal to about 35%). Therefore, the functionally active ligand-operated ionotropic P2Y receptors and metabotropic
G protein-related P2Y receptors do exist in acinar cells of the submandibular salivary gland and play an important role in
the control of functioning of this gland.
Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 395–402, September–December, 2005. 相似文献
20.
Mitsutoshi Tsukimoto Akihiro Tokunaga Hitoshi Harada 《Biochemical and biophysical research communications》2009,384(4):512-1495
Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y6 antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X7 antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y6 and P2X7 receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca2+ in T cells. The P2X7 and P2Y6 receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y6 and P2X7 receptors contributes to T cell activation via TCR. 相似文献