Cerebellar lesion up-regulates P2X1 and P2X2 purinergic receptors in precerebellar nuclei

ATP released in the extracellular space by neuronal injury can influence neighboring neurons via activation of purinergic receptors. In vitro data suggest the involvement of ATP and purinergic receptors as trophic agents in different biological events such as neuritogenesis and cell survival. Recently, in vivo studies have demonstrated modifications in the glial expression of ionotropic purinergic receptors after CNS lesions. In the present study, we investigated the effects of CNS lesion on the neuronal expression of P2X(1) and P2X(2) receptor subunits by immunohistochemistry and western blotting techniques. In the precerebellar structures of normal animals the expression of P2X(1) and P2X(2) was lower than previously reported. P2X(1) immunostaining was confined only to fibers, while P2X(2) immunostaining demonstrated a neuronal expression. After unilateral cerebellar lesion (hemicerebellectomy) axotomized precerebellar neurons underwent marked cell loss; however, some precerebellar neurons did not degenerate. Seven to 35 days after hemicerebellectomy, a transient, time-dependent, marked increase in the number of immunopositive P2X(1) and P2X(2) neurons was observed in the precerebellar nuclei of the experimental side. An even distribution of immunopositive neurons was present in almost all precerebellar nuclei examined, except for the inferior olive. In this latter structure, differences in the distribution of immunopositive neurons were evident among the subnuclei. Up-regulation of immunoreactivity over relatively long time periods, distribution selectivity and absence of degenerating morphological features in immunopositive neurons suggest that purinergic receptors may have a role in mediating the survival of neuronal responses to axotomy.The present findings are the first report in the CNS of P2X(1) and P2X(2) receptor subunit involvement in neuronal reaction to axotomy. They provide in vivo evidence of a correlation between purinergic receptor subunit up-regulation and survival of injured neurons.     

P2X_2和P2X_3受体在小鼠咽粘膜味蕾内的分布

应用免疫组织化学双标记技术研究ATP受体的P2X2和P2X3受体亚型在小鼠咽部粘膜味蕾和邻近组织内的分布,探究ATP在咽部味觉及一般感觉信号传导中的作用。在咽部各水平粘膜味蕾内均可观察到许多P2X2受体阳性味细胞和基细胞,未见P2X2受体阳性的神经纤维。许多降钙素基因相关肽(CGRP)阳性神经纤维围绕在味蕾周围,并发出分支与P2X2受体阳性味细胞和基细胞形成密切接触。P2X3受体多在咽部粘膜上皮味蕾内的神经终末表达,P2X3受体阳性纤维在粘膜基底部形成纤维束并与粘膜下P2X3受体阳性纤维相连,其分支在味蕾基底部形成神经丛,由神经丛发出神经终末到达顶部的味孔和味蕾各部,未见味蕾细胞表达P2X3受体。在无味蕾的粘膜上皮内,也观察到少量P2X3受体阳性神经终末。CGRP阳性纤维缠绕在味蕾周围,并发出分支与味蕾内的P2X3受体阳性纤维形成密切接触。上述结果表明ATP可能是咽粘膜内味觉信号感受与传导的神经递质。     

ATP elicits inward currents in isolated vasopressinergic neurohypophysial terminals via P2X2 and P2X3 receptors

Effects of extracellular adenosine tri-phosphate (ATP) on ionic currents were investigated using the perforated-patch whole-cell recording technique on isolated terminals of the Hypothalamic Neurohypophysial System (HNS). ATP induced a current response in 70% of these isolated terminals. This inwardly-rectifying, inactivating current had an apparent reversal near 0 mV and was dose-dependent on ATP with an EC₅₀=9.6±1.0 M. In addition, current amplitudes measured at maximal ATP concentrations and optimum holding potentials had a current density of 70.8 pA pF^–1 and were greatly inhibited by suramin and PPADS. Different purinergic receptor agonists were tested, with the following efficacy: ATP 2-methylthioATP > ATP--S > Bz-Bz-ATP > ,-methylene-ATP > ,-methylene-ATP. However, UTP and ADP were ineffective. These data suggest the involvement of a P2X purinergic receptor in the ATP-induced responses. Immunocytochemical labeling in vasopressinergic terminals indicates the existence of P2X_{2,3,4, and 7}, but not P2X₆ receptors. Additionally, P2X_{2 and 3} were not found in terminals which labeled for oxytocin. In summary, the EC₅₀, decay, inactivation, and pharmacology indicate that a functional mixture of P2X_{2 and 3} homomeric receptors mediate the majority of the ATP responses in vasopressinergic HNS terminals. We speculate that the characteristics of these types of receptors reflect the function of co-released ATP in the terminal compartment of these and other CNS neurons.     

大鼠舌咽和迷走神经感觉神经节内P2X2、P2X3受体与calretinin和IB4结合位点的共存

应用免疫组织化学荧光三标方法结合激光共聚焦显微镜技术研究了大鼠舌咽和神经迷走神经感觉神经节内ATP受体P2X2、P2X3与calretinin、植物凝集素(IB4)结合位点的共存。结果显示:舌咽和迷走神经感觉神经节内可见大量P2X2、P2X3阳性的神经元胞体和纤维,P2X2受体阳性细胞多为大、中、小型神经元,P2X3阳性细胞多为中、小型神经元;可见较多的P2X2/IB4及P2X3/IB4双标神经元。有(91.1±4.7)%(结状神经节)、(78.8±2.4)%(岩神经节)、(76.8±2.7)%(颈静脉神经节)为P2X2阳性细胞;(77.0±3.2)%(结状神经节)、(91.2±3.9)%(岩神经节)、(78.4±3.6)%(颈静脉神经节)的P2X3阳性细胞结合IB4;有8.9±1.6%(结状神经节)、7.7±1.4%(岩神经节)、9.1±1.1%(颈静脉神经节)的P2X2阳性细胞呈calretinin阳性;三神经节内均观察到少量P2X2/calretinin/IB4三标细胞。P2X3/calretinin双标或P2X3/calretinin/IB4三标神经元数量较少。这些结果提示,舌咽和迷走神经感觉神经节内ATP受体P2X2、P2X3与IB4结合位点存在广泛的共存,部分P2X2受体与calretinin存在着共存。     

Immunohistochemical identification of cells expressing ATP‐gated cation channels (P2X receptors) in the adult rat thyroid

We carried out immunohistochemistry and western blotting of fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were performed with P2X receptor antibodies from 2 different sources. P2X‐immunopositive cells were identified by fluorescence double labelling and confocal microscopy. Results of the western blotting experiments showed double bands of approximately 70 kDa and 140 kDa for all 7 P2X receptor subtypes with both sets of antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X₁, P2X₂ and P2X₆ receptors were detected exclusively in vascular smooth muscle; P2X₅ and P2X₇ receptors were also present on vascular smooth muscle. Endothelial cells stained for P2X₃, P2X₄ and P2X₇ receptors. Thyroid follicular cells displayed immunoreactivity for P2X₃, P2X₄ and P2X₅ receptors. No immunostaining for P2X receptors was observed on C‐cells. Possible roles for the broad expression of P2X receptor subtypes in the rat thyroid are discussed.     

Increase of intracellular Ca by P2X and P2Y receptor-subtypes in cultured cortical astroglia of the rat

Astrocytes express purinergic receptors that are involved in glial–neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed “astroglia” throughout. Astroglia expressed predominantly P2X_4,6,7 as well as P2Y_1,2 receptor-subtypes. Less intensive immunostaining was also found for P2X₅ and P2Y_4,6,13,14 receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca²⁺ ([Ca²⁺]_i). Of the agonists tested, only the P2X_1,3 receptor-selective α,β-methylene ATP was ineffective. Experiments with Ca²⁺-free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca²⁺-ATPase, indicated that the [Ca²⁺]_i response to most nucleotides, except for ATP and 2′,3′-O-(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca²⁺ from intracellular stores. A Gprotein–mediated release of Ca²⁺ is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y_1,2,6,14 and probably also P2Y₄) receptors. Several unidentified P2X receptors, including P2X₇, may also be present, although they appear to only moderately participate in the regulation of [Ca²⁺]_i. The rise of [Ca²⁺]_i is due in this case to the transmembrane flux of Ca²⁺ via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca²⁺]_i of cultured astroglia and thereby may play an important role in cell-to-cell signaling.     

Impaired P2X and P2Y receptor-mediated signaling in HPRT-deficient B103 neuroblastoma cells

Defect of hypoxanthine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND), but the link between HPRT deficiency and the self-injurious behavior of LND is unknown. In a previous study (Pinto et al., J. Neurochem. 72 (2005) 1579-1586) we reported on a decrease in nucleotidase activity in membranes of several HPRT⁻ cell lines and fibroblasts from LND patients. Since nucleotidases are involved in ATP-induced signal transduction, in the present study, we tested the hypothesis that P2X and P2Y receptor-mediated signal transduction is impaired in HPRT deficiency. As model we studied rat B103 neuroblastoma cells. Compared to control cells, in HPRT⁻ cells, NTP and NDP-induced Ca²⁺ influx across the membrane and Ca²⁺ mobilization from intracellular stores were impaired. Both P2X and P2Y receptors were involved in the responses. Quantitative real-time PCR revealed reduced expression of receptors P2X₃, P2X₅, P2Y₂, P2Y₄, P2Y₁₂, P2Y₁₃ and P2Y₁₄ in HPRT deficiency. Collectively, HPRT deficiency is associated with abnormal purinergic signaling, encompassing P2X and P2Y receptors and nucleotidases.     

Contribution from P2X and P2Y purinoreceptors to ATP-evoked changes in intracellular calcium concentration on cultured myotubes

Although the alteration of purinoreceptor pattern on skeletal muscle is known to accompany physiological muscle differentiation and the pathogenesis of muscle dystrophy, the exact identity of and the relative contribution from the individual receptor subtypes to the purinergic signal have been controversial. To identify these subtypes in cultured myotubes of 5–10 nuclei, changes in intracellular calcium concentration and surface membrane ionic currents were detected and calcium fluxes calculated after the application of the subtype-specific agonists 2′3′-O-(benzoyl-4-benzoyl)-ATP (BzATP), 2-methyltio-ADP and UTP. The effectiveness of these agonists together with positive immunocytochemical staining revealed the presence of P2X₄, P2X₅, P2X₇, P2Y₁ and P2Y₄ receptors. siRNA-reduced protein expression of P2X₅, P2X₇ and P2Y₁ receptors was accompanied by reduction in the ATP-evoked calcium transients. Furthermore, anti-P2X₇ siRNA caused a significant drop in the early peak and delayed steady component of the calculated calcium flux. The use of its antagonist, oxidized ATP, similarly to transfection with anti-P2X₇ siRNA caused significant reduction in the agonist-elicited ionic currents I _ATP and I _BzATP, with a greater drop in the latter. Our results demonstrate that the activation of ionotropic P2X₄, P2X₅ and P2X₇ and metabotropic P2Y₁ and P2Y₄ purinoreceptors participates in forming the calcium transients of multinucleated myotubes.     

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.     

P2X3 receptor mediates ectopic mechanical allodynia with inflamed lower lip in mice

Ectopic pain in other orofacial regions develops with local inflammation in separated orofacial structures. However, the basis for the spreading of pain to adjacent orofacial areas after local inflammation is still unknown. In the present study, we determined if the P2X₃ receptor (P2X₃R) was associated with altered mechanical sensitivity of the whisker pad skin following complete Freund's adjuvant (CFA) injection into the lower lip. Mice with local inflammation induced by CFA injection into the lower lip demonstrated significant mechanical allodynia of whisker pad skin. The mechanical allodynia was reversed by P2X₃R antagonist, A-317491 administration into whisker pad skin. The number of P2X₃R and calcitonin gene-related peptide (CGRP) positive trigeminal ganglion (TG) neurons that innervates the whisker pad skin and lower lip was increased after CFA injection into the lower lip. CGRP protein expression in TG ipsilateral to CFA injection was also significantly greater than that of the saline-injected mice. The present findings suggest that induced CGRP by local inflammation in the lower lip increases P2X₃R in TG neurons, the increased P2X₃Rs are involved in the sensitization of primary afferent neurons in the whisker pad skin. This P2X₃R overexpression may underlie ectopic mechanical allodynia in the whisker pad skin after CFA injection into the lower lip.     

Neuropeptides in the human dorsal vagal complex: An immunohistochemical study

The distribution of twelve biologically active neuropeptides, i.e., thyrotropin-releasing hormone, corticotropin-releasing factor, pro-opiomelanocortin-derived peptides (adrenocorticotropic hormone, β-endorphin, α-melanocyte-stimulating hormone), leucine-enkephalin, dynorphin A, dynorphin B, cholecystokinin, substance P, galanin and calcitonin gene-related peptide, was examined by immunohistochemistry in the human dorsal vagal complex including the nucleus of the solitary tract, the dorsal motor nucleus of the vagus and the area postrema. Immunoreactivity of all the twelve neuropeptides was found widely distributed in the various subdivisions of the nucleus of the solitary tract, showing a unique distribution for every peptide. Neuronal cell bodies immunostained with leucine-enkephalin, galanin and dynorphin B were found in this region. There were no immunopositive perikarya for any of the peptides in the other structures studied. Fibers containing galanin, corticotropin-releasing factor, substance P, dynorphin B, thyrotropin-releasing hormone and calcitonin gene-related peptide were observed at a relatively high density in the nucleus of the solitary tract. In the same structure, a moderately dense network of fibers immunostained with dynorphin A, cholecystokinin and leucine-enkephalin, but only solitary pro-opiomelanocortin-derived peptides-containing fiber fragments were observed. In the dorsal motor nucleus of the vagus the most prominent network of fibers was found to contain thyrotropin-releasing hormone, galanin and substance P. In contrast to these, no β-endorphin immunoreactivity was detected. The area postrema contained only moderate to low densities of galanin-, substance P-, calcitonin gene-related peptide-, dynorphin B- and cholecystokinin-immunoreactive fibers.     

P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding

The redistribution of purinergic P2X receptor subunits (P2X₁ to P2X₇) within the rabbit aorta wall three weeks after endothelial balloon injury/cholesterol feeding was examined. P2X₁ receptor cluster density was elevated in the media following balloon injury/cholesterol feeding by about 30% and these clusters appeared on smooth muscle cells throughout the greatly expanded neointima but they did not change significantly on the endothelial cells following balloon injury. P2X₄ clusters were found in high density throughout the media and in very high density in the enlarged neointima following balloon injury, particularly on the endothelial cells where the density increased about 10-fold after balloon injury. P2X₅ clusters were found in high density in the media of normal aorta but with little change following balloon injury. P2X₃, P2X₆ and P2X₇ cluster density was low in normal aorta and remained unchanged following balloon injury. All receptor subunits were found on endothelial cells. It is suggested that the release of ATP from damaged endothelial cells and from smooth muscle cells sufficient to activate P2X₄ receptors may contribute to neointimal proliferation.     

大鼠咽黏膜内ATP受体P2X3阳性感觉纤维的分布

目的 研究大鼠咽黏膜内三磷酸腺苷(ATP)受体P2X₃阳性感觉纤维的分布,为探究ATP在咽黏膜感觉信号传导中的作用提供形态学依据。 方法 成年Wistar大鼠12只,应用免疫荧光组织化学双标技术结合激光扫描共焦显微镜。 结果 1.在咽黏膜各部位均可观察到P2X₃阳性感觉纤维,纤维分为两类：一类为串珠样游离神经纤维;另一类纤维在黏膜内发出分支并形成复杂的树枝状末梢,纤维分支在黏膜内形成神经丛。2.可见许多降钙素基因相关肽（CGRP）阳性纤维缠绕在P2X₃阳性树枝状末梢周围,并有少量的串珠样P2X₃阳性纤维与CGRP共存。3.在岩神经节内可观察到许多P2X₃或CGRP免疫阳性神经元,其中有少量神经元同时呈P2X₃/CGRP免疫阳性。 结论 大鼠咽黏膜内不同类型的感觉纤维均表达ATP受体P2X₃阳性,提示ATP可能与咽黏膜伤害性刺激和其他生理性刺激向中枢的传递有关。     

Changes in the distribution of different subtypes of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the pregnant rat urinary bladder

Clusters of purinergic receptor subunits, about 1 m diameter, are found on the smooth muscle cell membrane beneath junctional varicosities in the detrusor muscle of the rat urinary bladder. We have examined the extent of redistribution of the six different subunit clusters, P2X₁ to P2X₆, with respect to junctional varicosities during pregnancy, as it is known that the detrusor muscle undergoes changes in purinergic innervation during this period. Before pregnancy, clusters at junctional varicosities are principally composed of the subtypes P2X₁, P2X₂, P2X₃ and P2X₅. However this subtype distribution changes dramatically during pregnancy, such that by day 14 of pregnancy, the extent of P2X₁, P2X₂, P2X₃ and P2X₅ junctional clusters has decreased by more than 80% whereas the extent of P2X₄ and P2X₆ junctional clusters has increased by more than 80%. These changes were confirmed with Western blots for different subtypes. It is suggested that the changes in the purinergic innervation of the detrusor muscle during pregnancy reflect changes in the P2X subtypes found on the smooth muscle membrane beneath junctional varicosities.     

Reversible Inhibition of Chlamydia trachomatis Infection in Epithelial Cells Due to Stimulation of P2X4 Receptors

Bacterial infections of the mucosal epithelium are a major cause of human disease. The prolonged presence of microbial pathogens stimulates inflammation of the local tissues, which leads to changes in the molecular composition of the extracellular milieu. A well-characterized molecule that is released to the extracellular milieu by stressed or infected cells is extracellular ATP and its ecto-enzymatic degradation products, which function as signaling molecules through ligation of purinergic receptors. There has been little information, however, on the effects of the extracellular metabolites on bacterial growth in inflamed tissues. Millimolar concentrations of ATP have been previously shown to inhibit irreversibly bacterial infection through ligation of P2X₇ receptors. We show here that the proinflammatory mediator, ATP, is released from Chlamydia trachomatis-infected epithelial cells. Moreover, further stimulation of the infected cells with micromolar extracellular ADP or ATP significantly impairs the growth of the bacteria, with a profile characteristic of the involvement of P2X₄ receptors. A specific role for P2X₄ was confirmed using cells overexpressing P2X₄. The chlamydiae remain viable and return to normal growth kinetics after removal of the extracellular stimulus, similar to responses previously described for persistence of chlamydial infection.     

P2X receptors are expressed on neurons containing luteinizing hormone-releasing hormone in the mouse hypothalamus

Expression of P2X₁, P2X₂, P2X₃, P2X₄, P2X₅ and P2X₆ receptors, members of a family of ATP-gated cation channels, on neurons containing luteinizing hormone-releasing hormone (LHRH) in the mouse hypothalamus was studied with double-labeling fluorescence immunohistochemistry. This study demonstrated that different combinations of P2X receptor subunits were expressed on the perikarya and axon terminals of LHRH-producing neurons. It was shown for the first time that P2X₂, P2X₄, P2X₅ and P2X₆ receptor subunits were expressed on the perikarya of LHRH-producing neurons and P2X₂ and P2X₆ on their axon terminals. These results suggest that activation of P2X receptors by ATP via different homomeric or heteromeric P2X receptors at both presynaptic and postsynaptic sites could be involved in the regulation of LHRH secretion at the forebrain level.     

P2X receptor stimulation amplifies complement-induced haemolysis

Activation of the complement system evokes cell damage by insertion of membrane attack complexes, which constitute the basis of the pathogenesis of various haemolytic disorders. Recently, we found that haemolysis caused by other types of membrane pore-forming proteins such as α-haemolysin (HlyA) from Escherichia coli, α-toxin from Staphylococcus aureus and leukotoxin from Aggregatibacter actinomycetemcomitans inflict their cytotoxic effects through P2 receptor activation. Here we show that similar to haemolysis induced by HlyA, leukotoxin and α-toxin, complement-induced haemolysis is amplified through ATP release and subsequent P2 receptor activation. Similar results were found both in murine, sensitised ovine and human erythrocytes, with either human plasma or guinea pig serum as complement donors. Non-selective P2 antagonists (PPADS and suramin) concentration-dependently inhibited complement-induced haemolysis. More specific P2 receptor antagonists imply that P2X₁ and P2X₇ are the main receptors involved in this response. Moreover, complement activation produces a sustained increase in [Ca²⁺]_i, which initially triggers significant erythrocyte shrinkage, most likely mediated by K_Ca3.1-dependent K⁺ efflux. These results indicate that complement, similar to HlyA and α-toxin, requires purinergic signalling for full haemolysis and that activation of erythrocyte volume regulation protracts the process. This finding points to several new pathways to interfere with haemolytic diseases and implies that P2 receptor antagonists potentially can be used to prevent intravascular haemolysis.     

Up-regulation of P2X7 receptor-immunoreactivity by in vitro ischemia on the plasma membrane of cultured rat cortical neurons

Mixed neuronal/astrocytic cortical cell cultures of the rat were incubated for 2 or 12 h under normoxic or ischemic conditions. Subsequent flow cytometric analysis with an anti-P2X₇ receptor antibody directed against an extracellular epitope indicated the up-regulation of these receptors at the plasma membrane by 12 h of ischemia. Labelling of MAP-2 immunopositive neurons by an anti-P2X₇ antibody directed against a C-terminal epitope, documented the selectivity of the ischemia-induced increase in receptor-density for the neuronal population. By contrast, staining of GFAP immunopositive astrocytes by the same anti-P2X₇ antibody excluded any effect of ischemia on the astrocytic density of P2X₇ receptors. The ischemic up-regulation of neuronal P2X₇ receptors is in perfect agreement with the previously reported facilitation of transmitter release from the GABAergic non-pyramidal cell type in such cultures [K. Wirkner, A. Köfalvi, W. Fischer, A. Günther, H. Franke, H. Gröger-Arndt, W. Nörenberg, E. Madarasz, E.S. Vizi, D. Schneider, B. Sperlagh, P. Illes, Supersensitivity of P2X₇ receptors in cerebrocortical cell cultures after in vitro ischemia, J. Neurochem. 95 (2005) 1421–1437].     

CGRP,but not substance P,induces an increased negativity of the interstitial fluid pressure in rat trachea

Neurogenic inflammation is mediated by neuropeptides released from sensory nerves following electrical stimulation of the vagal nerve or by capsaicin. The released neuropeptides are, among others, calcitonin gene-related peptide and substance P, which both induce vasodilation, while only substance P induces plasma extravasation. Electrical stimulation of the vagal nerve induces increased negativity of interstitial fluid pressure (P_if), which will contribute to enhance oedema formation. P_if was measured, on the abluminal side of the surgically exposed trachea, with sharpened glass capillaries (4–10 μm) connected to a servo-controlled counterpressure system. Measurements were performed after circulatory arrest, since the oedema formation associated with acute inflammation will increase P_if in a positive direction, which may potentially underestimate the increased negativity of P_if. Experiments were carried out in pentobarbital anaesthetized (50 mg kg^?1) Wistar–Møller rats. P_if in control rats averaged ?1.2 ± 0.9 (SD) mmHg (n=9). Intravenous injection of capsaicin (65.0 nmol) and calcitonin gene-related peptide (1.3 nmol) increased the negativity of P_if to ?4.0 ± 1.2 mmHg (n=8)(P < 0.01) and ?4.7 ± 2.0 mmHg (n=9) (P < 0.01), respectively. Intravenous injection of substance P (7.4 nmol, n=9; and 37.0 nmol, n=8) did not affect P_if compared to control (P > 0.05). Similarly, potentiation of the available substance P with thiorphan or captopril did not increase the negative P_if, nor did injection of stable substance P analogues. Thus, the present study seems to support the theory that, in rat trachea, the increased negativity of P_if after intravenous injection of capsaicin and after vagal stimulation is caused by calcitonin gene-related peptide.