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.     

The effects of NONRATT021972 lncRNA siRNA on PC12 neuronal injury mediated by P2X<Subscript>7</Subscript> receptor after exposure to oxygen-glucose deprivation

Adenosine triphosphate (ATP) participates in signal transmission by acting on P2X receptors, and the P2X₇ receptor is involved in the pathophysiological changes of ischemic injury. The PC12 cell line is a popular model system to study sympathetic neuronal function. Long noncoding RNAs (lncRNAs) are highly expressed in the nervous system and serve as regulatory RNAs. In this study, the effects of NONRATT021972 lncRNA siRNA on P2X₇-mediated PC12 neuronal injury after exposure to oxygen-glucose deprivation (OGD) were investigated. Our results showed that the viability of PC12 cells cultured with OGD or the P2X₇ agonist BzATP was significantly decreased. Treatment with NONRATT021972 siRNA reversed the decreased viability of PC12 cells under OGD conditions. The upregulated P2X₇ mRNA and protein levels in PC12 cells under OGD conditions or BzATP treatment were significantly decreased when pretreated with NONRATT021972 siRNA. Moreover, NONRATT021972 siRNA treatment effectively suppressed the increase in [Ca²⁺]_i induced by OGD or P2X₇ agonists (ATP or BzATP) in PC12 cells. Therefore, treatment with NONRATT021972 siRNA may decrease sympathetic neuronal injury induced by ischemia.     

Sensory–sympathetic coupling in superior cervical ganglia after myocardial ischemic injury facilitates sympathoexcitatory action via P2X7 receptor

P2X receptors participate in cardiovascular regulation and disease. After myocardial ischemic injury, sensory–sympathetic coupling between rat cervical DRG nerves and superior cervical ganglia (SCG) facilitated sympathoexcitatory action via P2X₇ receptor. The results showed that after myocardial ischemic injury, the systolic blood pressure, heart rate, serum cardiac enzymes, IL-6, and TNF-α were increased, while the levels of P2X₇ mRNA and protein in SCG were also upregulated. However, these alterations diminished after treatment of myocardial ischemic (MI) rats with the P2X₇ antagonist oxATP. After siRNA P2X₇ in MI rats, the systolic blood pressure, heart rate, serum cardiac enzymes, the expression levels of the satellite glial cell (SGC) or P2X₇ were significantly lower than those in MI group. The phosphorylation of ERK 1/2 in SCG participated in the molecular mechanism of the sympathoexcitatory action induced by the myocardial ischemic injury. Retrograde tracing test revealed the sprouting of CGRP or SP sensory nerves (the markers of sensory afferent fibers) from DRG to SCG neurons. The upregulated P2X₇ receptor promoted the activation of SGCs in SCG, resulting in the formation of sensory–sympathetic coupling which facilitated the sympathoexcitatory action. P2X₇ antagonist oxATP could inhibit the activation of SGCs and interrupt the formation of sensory–sympathetic coupling in SCG after the myocardial ischemic injury. Our findings may benefit the treatment of coronary heart disease and other cardiovascular diseases.     

Electrophysiological studies of upregulated P2X7 receptors in rat superior cervical ganglia after myocardial ischemic injury

Myocardial ischemic injury activates cardiac sympathetic afferent fibers and elicits a sympathoexcitatory reflex by exciting sympathetic efferent action, with resultant augmentation of myocardial oxygen consumption, leading to a vicious cycle of exaggerating myocardial ischemia. P2X₇ receptor participates in the neuronal functions and the neurological disorders. This study examined the role of P2X₇ receptor of superior cervical ganglia (SCG) in sympathoexcitatory reflex. Our results showed that the expression of P2X₇ receptor at both mRNA and protein in SCG was increased after myocardial ischemic injury. P2X₇ receptor agonists at the same concentration activated much larger amplitudes of the currents in the SCG neurons of myocardial ischemic rats than those in control rats. P2X₇ receptor antagonist (brilliant blue G, BBG) significantly inhibited P2X₇ receptor agonist-activated currents in the SCG neurons. Excessive phosphorylation of MAPK ERK1/2 upon the activation of P2X₇ receptor might be a mechanism mediating the signal transduction after myocardial ischemic injury. Therefore, the sensitized P2X₇ receptor in SCG was involved in the nociceptive transmission of sympathoexcitatory reflex induced by myocardial ischemic injury.     

P2X7 receptor inhibition attenuated sympathetic nerve sprouting after myocardial infarction via the NLRP3/IL‐1β pathway

Mounting evidence supports the hypothesis that inflammation modulates sympathetic sprouting after myocardial infarction (MI). The myeloid P2X₇ signal has been shown to activate the nucleotide‐binding and oligomerization domain‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome, a master regulator of inflammation. We investigated whether P2X₇ signal participated in the pathogenesis of sympathetic reinnervation after MI, and whether NLRP3/interleukin‐1β (IL‐1β) axis is involved in the process. We explored the relationship between P2X₇ receptor (P2X₇R) and IL‐1β in the heart tissue of lipopolysaccharide (LPS)‐primed naive rats. 3′‐O‐(4‐benzoyl) benzoyl adenosine 5′‐triphosphate (BzATP), a P2X₇R agonist, induced caspase‐1 activation and mature IL‐1β release, which was further neutralized by a NLRP3 inhibitor (16673‐34‐0). MI was induced by coronary artery ligation. Following infarction, a marked increase in P2X₇R was localized within infiltrated macrophages and observed in parallel with an up‐regulation of NLRP3 inflammasome levels and the release of IL‐1β in the left ventricle. The administration of A‐740003 (a P2X₇R antagonist) significantly prevented the NLRP3/IL‐1β increase. A‐740003 and/or Anakinra (an IL‐1 receptor antagonist) significantly reduced macrophage infiltration as well as macrophage‐based IL‐1β and NGF (nerve growth factor) production and eventually blunted sympathetic hyperinnervation, as assessed by the immunofluorescence of tyrosine hydroxylase (TH) and growth‐associated protein 43 (GAP 43). Moreover, the use of Anakinra partly attenuated sympathetic sprouting. This indicated that the effect of P2X₇ on neural remodelling was mediated at least partially by IL‐1β. The arrhythmia score of programmed electric stimulation was in accordance with the degree of sympathetic hyperinnervation. In vitro studies showed that BzATP up‐regulated secretion of nerve growth factor (NGF) in M1 macrophages via IL‐1β. Together, these data indicate that P2X₇R contributes to neural and cardiac remodelling, at least partly mediated by NLRP3/IL‐1β axis. Therapeutic interventions targeting P2X₇ signal may be a novel approach to ameliorate arrhythmia following MI.     

P2X<Subscript>7</Subscript> receptor and klotho expressions in diabetic nephropathy progression

Diabetes mellitus is characterized by increased levels of reactive oxygen species (ROS), leading to high levels of adenosine triphosphate (ATP) and the activation of purinergic receptors (P2X₇), which results in cell death. Klotho was recently described as a modulator of oxidative stress and as having anti-apoptotic properties, among others. However, the roles of P2X₇ and klotho in the progression of diabetic nephropathy are still unclear. In this context, the aim of the present study was to characterize P2X₇ and klotho in several stages of diabetes in rats. Diabetes was induced in Wistar rats by streptozotocin, while the control group rats received the drug vehicle. From the 1st to 8th weeks after the diabetes induction, the animals were placed in metabolic cages on the 1st day of each week for 24 h to analyze metabolic parameters and for the urine collection. Then, blood samples and the kidneys were collected for biochemical analysis, including Western blotting and qPCR for P2X₇ and klotho. Diabetic rats presented a progressive loss of renal function, with reduced nitric oxide and increased lipid peroxidation. The P2X₇ and klotho expressions were similar up to the 4th week; then, P2X₇ expression increased in diabetes mellitus (DM), but klotho expression presented an opposite behavior, until the 8th week. Our data show an inverse correlation between P2X₇ and klotho expressions through the development of DM, which suggests that the management of these molecules could be useful for controlling the progression of this disease and diabetic nephropathy.     

LncRNA NONRATT021972 siRNA attenuates P2X7 receptor expression and inflammatory cytokine production induced by combined high glucose and free fatty acids in PC12 cells

Diabetic neuropathy (DNP) is a frequent chronic complication of diabetes mellitus with potentially life-threatening outcomes. High glucose and elevated free fatty acids (FFAs) have been recently recognized as major causes of nervous system damage in diabetes. Our previous study has indicated extracellular stimuli, such as high glucose and/or FFA stress, may activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway and induce a p38 MAPK-dependent sensitization of the P2X7 receptor and release of inflammatory factors in PC12 cells, while the mechanisms underlying remain to be elucidated. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes, including activation of a series of pathway signalings. Here, we showed combined high d-glucose and FFAs (HGHF) induced an increment of lncRNA-NONRATT021972 (NONCODE ID, nc021972) in PC12 cells. Nc021972 small interference RNA (siRNA) alleviated HGHF-induced activation of p38 MAPK, expression of the P2X7 receptor, and [Ca²⁺]_i increment upon P2X7 receptor activation. Further experiments showed that there existed a crosstalk between nc021972 and the p38 MAPK signaling pathway. Inhibition of p38 MAPK signaling decreased nc021972-induced expression of the P2X7 receptor and [Ca²⁺]_i increment upon P2X7 receptor activation. Also, nc021972 siRNA inhibited HGHF-induced PC12 release of TNF-α and IL-6 and rescued decreased cell viability mediated by the P2X7 receptor. Therefore, inhibition of nc021972 may serve as a novel therapeutic strategy for diabetes complicated with nervous inflammatory diseases.     

The effect of sinomenine in diabetic neuropathic pain mediated by the P2X<Subscript>3</Subscript> receptor in dorsal root ganglia

Type 2 diabetes mellitus (T2DM) accounts for more than 90% of all cases of diabetes mellitus (DM). Diabetic neuropathic pain (DNP) is a common complication of T2DM. Sinomenine is a natural bioactive component extracted from the Sinomenium acutum and has anti-inflammatory effects. The aim of our study was to investigate the effects of sinomenine on DNP mediated by the P2X₃ receptor in dorsal root ganglia (DRG). The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in T2DM rats were lower than those of control rats. MWT and TWL in T2DM rats treated with sinomenine were higher compared with those in T2DM rats. The expression levels of the P2X₃ protein and mRNA in T2DM rat DRG were higher compared with those of the control, while those in T2DM rats treated with sinomenine were significantly lower compared with those of the T2DM rats. Sinomenine significantly inhibited P2X₃ agonist ATP-activated currents in HEK293 cells transfected with the P2X₃ receptor. Sinomenine decreased the phosphorylation and activation of P38MAPK in T2DM DRG. Therefore, sinomenine treatment may suppress the up-regulated expression and activation of the P2X₃ receptor and relieve the hyperalgesia potentiated by the activation of P38MAPK in T2DM rats.     

Expression of the apoptotic calcium channel P2X<Subscript>7</Subscript> in the glandular epithelium

In the current study, expression of the apoptotic calcium channel receptor P2X₇ and prostate-specific antigen (PSA) levels were studied in biopsy cores from 174 patients as well as 20 radical prostatectomy cases. In clinical biopsies, we have previously demonstrated that P2X₁ and P2X₂ calcium channel receptors are absent from normal prostate epithelium that does not progress to prostate cancer within 5 years. In cases that did progress to prostate cancer however, P2X₁ and P2X₂ labeling was observed in a stage-specific manner first in the nucleus, then the cytoplasm and finally on the apical epithelium, as prostate cancer developed. These markers were present up to 5years before cancer was detectable by the usual morphological criteria (Gleason grading) as determined by H&E staining. In the current study, the apoptotic calcium channel receptor P2X₇ yielded similar results to that of P2X₁ and P2X₂. Using radical prostatectomy tissue sections as well as biopsies, these changes in calcium channel metabolism were noted throughout the prostate, indicating a field effect. This finding suggests that the presence of a prostate tumor could be detected without the need for direct sampling of tumor tissue, leading to detection of false negative cases missed by H&E stain. The reliability of PSA levels as a prognostic indicator has been questioned in recent years. In the current study, PSA levels were correlated with the P2X₇ labeling results. All patients who exhibited no P2X₇ labeling had a prostatic serum antigen (PSA) level of <2. Patients who exhibited stage-specific P2X₇ expression, and who later developed obvious prostate cancer as diagnosed by H&E stain, all had a PSA > 2. This finding suggests that increasing PSA may be an accurate indicator of cancer development.     

Lack of β2‐adrenoceptors aggravates heart failure‐induced skeletal muscle myopathy in mice

Skeletal myopathy is a hallmark of heart failure (HF) and has been associated with a poor prognosis. HF and other chronic degenerative diseases share a common feature of a stressed system: sympathetic hyperactivity. Although beneficial acutely, chronic sympathetic hyperactivity is one of the main triggers of skeletal myopathy in HF. Considering that β₂‐adrenoceptors mediate the activity of sympathetic nervous system in skeletal muscle, we presently evaluated the contribution of β₂‐adrenoceptors for the morphofunctional alterations in skeletal muscle and also for exercise intolerance induced by HF. Male WT and β₂‐adrenoceptor knockout mice on a FVB genetic background (β₂KO) were submitted to myocardial infarction (MI) or SHAM surgery. Ninety days after MI both WT and β₂KO mice presented to cardiac dysfunction and remodelling accompanied by significantly increased norepinephrine and epinephrine plasma levels, exercise intolerance, changes towards more glycolytic fibres and vascular rarefaction in plantaris muscle. However, β₂KO MI mice displayed more pronounced exercise intolerance and skeletal myopathy when compared to WT MI mice. Skeletal muscle atrophy of infarcted β₂KO mice was paralleled by reduced levels of phosphorylated Akt at Ser 473 while increased levels of proteins related with the ubiquitin‐–proteasome system, and increased 26S proteasome activity. Taken together, our results suggest that lack of β₂‐adrenoceptors worsen and/or anticipate the skeletal myopathy observed in HF.     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

Identification and characterization of a novel variant of the human P2X<Subscript>7</Subscript> receptor resulting in gain of function

The P2X₇ receptor exhibits significant allelic polymorphism in humans, with both loss and gain of function variants potentially impacting on a variety of infectious and inflammatory disorders. At least five loss-of-function polymorphisms (G150R, R307Q, T357S, E496A, and I568N) and two gain-of-function polymorphisms (H155Y and Q460R) have been identified and characterized to date. In this study, we used RT-PCR cloning to isolate and characterize P2X₇ cDNA clones from human PBMCs and THP-1 cells. A previously unreported variant with substitutions of V80M and A166G was identified. When expressed in HEK293 cells, this variant exhibited heightened sensitivity to the P2X₇ agonist (BzATP) relative to the most frequent allele, as shown by pore formation measured by fluorescent dye uptake into cells. Mutational analyses showed that A166G alteration was critical for the gain-of-function change, while V80M was not. Full-length variants with multiple previously identified nonsynonymous SNPs (H155Y, H270R, A348T, and E496A) were also identified. Distinct functional phenotypes of the P2X₇ variants or mutants constructed with multiple polymorphisms were observed. Gain-of-function variations (A166G or H155Y) could not rescue the loss-of-function E496A polymorphism. Synergistic effects of the gain-of-function variations were also observed. We also identified the A348T alteration as a weak gain-of-function variant. Thus, these results identify the new gain-of-function variant A166G and demonstrate that multiple-gene polymorphisms contribute to functional phenotypes of the human P2X₇ receptor. Furthermore, the results demonstrate that the C-terminal of the cysteine-rich domain 1 of P2X₇ is critical for regulation of P2X₇-mediated pore formation.     

The P2X<Subscript>7</Subscript>receptor is a candidate product of murine and human lupus susceptibility loci: a hypothesis and comparison of murine allelic products

Systemic lupus erythematosus and its murine equivalent, modelled in the New Zealand Black and New Zealand White (NZB × NZW)F₁ hybrid strain, are polygenic inflammatory diseases, probably reflecting an autoimmune response to debris from cells undergoing programmed cell death. Several human and murine loci contributing to disease have been defined. The present study asks whether the proinflammatory purinergic receptor P2X₇, an initiator of a form of programmed cell death known as aponecrosis, is a candidate product of murine and human lupus susceptibility loci. One such locus in (NZB × NZW)F₁ mice is lbw3, which is situated at the distal end of NZW chromosome 5. We first assess whether NZB mice and NZW mice carry distinct alleles of the P2RX ₇ gene as expressed by common laboratory strains, which differ in sensitivity to ATP stimulation. We then compare the responses of NZB lymphocytes, NZW lymphocytes and (NZB × NZW)F₁ lymphocytes to P2X₇ stimulation. NZB and NZW parental strains express the distinct P2X₇-L and P2X₇-P alleles of P2RX ₇, respectively, while lymphocytes from these and (NZB × NZW)F₁ mice differ markedly in their responses to P2X₇ receptor stimulation. NZB mice and NZW mice express functionally distinct alleles of the proinflammatory receptor, P2X₇. We show that current mapping suggests that murine and human P2RX ₇ receptor genes lie within lupus susceptibility loci lbw3 and SLEB4, and we argue that these encode a product with the functional characteristics consistent with a role in lupus. Furthermore, we argue that aponecrosis as induced by P2X₇ is a cell death mechanism with characteristics that potentially have particular relevance to disease pathogenesis.     

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.     

Distribution of P2X<Subscript>3</Subscript> receptor immunoreactivity in myenteric ganglia of the mouse esophagus

Intraganglionic laminar endings (IGLEs) represent the major vagal afferent terminals throughout the gut. Electrophysiological experiments revealed a modulatory role of ATP in the IGLE-mechanotransduction process and the P2X₂-receptor has been described in IGLEs of mouse, rat and guinea pig. Another purinoceptor, the P2X₃-receptor, was found in IGLEs of the rat esophagus. These findings prompted us to investigate occurrence and distribution of the P2X₃-receptor in the mouse esophagus. Using multichannel immunofluorescence and confocal microscopy, P2X₃-immunoreactivity (-iry) was found colocalized with the vesicular glutamate transporter 2 (VGLUT2), a specific marker for IGLEs, on average in three-fourths of esophageal IGLEs. The distribution of P2X₃ immunoreactive (-ir) IGLEs was similar to that of P2X₂-iry and showed increasing numbers towards the abdominal esophagus. P2X₃/P2X₂-colocalization within IGLEs suggested the occurrence of heteromeric P2X_2/3 receptors. In contrast to the rat, where only a few P2X₃-ir perikarya were described, P2X₃ stained perikarya in ~80% of myenteric ganglia in the mouse. Detailed analysis revealed P2X₃-iry in subpopulations of nitrergic (nNOS) and cholinergic (ChAT) myenteric neurons and ganglionic neuropil of the mouse esophagus. We conclude that ATP might act as a neuromodulator in IGLEs via a (P2X₂)-P2X₃ receptor-mediated pathway especially in the abdominal portion of the mouse esophagus.     

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.     

P2X<Subscript>7</Subscript>R modulation of visually evoked synaptic responses in the retina

P2X₇Rs are distributed throughout all layers of the retina, and thus, their localisation on various cell types puts into question their specific site(s) of action. Using a dark-adapted, ex vivo mouse retinal whole mount preparation, the present study aimed to characterise the effect of P2X₇R activation on light-evoked, excitatory RGC ON-field excitatory post-synaptic potentials (fEPSPs) and on outer retinal electroretinogram (ERG) responses under comparable conditions. The pharmacologically isolated NMDA receptor-mediated RGC ON-fEPSP was reduced in the presence of BzATP, an effect which was significantly attenuated by A438079 and other selective P2X₇R antagonists A804598 or AF27139. In physiological Krebs medium, BzATP induced a significant potentiation of the ERG a-wave, with a concomitant reduction in the b-wave and the power of the oscillatory potentials. Conversely, in the pharmacologically modified Mg²⁺-free perfusate, BzATP reduced both the a-wave and b-wave. The effects of BzATP on the ERG components were suppressed by A438079. A role for P2X₇R function in visual processing in both the inner and outer retina under physiological conditions remains controversial. The ON-fEPSP was significantly reduced in the presence of A804598 but not by A438079 or AF27139. Furthermore, A438079 did not have any effect on the ERG components in physiological Krebs but potentiated and reduced the a-wave and b-wave, respectively, when applied to the pharmacologically modified medium. Therefore, activation of P2X₇Rs affects the function in the retinal ON pathway. The presence of a high concentration of extracellular ATP would most likely contribute to the modulation of visual transmission in the retina in the pathophysiological microenvironment.     

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.     

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.