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Objectives: This systematic review and network meta-analysis aimed to compare different PMMA (polymethyl methacrylate) complete denture base manufacturing techniques by evaluating their mechanical properties. The objective was to determine which method-compression molding, injection molding, milling, or 3D printing-offers the best performance., Data: In vitro studies investigating mechanical properties of PMMA denture base resins., Sources: Four electronic databases such as PubMed, Embase, Web of Science, and the Cochrane Library were screened for English language articles. Two independent researchers selected studies, extracted data, assessed risk of bias, and evaluated evidence certainty., Results: A total of 17152 articles were found by electronic databases. Finally, 63 studies were analyzed, using random-effects model for network meta-analysis. The outcomes investigated were flexural strength, flexural modulus, surface roughness, impact strength, and Vickers hardness. Milling consistently ranked first or second across outcomes, excelling in flexural strength, modulus, and surface roughness. In contrast, 3D-printed denture bases demonstrated the lowest mechanical performance, highlighting the limitations of this technique at present., Conclusion: Milling is generally recommended for PMMA denture bases due to its superior mechanical properties across most outcomes, supporting its use in clinical settings. However, while promising, 3D-printed PMMA denture bases require further improvement to meet clinical performance standards., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.) more...

Background: N-methyl-D-aspartate type glutamate receptors (NMDARs) are fundamental to neuronal physiology and pathophysiology. The prefrontal cortex (PFC), a key region for cognitive function, is heavily implicated in neuropsychiatric disorders, positioning the modulation of its glutamatergic neurotransmission as a promising therapeutic target. Our recently published findings indicate that AT 1 receptor activation enhances NMDAR activity in layer V pyramidal neurons of the rat PFC. At the same time, it suggests that alternative angiotensin pathways, presumably involving AT 4 receptors (AT4Rs), might exert inhibitory effects. Angiotensin IV (Ang IV) and its analogs have demonstrated cognitive benefits in animal models of learning and memory deficits., Methods: Immunohistochemistry and whole-cell patch-clamp techniques were used to map the cell-type-specific localization of AT4R, identical to insulin-regulated aminopeptidase (IRAP), and to investigate the modulatory effects of Ang IV on NMDAR function in layer V pyramidal cells of the rat PFC., Results: AT4R/IRAP expression was detected in pyramidal cells and GABAergic interneurons, but not in microglia or astrocytes, in layer V of the PFC in 9-12-day-old and 6-month-old rats. NMDA (30 μM) induced stable inward cation currents, significantly inhibited by Ang IV (1 nM-1 µM) in a subset of pyramidal neurons. This inhibition was reproduced by the IRAP inhibitor LVVYP-H7 (10-100 nM). Synaptic isolation of pyramidal neurons did not affect the Ang IV-mediated inhibition of NMDA currents., Conclusions: Ang IV/IRAP-mediated inhibition of NMDA currents in layer V pyramidal neurons of the PFC may represent a way of regulating cognitive functions and thus a potential pharmacological target for cognitive impairments and related neuropsychiatric disorders. more...

NMDA receptors in the prefrontal cortex (PFC) play a crucial role in cognitive functions. Previous research has indicated that angiotensin II (Ang II) affects learning and memory. This study aimed to examine how Ang II impacts NMDA receptor activity in layer V pyramidal cells of the rat PFC. Whole-cell patch-clamp experiments were performed in pyramidal cells in brain slices of 9-12-day-old rats. NMDA (30 μM) induced inward currents. Ang II (0.001-1 µM) significantly enhanced NMDA currents in about 40% of pyramidal cells. This enhancement was reversed by the AT 1 antagonist eprosartan (1 µM), but not by the AT 2 receptor antagonist PD 123319 (5 μM). When pyramidal neurons were synaptically isolated, the increase in NMDA currents due to Ang II was eliminated. Additionally, the dopamine D1 receptor antagonist SCH 23390 (10 μM) reversed the Ang II-induced enhancement, whereas the D2 receptor antagonist sulpiride (20 μM) had no effect. The potentiation of NMDA currents in a subpopulation of layer V pyramidal neurons by Ang II, involving AT1 receptor activation and dopaminergic signaling, may serve as an underlying mechanism for the effects of the renin-angiotensin system (RAS) elements on neuronal functions. more...

The development of opioid tolerance in patients on long-term opioid analgesic treatment is an unsolved matter in clinical practice thus far. Dose escalation is required to restore analgesic efficacy, but at the price of side effects. Intensive research is ongoing to elucidate the underlying mechanisms of opioid analgesic tolerance in the hope of maintaining opioid analgesic efficacy. N-Methyl-D-aspartate receptor (NMDAR) antagonists have shown promising effects regarding opioid analgesic tolerance; however, their use is limited by side effects (memory dysfunction). Nevertheless, the GluN2B receptor remains a future target for the discovery of drugs to restore opioid efficacy. Mechanistically, the long-term activation of µ-opioid receptors (MORs) initiates receptor phosphorylation, which triggers β-arrestin-MAPKs and NOS-GC-PKG pathway activation, which ultimately ends with GluN2B receptor overactivation and glutamate release. The presence of glutamate and glycine as co-agonists is a prerequisite for GluN2B receptor activation. The extrasynaptic localization of the GluN2B receptor means it is influenced by the glycine level, which is regulated by astrocytic glycine transporter 1 (GlyT1). Enhanced astrocytic glycine release by reverse transporter mechanisms as a consequence of high glutamate levels or unconventional MOR activation on astrocytes could further activate the GluN2B receptor. GlyT1 inhibitors might inhibit this condition, thereby reducing opioid tolerance. more...

This review summarizes the current understanding of the role of transient receptor potential melastatin-subfamily member 7 (TRPM7) channels in the pathophysiology of neoplastic diseases. The TRPM family represents the largest and most diverse group in the TRP superfamily. Its subtypes are expressed in virtually all human organs playing a central role in (patho)physiological events. The TRPM7 protein (along with TRPM2 and TRPM6) is unique in that it has kinase activity in addition to the channel function. Numerous studies demonstrate the role of TRPM7 chanzyme in tumorigenesis and in other tumor hallmarks such as proliferation, migration, invasion and metastasis. Here we provide an up-to-date overview about the possible role of TRMP7 in a broad range of malignancies such as tumors of the nervous system, head and neck cancers, malignant neoplasms of the upper gastrointestinal tract, colorectal carcinoma, lung cancer, neoplasms of the urinary system, breast cancer, malignant tumors of the female reproductive organs, prostate cancer and other neoplastic pathologies. Experimental data show that the increased expression and/or function of TRPM7 are observed in most malignant tumor types. Thus, TRPM7 chanzyme may be a promising target in tumor therapy. more...

ATP, as a paracrine signalling molecule, induces intracellular Ca 2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca 2+ waves via diffusion of Ca 2+ mobilising second messenger IP 3 between neighbouring cells. We have compared the ATP-evoked Ca 2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca 2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB / c mice (P14-19). Octanol had no effect on Ca 2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca 2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca 2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca 2+ pump expressions and in IP 3 -Ca 2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP 3 -mediated Ca 2+ signalling inhibition by CBX cannot be excluded. more...

Stress disorders impair sleep and quality of life; however, their pathomechanisms are unknown. Prolactin-releasing peptide (PrRP) is a stress mediator; we therefore hypothesized that PrRP may be involved in the development of stress disorders. PrRP is produced by the medullary A1/A2 noradrenaline (NA) cells, which transmit stress signals to forebrain centers, and by non-NA cells in the hypothalamic dorsomedial nucleus. We found in male rats that both PrRP and PrRP-NA cells innervate melanin-concentrating hormone (MCH) producing neurons in the dorsolateral hypothalamus (DLH). These cells serve as a key hub for regulating sleep and affective states. Ex vivo , PrRP hyperpolarized MCH neurons and further increased the hyperpolarization caused by NA. Following sleep deprivation, intracerebroventricular PrRP injection reduced the number of REM sleep-active MCH cells. PrRP expression in the dorsomedial nucleus was upregulated by sleep deprivation, while downregulated by REM sleep rebound. Both in learned helplessness paradigm and after peripheral inflammation, impaired coping with sustained stress was associated with (1) overactivation of PrRP cells, (2) PrRP protein and receptor depletion in the DLH, and (3) dysregulation of MCH expression. Exposure to stress in the PrRP-insensitive period led to increased passive coping with stress. Normal PrRP signaling, therefore, seems to protect animals against stress-related disorders. PrRP signaling in the DLH is an important component of the PrRP's action, which may be mediated by MCH neurons. Moreover, PrRP receptors were downregulated in the DLH of human suicidal victims. As stress-related mental disorders are the leading cause of suicide, our findings may have particular translational relevance. SIGNIFICANCE STATEMENT Treatment resistance to monoaminergic antidepressants is a major problem. Neuropeptides that modulate the central monoaminergic signaling are promising targets for developing alternative therapeutic strategies. We found that stress-responsive prolactin-releasing peptide (PrRP) cells innervated melanin-concentrating hormone (MCH) neurons that are crucial in the regulation of sleep and mood. PrRP inhibited MCH cell activity and enhanced the inhibitory effect evoked by noradrenaline, a classic monoamine, on MCH neurons. We observed that impaired PrRP signaling led to failure in coping with chronic/repeated stress and was associated with altered MCH expression. We found alterations of the PrRP system also in suicidal human subjects. PrRP dysfunction may underlie stress disorders, and fine-tuning MCH activity by PrRP may be an important part of the mechanism., (Copyright © 2023 Vas et al.) more...

Neurodegenerative-neuroinflammatory disorders of the retina seriously hamper human vision. In searching for key factors that contribute to the development of these pathologies, we considered potential interactions among purinergic neuromodulation, glycinergic neurotransmission, and microglia activity in the retina. Energy deprivation at cellular levels is mainly due to impaired blood circulation leading to increased release of ATP and adenosine as well as glutamate and glycine. Interactions between these modulators and neurotransmitters are manifold. First, P2Y purinoceptor agonists facilitate reuptake of glycine by glycine transporter 1, while its inhibitors reduce reverse-mode operation; these events may lower extracellular glycine levels. The consequential changes in extracellular glycine concentration can lead to parallel changes in the activity of NR1/NR2B type NMDA receptors of which glycine is a mandatory agonist, and thereby may reduce neurodegenerative events in the retina. Second, P2Y purinoceptor agonists and glycine transporter 1 inhibitors may indirectly inhibit microglia activity by decreasing neuronal or glial glycine release in energy-compromised retina. These inhibitions may have a role in microglia activation, which is present during development and progression of neurodegenerative disorders such as glaucomatous and diabetic retinopathies and age-related macular degeneration or loss of retinal neurons caused by thromboembolic events. We have hypothesized that glycine transporter 1 inhibitors and P2Y purinoceptor agonists may have therapeutic importance in neurodegenerative-neuroinflammatory disorders of the retina by decreasing NR1/NR2B NMDA receptor activity and production and release of a series of proinflammatory cytokines from microglial cells. more...

TRPM7 plays an important role in cellular Ca 2+ , Zn 2+ and Mg 2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca 2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg 2+ ([Mg 2+ ] i ), were reduced by elevated [Mg 2+ ] i , and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg 2+ ] i , reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca 2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca 2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca 2+ uptake and as an independent Ca 2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca 2+ transport in amelogenesis. more...

Age-related hearing loss (ARHL), a sensorineural hearing loss of multifactorial origin, increases its prevalence in aging societies. Besides hearing aids and cochlear implants, there is no FDA approved efficient pharmacotherapy to either cure or prevent ARHL. We hypothesized that selegiline, an antiparkinsonian drug, could be a promising candidate for the treatment due to its complex neuroprotective, antioxidant, antiapoptotic, and dopaminergic neurotransmission enhancing effects. We monitored by repeated Auditory Brainstem Response (ABR) measurements the effect of chronic per os selegiline administration on the hearing function in BALB/c and DBA/2J mice, which strains exhibit moderate and rapid progressive high frequency hearing loss, respectively. The treatments were started at 1 month of age and lasted until almost a year and 5 months of age, respectively. In BALB/c mice, 4 mg/kg selegiline significantly mitigated the progression of ARHL at higher frequencies. Used in a wide dose range (0.15-45 mg/kg), selegiline had no effect in DBA/2J mice. Our results suggest that selegiline can partially preserve the hearing in certain forms of ARHL by alleviating its development. It might also be otoprotective in other mammals or humans. more...

The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30-60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP. more...

The administration of immune checkpoint inhibitors (ICIs) often leads to immune-related adverse events. However, their effect on auditory function is largely unexplored. Thorough preclinical studies have not been published yet, only sporadic cases and pharmacovigilance reports suggest their significance. Here we investigated the effect of anti-PD-1 antibody treatment (4 weeks, intraperitoneally, 200 μg/mouse, 3 times/week) on hearing function and cochlear morphology in C57BL/6J mice. ICI treatment did not influence the hearing thresholds in click or tone burst stimuli at 4-32 kHz frequencies measured by auditory brainstem response. The number and morphology of spiral ganglion neurons were unaltered in all cochlear turns. The apical-middle turns (<32 kHz) showed preservation of the inner and outer hair cells (OHCs), whilst ICI treatment mitigated the age-related loss of OHCs in the basal turn (>32 kHz). The number of Iba1-positive macrophages has also increased moderately in this high frequency region. We conclude that a 4-week long ICI treatment does not affect functional and morphological integrity of the inner ear in the most relevant hearing range (4-32 kHz; apical-middle turns), but a noticeable preservation of OHCs and an increase in macrophage activity appeared in the >32 kHz basal part of the cochlea. more...

Exploring the development of the hearing organ helps in the understanding of hearing and hearing impairments and it promotes the development of the regenerative approaches-based therapeutic efforts. The role of supporting cells in the development of the organ of Corti is much less elucidated than that of the cochlear sensory receptor cells. The use of our recently published method of single-cell electroporation loading of a fluorescent Ca 2+ probe in the mouse hemicochlea preparation provided an appropriate means to investigate the Deiters' cells at the subcellular level in two different cochlear turns (apical, middle). Deiters' cell's soma and process elongated, and the process became slimmer by maturation without tonotopic preference. The tonotopically heterogeneous spontaneous Ca 2+ activity less frequently occurred by maturation and implied subcellular difference. The exogenous ATP- and UTP-evoked Ca 2+ responses were maturation-dependent and showed P2Y receptor dominance in the apical turn. By monitoring the basic structural dimensions of this supporting cell type as well as its spontaneous and evoked purinergic Ca 2+ signaling in the hemicochlea preparation in different stages in the critical postnatal P5-25 developmental period for the first time, we showed that the soma and the phalangeal process of the Deiters' cells go through age- and tonotopy-dependent changes in the morphometric parameters and purinergic signaling. more...

Interneurons operating with glycine neurotransmitter are involved in the regulation of pain transmission in the dorsal horn of the spinal cord. In addition to interneurons, glycine release also occurs from glial cells neighboring glutamatergic synapses in the spinal cord. Neuronal and glial release of glycine is controlled by glycine transporters (GlyTs). Inhibitors of the two isoforms of GlyTs, the astrocytic type-1 (GlyT-1) and the neuronal type-2 (GlyT-2), decrease pain sensation evoked by injuries of peripheral sensory neurons or inflammation. The function of dorsal horn glycinergic interneurons has been suggested to be reduced in neuropathic pain, which can be reversed by GlyT-2 inhibitors (Org-25543, ALX1393). Several lines of evidence also support that peripheral nerve damage or inflammation may shift glutamatergic neurochemical transmission from N-methyl-D aspartate (NMDA) NR1/NR2A receptor- to NR1/NR2B receptor-mediated events (subunit switch). This pathological overactivation of NR1/NR2B receptors can be reduced by GlyT-1 inhibitors (NFPS, Org-25935), which decrease excessive glycine release from astroglial cells or by selective antagonists of NR2B subunits (ifenprodil, Ro 25-6981). Although several experiments suggest that GlyT inhibitors may represent a novel strategy in the control of neuropathic pain, proving this concept in human beings is hampered by lack of clinically applicable GlyT inhibitors. We also suggest that drugs inhibiting both GlyT-1 and GlyT-2 non-selectively and reversibly, may favorably target neuropathic pain. In this paper we overview inhibitors of the two isoforms of GlyTs as well as the effects of these drugs in experimental models of neuropathic pain. In addition, the possible mechanisms of action of the GlyT inhibitors, i.e. how they affect the neurochemical and pain transmission in the spinal cord, are also discussed. The growing evidence for the possible therapeutic intervention of neuropathic pain by GlyT inhibitors further urges development of drugable compounds, which may beneficially restore impaired pain transmission in various neuropathic conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.) more...

Purinergic signaling is deeply involved in the development, functions and protective mechanisms of the cochlea. Release of ATP and activation of purinergic receptors on sensory and supporting/epithelial cells play a substantial role in cochlear (patho)physiology. Both the ionotropic P2X and the metabotropic P2Y receptors are widely distributed on the inner and outer hair cells as well as on the different supporting cells in the organ of Corti and on other epithelial cells in the scala media. Among others, they are implicated in the sensitivity adjustment of the receptor cells by a K + shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics acting on outer hair cells and supporting cells. Cochlear blood flow is also regulated by purines. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy. Decreasing hearing sensitivity and increasing cochlear blood supply by pharmacological targeting of purinergic signaling in the cochlea are potential new therapeutic approaches in these hearing disabilities, especially in the noise-induced ones., (Copyright © 2019 Elsevier Inc. All rights reserved.) more...

Hearing impairment is the most common sensory deficit, affecting more than 400 million people worldwide. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy largely due to the insufficient knowledge of the pathomechanism. Purinergic signaling plays a substantial role in cochlear (patho)physiology. P2 (ionotropic P2X and the metabotropic P2Y) as well as adenosine receptors expressed on cochlear sensory and non-sensory cells are involved mostly in protective mechanisms of the cochlea. They are implicated in the sensitivity adjustment of the receptor cells by a K + shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics. Cochlear blood flow is also regulated by purines. Here, we propose to comprehend this field with the purine-immune interactions in the cochlea. The role of harmful immune mechanisms in sensorineural hearing losses has been emerging in the horizon of cochlear pathologies. In addition to decreasing hearing sensitivity and increasing cochlear blood supply, influencing the immune system can be the additional avenue for pharmacological targeting of purinergic signaling in the cochlea. Elucidating this complexity of purinergic effects on cochlear functions is necessary and it can result in development of new therapeutic approaches in hearing disabilities, especially in the noise-induced ones. more...

Ca 2+ is an important intracellular messenger and regulator in both physiological and pathophysiological mechanisms in the hearing organ. Investigation of cellular Ca erscript>2+ homeostasis in the mature cochlea is hampered by the special anatomy and high vulnerability of the organ. A quick, straightforward and reliable Ca 2+ imaging method with high spatial and temporal resolution in the mature organ of Corti is missing. Cell cultures or isolated cells do not preserve the special microenvironment and intercellular communication, while cochlear explants are excised from only a restricted portion of the organ of Corti and usually from neonatal pre-hearing murines. The hemicochlea, prepared from hearing mice allows tonotopic experimental approach on the radial perspective in the basal, middle and apical turns of the organ. We used the preparation recently for functional imaging in supporting cells of the organ of Corti after bulk loading of the Ca 2+ indicator. However, bulk loading takes long time, is variable and non-selective, and causes the accumulation of the indicator in the extracellular space. In this study we show the improved labeling of supporting cells of the organ of Corti by targeted single-cell electroporation in mature mouse hemicochlea. Single-cell electroporation proved to be a reliable way of reducing the duration and variability of loading and allowed subcellular Ca 2+ imaging by increasing the signal-to-noise ratio, while cell viability was retained during the experiments. We demonstrated the applicability of the method by measuring the effect of purinergic, TRPA1, TRPV1 and ACh receptor stimulation on intracellular Ca 2+ concentration at the cellular and subcellular level. In agreement with previous results, ATP evoked reversible and repeatable Ca 2+ transients in Deiters', Hensen's and Claudius' cells. TRPA1 and TRPV1 stimulation by AITC and capsaicin, respectively, failed to induce any Ca 2+ response in the supporting cells, except in a single Hensen's cell in which AITC evoked transients with smaller amplitude. AITC also caused the displacement of the tissue. Carbachol, agonist of ACh receptors induced Ca 2+ transients in about a third of Deiters' and fifth of Hensen's cells. Here we have presented a fast and cell-specific indicator loading method allowing subcellular functional Ca 2+ imaging in supporting cells of the organ of Corti in the mature hemicochlea preparation, thus providing a straightforward tool for deciphering the poorly understood regulation of Ca 2+ homeostasis in these cells., (Copyright © 2018 Elsevier B.V. All rights reserved.) more...

P2X7 receptors (P2X7Rs) are ligand-gated ion channels sensitive to extracellular ATP. Here we examined for the first time the role of P2X7R in an animal model of schizophrenia. Using the PCP induced schizophrenia model we show that both genetic deletion and pharmacological inhibition of P2X7Rs alleviate schizophrenia-like behavioral alterations. In P2rx7+/+ mice, PCP induced hyperlocomotion, stereotype behavior, ataxia and social withdrawal. In P2X7 receptor deficient mice (P2rx7-/-), the social interactions were increased, whereas the PCP induced hyperlocomotion and stereotype behavior were alleviated. The selective P2X7 receptor antagonist JNJ-47965567 partly replicated the effect of gene deficiency on PCP-induced behavioral changes and counteracted PCP-induced social withdrawal. We also show that PCP treatment upregulates and increases the functional responsiveness of P2X7Rs in the prefrontal cortex of young adult animals. The amplitude of NMDA evoked currents recorded from layer V pyramidal neurons of cortical slices were slightly decreased by both genetic deletion of P2rx7 and by JNJ-47965567. PCP induced alterations in mRNA expression encoding schizophrenia-related genes, such as NR2A, NR2B, neuregulin 1, NR1 and GABA α1 subunit were absent in the PFC of young adult P2rx7-/- animals. Our findings point to P2X7R as a potential therapeutic target in schizophrenia. more...

Background: Allyphenyline, a novel α2-adrenoceptor (AR) ligand, has been shown to selectively activate α2C-adrenoceptors (AR) and 5HT1A receptors, but also to behave as a neutral antagonist of α2A-ARs. We exploited this unique pharmacological profile to analyze the role of α2C-ARs and 5HT1A receptors in the regulation of gastric mucosal integrity and gastrointestinal motility., Methods: Gastric injury was induced by acidified ethanol in Wistar rats. Mucosal catalase and superoxide dismutase levels were measured by assay kits. The effect of allyphenyline on electrical field stimulation (EFS)-induced fundic and colonic contractions was determined in C57BL/6 mice., Results: Intracerebroventricularly injected allyphenyline (3 and 15 nmol/rat) dose dependently inhibited the development of mucosal damage, which was antagonized by ARC 239 (α2B/C-AR and 5HT1A receptor antagonist), (S)-WAY 100135 (selective 5HT1A receptor antagonist), and JP-1302 (selective α2C-AR antagonist). This protection was accompanied by significant elevation of mucosal catalase and superoxide dismutase levels. Allyphenyline (10(-9)-10(-5) M) also inhibited EFS-induced fundic contractions, which was antagonized by ARC 239 and (S)-WAY 100135, but not by JP-1302. Similar inhibition was observed in the colon; however, in this case only ARC 239 reduced this effect, while neither selective inhibition of α2C-ARs and 5HT1A receptors nor genetic deletion of α2A- and α2B-ARs influenced it., Conclusions: Activation of both central α2C-ARs and 5HT1A receptors contributes to the gastroprotective action of allyphenyline in rats. Its inhibitory effect on fundic contractions is mediated by 5HT1A receptors, but neither α2-ARs nor 5HT1A receptors take part in its inhibitory effect on colonic contractility in mice. more...

Glutamate is the main excitatory neurotransmitter of the central nervous system (CNS), released both from neurons and glial cells. Acting via ionotropic (NMDA, AMPA, kainate) and metabotropic glutamate receptors, it is critically involved in essential regulatory functions. Disturbances of glutamatergic neurotransmission can be detected in cognitive and neurodegenerative disorders. This paper summarizes the present knowledge on the modulation of glutamate-mediated responses in the CNS. Emphasis will be put on NMDA receptor channels, which are essential executive and integrative elements of the glutamatergic system. This receptor is crucial for proper functioning of neuronal circuits; its hypofunction or overactivation can result in neuronal disturbances and neurotoxicity. Somewhat surprisingly, NMDA receptors are not widely targeted by pharmacotherapy in clinics; their robust activation or inhibition seems to be desirable only in exceptional cases. However, their fine-tuning might provide a promising manipulation to optimize the activity of the glutamatergic system and to restore proper CNS function. This orchestration utilizes several neuromodulators. Besides the classical ones such as dopamine, novel candidates emerged in the last two decades. The purinergic system is a promising possibility to optimize the activity of the glutamatergic system. It exerts not only direct and indirect influences on NMDA receptors but, by modulating glutamatergic transmission, also plays an important role in glia-neuron communication. These purinergic functions will be illustrated mostly by depicting the modulatory role of the purinergic system on glutamatergic transmission in the prefrontal cortex, a CNS area important for attention, memory and learning. more...

The therapeutic use of opioids is limited by the development of tolerance to the analgesic effect and the cellular and molecular mechanisms underlying this phenomenon are still not completely understood. For this reason the search for new analgesic derivatives, endowed with lower tolerance, is always an active field. The newly synthesized 14-O-Methylmorphine-6-sulfate (14-O-MeM6SU) shows high efficacy in in vitro assays and a strong analgesic action in the rat tail flick test. The aim of present work was to investigate: the analgesic effect of 14-O-MeM6SU in mouse tail-flick test; the tolerance to analgesic effect of 14-O-MeM6SU compared to morphine in mice, the effects of test compounds on glutamatergic neurotransmission by measuring spontaneous excitatory postsynaptic currents (sEPSCs) of layer V pyramidal cells from rat prefrontal cortices; and the effect of acute and chronic 14-O-MeM6SU treatments on opioid receptor gene expression in SH-SY5Y neuroblastoma cells expressing μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. 14-O-MeM6SU was 17 times more potent than morphine in analgesia and had long duration of action in analgesic dose equipotent to morphine. Mice were treated subcutaneously (s.c.) either with 200 μmol/kg morphine or with 14-O-MeM6SU (12 μmol/kg) twice daily for three days. The magnitude of tolerance or cross-tolerance indicated by the shift in antinociceptive ED50 measured was greater for morphine compared to 14-O-MeM6SU. Subsequent to behavioral testing, patch-clamp experiments in layer V pyramidal neurons of rat prefrontal cortical slices in the presence of bicuculline were performed. Both 14-O-MeM6SU (0.1 μM) and morphine (1 μM) decreased the frequency of sEPSCs, indicating reduction of glutamate release. The effect of the novel compound was reversed by the opioid receptor antagonist naloxone, indicating an opioid mediated action. In contrast, the amplitude was not affected. Finally, gene expression data showed a dose dependent down-regulation of MOP receptor after 24h and 48 h exposure to 14-O-MeM6SU. Interestingly, no changes were detected for NOP receptor gene expression. The specific lack of this effect could be related to the lower tolerance development to analgesic effect of 14-O-MeM6SU. Furthermore, 14-O-MeM6SU displayed high intrinsic efficacy possibly an important factor in the observed effects. Further, the observed inhibition of glutamatergic signaling might be attributed also to the reduction of opioid tolerance. Based on our results the development of a new clinically important, safe analgesic agent might be possible., (Copyright © 2015 Elsevier Inc. All rights reserved.) more...

The medial prefrontal cortex (PFC) is thought to be the highest order association area in the mammalian cortex which is involved in cognitive functions. Especially, layer V pyramidal cells integrating afferent innervations from dopaminergic cell groups in the ventral tegmental area, glutamatergic inputs from the thalamus and neighbouring PFC pyramical cells, as well as GABAergic inputs from local interneurons are crucial for processing short-term working memory. These neurons are endowed with the NMDA- and AMPA-type excitatory amino acid receptors, described to be involved in the regulation of synaptic plasticity, the apparent basis of elementary learning processes. NMDA receptor currents were in fact regulated on the one hand by dopamine D1 receptors and on the other hand by ATP-sensitive receptors of the P2Y-type. P2Y4 receptors acted indirectly to potentiate NMDA receptor-currents by releasing vesicular glutamate from astrocytes, or attenuated these currents directly by stimulating P2Y1 receptors located at the PFC cells themselves. Long-term depression (LTD) induced in PFC pyramidal neurons could be blocked by P2Y1 receptors in a manner not depending on NMDA receptors but targeting voltage-sensitive dendritic Ca2+ channels. In vivo data also support the notion that P2Y1 receptors participate in the regulation of cognitive processes and addiction. For example, in a spatial delayed win-shift task, P2Y1 receptor-activation has been shown to deteriorate not the primary storage of information but its processing during and after a delay. Further, it is widely accepted that behavioural sensitization in animals provides a model for the intensification of drug craving believed to underlie addiction in humans. In fact, sensitization to amphetamine was interrupted by the blockade of P2Y1 receptors in the mesocortico-limbic dopaminergic system. more...

Neocortical and striatal TRPV1 (vanilloid or capsaicin) receptors (TRPV1Rs) are excitatory ligand-gated ion channels, and are implicated in psychiatric disorders. However, the purported presynaptic neuromodulator role of TRPV1Rs in glutamatergic, serotonergic or dopaminergic terminals of the rodent forebrain remains little understood. With the help of patch-clamp electrophysiology and neurochemical approaches, we mapped the age-dependence of presynaptic TRPV1R function, and furthermore, we aimed at exploring whether the presence of CB1 cannabinoid receptors (CB1Rs) influences the function of the TRPV1Rs, as both receptor types share endogenous ligands. We found that the major factor which affects presynaptic TRPV1R function is age: by post-natal day 13, the amplitude of capsaicin-induced release of dopamine and glutamate is halved in the rat striatum, and two weeks later, capsaicin already loses its effect. However, TRPV1R receptor function is not enhanced by chemical or genetic ablation of the CB1Rs in dopaminergic, glutamatergic and serotonergic terminals of the mouse brain. Altogether, our data indicate a possible neurodevelopmental role for presynaptic TRPV1Rs in the rodent brain, but we found no cross-talk between TRPV1Rs and CB1Rs in the same nerve terminal., (Copyright © 2013 Elsevier Inc. All rights reserved.) more...

Background/aims: ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs., Methods: We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs., Results: Our data show that ATP (< 1 μM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs., Conclusions: Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different., (Copyright © 2013 S. Karger AG, Basel.) more...

Purine and pyrimidine nucleotides are extracellular signaling molecules in the central nervous system (CNS) leaving the intracellular space of various CNS cell types via nonexocytotic mechanisms. In addition, ATP is a neuro-and gliotransmitter released by exocytosis from neurons and neuroglia. These nucleotides activate P2 receptors of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) types. In mammalians, seven P2X and eight P2Y receptor subunits occur; three P2X subtypes form homomeric or heteromeric P2X receptors. P2Y subtypes may also hetero-oligomerize with each other as well as with other G protein-coupled receptors. P2X receptors are able to physically associate with various types of ligand-gated ion channels and thereby to interact with them. The P2 receptor homomers or heteromers exhibit specific sensitivities against pharmacological ligands and have preferential functional roles. They may be situated at both presynaptic (nerve terminals) and postsynaptic (somatodendritic) sites of neurons, where they modulate either transmitter release or the postsynaptic sensitivity to neurotransmitters. P2 receptors exist at neuroglia (e.g., astrocytes, oligodendrocytes) and microglia in the CNS. The neuroglial P2 receptors subserve the neuron-glia cross talk especially via their end-feets projecting to neighboring synapses. In addition, glial networks are able to communicate through coordinated oscillations of their intracellular Ca(2+) over considerable distances. P2 receptors are involved in the physiological regulation of CNS functions as well as in its pathophysiological dysregulation. Normal (motivation, reward, embryonic and postnatal development, neuroregeneration) and abnormal regulatory mechanisms (pain, neuroinflammation, neurodegeneration, epilepsy) are important examples for the significance of P2 receptor-mediated/modulated processes., (Copyright © 2011 Elsevier Inc. All rights reserved.) more...

Locus coeruleus (LC) neurons in a rat brain slice preparation were superfused with a Mg(2+)-free and bicuculline-containing external medium. Under these conditions, glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) were recorded by means of the whole-cell patch-clamp method. ATP, as well as its structural analogue 2-methylthio ATP (2-MeSATP), both caused transient inward currents, which were outlasted by an increase in the frequency but not the amplitude of the sEPSCs. PPADS, but not suramin or reactive blue 2 counteracted both effects of 2-MeSATP. By contrast, α,β-methylene ATP (α,β-meATP), UTP and BzATP did not cause an inward current response. Of these latter agonists, only BzATP slightly facilitated the sEPSC amplitude and strongly potentiated its frequency. PPADS and Brilliant Blue G, as well as fluorocitric acid and aminoadipic acid prevented the activity of BzATP. Furthermore, BzATP caused a similar facilitation of the miniature (m)EPSC (recorded in the presence of tetrodotoxin) and sEPSC frequencies (recorded in its absence). Eventually, capsaicin augmented the frequency of the sEPSCs in a capsazepine-, but not PPADS-antagonizable, manner. In conclusion, the stimulation of astrocytic P2X7 receptors appears to lead to the outflow of a signalling molecule, which presynaptically increases the spontaneous release of glutamate onto LC neurons from their afferent fibre tracts. It is suggested, that the two algogenic compounds ATP and capsaicin utilise separate receptor systems to potentiate the release of glutamate and in consequence to increase the excitability of LC neurons. more...

Nociceptin (N/OFQ) and nocistatin (NST) are two endogenous neuropeptides derived from the same precursor protein, preproN/OFQ. The aim of the present work was to study the effect of NST on the ethanol-induced mucosal damage compared with that of N/OFQ following intracerebroventricular (i.c.v.) administration in the rat and to analyze the mechanism of the gastroprotective action. It was found that both NST and N/OFQ reduced the mucosal lesions in the same dose range (0.2-1 nmol i.c.v.), but in higher doses (2-5 nmol i.c.v.) the gastroprotective effect of both peptides was highly diminished. The gastroprotective effect of N/OFQ (1 nmol), but not that of NST (1 nmol), was reduced by the selective nociceptin receptor antagonist J-113397 (69 nmol i.c.v.). Similarly, decrease of the gastroprotective effect was observed after the combination of NST (1 nmol) with N/OFQ (0.6 or 1 nmol). However, addition of the gastroprotective effects was observed, when lower dose (0.2 nmol) of NST was given prior to N/OFQ (0.6 nmol). The gastroprotective effect of both N/OFQ and NST was antagonized by naloxone (27 nmol), beta-funaltrexamine (20 nmol), naltrindole (5 nmol) and norbinaltorphimine (14 nmol), the mu-, delta- and kappa-opioid receptor antagonists, respectively, given i.c.v. The mucosal protection was significantly decreased after bilateral cervical vagotomy. The present findings suggest that NST similar to N/OFQ, may also induce gastric mucosal protective action initiated centrally in a vagal-dependent mechanism. Opioid component is likely to be involved in the gastroprotective effect of both NST and N/OFQ. more...

Membrane currents of layer V pyramidal cells in slices of the rat prefrontal cortex (PFC) were recorded with the patch-clamp technique. In an Mg2+-free superfusion medium l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a preferential blocker of astrocytic glutamate transporters, caused inward current due to the activation of NMDA receptors. The blockade of conducted action potentials by tetrodotoxin did not interfere with this effect. ATP was inactive when given alone and potentiated the NMDA-induced current in an Mg2+-containing but not Mg2+-free superfusion medium. Agonists of group I ((S)-3,5-dihydroxyphenylglycine; DHPG) and II ((1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid; LY 379268) metabotropic glutamate receptors (mGluRs) also potentiated responses to NMDA, whereas the group III mGluR agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) did not affect them. In contrast to ATP, PDC evoked inward current in the absence but not in the presence of external Mg2+, when given alone, and facilitated the NMDA effect Mg2+-independently. The PDC-induced facilitation of NMDA responses was blocked by group II ((2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; LY 341495), but not group I ((RS)-1-aminoindan-1,5-dicarboxylic acid; AIDA) or III (alpha-methyl-3-methyl-4-phosphonophenylglycine; UBP 1112) mGluR antagonists. In conclusion, the blockade of astrocytic glutamate uptake by PDC may lead to a stimulation of group II mGluRs, while the triggering of exocytotic glutamate release from astrocytes by ATP may cause activation of group I mGluRs, both situated postsynaptically at layer V PFC pyramidal cells. Either group of mGluRs may interact with NMDA receptors in a positive manner. more...

The ventral tegmental area (VTA), the prefrontal cortex and the nucleus accumbens (NAc) are key elements of the mesolimbic dopaminergic system. Dopaminergic neurotransmission in the NAc is essential in the regulation of motor activity and reward. Extracellular ATP by activating P2 receptors may function as a neurotransmitter or a neuromodulator. We showed that P2 receptors are expressed both in the NAc and VTA, and their activation (probably of the P2Y1 subtype) results in increased dopamine release. It leads to complex neurophysiologic and behavioral changes. We observed activation of the EEG: an elevation of the absolute EEG power and the power in the alpha-frequency band as well as decrease in the delta-frequency band. Behavioral studies demonstrated that activation of P2 receptors elicited more consistent and stronger goal-directed locomotor activity in response to the stimulus of a novel environment. P2Y receptors were also involved in regulation of feeding, their inhibition decreased the amount and the duration of feeding. On the other hand, in various behavioral functions, P2 receptor-mediated glutamate release or the activation of the adenosine receptors counterbalanced the actions mediated by ATP-induced dopamine release. We also showed that enhancement of the P2Y1 receptor expression may be involved in adaptive changes of the mesolimbic system such as behavioral sensitization to repeated amphetamine administration. In summary, the mesolimbic dopaminergic system is modulated via P2Y purinergic receptors, and it may lead to complex behavioral pharmacological changes. more...

Ionotropic P2X and metabotropic P2Y receptors interact with a number of macromolecules in the cell membrane which may contribute to their functional plasticity. P2X receptors are homomeric or heteromeric assemblies of three subunits. P2Y receptors may form oligomeric complexes either with the same or with other P2Y receptor types. Although the signalling mechanism of P2X receptor channels is fast (within milliseconds) and relatively simple, by originating from the opening of an ion channel permeable to mono- and divalent cations, various macromolecules may modify the trafficking of these receptors to and from the cell membrane, as well as their activation and desensitization kinetics, and the possible opening of membrane pores induced by long-lasting exposure to agonists. P2X and Cys-loop receptors may physically interact with each other, resulting in mutual current occlusion. Heteromeric P2Y receptors may, via G(s), G(q/11) or G(i/o) protein-coupling and activation of the respective transduction mechanisms, mediate responses in the range of a few seconds. However, P2Y receptors may also interact with the signalling cascade of, e.g. receptor tyrosine kinases, and thereby mediate responses on a much slower time scale (within hours to days). In addition, P2Y receptors may interact with small, homomeric G proteins, integrins, and PDZ proteins. Eventually, P2Y receptors may cross-talk via Galpha-dependent signalling with other G protein-coupled receptors and via Gbetagamma (or indirectly Galpha)-dependent signalling with various ion channels. Thus, the activation of P2X and P2Y receptors by extracellular adenosine triphosphate/adenosine diphosphate or uridine triphosphate/uridine diphosphate may trigger specific chains of events which interact at the level of the individual elements both with each other and with the transduction mechanisms of other receptors, creating a huge diversity of the possible effects. more...

Whole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X(3) receptors. In HEK293 cells permanently transfected with human (h) P2X(3) receptors (HEK293-hP2X(3) cells), an acidic pH shifted the concentration-response curve for alpha,beta-methylene ATP (alpha,beta-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of alpha,beta-meATP. Further, a low pH value increased the activation time constant (tau(on)) of the alpha,beta-meATP current; the fast and slow time constants of desensitization (tau(des1), tau(des2)) were at the same time also increased. Finally, acidification accelerated the recovery of P2X(3) receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X(3) receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the alpha,beta-meATP currents. The voltage sensitivity and reversal potential of the currents activated by alpha,beta-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X(3) receptor channels. It is suggested that acidification may have a dual effect on P2X(3) channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues. more...

Current responses to N-methyl-D-aspartate (NMDA) in layer V pyramidal neurons of the rat prefrontal cortex were potentiated by the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP). The failure of these nucleotides to induce inward current on fast local superfusion suggested the activation of P2Y rather than P2X receptors. The potentiation by ATP persisted in a Ca(2+)-free superfusion medium but was abolished by 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester, cyclopiazonic acid, 7-nitroindazole, fluoroacetic acid, bafilomycin, and tetanus toxin, indicating that an astrocytic signaling molecule may participate. Because the metabotropic glutamate receptor (mGluR) agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (group I/II) and (RS)-3,5-dihydroxyphenylglycine (group I) both imitated the effect of ATP and the group I mGluR antagonist 1-aminoindan-1,5-dicarboxylic acid or a combination of selective mGluR(1) (7-(hydroxyimino)-cyclopropa[b]chromen-1a-carboxylate) and mGluR(5) (2-methyl-6-(phenylethynyl)pyridine) antagonists abolished the facilitation by ATP, it was concluded that the signaling molecule may be glutamate. Pharmacological tools known to interfere with the transduction cascade of type I mGluRs (guanosine 5'-O-(3-thiodiphosphate), U-73122, xestospongin C, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin kinase II [CAMKII] inhibitor peptide) depressed the actions of both ATP and ACPD. Characterization of the P2Y receptor by agonists (ATP and UTP), antagonists (suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid), and knockout mice (P2Y(2)(-/-)) suggested that the nucleotides act at the P2Y(4) subtype. In conclusion, we propose that exogenous and probably also endogenous ATP release vesicular glutamate from astrocytes by P2Y(4) receptor activation. This glutamate then stimulates type I mGluRs of layer V pyramidal neurons and via the G(q)/phospholipase C/inositol 1,4,5-trisphosphate/Ca(2+)/CAMKII transduction pathway facilitates NMDA receptor currents. more...

Purinergic signaling is involved in the proper functioning of virtually all organs of the body. Although in some cases purines have a major influence on physiological functions (e.g. thrombocyte aggregation), more often they are just background modulators contributing to fine tuning of biological events. However, under pathological conditions, when a huge amount of adenosine 5'-triphosphate (ATP) can reach the extracellular space, their significance is increasing. ATP and its various degradation products activate membrane receptors divided into two main classes: the metabotropic P2Y and the ionotropic P2X family. This latter group, the purine ionotropic receptor, is the object of this review. After providing a description about the distribution and functional properties of P2X receptors in the body, their pharmacology will be summarized. In the second part of this review, the role of purines in those organ systems and body functions will be highlighted, where the (patho)physiological role of P2X receptors has been suggested or is even well established. Besides the regulation of organ systems, for instance in the cardiovascular, respiratory, genitourinary or gastrointestinal system, some special issues will also be discussed, such as the role of P2X receptors in pain, tumors, central nervous system (CNS) injury and embryonic development. Several examples will indicate that purine ionotropic receptors might serve as attractive targets for pharmacological interventions in various diseases, and that selective ligands for these receptors will probably constitute important future therapeutic tools in humans. more...

The whole-cell patch-clamp technique was used to record current responses to nucleotides and nucleosides in human embryonic kidney HEK293 cells transfected with the human purinergic P2X3 receptor. When guanosine 5'-O-(3-thiodiphosphate) was included into the pipette solution, UTP at concentrations that did not alter the holding current facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. ATP and GTP, but not UDP or uridine, had an effect similar to that of UTP. Compounds known to activate protein kinase C (PKC) acted like the nucleoside triphosphates investigated, whereas various PKC inhibitors invariably reduced the effects of both PKC activators and UTP. The substitution by Ala of Ser/Thr residues situated within PKC consensus sites of the P2X3 receptor ectodomain either abolished (PKC2 and PKC3; T134A, S178A) or did not alter (PKC4 and PKC6; T196A, S269A) the UTP-induced potentiation of the alpha,beta-meATP current. Both the blockade of ecto-protein kinase C activity and the substitution of Thr-134 or Ser-178 by Ala depressed the maximum of the concentration-response curve for alpha,beta-meATP without altering the EC50 values. Molecular simulation of the P2X3 receptor structure indicated no overlap between assumed nucleotide binding domains and the relevant phosphorylation sites PKC2 and PKC3. alpha,beta-meATP-induced currents through native homomeric P2X3 receptors of rat dorsal root ganglia were also facilitated by UTP. In conclusion, it is suggested that low concentrations of endogenous nucleotides in the extracellular space may prime the sensitivity of P2X3 receptors toward the effect of subsequently applied (released) higher agonistic concentrations. The priming effect of nucleotides might be attributable to a phosphorylation of PKC sites at the ectodomain of P2X3 receptors. more...

Adenine and/or uridine nucleotide-sensitive receptors are classified into two types belonging to the ligand-gated ionotropic family (P2X) and the metabotropic, G-protein-coupled family (P2Y). In humans, seven different P2X receptors (P2X(1-7)) and eight different P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11-14)) have been detected hitherto. All P2 receptors are expressed in the CNS, with the preferential expression of the P2X(2), P2X(4), P2X(6) and P2Y(1) receptors in neurons. In addition to the neurotransmitter and modulator functions, neurite outgrowth, proliferation of glial cells and the expression of transmitter receptors at target cells have also been suggested to be regulated by extracellular nucleotides in the nervous system. In spite of the expanding knowledge in the purinergic research field, the present therapeutic utilization of P2 receptor ligands is mostly related to peripheral diseases such as thromboembolic disorders and cystic fibrosis. In this review we provide some evidence that P2 receptors play an important role in the regulation of CNS functions related to hippocampal activity, the mesolimbic dopaminergic system and the nociceptive system. The role of purinergic receptors located on astrocytes/microglia and implications of these receptors for neurodegenerative/neuroinflammatory disorders, CNS injury and epilepsy will be highlighted as well., ((c) 2005 Prous Science. All rights reserved.) more...

Dopamine-glutamate interactions in the prefrontal cortex (PFC) are associated with higher order cognitive functions, and are involved in the pathophysiology of schizophrenia and addiction. Recordings with intracellular sharp microelectrodes and patch-clamp pipettes were used to investigate these interactions in layer V pyramidal cells of brain slices obtained from the rat PFC. Dopamine (100 microM) potentiated N-methyl-d-aspartate (NMDA; 10mM)-evoked depolarizations, but did not change those elicited by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA; 1mM). Dopamine (100 microM) increased the amplitude of the NMDA (30 microM)-induced currents as well, and 1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF 38393; 1, 10 microM), a D(1) receptor agonist, concentration-dependently reproduced this effect. Furthermore, 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzapine hydrochloride (SCH 23390; 10 microM), a D(1) receptor antagonist, reversed both the dopamine- and the SKF 38393-evoked potentiation. The D(2) receptor agonists lisuride and quinpirole (10 microM both), as well as noradrenaline (100 microM) failed to mimic the stimulatory effect of dopamine. Isoproterenol (1, 10 microM) concentration-dependently facilitated NMDA responses. However, neither this effect at 10 microM nor that of dopamine at 100 microM could be antagonized by propranolol (10 microM), a non-selective beta adrenoceptor blocker. The isoproterenol-induced facilitation of NMDA currents was abolished by SCH 23390 (10 microM). The results indicate that dopamine potentiates NMDA responses in layer V pyramidal cells of the PFC solely by activating D(1) receptors. D(2) receptors and alpha or beta adrenoceptors are not involved in the dopamine-NMDA interaction. more...

The function of adenosine A(2A) receptors, localized at the enkephalin-containing GABAergic medium spiny neurons of the striatum, has been discussed controversially. Here we show that, in the absence of external Mg(2+), the adenosine A(2A) receptor agonist CGS 21680 postsynaptically depressed the NMDA, but not the non-NMDA (AMPA/kainate) receptor-mediated fraction of the electrically evoked EPSCs in a subpopulation of striatal neurons. Current responses to locally applied NMDA but not AMPA were also inhibited by CGS 21680. However, in the presence of external Mg(2+), the inhibition by CGS 21680 of the GABA(A) receptor-mediated IPSCs led to a depression of the EPSC/IPSC complexes. The current response to the locally applied GABA(A) receptor agonist muscimol was unaltered by CGS 21680. Whereas, the frequency of spontaneous (s)IPSCs was inhibited by CGS 21680, their amplitude was not changed. Hence, it is suggested that under these conditions the release rather than the postsynaptic effect of GABA was affected by CGS 21680. In conclusion, under Mg(2+)-free conditions, CGS 21680 appeared to postsynaptically inhibit the NMDA receptor-mediated component of the EPSC, while in the presence of external Mg(2+) this effect turned into a presynaptic inhibition of the GABA(A) receptor-mediated IPSC. more...

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene. more...

In the first part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex were evoked by electrical stimulation of layer I. Recordings by intracellular sharp microelectrodes showed that EPSPs were concentration-dependently facilitated by the P2 receptor antagonistic ATP analogue 2-methylthio ATP (2-MeSATP), while ATP itself depressed the synaptic potentials. The inhibitory effect of ATP turned into facilitation in the presence of the adenosine A(1) receptor antagonist DPCPX. The 2-MeSATP-induced potentiation of EPSP amplitudes were prevented by the P2 receptor antagonists PPADS and Suramin. The EPSP was almost abolished by coapplication of the NMDA receptor antagonist AP-5 and the AMPA/kainate receptor antagonist CNQX. After blockade of the NMDA receptor-mediated part of the EPSP by AP-5, the stimulatory effect of 2-MeSATP disappeared. When NMDA or AMPA were pressure-applied onto pyramidal cells, only the NMDA-induced depolarization was potentiated by 2-MeSATP. In the second part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ATP, 2-MeSATP, UDP and UTP potentiated the response to NMDA, while ADP-beta-S was inactive. PPADS antagonized the effect of ATP. Synaptic isolation of pyramidal neurons by a Ca(2+)-free medium or tetrodotoxin did not alter the effect of ATP which, however, was markedly depressed when GTP in the micropipette was replaced by GDP-beta-S. These observations suggest that in layer V pyramidal neurons of the prefrontal cortex postsynaptically localized P2Y receptors interact with NMDA receptor-channels. more...

Membrane currents in response to the application of alpha, beta-methylene ATP (alpha,beta-meATP) were recorded by the whole-cell patch-clamp technique in human embryonic kidney 293 cells transfected with the human P2X3 receptor (HEK 293-hP2X3 cells). Trichloroethanol, the biologically active metabolite of chloral hydrate, but not ethanol itself concentration-dependently and reversibly inhibited the current responses. It was concluded that the reported analgesic effect of chloral hydrate may be due to the interruption of pain transmission in dorsal root ganglia expressing P2X3 receptors. more...