The P2X7 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 P2X₇ 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 A₃ receptors can attenuate the rise in calcium and death accompanying P2X₇ 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 P2X₇ receptors, implicating a role for the receptor in the loss of ganglion cell activity in glaucoma. In summary, the activation of P2X₇ receptors has both physiologic and pathophysiologic implications for ganglion cell function. These characteristics may also provide an insight into the contributions the P2X₇ receptor makes to neurons elsewhere.     

P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> purinoceptors in the rat enteric nervous system

Adenosine 5-triphosphate receptors are known to be involved in fast excitatory postsynaptic currents in myenteric neurons of the digestive tract. In the present study, the distribution of P2X₂ and P2X₃ receptor mRNA was examined by in situ hybridisation while P2X₂ and P2X₃ receptor protein was localised by immunohistochemical methods. In addition, P2X₂ and P2X₃ receptors were colocalised with calbindin and calretinin in the myenteric and submucosal plexus. P2X₂- and P2X₃-immunoreactive neurons were found in the myenteric and submucosal plexuses throughout the entire length of the rat digestive tract from the stomach to the colon. Approximately 60%, 70% and 50% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 56% and 45% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₂ receptor. Approximately 10%, 2% and 15% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 62% and 40% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₃ receptor. Double-labelling studies showed that about 10–25% of the neurons with P2X₂ immunoreactivity in myenteric plexus and 30–50% in the submucosal plexus were found to express calbindin or calretinin. About 80% of the neurons with P2X₃ receptor immunoreactivity in the myenteric plexus and about 40% in the submucosal plexus expressed calretinin. Approximately 30–75% of the neurons with P2X₃ receptor immunoreactivity in the submucosal plexus expressed calbindin, while none of them were found to express calbindin in the myenteric plexus.     

P2X<Subscript>3</Subscript> Receptor Involvement in Pain States

The understanding of how pain is processed at each stage in the peripheral and central nervous system is the precondition to develop new therapies for the selective treatment of pain. In the periphery, ATP can be released from various cells as a consequence of tissue injury or visceral distension and may stimulate the local nociceptors. The highly selective distribution of P2X₃ and P2X_2/3 receptors within the nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. Depolarization by ATP of neurons in pain–relevant neuronal structures such as trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn neurons are well investigated. P2X receptor-mediated afferent activation appears to have been implicated in visceral and neuropathic pain and even in migraine and cancer pain. This article reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X₃ receptor in different states of pain.     

<Emphasis Type="Italic">N</Emphasis>-Arylpiperazine modified analogues of the P2X<Subscript>7</Subscript> receptor KN-62 antagonist are potent inducers of apoptosis of human primary osteoclasts

Summary The P2X₇ nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L-tyrosine derivatives 1–3 as potent P2X₇ antagonists on human primary osteoclasts. We found that the level of expression of P2X₇ receptor increased after treatment with the derivatives 1–3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1–3 was found on human osteoblasts. Our results suggest that the development of specific P2X₇ receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies.     

Development of nerves expressing P2X<Subscript>3</Subscript> receptors in the myenteric plexus of rat stomach

Development of neurones and fibres expressing P2X₃ receptors in the myenteric plexus of rat stomach and coexistence of the P2X₃ receptor with calbindin, calretinin and NOS during postnatal development, were investigated with immunostaining methods. Extrinsic nerves expressing P2X₃ receptors appeared as early as E12 and were localised in the trunk and branches of the vagus nerve, which extended rapidly onto the whole rat stomach from E12 to E14. Intrinsic neurone cell bodies with P2X₃-immunoreactivity in the myenteric ganglia were first demonstrated postnatally at P1, and at P14, when the number of neurones expressing the P2X₃ receptor peaked at 45%. P2X₃ receptor-immunoreactivity decreased subsequently, and at P60 only about 11% were P2X₃-immunoreactive. Intraganglionic laminar nerve endings and intramuscular arrays were first demonstrated postnatally at P1 and P7, respectively. In the early postnatal days, there were many growth cone-like structures with strong P2X₃ immunostaining associated with these endings and arrays. Double-immunostaining showed that 9–15% of P2X₃-immunoreactive neurones in the gastric myenteric plexus expressed calbindin D-28 k only in the early postnatal days, while 14–21% of neurones from P1 to P60 increasingly expressed calretinin. About 20% of neurones with P2X₃ immunoreactivity coexpressed NOS throughout perinatal development.     

LncRNA NONRATT021972 involved the pathophysiologic processes mediated by P2X<Subscript>7</Subscript> receptors in stellate ganglia after myocardial ischemic injury

Adenosine triphosphate (ATP) acts on P2X receptors to initiate signal transmission. P2X₇ receptors play a role in the pathophysiological process of myocardial ischemic injury. Long noncoding RNAs (lncRNAs) participate in numerous biological functions independent of protein translation. LncRNAs are implicated in nervous system diseases. This study investigated the effects of NONRATT021972 small interference RNA (siRNA) on the pathophysiologic processes mediated by P2X₇ receptors in stellate ganglia (SG) after myocardial ischemic injury. Our results demonstrated that the expression of NONRATT021972 in SG was significantly higher in the myocardial ischemic (MI) group than in the control group. Treatment of MI rats with NONRATT021972 siRNA, the P2X₇ antagonist brilliant blue G (BBG), or P2X₇ siRNA improved the histology of injured ischemic cardiac tissues and decreased the elevated concentrations of serum myocardial enzymes, creatine kinase (CK), CK isoform MB (CK-MB), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) compared to the MI rats. NONRATT021972 siRNA, BBG, or P2X₇ siRNA treatment in MI rats decreased the expression levels of P2X₇ immunoreactivity, P2X₇ messenger RNA (mRNA), and P2X₇ protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) in the SG compared to MI rats. NONRATT021972 siRNA treatment prevented the pathophysiologic processes mediated by P2X₇ receptors in the SG after myocardial ischemic injury.     

Tissue distribution of P2X<Subscript>4</Subscript> receptors studied with an ectodomain antibody

A monoclonal antibody was developed to the extracellular domain of the rat P2X₄ receptor. The antibody was highly selective among all rat P2X receptor subunits, and recognised only the oligomeric, non-denatured form of the P2X₄ receptor. Immunohistochemistry showed an extensive pattern of distribution throughout the central and peripheral nervous systems, the epithelia of ducted glands and airways, smooth muscle of bladder, gastrointestinal tract, uterus, and arteries, uterine endometrium and fat cells. The protein was identified by Western blotting in membrane extracts of these tissues, and the ectodomain antibody immunoprecipitated a protein that was recognised with a P2X₄ receptor C terminus antibody. The findings indicate that the P2X₄ receptor subunit has a very extensive distribution among mammalian tissues, and this suggests possible new functional roles.The work was supported by the Wellcome Trust (R.S., R.A.N.) and the British Heart Foundation (G.B.)     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Balance of purines may determine life or death of retinal ganglion cells as A3 adenosine receptors prevent loss following P2X7 receptor stimulation

The purines ATP and adenosine can act as a coordinated team of transmitters. As extracellular adenosine is frequently derived from the enzymatic dephosphorylation of released ATP, the distinct actions of the two purines can be synchronized. In retinal ganglion cells (RGCs), stimulation of the P2X7 receptor for ATP leads to increased intracellular Ca2+ and death. Here we define the contrasting effects of adenosine and identify protective actions mediated by the A3 receptor. Adenosine attenuated the rise in Ca2+ produced by the P2X7 agonist 3'-O-(4-benzoylbenzoyl)ATP (BzATP). Adenosine was also neuroprotective, increasing the survival of ganglion cells exposed to BzATP. The A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronimide (Cl-IB-MECA) mimicked the inhibition of the Ca2+ rise, whereas the A3 antagonist 3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191) reduced the protective effects of adenosine. Both Cl-IB-MECA and a second A3 receptor agonist IB-MECA reduced the cell loss triggered by BzATP. The actions of BzATP were mimicked by ATPgammaS, but not by ATP. In summary, adenosine can stop the rise in Ca2+ and cell death resulting from stimulation of the P2X7 receptor on RGCs, with the A3 adenosine receptor contributing to this protection. Hydrolysis of ATP into adenosine and perhaps inosine shifts the balance of purinergic action from that of death to the preservation of life.     

Changes in P2X<Subscript>3</Subscript> purinoceptors in sensory ganglia of the mouse during embryonic and postnatal development

The expression of the P2X₃ nucleotide receptor in embryonic day 14–18, postnatal day 1–14 and adult mouse sensory ganglia was examined using immunohistochemistry. Nearly all sensory neurons in dorsal root ganglia, trigeminal ganglia and nodose ganglia in embryos at embryonic day 14 expressed P2X₃ receptors, but after birth there was a gradual decline to about 50% of neurons showing positive immunostaining for P2X₃. In embryos there were only small neurons, while from postnatal day 7 both large and small neurons were present. Isolectin B₄ (IB₄)-positive neurons in dorsal, trigeminal and nodose ganglia did not appear until birth, but the numbers increased to about 50% by postnatal day 14 when a high proportion of IB₄-positive neurons were also positively labelled for the P2X₃ receptor. About 10% of neurons in dorsal, trigeminal and nodose ganglia were positive for calcitonin gene-related peptide in embryos, nearly all of which stained for P2X₃ receptors. This increased postnatally to about 35–40% in adults, although only a few colocalised with P2X₃ receptors. Neurofilament 200 was expressed in about 50% of neurons in trigeminal ganglia in the embryo, and this level persisted postnatally. All neurofilament 200-positive neurons stained for P2X₃ in embryonic dorsal root ganglia, trigeminal ganglia and nodose ganglia, but by adulthood this was significantly reduced. The neurons that were positive for calbindin in embryonic dorsal, trigeminal and nodose ganglia showed colocalisation with P2X₃ receptors, but few showed colocalisation postnatally.     

Trehalose uptake through P2X7 purinergic channels provides dehydration protection

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.     

P2X<Subscript>7</Subscript> Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X₇ receptor activated a reversible, Ca²⁺-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C_m) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C_m without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu²⁺. The capacitance decrease persisted in Na⁺-, Ca²⁺- and Cl⁻-free external saline or with Ca²⁺-free pipette solution. It is concluded that ATP activation of the inotropic P2X₇ receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca²⁺ or influx of Na⁺ or Ca²⁺ through the receptor-gated channel.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes

Contact of T lymphocytes with nicotinamide adenine dinucleotide (NAD) or ATP causes cell death that requires expression of purinergic receptor P2X(7) (P2X(7)R). T cell subsets differ in their responses to NAD and ATP, which awaits a mechanistic explanation. Here, we show that sensitivity to ATP correlates with P2X(7)R expression levels in CD4 cells, CD8 cells and CD4(+)CD25(+) cells from both C57BL/6 and BALB/c mice. But P2X(7)R ligands do not only induce cell death but also shedding of CD62L. It is shown here that in CD62L(high) T cells, CD62L shedding correlates with low expression of P2X(7)Rs and lower cell death, whereas in CD62L(low) cells P2X(7)R expression and death are higher. The possibility is therefore investigated that P2X(7)Rs induce T cell activation. Experiments show that spontaneous T cell proliferation is somewhat higher in cells expressing P2X(7)Rs, but this effect we suggest is caused by P2X(7)R expression on accessory cells.     

The role of P2X7 receptors in tissue fibrosis: a brief review

Many previous studies have demonstrated that P2X₇ receptors (P2X₇Rs) have a pleiotropic function in different pathological conditions and could represent a novel target for the treatment of a range of diseases. In particular, recent studies have explored the role of P2X₇R in fibrosis, the pathological outcome of most chronic inflammatory diseases. The aim of this review is to discuss the biological features of P2X₇R and summarize the current knowledge about the putative role of the P2X₇R in triggering fibrosis in a wide spectrum of organs such as the lung, kidney, liver, pancreas, and heart.