Types Of Purinergic Receptors Research Articles

Purinergic receptor types in the hypothalamic-neurohypophysial system.

Many different types of purinergic receptors are present in the Hypothalamic-Neurohypophysial System (HNS), which synthesizes and releases vasopressin and oxytocin. The specific location of purinergic receptor subtypes has important functional repercussions for neuronal activity and synaptic output. Yet, until the advent of receptor KOs, this had been hindered by the low selectivity of the available pharmacological tools. The HNS offers an excellent opportunity to differentiate the functional properties of these purinergic receptors in cell bodies vs. terminals of the same physiological system. P2X2, P2X3, P2X4 and P2X7 receptors are present in vasopressin terminals while oxytocin terminals exclusively express the P2X7 subtype. The latter is not functional in the cell bodies of the HNS. These purinergic receptor subtypes are permeable to sodium vs. calcium in varying amounts and this could play an important role in the release of vasopressin vs. oxytocin during bursting activity. Endogenous ATP and its metabolite, adenosine, have autocrine and paracrine modulatory effects on the release of these neuropeptides during physiological stimulation. Finally, we hypothesize that during such action potential bursts, ATP potentiates the release of vasopressin but not of oxytocin, and that adenosine, via A1 receptors, inhibits the release of both neuropeptides. This article is protected by copyright. All rights reserved.

P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction

It is known that adenosine 5′-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12–13 receptors, 2-methylthioadenosine 5′-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12–13 antagonists AR-C69931MX or 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5′-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.

The role of purinergic signaling in the etiology of migraine and novel antimigraine treatment.

Etiopathogenesis of migraine involves different structures of the central nervous system: the trigeminal nerve with nuclei located in the brain stem, vascular system, and the cerebral cortex as well as diverse mechanisms and pathological processes. The multidirectional action of purines in different cell types (blood vessels, neurons, and satellite glial cells) and through different types of purinergic receptors contributes to the etiopathogenesis of migraine pain. Adenosine triphosphate (ATP) and its derivatives are involved in initiation and propagation of migrenogenic signals in several ways: they participate in vasomotor mechanism, cortical spreading depression, and in fast transmission or cross-excitation based on the satellite glial cells in trigeminal ganglion. Contribution of purinergic signaling in the conduction of pain is realized through the activation of P1 and P2 receptors expressed widely in the central nervous system: on the neurons and glial cells as well as on the smooth muscles and endothelium in the vascular system. Therefore, the purinergic receptors can be an excellent target for pharmacologists constructing new antimigraine therapeutics. Moreover, the mechanisms facilitating ATP and adenosine degradation may prevent vasodilatation and thus avoid a secondary central sensitization during a migraine attack. Thus, agonists and antagonists of P receptors as well as ecto-enzymes metabolizing nucleotides/nucleosides could gain the growing attention as therapeutic agents.

Ectonucleotidases in Cancer and Inflammation

In 1970, Geoffrey Burnstock described the release of extracellular adenosine triphosphate (ATP) as a transmitter substance by nonadrenergic inhibitory nerves, and later in 1972, he formulated the hypothesis of purinergic neurotransmission [1]. Together with Che Su and John Bevan, Geoffrey Burnstock built the foundation to what was to become an entirely new field of research in biology [2]. While early studies focused on the role of purinergic receptors in neurotransmission, it soon became obvious that extracellular ATP and its hydrolyzed derivative adenosine had important roles in immune regulation. It is now recognized that purinergic signaling not only regulates neurotransmission and inflammation, but also influences diverse biological pathways, such as cell survival, proliferation, differentiation, lipid synthesis, and cell motility. In 1978, Burnstock proposed two types of purinergic receptors: P1 receptors selective for adenosine and P2 receptors selective for ATP and ADP. In 1985, a pharmacological approach was proposed to distinguish between two types of P2 receptors: ionotropic P2X and metabotropic P2Y receptors [3]. Nucleotides, such as ATP, are released by a variety of cell types especially under stress conditions. Cancer cells, for instance, are known to release ATP in the tumor microenvironment. Nucleotides that are released in response to a stress signal can be hydrolyzed by membrane-bound enzymes called ectonucleotidases. By regulating the levels of extracellular nucleotides and nucleosides, ectonucleotidases are thus involved in numerous physiological and pathological conditions. In this special issue, P. Chernogorova and R. Zeiser discuss the role of ectonucleotidases, in particular CD39 (NTPDase1) and CD73 (ecto-5′-nucleotidase) on allograft rejection, acute graft-versus-host disease (GvHD), and graft-versus-leukemia (GvL) effect. Interestingly, recent evidence suggests that purinergic signaling influences the severity of alloimmune responses. As highlighted by P. Chernogorova and R. Zeiser, these studies suggest potential clinical use of recombinant ectonucleotidases or adenosine receptor agonists for regulation of alloimmune responses which can be tailored according to the clinical situation. F. Ghiringhelli et al. review the current literature on the role of ectonucleotidases in tumor immune escape, with a focus on the immune regulatory function of Th17 cells. Th17 cells have emerged as key participants in a wide range of immune disorders and cancers. While the role of Th17 cells in cancer immunity remains controversial, F. Ghiringhelli et al. describe their recent seminal observation that ectonucleotidases are expressed on Th17 cells and that this is relevant in the context of tumor growth. A. Battastini and colleagues present an overview of the various roles of purinergic signaling in gliomas. In gliomas, the presence of an inflammatory infiltrate is directly correlated with tumor malignancy, and this appears to be regulated in part by purinergic signaling. The authors discuss the observation that ATP triggers glioma cells to release proinflammatory factors important for the recruitment of monocytes and neutrophils, thereby favoring tumor growth. The authors discuss their own work that aims to establish whether ectonucleotidases are involved in macrophage polarization in gliomas. A. Czopik and colleagues present a research article describing the protective role of CD73 in a model of colitis induced by administration of dextran sulfate sodium salt (DSS). The authors demonstrate that compared to wild-type mice, CD73-deficient gene-targeted mice are highly susceptible to DSS-induced colitis. The authors conclude that CD73 expression in the colon is critical for regulating the magnitude and the resolution of colonic immune responses. K. M. Dwyer and colleagues discuss the role of the CD39-adenosinergic axis in the pathogenesis of type 1 and type 2 diabetes. They discuss recent work suggesting that CD39 is involved in the pathophysiology of pancreatic dysfunction. This has importance clinical consequences, as drug development targeting different components of the pathway may be of relevance in the treatment of both type 1 and type 2 diabetes. The authors also discuss unanswered questions, such as what is the source of ecto-5′-nucleotidase activity given the lack of CD73 expression within the pancreas. J. Stagg and colleagues describe the role of CD73 and extracellular adenosine signaling in promoting tumor growth through paracrine and autocrine action. The authors discuss their recent work on the role of CD73 in spontaneous tumor growth and on the role of CD73 and adenosine on endothelial and immune cells. In conclusion, this special issue on ectonucleotidases aims to acquaint investigators not familiar with this field with important recent advances with the hope of attracting scientists to join the effort of exploiting modulation of purinergic signaling for therapeutic purposes to treat cancer and inflammatory diseases. John Stagg Linda F. Thompson Karen M. Dwyer

Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation

In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1-GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway.

CD73-Generated Extracellular Adenosine Creates Microenvironmental Conditions Favoring Growth and Survival of Chronic Lymphocytic Leukemia Cells

Abstract 621CD39 (ecto-nucleoside-triphosphate-diphosphohydrolase-1) and CD73 (5'-nucleotidase) are surface enzymes with extracellular catalytic sites. CD39 hydrolyses ATP/ADP to AMP, which is then converted to adenosine (ADO) by CD73. Once ADO is released in the extracellular milieu, it may re-enter the cell or engage different types of purinergic receptors, eliciting potent autocrine and paracrine cytoprotective, anti-inflammatory and immunosuppressive effects. Several lines of evidence suggest that the tumor microenvironment is marked by increased turnover of extracellular nucleotides and nucleosides, as well as by upregulation of ecto-enzymes that dismantle them. These alterations are believed to contribute to increasing local concentrations of ADO, creating local conditions that favor tumor expansion. We have investigated expression of CD39/CD73 by chronic lymphocytic leukemia (CLL) cells and evaluated the functional significance of the autocrine ADO production in supporting the survival of the leukemic cells and their expansion.Immunohistochemical analyses of lymph node biopsies indicated that CD39 was widely expressed by neoplastic cells, as well as by surrounding non-neoplastic stroma. On the contrary, CD73 was largely restricted to CLL proliferation centers and to cells with a prolymphocyte / para-immunoblast morphology, suggesting that ADO generation is confined to specific tissue niches and to specific cell subpopulations. The highest intensity of expression was scored by Ki-67+ CLL cells, in close association with T lymphocytes and localized to perivascular areas, where recirculation to and from lymphoid organs occurs.When analyzed in the peripheral blood, CD39 was constantly expressed, while CD73 was present in approximately 30% of cases and associated with markers of aggressive disease. CD39+/CD73+ CLL cells could selectively produce ADO from ADP, in a time- and concentration-dependent way. RT-PCR data indicated that CLL express high levels of A2A ADO receptor, further elevated in proliferating leukemic cells. Ligation of the A2A receptor by extracellular ADO or by the selective agonist CGS21680 increased cytoplasmic cAMP levels. The functional consequence was the inhibition of CXCL12-mediated chemotaxis, suggesting that the adenosinergic axis contributes to keeping CLL cells in a growth-favorable environment where they were attracted by CXCL12. Furthermore, activation of the axis conferred significant protection from spontaneous and/or fludarabine-induced apoptosis. The contribution of CD73 in regulating ADO production was confirmed by using a selective enzyme inhibitor.These results are consistent with the existence of an adenosinergic loop, active in selected CLL patients, creating conditions in the lymph node microenvironment that favor the development, maintenance and progression of the leukemia. Disclosures:No relevant conflicts of interest to declare.

2.35 CD73-Generated Extracellular Adenosine Creates Microenvironmental Conditions Favoring Growth and Survival of Chronic Lymphocytic Leukemia Cells

Abstract Abstract 621 CD39 (ecto-nucleoside-triphosphate-diphosphohydrolase-1) and CD73 (5'-nucleotidase) are surface enzymes with extracellular catalytic sites. CD39 hydrolyses ATP/ADP to AMP, which is then converted to adenosine (ADO) by CD73. Once ADO is released in the extracellular milieu, it may re-enter the cell or engage different types of purinergic receptors, eliciting potent autocrine and paracrine cytoprotective, anti-inflammatory and immunosuppressive effects. Several lines of evidence suggest that the tumor microenvironment is marked by increased turnover of extracellular nucleotides and nucleosides, as well as by upregulation of ecto-enzymes that dismantle them. These alterations are believed to contribute to increasing local concentrations of ADO, creating local conditions that favor tumor expansion. We have investigated expression of CD39/CD73 by chronic lymphocytic leukemia (CLL) cells and evaluated the functional significance of the autocrine ADO production in supporting the survival of the leukemic cells and their expansion. Immunohistochemical analyses of lymph node biopsies indicated that CD39 was widely expressed by neoplastic cells, as well as by surrounding non-neoplastic stroma. On the contrary, CD73 was largely restricted to CLL proliferation centers and to cells with a prolymphocyte / para-immunoblast morphology, suggesting that ADO generation is confined to specific tissue niches and to specific cell subpopulations. The highest intensity of expression was scored by Ki-67+ CLL cells, in close association with T lymphocytes and localized to perivascular areas, where recirculation to and from lymphoid organs occurs. When analyzed in the peripheral blood, CD39 was constantly expressed, while CD73 was present in approximately 30% of cases and associated with markers of aggressive disease. CD39+/CD73+ CLL cells could selectively produce ADO from ADP, in a time- and concentration-dependent way. RT-PCR data indicated that CLL express high levels of A2A ADO receptor, further elevated in proliferating leukemic cells. Ligation of the A2A receptor by extracellular ADO or by the selective agonist CGS21680 increased cytoplasmic cAMP levels. The functional consequence was the inhibition of CXCL12-mediated chemotaxis, suggesting that the adenosinergic axis contributes to keeping CLL cells in a growth-favorable environment where they were attracted by CXCL12. Furthermore, activation of the axis conferred significant protection from spontaneous and/or fludarabine-induced apoptosis. The contribution of CD73 in regulating ADO production was confirmed by using a selective enzyme inhibitor. These results are consistent with the existence of an adenosinergic loop, active in selected CLL patients, creating conditions in the lymph node microenvironment that favor the development, maintenance and progression of the leukemia. Disclosures: No relevant conflicts of interest to declare.

Clarifying the role of (apical) K+ channels in Na+ and Cl− transport

Clarifying the role of (apical) K⁺ channels in Na⁺ and Cl⁻ transport

An adenosinergic loop contributes to the generation of a microenvironment favoring growth and expansion of CLL cells (165.39)

Abstract CD39 and CD73 are surface enzymes with extracellular catalytic sites. CD39 hydrolyses ATP/ADP to AMP, which is then converted to adenosine (ADO) by CD73. ADO may re-enter the cell or engage different types of purinergic receptors, eliciting potent immunosuppressive responses. The working hypothesis is that CD39+/CD73+ leukemic cells from chronic lymphocytic leukemia (CLL) patients produce ADO, generating environmental conditions favoring growth, through the inhibition of the host immune response. Results indicate that while CD39 is constantly expressed, CD73 is present in 30% of a cohort of 300 CLL patients, where it associates with markers of aggressive disease. Differential CD73 expression is confirmed by immunohistochemical analyses of BM and LN biopsies, with the highest intensity scored by cells within the lymph nodes, where the proliferative core of the leukemia resides. Furthermore, CD73 is expressed at high levels in proliferation centers and by CLL cells surrounded by CD3+ T lymphocytes. CD39+/CD73+ CLL cells produce ADO from ADP, in a time- and dose-dependent way. RT-PCR data indicate that T and CLL lymphocytes express higher levels of a functional A2A receptor, as compared to age-matched healthy controls. These results suggest the existence of an adenosinergic loop, active in selected CLL patients, which likely contributes to determining conditions that favour the development, maintenance and progression of the leukemia.

Immune cell regulation by autocrine purinergic signalling

Stimulation of almost all mammalian cell types leads to the release of cellular ATP and autocrine feedback through a diverse array of purinergic receptors. Depending on the types of purinergic receptors that are involved, autocrine signalling can promote or inhibit cell activation and fine-tune functional responses. Recent work has shown that autocrine signalling is an important checkpoint in immune cell activation and allows immune cells to adjust their functional responses based on the extracellular cues provided by their environment. This Review focuses on the roles of autocrine purinergic signalling in the regulation of both innate and adaptive immune responses and discusses the potential of targeting purinergic receptors for treating immune-mediated disease.

Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP >> ATP > ADP >> UTP, and removal of external Ca(2+) dramatically suppressed the initial ATP-induced fluid secretion ( approximately 85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X(7) gene were used to evaluate the role of the P2X(7) receptor in nucleotide-evoked fluid secretion. P2X(7) receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X(7)-null mice. Consistent with these observations, the ATP-induced increases in [Ca(2+)](i) were nearly abolished in P2X(7)(-/-) submandibular acinar and duct cells. ATP appeared to also act through the P2X(7) receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X(7) receptor regulates fluid secretion in the mouse submandibular gland.

Ca 2+ influx through P2X receptors induces actin cytoskeleton reorganization by the formation of cofilin rods in neurites

In physiological and pathological events, extracellular ATP plays an important role by controlling several types of purinergic receptors and changing cytoskeleton dynamics. To know the process of ATP-dependent cytoskeleton remodeling, we focused on cofilin, a key regulator of actin cytoskeleton, and investigated the dynamics of cofilin in PC12 cells through fluorescent protein-labeled cofilin and actin, Ca 2+ imaging, and fluorescence resonance energy transfer (FRET) techniques. As a result, ATP induced intracellular Ca 2+ increase, following cofilin rods' formation. ATP-induced cofilin rods' formation was not observed in cells expressing unphosphorylatable variant of cofilin. A P2X receptor agonist, but not P2Y, induced the formation of cofilin rods, whereas calmodulin and calcineurin inhibitors suppressed it. These results indicate that Ca 2+ influx through P2X receptors induces the formation of cofilin rods via calcineurin-dependent dephosphorylation of cofilin. This pathway might be one candidate to explain the effects of ATP on neuronal development and injury.

Co‐localization and functional cross‐talk between A1 and P2Y1 purine receptors in rat hippocampus

Adenosine and ATP, via their specific P1 and P2 receptors, modulate a wide variety of cellular and tissue functions, playing a neuroprotective or neurodegenerative role in brain damage conditions. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, recent data suggest the existence of a heterodimerization and a functional interaction between P1 and P2 receptors in the brain. In particular, interactions of adenosine A1 and P2Y1 receptors may play important roles in the purinergic signalling cascade. In the present work, we investigated the subcellular localization/co-localization of the receptors and their functional cross-talk at the membrane level in Wistar rat hippocampus. This is a particularly vulnerable brain area, which is sensitive to adenosine- and ATP-mediated control of glutamatergic transmission. The postembedding immunogold electron microscopy technique showed that the two receptors are co-localized at the synaptic membranes and surrounding astroglial membranes of glutamatergic synapses. To investigate the functional cross-talk between the two types of purinergic receptors, we evaluated the reciprocal effects of their activation on their G protein coupling. P2Y1 receptor stimulation impaired the potency of A1 receptor coupling to G protein, whereas the stimulation of A1 receptors increased the functional responsiveness of P2Y1 receptors. The results demonstrated an A1–P2Y1 receptor co-localization at glutamatergic synapses and surrounding astrocytes and a functional interaction between these receptors in hippocampus, suggesting ATP and adenosine can interact in purine-mediated signalling. This may be particularly important during pathological conditions, when large amounts of these mediators are released.

Regulation of expression of early growth response transcription factors in rat primary cortical neurons by extracellular ATP

The zinc finger transcription factor early growth response-1 (Egr-1, NGFI-A, zif268, Krox 24, TIS8, ZENK) is upregulated immediately in the brain by cortical spreading depression (CSD) and other preconditioning stimuli and thus might participate in regulation of the overall genomic response to preconditioning. In the present study, the induction of expression of Egr-1 and other early growth response family members was characterized in rat primary cortical neuronal cultures. In neuronal cultures in vitro, depolarization or exposure to extracellular glutamate caused a 4-fold increase in egr-1 mRNA while exposure to extracellular ATP caused a 10-fold increase. The presence of mRNA encoding for multiple types of purinergic receptors was confirmed by RT-PCR. A number of nucleotide agonists proved effective in eliciting an increase in egr-1 mRNA. Over a limited range of concentration, the most effective agonists were ATP > ADP > α, β-methylene ATP > UTP > cAMP > UDP > AMP > adenosine. Pertussis toxin, suramin, reactive blue 2, PPADS, DPCPX and inhibitors of Protein Kinase C, Protein Kinase A and PI3 kinase significantly reduced the upregulation of egr-1 by exposure to extracellular ATP. These findings suggest that neuronal metabotropic purinergic receptor activation contributes to the induction of early growth response transcription factors and may provide a target that can be manipulated to increase ischemic tolerance of the brain in vivo.

Axon–glia interactions modulate axonal excitability in mammalian unmyelinated nerves

A period of electrical activity in unmyelinated nerve fibers is followed by a post-tetanic hyperpolarization (PTH), generated by the hyperactivity of the electrogenic Na +–K + pump. In order to protect the membrane potential against these strong hyperpolarizations, different types of axonal inward currents are activated during the PTH. We investigated in the rabbit vagus nerve one of these currents, which was activated by carbamylcholine (CCh). We observed that the effect of CCh on the PTH amplitude could be blocked or reversed with scopolamine. Moreover, the PTH amplitude increased when scopolamine alone was added to the perfusate, indicating that an endogenous muscarinic agonist was liberated in the preparation during the period of electrical activity. This CCh-activated current was TEA but not Ba 2+ or Cs + sensitive. It has been shown previously that muscarinic acetylcholine receptors (mAChRs) in the rabbit vagus nerve are located on the axonal but not glial membrane and that Schwann cells express several types of purinergic receptors, which activation evoke Ca 2+ transients in Schwann cells. We hypothesise that during electrical activity axons release a transmitter, presumably ATP. This transmitter evoke in the neighbouring Schwann cells a Ca 2+-dependent liberation of a endogenous muscarinic agonist, which in turn activates a TEA-sensitive inward current in axons. We suggest that the major purpose of this mechanism is the control of the membrane potential during and after a period of intense electrical activity when the Na +–K + pump generates a robust PTH.

Different purinergic receptors lead to intracellular calcium increases in pancreatic ducts.

Extracellular adenosine 5'-triphosphate (ATP) has been described to act as a regulator in many cells and tissues, including epithelia, and in the gastrointestinal tract ATP is one of the substances involved in non-cholinergic non-adrenergic control. However, very little is known about the effect of ATP on pancreatic ducts, which normally secrete bicarbonate-rich fluid in response to secretin. Hence, the aim of our present study was to test the effect of ATP and other nucleotides on intracellular Ca2+ activity ([Ca2+]i) of pancreatic ducts, and thereby get information about purinergic receptors that might play a role in the regulation of pancreatic bicarbonate transport. Native intralobular ducts were obtained from rat pancreas and [Ca2+]i in 10-20 cells was measured using the fura-2 method. ATP (10(-4) mol/l) evoked a characteristic biphasic Ca2+ transient in duct cells. Nucleotides, used to classify the P2 receptors, acted with the following potency on the peak Ca2+ in many ducts: uridine 5'-triphosphate (UTP) >/= ATP >inosine 5'-triphosphate >/= 2-methylthio-ATP > beta,gamma-methyl-ATP > adenosine. However, although the peak [Ca2+]i responses to ATP and UTP were similar, the plateau [Ca2+]i was nearly doubled with UTP. Moreover, in about one-third of the ducts studied, UTP had no effect on cell Ca2+, while the response to ATP was normal. In further experiments we found that removal of extracellular Mg2+ increased the peak [Ca2+]i evoked in response to ATP. 2'&3'-O-(4-benzoylbenzoyl) ATP (BzATP) evoked a monophasic and slower increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+, or by addition of 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS). Taken together, our data indicate that there are two types of purinergic receptors on pancreatic ducts through which ATP can act. These are pharmacologically known as P2U and P2Z receptors and may correspond to P2Y2 and P2X7 receptors.

Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor.

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.

Presence of a metabotropic and an ionotropic purinergic receptor on rat submandibular ductal cells.

ATP (1 microM-1 mM) increased the intracellular Ca2+ concentration ([Ca2+]i) in rat submandibular ductal cells. The dose-response curve was biphasic with a first plateau approximately 10 microM and a second increase at concentrations higher than 100 microM. This second increase was abolished in the absence of extracellular calcium or in the presence of Coomassie blue and could be mimicked by benzoyl-ATP. The activation of this response increased the uptake of extracellular manganese and the release of kallikrein, a ductal cell marker. The response at low concentrations of ATP was mimicked by ADP, 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP; which was the most potent agonist), adenosine 5'-O-(2-thiodiphosphate), and UTP. Removal of extracellular calcium did not abolish this response, but ADP or 2-MeS-ATP had no effect after a preincubation with thapsigargin. A preincubation with 2-MeS-ATP desensitized the receptor involved in the response to both ADP and UTP. Similarly, a pretreatment with UTP prevented a further response to ADP. ADP increased the intracellular concentration of inositol 1,4,5-trisphosphate but did not affect the secretion of kallikrein. In conclusion, rat submandibular ductal cells possess two types of purinergic receptors: a metabotropic (P2y1) receptor and a P2x ionotropic purinergic receptor coupled to a manganese-permeant calcium channel and to kallikrein secretion.

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P.

The effect of extracellular ATP on the intracellular calcium concentration ([Ca2+]i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1-30 microM concentration range and a further increase at concentrations higher than 100 microM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATP tau S blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca2+]i in response to 100 microM carbachol. By itself, substance P (100 pM-1 microM) increased the [Ca2+]i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 microM and substance P (100 pM-1 microM) increased the release of inositol trisphosphate (IP3) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 microM carbachol (-50%) and by 100 nM substance P (-60% at 1 nM substance P and -40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 microM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P2z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca2+]i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca2+]i.

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca2+]i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca2+]i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca2+]i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP3) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca2+]i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca2+]i. © 1996 Wiley-Liss, Inc.