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.     

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.     

Adenosine and blood platelets

Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. Adenosine released from different cells or generated through the activity of cell-surface ectoenzymes exerts its effects through the binding of four different G-protein-coupled adenosine receptors. In platelets, binding of A₂ subtypes (A_2A or A_2B) leads to consequent elevation of intracellular cyclic adenosine monophosphate, an inhibitor of platelet activation. The significance of this ligand and its receptors for platelet activation is addressed in this review, including how adenosine metabolism and its A₂ subtype receptors impact the expression and activity of adenosine diphosphate receptors. The expression of A₂ adenosine receptors is induced by conditions such as oxidative stress, a hallmark of aging. The effect of adenosine receptors on platelet activation during aging is also discussed, as well as potential therapeutic applications.     

Purinergic receptors and synaptic transmission in enteric neurons

Purines such as ATP and adenosine participate in synaptic transmission in the enteric nervous system as neurotransmitters or neuromodulators. Purinergic receptors are localized on the cell bodies or nerve terminals of different functional classes of enteric neurons and, with other receptors, form unique receptor complements. Activation of purinergic receptors can regulate neuronal activity by depolarization, by regulating intracellular calcium, or by modulating second messenger pathways. Purinergic signaling between enteric neurons plays an important role in regulating specific enteric reflexes and overall gastrointestinal function. In the present article, we review evidence for purine receptors in the enteric nervous system, including P1 (adenosine) receptors and P2 (ATP) receptors. We will explore the role they play in mediating fast and slow synaptic transmission and in presynaptic inhibition of transmission. Finally, we will examine the molecular properties of the native receptors, their signaling mechanisms, and their role in gastrointestinal pathology.     

Receptor crosstalk: haloperidol treatment enhances A2A adenosine receptor functioning in a transfected cell model

A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.     

Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson’s disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 μM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A₁ (A₁R) and/or A_2A receptors (A_2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A₁R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A₁R agonist, did not alter GUO effects. Regarding A_2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A_2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A₁R antagonist DPCPX, and the A_2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A₁R-A_2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

     

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.     

The P2X<Subscript>7</Subscript> receptor in retinal ganglion cells: A neuronal model of pressure-induced damage and protection by a shifting purinergic balance

Retinal ganglion cells process the visual signal and transmit it along their axons in the optic nerve to the brain. Molecular, immunohistochemical, and functional analyses indicate that the majority of retinal ganglion cells express the ionotropic P2X(7) receptor. Stimulation of the receptor can lead to a rise in intracellular calcium and cell death, although death does not involve the opening of a large diameter pore. Adenosine acting at A(3) receptors can attenuate the rise in calcium and death accompanying P2X(7) receptor activation, suggesting that dephosphorylation of ATP into adenosine is neuroprotective and that the balance of extracellular purines can influence neuronal survival. Increased intraocular pressure can lead to release of excessive extracellular ATP in the retina and damage ganglion cells by acting on P2X(7) receptors, implicating a role for the receptor in the loss of ganglion cell activity in glaucoma. In summary, the activation of P2X(7) receptors has both physiologic and pathophysiologic implications for ganglion cell function. These characteristics may also provide an insight into the contributions the P2X(7) receptor makes to neurons elsewhere.     

Limonene, a natural cyclic terpene, is an agonistic ligand for adenosine A(2A) receptors

Limonene is a major aromatic compound in essential oils extracted from citrus rind. The application of limonene, especially in aromatherapy, has expanded significantly, but its potential effects on cellular metabolism have been elusive. We found that limonene directly binds to the adenosine A_2A receptor, which may induce sedative effects. Results from an in vitro radioligand binding assay showed that limonene exhibits selective affinity to A_2A receptors. In addition, limonene increased cytosolic cAMP concentration and induced activation of protein kinase A and phosphorylation of cAMP-response element-binding protein in Chinese hamster ovary cells transfected with the human adenosine A_2A receptor gene. Limonene also increased cytosolic calcium concentration, which can be achieved by the activation of adenosine A_2A receptors. These findings suggest that limonene can act as a ligand and an agonist for adenosine A_2A receptors.     

腺苷及其衍生物的心血管效应和作用机制

在实验中观察了腺苷及其衍生物的心血管效应和作用机制,结果表明：（１）腺苷和２－氯腺苷先引起由颈动脉体化学感受器内的Ａ２受体所中介的血压短暂升高,随之为心血管系统Ａ１和Ａ２受体中介的持久而明显的血压降低;（２）腺苷受体激动剂环戊腺苷抑制窦房结起搏细胞的电生理活动;（３）环戊腺苷减弱异丙肾上腺素诱发的早发和迟发性后除极及触发电活动;（４）内源性腺苷参与无氧所致的心率减慢;（５）预缺血时腺苷受体的激活及     

Cell-autonomous regulation of hematopoietic stem cell cycling activity by ATP

Extracellular nucleotides regulate many cellular functions through activation of purinergic receptors in the plasma membrane. Here, we show that in hematopoietic stem cell (HSC), ATP is stored in vesicles and released in a calcium-sensitive manner. HSC expresses ATP responsive P2X receptors and in vitro pharmacological P2X antagonism restrained hematopoietic progenitors proliferation, but not myeloid differentiation. In mice suffering from chronic inflammation, HSCs were significantly expanded and their cycling activity was sensitive to treatment with the P2X antagonist periodate-oxidized 2,3-dialdehyde ATP. Our results indicate that ATP acts as an autocrine stimulus in regulating HSCs pool size.     

Activation of adenosine A1 and A2A receptors modulates dopamine D2 receptor-induced responses in stably transfected human neuroblastoma cells

Adenosine can influence dopaminergic neurotransmission in the basal ganglia via postsynaptic interaction between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (approximately 100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH-SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCI treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCI-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCI, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS-21680 counteracted the D2 receptor-induced [Ca2+]i responses. The present study shows that SH-SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor-mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.     

G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions

The medicinal chemistry and pharmacology of the four subtypes of adenosine receptors (ARs) and the eight subtypes of P2Y receptors (P2YRs, activated by a range of purine and pyrimidine mono- and dinucleotides) has recently advanced significantly leading to selective ligands. X-ray crystallographic structures of both agonist- and antagonist-bound forms of the A(2A)AR have provided unprecedented three-dimensional detail concerning molecular recognition in the binding site and the conformational changes in receptor activation. It is apparent that this ubiquitous cell signaling system has implications for understanding and treating many diseases. ATP and other nucleotides are readily released from intracellular sources under conditions of injury and organ stress, such as hypoxia, ischemia, or mechanical stress, and through channels and vesicular release. Adenosine may be generated extracellularly or by cellular release. Therefore, depending on pathophysiological factors, in a given tissue, there is often a tonic activation of one or more of the ARs or P2YRs that can be modulated by exogenous agents for a beneficial effect. Thus, this field has provided fertile ground for pharmaceutical development, leading to clinical trials of selective receptor ligands as imaging agents or for conditions including cardiac arrhythmias, ischemia/reperfusion injury, diabetes, pain, thrombosis, Parkinson's disease, rheumatoid arthritis, psoriasis, dry eye disease, pulmonary diseases such as cystic fibrosis, glaucoma, cancer, chronic hepatitis C, and other diseases.     

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.     

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.     

CNS Adenosine A1 Receptors Are Altered After the Administration of Convulsant 3-Mercaptopropionic Acid and Cyclopentyladenosine: An Autoradiographic Study

Rat CNS adenosine A₁ receptors were studied by quantitative autoradiography after the administration of convulsant 3-mercaptopropionic acid (MP) and an adenosine analogue cyclopentyladenosine (CPA), using 2-chloro-N⁶-[cyclopentyl-2,3,4,5-³H adenosine]-([³H]CCPA) as radioactive ligand. Specific binding was quantified in hippocampus, cerebellum, cerebral cortex, thalamic nuclei, superior colliculus and striatum, and the highest densities were found in CA1, CA2, and CA3 hippocampus subareas and the lowest levels in superior colliculus and striatum. MP administration (150 mg/kg, i.p.) produced significant increases in [³H]CCPA binding in CA1 subarea at seizure (15%) and postseizure (21%) and in CA2 at seizure (15%) but a tendency to decrease in dentate gyrus. There was an increase in cerebellum at seizure (18%) but no significant changes in the other studied regions. CPA injection (2 mg/kg, i.p.) enhanced [³H]CCPA binding in CA1 and CA2 areas (17–18%) but not in CA3 area of the hippocampus. When CPA was administered before MP, which delayed seizure onset, an increase in [³H]CCPA binding in CA1 hippocampus subarea (19%) and cerebellum (28%) was also observed. Results showed that the administration of convulsant MP and adenosine analogue CPA exerts differential effects on adenosine A₁ receptors in CNS areas; hippocampus is the most affected area with all treatments, specially CA1 subarea, supporting an essential role in convulsant activity as well as in seizure prevention.     

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.