Coexpression of several types of metabotropic nucleotide receptors in single cerebellar astrocytes

We have examined the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes could be responsible for ATP metabotropic calcium responses in single type 1 astrocytes. To identify these subtypes we have studied the pharmacological profile of ATP calcium responses using fura-2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astrocytes also responded to 2-methylthioATP and ADP challenges. The agonist potency order was 2-methylthioATP > ADP > ATP = UTP. Cross-desensitization experiments carried out with ATP, UTP, and 2-methylthioATP showed that 2-methylthioATP and UTP interact with different receptors, P2Y(1) and P2Y(2) or P2Y(4). In a subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca(2+) concentration responses at very low concentrations. 2-MethylthioATP and UTP calcium responses exhibited different sensitivity to pertussis toxin and different inhibition patterns in response to P2 antagonists. The P2Y(1)-specific antagonist N:(6)-methyl-2'-deoxyadenosine 3', 5'-bisphosphate (MRS 2179) specifically blocked the 2-methylthio-ATP responses. We can conclude that all single astrocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y(1) and either P2Y(2) or P2Y(4) receptors. Moreover, 30-40% of astrocytes also coexpressed specific pyrimidine receptors of the P2Y(6) subtype, highly selective for UDP coupled to pertussis-toxin insensitive G protein.     

P2Y receptors play a critical role in epithelial cell communication and migration

Cellular injury induces a complex series of events that involves Ca2+ signaling, cell communication, and migration. One of the first responses following mechanical injury is the propagation of a Ca2+ wave (Klepeis et al. [2001] J Cell Sci 114(Pt 23):4185-4195). The wave is generated by the extracellular release of ATP, which also induces phosphorylation of ERK (Yang et al. [2004] J Cell Biochem 91(5):938-950). ATP and other nucleotides, which bind to and activate specific purinergic receptors were used to mimic injury. Our goal was to determine which of the P2Y purinergic receptors are expressed and stimulated in corneal epithelial cells and which signaling pathways are activated leading to changes in cell migration, an event critical for wound closure. In this study, we demonstrated that the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors were present in corneal epithelial cells. A potency profile was determined by Ca2+ imaging for nucleotide agonists as follows: ATP > or = UTP > ADP > or = UDP. In contrast, negligible responses were seen for beta,gamma-meATP, a general P2X receptor agonist and adenosine, a P1 receptor agonist. Homologous desensitization of the Ca2+ response was observed for the four nucleotides. However, P2Y receptor internalization and degradation was not detected following stimulation with ATP, which is in contrast to EGFR internalization observed in response to EGF. ATP induced cell migration was comparable to that of EGF and was maximal at 1 microM. Cells exposed to ATP, UTP, ADP, and UDP demonstrated a rapid twofold increase in phosphorylation of paxillin at Y31 and Y118, however, there was no activation elicited by beta,gamma-meATP or adenosine. Additional studies demonstrated that wound closure was inhibited by reactive blue 2. These results indicate that P2Y receptors play a critical role in the injury repair process.     

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPβS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αβMeATP, and βγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.     

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.     

UDP Facilitates Microglial Phagocytosis Through P2Y6 Receptors

Microglia engage in the clearance of dead cells or dangerous debris. When neighboring cells are injured, the cells release or leak ATP into extracellular space and microglia rapidly move toward or extend a process to the nucleotides as chemotaxis through P2Y12 receptors. In the meanwhile, microglia express the metabotropic P2Y6 receptors, the activation of which by uridine 5’-diphosphate (UDP) triggers microglial phagocytosis in a concentration-dependent fashion. UDP/UTP was leaked when hippocampal neurons were damaged by kainic acid in vivo and in vitro. Systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in mRNA for P2Y6 receptors in activated microglia. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and would function as a sensor for phagocytosis by sensing diffusible UDP signals.     

P2Y purine receptor responses and expression in the pulmonary circulation of juvenile rabbits

The purine nucleotide ATP mediates pulmonary vasodilation at birth by stimulation of P2Y purine receptors in the pulmonary circulation. The specific P2Y receptors in the pulmonary circulation and the segmental distribution of their responses remain unknown. We investigated the effects of purine nucleotides, ATP, ADP, and AMP, and pyrimidine nucleotides, UTP, UDP, and UMP, in juvenile rabbit pulmonary arteries for functional characterization of P2Y receptors. We also studied the expression of P2Y receptor subtypes in pulmonary arteries and the role of nitric oxide (NO), prostaglandins, and cytochrome P-450 metabolites in the response to ATP. In conduit size arteries, ATP, ADP, and AMP caused greater relaxation responses than UTP, UDP, and UMP. In resistance vessels, ATP and UTP caused comparable vasodilation. The response to ATP was attenuated by the P2Y antagonist cibacron blue, the NO synthase antagonist N(omega)-nitro-l-arginine methyl ester (l-NAME), and the cytochrome P-450 inhibitor 17-octadecynoic acid but not by the P2X antagonist alpha,beta-methylene ATP or the cyclooxygenase inhibitor indomethacin in conduit arteries. In the resistance vessels, l-NAME caused a more complete inhibition of the responses to ATP and UTP. Responses to AMP and UMP were NO and endothelium dependent, whereas responses to ADP and UDP were NO and endothelium independent in the conduit arteries. RT-PCR showed expression of P2Y(1), P2Y(2), and P2Y(4) receptors, but not P2Y(6) receptors, in lung parenchyma, pulmonary arteries, and pulmonary artery endothelial cells. These data suggest that distinct P2Y receptors mediate the vasodilator responses to purine and pyrimidine nucleotides in the juvenile rabbit pulmonary circulation. ATP appears to cause NO-mediated vasodilation predominantly through P2Y2 receptors on endothelium.     

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5′-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5′-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca²⁺]_i) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca²⁺]_i transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca²⁺]_i transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca²⁺]_i response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca²⁺]_i transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9468-1) contains supplementary material, which is available to authorized users.     

The role of nucleotides in the neuron--glia communication responsible for the brain functions

Accumulating findings indicate that nucleotides play an important role in cell-to-cell communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2X receptors (7 types; P2X(1)-P2X(7)) contain intrinsic pores that open by binding with ATP. P2Y (8 types; P2Y(1, 2, 4, 6, 11, 12, 13,) and (14)) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins. Nucleotides are released or leaked from non-excitable cells as well as neurons in physiological and pathophysiological conditions. One of the most exciting cells in non-excitable cells is the glia cells, which are classified into astrocytes, oligodendrocytes, and microglia. Astrocytes express many types of P2 purinoceptors and release the 'gliotransmitter' ATP to communicate with neurons, microglia and the vascular walls of capillaries. Microglia also express many types of P2 purinoceptors and are known as resident macrophages in the CNS. ATP and other nucleotides work as 'warning molecules' especially through activating microglia in pathophysiological conditions. Microglia play a key role in neuropathic pain and show phagocytosis through nucleotide-evoked activation of P2X(4) and P2Y(6) receptors, respectively. Such strong molecular, cellular and system-level evidence for extracellular nucleotide signaling places nucleotides in the central stage of cell communications in glia/CNS.     

Purinergic receptors and gastrointestinal secretomotor function

Secretomotor reflexes in the gastrointestinal (GI) tract are important in the lubrication and movement of digested products, absorption of nutrients, or the diarrhea that occurs in diseases to flush out unwanted microbes. Mechanical or chemical stimulation of mucosal sensory enterochromaffin (EC) cells triggers release of serotonin (5-HT) (among other mediators) and initiates local reflexes by activating intrinsic primary afferent neurons of the submucous plexus. Signals are conveyed to interneurons or secretomotor neurons to stimulate chloride and fluid secretion. Inputs from myenteric neurons modulate secretory rates and reflexes, and special neural circuits exist to coordinate secretion with motility. Cellular components of secretomotor reflexes variably express purinergic receptors for adenosine (A1, A2a, A2b, or A3 receptors) or the nucleotides adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), uridine 5'-triphosphate (UTP), or uridine diphosphate (UDP) (P2X(1-7), P2Y(2), P2Y(4), P2Y(6), P2Y(12) receptors). This review focuses on the emerging concepts in our understanding of purinergic regulation at these receptors, and in particular of mechanosensory reflexes. Purinergic inhibitory (A(1), A(3), P2Y(12)) or excitatory (A(2), P2Y(1)) receptors modulate mechanosensitive 5-HT release. Excitatory (P2Y(1), other P2Y, P2X) or inhibitory (A(1), A(3)) receptors are involved in mechanically evoked secretory reflexes or "neurogenic diarrhea." Distinct neural (pre- or postsynaptic) and non-neural distribution profiles of P2X(2), P2X(3), P2X(5), P2Y(1), P2Y(2), P2Y(4), P2Y(6), or P2Y(12) receptors, and for some their effects on neurotransmission, suggests their role in GI secretomotor function. Luminal A(2b), P2Y(2), P2Y(4), and P2Y(6) receptors are involved in fluid and Cl(-), HCO(3) (-), K(+), or mucin secretion. Abnormal receptor expression in GI diseases may be of clinical relevance. Adenosine A(2a) or A(3) receptors are emerging as therapeutic targets in inflammatory bowel diseases (IBD) and gastroprotection; they can also prevent purinergic receptor abnormalities and diarrhea. Purines are emerging as fundamental regulators of enteric secretomotor reflexes in health and disease.     

ATP-induced calcium oscillations and change of P2Y subtypes with culture conditions in HeLa cells

ATP, UTP, ADP and UDP induced intracellular Ca(2+) responses and oscillations in HeLa cells that sometimes lasted over 1 h. The response is due to the activation of P2Ys, G-protein coupled ATP receptors, because the oscillations persisted for several minutes even in Ca(2+)-free solution, and suramin and PPADS, antagonists of ATP receptors, partially inhibited the response. The potency of these nucleotides varied with the culture or cell conditions, i.e. UTP was generally most potent but in some cases UDP was more potent; responses to UDP were variable while those to ATP were constant. In addition, Ca(2+) responses to ATP and UDP were additive. These findings suggested the existence of two or more subtypes of P2Ys in HeLa cells. RT-PCR experiments revealed the existence of P2Y(2), P2Y(4) and P2Y(6). Recovery from starvation (culture in FBS-free medium overnight and re-addition of FBS) increased the responses to UTP and UDP but not to ATP, suggesting that the number or activity of P2Y(6) and/or P2Y(4) receptors may increase with cell proliferation in HeLa cells.     

P2X7 nucleotide receptor activation enhances IFN gamma-induced type II nitric oxide synthase activity in BV-2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFN gamma-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFN gamma-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFN gamma-induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFN gamma-induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate-oxidized ATP, a P2X7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFN gamma-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.     

P2Y<Subscript>2</Subscript> and P2Y<Subscript>6</Subscript> receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y₁₂ receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N?=?7–9 donors), with a potency ranking ADP (EC₅₀ 1.3 ± 1.0 μM)?>?ATP (EC₅₀ 2.2 ± 1.1 μM)?=?UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (<?30 μM) and UDP-glucose (<?30 μM). ATP responses were attenuated by selective P2Y₂ receptor antagonism (AR-C118925XX; IC₅₀ 1.1 ± 0.8 μM, 73.0?±?8.5% max inhibition; N?=?7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y₆ receptor antagonist, MRS2587 (IC₅₀ 437 ± 133nM, 81.0?±?8.4% max inhibition; N?=?6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y₂ and P2Y₆ receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.     

P2X and P2Y purinergic receptors on human intestinal epithelial carcinoma cells: effects of extracellular nucleotides on apoptosis and cell proliferation

Extracellular nucleotides interact with purinergic receptors, which regulate ion transport in a variety of epithelia. With the use of two different human epithelial carcinoma cell lines (HCT8 and Caco-2), we have shown by RT-PCR that the cells express mRNA for P2X1, P2X3, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors. Protein expression for P2Y1 and P2Y2 receptors was also demonstrated immunohistochemically, and P2X receptor subtype protein was present in the following decreasing order: P2X4 > P2X7 > P2X1 > P2X3 > P2X6 > P2X5 > P2X2. The functional presence of P2X7, P2Y1, P2Y2, and P2Y4 receptors was shown based on the effect of extracellular nucleotides on apoptosis or cell proliferation, and measurement of nucleotide-dependent calcium fluxes using a fluorometric imaging plate reader in the presence of different selective agonists and antagonists. ATP, at high concentrations, induced apoptosis through ligation of P2X7 and P2Y1 receptors; conversely, ATP, at lower concentrations, and UTP stimulated proliferation, probably acting via P2Y2 receptors. We therefore propose that stimulation or dysfunction of purinergic receptors may contribute at least partially to modulation of epithelial carcinoma cell proliferation and apoptosis.     

Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells

The purinergic regulation of ciliary activity was studied using small, continuously superfused explants of human nasal epithelium. The P2Y(2) purinoceptor (P2Y(2)-R) was identified as the major purinoceptor regulating ciliary beat frequency (CBF); UTP (EC(50) = 4.7 microM), ATP, and adenosine-5'-O-(3-thiotriphosphate) elicited similar maximal responses, approximately twofold over baseline. ATP, however, elicited a post-peak sustained plateau in CBF (1.83 +/- 0.1-fold), whereas the post-peak CBF response to UTP declined over 15 min to a low-level plateau (1.36 +/- 0.16-fold). UDP also stimulated ciliary beating, probably via P2Y(6)-R, with a maximal effect approximately one-half that elicited by P2Y(2)-R stimulation. Not indicated were P2Y(1)-R-, P2Y(4)-R-, or P2Y(11)-R-mediated effects. A(2B)-receptor agonists elicited sustained responses in CBF approximately equal to those from UTP/ATP [5'-(N-ethylcarboxamido)adenosine, EC(50) = 0.09 microM; adenosine, EC(50) = 0.7 microM]. Surprisingly, ADP elicited a sustained stimulation in CBF. The ADP effect and the post-peak sustained portion of the ATP response in CBF were inhibited by the A(2)-R antagonist 8-(p-sulfophenyl)theophylline. Hence, ATP affects ciliary activity through P2Y(2)-R and, after an apparent ectohydrolysis to adenosine, through A(2B)AR.     

Purinergic receptors in the splanchnic circulation

There is considerable evidence that purines are vasoactive molecules involved in the regulation of blood flow. Adenosine is a well known vasodilator that also acts as a modulator of the response to other vasoactive substances. Adenosine exerts its effects by interacting with adenosine receptors. These are metabotropic G-protein coupled receptors and include four subtypes, A(1), A(2A), A(2B) and A(3). Adenosine triphosphate (ATP) is a co-transmitter in vascular neuroeffector junctions and is known to activate two distinct types of P2 receptors, P2X (ionotropic) and P2Y (metabotropic). ATP can exert either vasoconstrictive or vasorelaxant effects, depending on the P2 receptor subtype involved. Splanchnic vascular beds are of particular interest, as they receive a large fraction of the cardiac output. This review focus on purinergic receptors role in the splanchnic vasomotor control. Here, we give an overview on the distribution and diversity of effects of purinergic receptors in splanchnic vessels. Pre- and post-junctional receptormediated responses are summarized. Attention is also given to the interactions between purinergic receptors and other receptors in the splanchnic circulation.     

Microglial neurotransmitter receptors trigger superoxide production in microglia; consequences for microglial-neuronal interactions

Microglia express three isoforms of the NADPH oxidase, Nox1, Nox2 and Nox4, with the potential to produce superoxide (O(2) ˙(-) ). Microglia also express neurotransmitter receptors, which can modulate microglial responses. In this study, microglial activity of Nox1, Nox2 and Nox4 in primary rat cultured microglia or the rodent BV2 cell line were altered by microglial neurotransmitter receptor modulation. Glutamate, GABA or ATP triggered microglial O(2) ˙(-) production via Nox activation. Nox activation was elicited by agonists of metabotropic mGlu3 receptors and by group III receptors, by GABA(A) but not GABA(B) receptors, and by purinergic P2X(7) or P2Y(2/4) receptors but not P2Y(1) receptors, and inhibited by metabotropic glutamate receptor 5 antagonists. The neurotransmitters also modulated Nox mRNA expression and NADPH activity. The activation of Nox by BzATP or GABA promoted a neuroprotective phenotype whilst the activation of Nox by glutamate promoted a neurotoxic phenotype. Taken together, these data indicate that microglial neurotransmitter receptors can signal via Nox to promote neuroprotection or neurotoxicity. This has implications for the subsequent neurotoxic profile of microglia when neurotransmitter levels may become skewed in neurodegeneration.     

UDP Facilitates Microglial Phagocytosis Through P2Y6 Receptors

Microglia engage in the clearance of dead cells or dangerous debris. When neighboring cells are injured, the cells release or leak ATP into extracellular space and microglia rapidly move toward or extend a process to the nucleotides as chemotaxis through P2Y12 receptors. In the meanwhile, microglia express the metabotropic P2Y6 receptors, the activation of which by uridine 5′-diphosphate (UDP) triggers microglial phagocytosis in a concentration-dependent fashion. UDP/UTP was leaked when hippocampal neurons were damaged by kainic acid in vivo and in vitro. Systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in mRNA for P2Y6 receptors in activated microglia. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and would function as a sensor for phagocytosis by sensing diffusible UDP signals.Key Words: microglia, phagocytosis, P2Y6 receptors, UDPAccumulating findings indicate that nucleotides play an important role in neuron to glia communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y) (Fig. 1). P2X receptors (seven types; P2X₁-P2X₇) contain intrinsic pores that open by binding with ATP. P2Y (eight types; P2Y_{1,2,4,6 and 11–14}) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins.¹ Microglia express P2X₄, P2X₇, P2Y₂, P2Y₆ and P2Y₁₂¹ and are known as resident macrophages in CNS, accounting for 5–10% of the total population of glia.^2,3 When neurons are injured or dead, microglia are activated, resulting in their interaction with immune cells, active migration to the site of injury, release of pro-inflammatory substances and the phagocytosis of damaged cells or debris. For such activation of microglial motilities, extracellular nucleotides have a central role. Extracellular ATP functions as a chemoattractant. Microglial chemotaxis by ATP via P2Y₁₂ receptors was originally found by Honda et al.,⁴ and has recently been confirmed in vivo in P2Y₁₂ receptor knockout animals.⁵ Neuronal injury results in the release or leakage of ATP that appears to be a “find-me” signal from damaged neurons to microglia to cause chemotaxis. In addition to microglial migration by ATP, another nucleotide, UDP, an endogenous agonist of the P2Y₆ receptor, greatly activates the motility of microglia and orders microglia to engulf damaged neurons.⁶Open in a separate windowFigure 1P2 purinergic receptors (ATP receptors).Phagocytosis is a specialized form of endocytosis taking relatively large particles (> 1.0 µm) into vacuoles and has a central role in tissue remodeling, inflammation and the defense against infectious agents.⁷ Phagocytosis is initiated by the activation of cell-surface phagocytosis receptors, including Fc receptors, complement receptors, integrins, endotoxin receptors (CD18, CD14), mannose receptors and scavenger receptors⁸ which are activated by corresponding extracellular ligands called as “eat-me” signals. Since recognition is the most important step for phagocytosis, extensive studies on phagocytosis receptors have been reported. With regard to apoptotic cells, it is well known that dying cells express so called “eat-me” signals such as phosphatidylserine (PS) on their surface membrane,⁸ by which microglia recognize the apoptotic cells in order to catch and remove them.⁸ As for amyloid β protein (Aβ), a key molecule that mediates Alzheimer''s disease, microglia remove Aβ presumably via Fc receptor-dependent phagocytosis.^9,10 It, however, is unclear how phagocytotic cells come to the target cells or debris. Our findings suggest that nucleotides might be the molecules to guide phagocytotic cells to the targets.We found that exogenously applied UDP caused microglial phagocytosis through P2Y₆ in a concentration-dependent manner, and that neuronal injury caused by kainic acid (KA) upregulated P2Y₆ receptors in microglia, the KA evoked neuronal injury resulted in an increase in extracellular UTP, which was immediately metabolized into UDP in vivo and in vitro. We also found that UDP leaked from injured neurons caused P2Y₆ receptor-dependent phagocytosis in vivo and in vitro. Thus, UDP could be a diffusible molecule that signals the crisis of damaged neurons to microglia, triggering phagocytosis. Nucleotides seem to have the ability to act as “eat-us” signals for necrotic cells suffering traumatic or ischemic injury because such necrotic cells cause swelling, followed by shrinkage, leading to the leakage of cytoplasmic molecules including a large amount of ATP and UTP and extracellular nucleotides are immediately degraded by ecto-nucleotideases, suggesting that leaked nucleotides could be transient and localized signals that alert to the crisis created by the presence of the necrotic cells. These findings suggest that microglia might be attracted by ATP/ADP^4,5,11,12 and subsequently recognize UDP, starting to recognize “eat-me” signals attached to the targets and engulf them (Fig. 2). It is interesting that ATP/ADP is not able to efficiently activate P2Y₆ receptors, nor can UDP act on P2Y₁₂ receptors. Thus, adenine and uridine nucleotides would regulate microglial motilities, i.e. chemotaxis and phagocytosis, in a coordinated fashion.Open in a separate windowFigure 2Illustration of nucleotide-activated microglial chemotaxix and phagocytosis. Activated microglia might be attracted by ATP/ADP is not able to efficiently activate P2Y6 receptors, nor ca UDP act on P2Y12 receptors.