P2X receptors in autonomic and sensory neurons

ATP-gated ion channels (P2X receptors) are ubiquitously present in autonomic and sensory neurons as well as in smooth muscle; they mediate fast excitatory synaptic transmission at sympathetic neuromuscular junctions, at some neuro-neuronal synapses and may be involved in the generation and transmission of primary afferent information. Five subtypes of native P2X receptors can be distinguished by their kinetics and their agonist and antagonist profile. Six distinct P2X receptors have been cloned; all are present in sensory neurons and most are present in autonomic neurons; homomeric or heteromeric forms of these cloned receptors reproduce the five phenotypes observed in native cells.     

Localization of P2X2 and P2X3 receptors in rat trigeminal ganglion neurons

Purine receptors have been implicated in central neurotransmission from nociceptive primary afferent neurons, and ATP-mediated currents in sensory neurons have been shown to be mediated by both P2X3 and P2X2/3 receptors. The aim of the present study was to quantitatively examine the distribution of P2X2 and P2X3 receptors in primary afferent cell bodies in the rat trigeminal ganglion, including those innervating the dura. In order to determine the classes of neurons that express these receptor subtypes, purine receptor immunoreactivity was examined for colocalization with markers of myelinated (neurofilament 200; NF200) or mostly unmyelinated, non-peptidergic fibers (Bandeiraea simplicifolia isolectin B4; IB4). Forty percent of P2X2 and 64% of P2X3 receptor-expressing cells were IB4 positive, and 33% of P2X2 and 31% of P2X3 receptor-expressing cells were NF200 positive. Approximately 40% of cells expressing P2X2 receptors also expressed P2X3 receptors and vice versa. Trigeminal ganglion neurons innervating the dura mater were retrogradely labeled and 52% of these neurons expressed either P2X2 or P2X3 or both receptors. These results are consistent with electrophysiological findings that P2X receptors exist on the central terminals of trigeminal afferent neurons, and provide evidence that afferents supplying the dura express both receptors. In addition, the data suggest specific differences exist in P2X receptor expression between the spinal and trigeminal nociceptive systems.     

Intraepithelial vagal sensory nerve terminals in rat pulmonary neuroepithelial bodies express P2X(3) receptors

The neurotransmitters/modulators involved in the interaction between pulmonary neuroepithelial bodies (NEBs) and the vagal sensory component of their innervation have not yet been elucidated. Because P2X(3) purinoreceptors are known to be strongly expressed in peripheral sensory neurons, the aim of the present study was to examine the localization of nerve endings expressing P2X(3) purinoreceptors in the rat lung in general and those contacting pulmonary NEBs in particular. Most striking were intraepithelial arborizations of P2X(3) purinoceptor-immunoreactive (IR) nerve terminals, which in all cases appeared to ramify between calcitonin gene-related peptide (CGRP)- or calbindin D28k (CB)-labeled NEB cells. However, not all NEBs received nerve endings expressing P2X(3) receptors. Using CGRP and CB staining as markers for two different sensory components of the innervation of NEBs, it was revealed that P2X(3) receptor and CB immunoreactivity were colocalized, whereas CGRP-IR fibers clearly formed a different population. The disappearance of characteristic P2X(3) receptor-positive nerve fibers in contact with NEBs after infranodosal vagal crush and colocalization of tracer and P2X(3) receptor immunoreactivity in vagal nodose neuronal cell bodies in retrograde tracing experiments further supports our hypothesis that the P2X(3) receptor-IR nerve fibers contacting NEBs have their origin in the vagal sensory nodose ganglia. Combination of quinacrine accumulation in NEBs, suggestive of the presence of high concentrations of adenosine triphosphate (ATP) in their secretory vesicles, and P2X(3) receptor staining showed that the branching intraepithelial P2X(3) receptor-IR nerve terminals in rat lungs were exclusively associated with quinacrine-stained NEBs. We conclude that ATP might act as a neurotransmitter/neuromodulator in the vagal sensory innervation of NEBs via a P2X(3) receptor-mediated pathway. Further studies are necessary to determine whether the P2X(3) receptor-expressing neurons, specifically innervating NEBs in the rat lung, belong to a population of P2X(3) receptor-IR nociceptive vagal nodose neurons.     

P2X receptors in peripheral neurons

P2X receptors are a family of ligand-gated ion channels, activated by extracellular ATP. The seven subunits cloned (P2X1-7) can assemble to form homomeric and heteromeric receptors. Peripheral neurons of neural crest origin (e.g. those in dorsal root, trigeminal, sympathetic and enteric ganglia) and placodal origin (e.g. those in nodose and petrosal ganglia) express mRNAs for multiple P2X subunits. In this review, we summarize the molecular biological, electrophysiological and immunohistochemical evidence for P2X receptor subunits in sensory, sympathetic, parasympathetic, pelvic and myenteric neurons and adrenomedullary chromaffin cells. We consider the pharmacological properties of these native P2X receptors and their physiological roles. The responses of peripheral neurons to ATP show considerable heterogeneity between cells in the same ganglia, between ganglia and between species. Nevertheless, these responses can all be accounted for by the presence of P2X2 and P2X3 subunits, giving rise to varying proportions of homomeric and heteromeric receptors. While dorsal root ganglion neurons express predominantly P2X3 and rat sympathetic neurons express mainly P2X2 receptors, nodose and guinea-pig sympathetic neurons express mixed populations of P2X2 and heteromeric P2X2/3 receptors. P2X receptors are important for synaptic transmission in enteric ganglia, although their roles in sympathetic and parasympathetic ganglia are less clear. Their presence on sensory neurons is essential for some processes including detection of filling of the urinary bladder. The regulation of P2X receptor expression in development and in pathological conditions, along with the interactions between purinergic and other signalling systems, may reveal further physiological roles for P2X receptors in autonomic and sensory ganglia.     

TNP-ATP-resistant P2X ionic current on the central terminals and somata of rat primary sensory neurons

P2X receptors have been suggested to be expressed on the central terminals of A delta-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X2+3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X1, P2X3 homomers, and P2X2+3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined alpha,beta-methylene-ATP (alpha beta meATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. alpha beta meATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 microM TNP-ATP (n = 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 microM TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 microM alpha beta meATP could be largely inhibited by 10 microM iso-pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive A delta-afferent fibers and play a role in modulating sensory transmission to lamina V neurons.     

Cyclophosphamide-induced bladder inflammation sensitizes and enhances P2X receptor function in rat bladder sensory neurons

We studied sensitization of retrogradely labeled bladder sensory neurons and plasticity of P2X receptor function in a model of cystitis using patch-clamp techniques. Saline (control) or cyclophosphamide (CYP) was given intraperitoneally to rats on days 0, 2, and 4. On day 5, lumbosacral (LS, L6-S2) or thoracolumbar (TL, T12-L2) dorsal root ganglia were removed and dissociated. Bladders from CYP-treated rats showed partial loss of the urothelium and greater myeloperoxidase activity compared with controls. Bladder neurons from CYP-treated rats were increased in size (based on whole cell capacitance) compared with controls and exhibited lower activation threshold, increased action potential width, and greater number of action potentials in response to current injection or application of purinergic agonists. Most control LS bladder neurons (>85%) responded to ATP or alpha,beta-metATP with a slowly desensitizing current; these agonists affected only half of TL neurons, producing predominantly fast/mixed desensitizing currents. CYP treatment increased the fraction of TL bladder neurons sensitive to purinergic agonists (>80%) and significantly increased current density in both LS and TL bladder neurons compared with control. Importantly, LS and TL neurons from CYP-treated rats showed a selective increase in the functional expression of heteromeric P2X(2/3) and homomeric P2X(3) receptors, respectively. Although desensitizing kinetics were slower in LS neurons from CYP-treated compared with control rats, recovery kinetics were similar. The present results demonstrate that bladder inflammation sensitizes and increases P2X receptor expression and/or function for both pelvic and lumbar splanchnic pathways, which contribute, in part, to the hypersensitivity associated with cystitis.     

Gastric ulcers evoke hyperexcitability and enhance P2X receptor function in rat gastric sensory neurons

Tissue inflammation contributes to the development of hyperalgesia, which is at least in part due to altered properties of primary afferent neurons. We hypothesized that gastric ulcers enhance the excitability of gastric sensory neurons and increase their response to purinergic agonists. The rat stomach was surgically exposed, and a retrograde tracer [1.1'-dioctadecyl-3,3,3,'3-tetramethylindocarbocyanine methanesulfonate (DiI)] was injected into the wall of the distal stomach. Kissing ulcers (KUs) were produced by a single injection of acetic acid (0.1 ml for 45 s; 60%) into the clamped gastric lumen. Saline injection served as control. Gastric nodose ganglion (NG) or dorsal root ganglion (DRG) cells were harvested 7 days later and acutely dissociated for whole cell recordings. Based on whole cell capacitance, gastric DRG neurons exhibited larger cell size than NG neurons. Significantly more control gastric DRG neurons compared with NG counterparts had TTX-resistant action potentials. Almost all control NG neurons (90%) compared with significantly less DRG neurons (< or =38%) responded to ATP or alpha,beta-metATP. Whereas none of the control cells exhibited spontaneous activity, about 20% of the neurons from KU animals generated spontaneous action potentials. KUs enhanced excitability as shown by a decrease in threshold for action potential generation, which was in part due to an increased input resistance. This was associated with an increase in the fraction of neurons with TTX-resistant action potentials and cells responding to capsaicin and purinergic agonists. KU doubled the current density evoked by the P2X receptor agonist alpha,beta-metATP and slowed decay of the slowly desensitizing component of the current without affecting the concentration dependence of the response. These data show that KU sensitizes vagal and spinal gastric afferents by affecting both voltage- and ligand-gated channels, thereby potentially contributing to the development of dyspeptic symptoms.     

Distinct roles of P2X receptors in modulating glutamate release at different primary sensory synapses in rat spinal cord

Using spinal cord slice preparations and patch-clamp recordings in lamina II and lamina V regions, we tested a hypothesis that P2X receptor subtypes differentially modulate glutamate release from primary afferent terminals innervating different sensory regions. We found that activation of P2X receptors by alpha,beta-methylene-ATP increased glutamate release onto >80% of DH neurons in both lamina regions. However, two distinct types of modulation, a transient and a long-lasting enhancement of glutamate release were observed. In lamina II recordings, >70% of the modulation was transient. In contrast, P2X receptor-mediated modulation was always long-lasting in lamina V. Pharmacologically, both transient and long-lasting types of modulation were blocked by 10 microM pyridxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium, a broad-spectrum P2X receptor antagonist. Transient modulation was not observed in the presence of 1 microM trinitrophenyl-ATP (TNP-ATP), a subtype-selective P2X receptor antagonist, suggesting that homomeric P2X3 receptors may be involved in the transient modulation in lamina II. The long-lasting modulation remained in the presence of 1 microM TNP-ATP. Selective removal of P2X3-expressing afferent terminals by the targeting toxin saporin-conjugated isolectin B4 or surgical removal of superficial DH did not affect P2X receptor-mediated long-lasting modulation in lamina V. Taken together, these results suggest that P2X receptor subtypes play distinct roles in sensory processing in functionally different sensory regions.     

Expression of glycine receptors in rat sensory neurons vs. HEK293 cells yields different functional properties

In neurodegenerative diseases, such as Alzheimer's disease or HIV encephalitis, neuronal DNA fragmentation has been observed at unexpected high frequencies, without definitive evidence for activation of an irreversible apoptotic pathway. The wobbler mouse is a suggested genetic model of neurodegenerative disease. The mutant mouse develops normally until the fourth week of age when atrophy and weakness of forelimb muscles become apparent. There is a slow progression of the disease and wobbler mice may survive for several months. Spinal cord examination reveals the presence of several motoneurons with perikaryal vacuolar degeneration. In this study, we observed, using terminal dUTP nick-end-labelling staining in mutant spinal cord sections, a massive although very transient DNA fragmentation in different cell types, including glial cells and motoneurons, before the apparition of any clinical symptoms. In older wobbler mice, this DNA fragmentation had completely disappeared and the majority of motoneurons survived. To our knowledge, this is the first example of a massive and transient DNA fragmentation in the central nervous system during the early course of a neurodegenerative disease.     

外周神经元P2X受体的研究进展

早在1948年Feldberg和Hebb既已证明：三磷酸腺苷(ATP)在自主神经节产生效应，动脉内注射ATP可兴奋猫颈上交感神经节神经元。1954年，Feldberg和Sherwood证实脑室内注射ATP产生共济失调及睡眠的现象。以后的化学及电生理研究也显示嘌呤类物质(包括ATP和腺苷等)导致大脑皮层神经元兴奋。这是ATP在外周和中枢神经系统具有作用的早期报道。1972年Burnstock推测ATP是介导胃肠道平滑肌非肾上腺素能和非胆碱能神经反应的递质，从而首次提出了嘌呤能神经(purmergic nerve)的概念。     

Extracellular cAMP inhibits P2X3 receptors in rat sensory neurones through G protein-mediated mechanism

Aim: To identify the mechanisms of P2X₃ receptor inhibition by extracellular cyclic adenosine monophosphate (cAMP) in rat dorsal root ganglion (DRG) neurones. Methods: Whole-cell currents were measured in cultured DRG neurones using the combination of voltage and concentration clamp. Results: We have found that extracellular cAMP inhibits P2X₃-mediated currents in a concentration- and use-dependent manner. The P2X₃ currents, activated by ATP applied every 4 min, were inhibited by 55% in the presence of 10 μm cAMP and by 81% in the presence of 30 μm cAMP. At 8 min interval between ATP applications the same concentration of cAMP did not alter the currents. Addition of 0.5 mm of guanosine 5′-O-(2-thiodiphosphate) to intracellular solution blocked the inhibitory action of cAMP. The inhibitory effects of cAMP were not mimicked by extracellular application of 30 μm adenosine. Conclusions: In this paper, we demonstrate, for the first time, that extracellular application of cAMP to rat sensory neurones inhibits P2X₃ receptors via a G protein-coupled mechanism in a use-dependent manner, thus indicating the neuronal expression of specific plasmalemmal cAMP receptor.     

Subsets of retinal neurons and glia express P2Y1 receptors

Recent evidence suggests that extracellular ATP modulates retinal processing and could play a role in modulating glial cells during retinal diseases. Here, we evaluated the localization of P2Y₁ receptors in the rat retina using indirect immunofluorescence immunocytochemistry. We observed labeling within defined populations of inner retinal neurons and Müller cell processes and end feet. Double labeling of P2Y₁ receptor with choline acetyltransferase revealed extensive colocalization indicating the expression of this receptor by cholinergic amacrine cells. Ganglion cell labeling for P2Y₁ receptors was also observed. Having established the normal pattern of immunolabeling within the retina, we next examined whether immunolabeling was altered by retinal disease. P2Y₁ receptor immunolabeling of Müller cells was of greater intensity following light-induced retinal degeneration, suggesting that Müller cell gliosis is accompanied by changes in P2Y₁ receptor expression. Overall, these data provide further evidence for a role of extracellular ATP in retinal signaling within subsets of retinal neurons as well as glia.     

P2X purinoceptors and sensory transmission

The involvement of P2X purinoreceptors (P2X receptors) in somatosensory transmission is herein reviewed with a focus on those receptors that are expressed on sensory neurons to elucidate their roles in the initiation of sensory excitation from primary afferent neurons, in modulating synaptic transmission at the first sensory synapses formed between primary afferent central terminals and dorsal horn neurons, in directly mediating sensory synaptic transmission to the spinal cord dorsal horn, and in modulating synaptic transmission among spinal cord dorsal horn neurons. Research on P2X receptors has indicated that these receptors play a significant role in both physiological and pathological pain states. As a result, P2X receptors may serve as therapeutic targets for the treatment of pathological pain conditions associated with nerve injury, tissue inflammation, cancer, and other diseases.     

Nerve growth factor induces functional nicotinic acetylcholine receptors on rat sensory neurons in culture

Neonatal sensory neurons from rat nodose ganglia express nicotinic acetylcholine receptors when grown in tissue culture without other cell types. The present study investigates the role of nerve growth factor in inducing these receptors. Nerve growth factor has little effect on the growth and survival of nodose neurons in culture, although most neurons were found by quantitative radioautography to have high-affinity nerve growth factor receptors. Nerve growth factor strongly influenced the expression of nicotinic receptors on these neurons: the proportion of acetylcholine-sensitive neurons was approximately 60% in cultures with nerve growth factor compared with 15% in cultures grown without nerve growth factor. The proportion of acetylcholine-sensitive neurons increased over the first week, plateaued by day 12 and remained high for at least three weeks. In contrast, without NGF, the proportion of acetylcholine-sensitive neurons was low throughout the three-week period. The results indicate that nerve growth factor is an important factor in promoting nicotinic receptors on these neurons in culture.     

Depletion of substance P from rat primary sensory neurons by ATP, an implication of P2X receptor-mediated release of substance P.

Effects of ATP on substance P immunoreactivity were examined in cultured dorsal root ganglion neurons. We found that treatment of dorsal root ganglion neurons with ATP significantly depleted substance P immunoreactivity on the neurites and somata of the neurons. The effects of ATP were significantly inhibited by the purinergic P2 receptor antagonists suramin (30 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (10 microM). We also showed that ATP-induced depletion of substance P immunoreactivity from dorsal root ganglion neurons depended on the entry of Ca(2+). In a spinal cord slice preparation, we also found the internalization of neurokinin-1/substance P receptors in many dorsal horn neurons following the application of ATP or alpha,beta-methylene-ATP.Together these results indicate that activation of P2X receptors may result in release of substance P from primary afferent neurons.     

Role of P2X and P2Y receptors in rat myocardial contractility during ontogeny

Stable agonist of P2 receptors 2-methylthio-ATP and selective antagonists of P2X and P2Y receptors PPADS and reactive blue-2 were used for evaluation of the role of P2 receptors in positive contractile reaction of atrial and ventricular myocardium in rats. PPADS significantly moderated the effects of 2-methylthio-ATP in 14-, 21-, and 56-day-old rat pups, but potentiated them in 100-day-old rats. Under conditions of reactive blue-2 treatment, the positive effect of the agonist was preserved in the atria and ventricles in all age groups and was age-dependent. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 6, pp. 637–640, June, 2007     

Dopaminergic substantia nigra neurons express functional nmda receptors in postnatal rats

Activation of N-methyl-D-aspartate (NMDA) receptors in the Substantia Nigra zona compacta (SNc) may determine the degree of physiological apoptosis during the early postnatal period. However, the expression of these receptors during this stage of development is uncertain, as a recent study failed to detect responses to NMDA in unidentified SNc neurons isolated from 2-wk-old rats. Using conventional or perforated-patch whole cell recordings, we examined the presence of NMDA-evoked responses in SNc neurons acutely dissociated from P4 to P16 rats, applying strict criteria for identification of these neurons as nigrostriatal and dopaminergic. The SNc neurons were identified by retrograde labeling after striatal injection of Fluoro-Gold; the presence of I(h) current; and the inhibition of firing by dopamine (50 microM). NMDA (100 microM, V(hold) = -60 mV) evoked inward, APV-sensitive currents (56.4 +/- 8.6 pA) in all tested neurons (n = 29). Strong depolarizing responses were observed under current-clamp. These results indicate that NMDA receptors play a functional role in SNc neurons during the first two postnatal weeks.