Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening.

BACKGROUND AND PURPOSE: It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. EXPERIMENTAL APPROACH: A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. KEY RESULTS: A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,ß-methylene ATP (α,ß-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,ß-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. CONCLUSIONS AND IMPLICATIONS: In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.

Saffron extract and trans-crocetin inhibit glutamatergic synaptic transmission in rat cortical brain slices.

Saffron, the dried stigmata of Crocus sativus L., is used in traditional medicine for a wide range of indications including cramps, asthma, and depression. To investigate the influence of hydro-ethanolic saffron extract (CSE) and trans-crocetin on synaptic transmission, postsynaptic potentials (PSPs) were elicited by focal electrical stimulation and recorded using intracellular placed microelectrodes in pyramidal cells from rat cingulate cortex. CSE (10-200 µg/ml) inhibited evoked PSPs as well as the isolated NMDA and non-NMDA component of PSPs. Glutamate (500 µM) added into the organ bath induced membrane depolarization. CSE decreased glutamate-induced membrane depolarization. Additionally, CSE at 100 µg/ml decreased NMDA (20 µM) and kainate (1 µM)-induced depolarization, whereas AMPA (1 µM)-induced depolarization was not affected. Trans-crocetin (1-50 µM) showed inhibition of evoked PSPs and glutamate-induced membrane depolarization comparable to CSE. Trans-crocetin at 10 µM decreased NMDA (20 µM)-induced membrane depolarization, but did not inhibit the isolated non-NMDA component of PSPs. We conclude that trans-crocetin is involved in the antagonistic effect of CSE on NMDA but not on kainate receptors.

Alterations of cholinergic receptors and the vesicular acetylcholine transporter after lateral fluid percussion injury in newborn piglets.

AIMS: Traumatic brain injury (TBI) is one of the leading causes of death and disability in children. Adult animal models of TBI showed cholinergic alterations. However, there is no comparable data on immature animals. Therefore, this study investigates cholinergic markers in a large animal model of juvenile TBI. METHODS: Twenty-seven female newborn piglets were subjected to lateral fluid percussion (FP) injury and compared with 12 untreated animals. After 6 h, animals were sacrificed and the brains removed. The hemispheres ipsilateral to FP-TBI from seven piglets and corresponding hemispheres from six control animals were used for autoradiography. Receptor density was determined with [(3)H]epibatidine (nicotinic acetylcholine receptors) or [(3)H]QNB (muscarinic acetylcholine receptors). The density of the vesicular acetylcholine transporter (vAChT) was assessed with (-)-[(3)H]vesamicol. Cerebral blood flow was measured by coloured microsphere method. RESULTS: Cerebral blood flow and brain oxygen delivery were transiently reduced early after FP-TBI (P < 0.05). TBI caused reductions of muscarinic acetylcholine receptor density (fmol/mg) in the basal forebrain (sham: 10797 +/- 1339, TBI: 8791 +/- 1031), while nicotinic acetylcholine receptor remained stable. Significant increases in vAChT density (fmol/mg) were observed in the basal forebrain (sham: 2347 +/- 171, TBI: 2884 +/- 544), putamen (sham: 2276 +/- 181, TBI: 2961 +/- 386), cortex (sham: 1928 +/- 262, TBI: 2377 +/- 294), thalamic areas (sham: 2133 +/- 272, TBI: 2659 +/- 413), hippocampus (sham: 2712 +/- 145, TBI: 3391 +/- 501) and hypothalamus (sham: 2659 +/- 139, TBI: 3084 +/- 304). CONCLUSIONS: Cholinergic markers are altered after mild-to-moderate TBI in the immature brain. Whereas the ACh receptors are stable in almost any brain region after TBI, vAChT expression increases after trauma at the employed severity of this specific trauma model.

Release of MCP-1 and IL-8 from lung epithelial cells exposed to volatile organic compounds.

Increased indoor air concentrations of volatile organic compounds (VOC) have been shown to contribute to the risk of respiratory and allergic diseases. The aim of this study was to investigate the inflammatory potential of single VOC and mixtures using an in vitro model. TNF-alpha stimulated human lung epithelial cells (A549) were exposed to VOC (1 ng/m3-100g/m3) via gas phase. After 20 h of exposure cytotoxicity and the release of the pro-inflammatory molecules monocyte chemoattractant protein-1 (MCP-1), Interleukin-6 (IL-6) and IL-8 was analysed. Exposure of A549 cells to chlorobenzene, styrene or m-xylene increased the MCP-1 production within the indoor relevant concentration range (1-25,000 microg/m3), higher concentrations increased the secretion of IL-8. Mixtures of aromatic compounds caused comparable effects to the single compounds on MCP-1 and IL-8 with a shift to lower concentration ranges. Neither the aliphatic compounds n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, and methylcyclopentane nor the mixture of these VOC showed any effects on MCP-1 and IL-8 production. Cytotoxic effects were not observed. These results show that aromatic, but no aliphatic compounds stimulate the release of pro-inflammatory mediators from lung epithelial cells. When aromatic compounds were mixed the sensitivity of lung cells to these compounds was increased.

Modulation of postsynaptic potentials in rat cortical neurons by valerian extracts macerated with different alcohols: involvement of adenosine A(1)- and GABA(A)-receptors.

Valeriana officinalis (valerian) is used traditionally as a mild sedative. Research into valerian is sparse, and studies differ greatly with respect to design, measures and preparations used. This study compares the action of a methanol (M-E), ethanol (E-E) and an extract macerated with ethylacetate (EA-E) from roots of valerian (Valeriana officinalis L., Valerianaceae) on postsynaptic potentials (PSPs) in cortical neurons. Intracellular recordings were performed in rat brain slice preparations containing pyramidal cells of the cingulate cortex. PSPs were induced by electrical field stimulation. The M-E induced strong inhibition in the concentration range 0.1-15 mg/mL, whereas the E-E (1-10 mg/mL) did not influence significantly the PSPs. The maximum inhibition induced by the M-E was completely antagonized by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microm), an antagonist on the adenosine A(1) receptor. Contrary to the M-E, the EA-E (10 mg/mL) induced an increase of the PSPs, which was completely blocked by the GABA(A) receptor antagonist picrotoxin (100 microm). The data suggest that activation of adenosine A(1) and GABA(A) receptors is mediated by different components within the valerian extract. The two mechanisms may contribute independently to the sleep-inducing effect of valerian.

Valerian extract Ze 911 inhibits postsynaptic potentials by activation of adenosine A1 receptors in rat cortical neurons.

In this study we evaluated the adenosine A1 receptor-mediated effect of valerian extract (Ze 911) on postsynaptic potentials (PSPs) in pyramidal cells of the rat cingulate cortex in a slice preparation. We first observed that N6-cyclopentyladenosine (CPA, 0.01 - 10 microM), an adenosine A1 receptor agonist, inhibited PSPs in a concentration-dependent manner. The CPA (10 microM)-induced inhibition was antagonized by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM), an adenosine A1 receptor antagonist. Ze 911 concentration dependently (0.1 - 15 mg/mL) inhibited PSPs in the presence of the adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC, 0.2 microM) and adenosine deaminase (1 U/mL). The maximal inhibition induced by 10 mg/mL was completely antagonised by DPCPX (0.1 microM), an A1 receptor blocker. The data suggest that activation of adenosine A1 receptors is involved in the pharmacological effects of the valerian extract Ze 911.

Pentoxyphylline and propentophylline are inhibitors of TNF-alpha release in monocytes activated by advanced glycation endproducts.

Non-enzymatic glycation of proteins with reducing sugars and subsequent transition metal-catalyzed oxidation leads to the formation of protein-bound "advanced glycation endproducts" (AGEs). They accumulate on long-lived protein deposits inducing senile plaques in Alzheimer's disease. AGE-modified proteins are able to activate microglia and astroglia and can cause chronic inflammation. The aim of the present study was to confirm the stimulatory effect of different AGEs on TNF-alpha release in human monocytes. Furthermore, the effects of four xanthine derivatives on AGE-induced TNF-alpha release were investigated. We show that chicken egg albumin-AGEs prepared with glucose and chicken egg albumin-AGEs prepared with methylglyoxal dose-dependently induce TNF-alpha release. The xanthine derivatives pentoxyphylline and propentophylline attenuate AGE-induced TNF-alpha release in a dose-dependent manner. Theophylline at low concentrations slightly stimulated TNF-alpha release whereas caffeine had no effect. The inhibition of the AGE-induced TNF-alpha release by pentoxyphylline and propentophylline provides interesting pharmacological strategies for diseases with local neuroinflammation such as Alzheimer's disease.

Activation of A(3) receptors by endogenous adenosine inhibits synaptic transmission during hypoxia in rat cortical neurons.

PURPOSE: The aim of our study was to characterize the influence of A(3) receptors on synaptic potentials (PSPs) in pyramidal cells from the rat cingulate cortex during hypoxia. METHODS: Intracellular recordings (n=49) were taken from slice preparations. PSPs were evoked by electrical stimulation. RESULTS: Hypoxia (95%N(2)-5%CO(2), 5 min) reduced the amplitude of the PSPs significantly. The effect was more pronounced in the presence of adenosine re-uptake inhibitor S-(p-nitrobenzyl)-6-thioguanosine (NBTG) and deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA); the effect was completely reversed by bovine adenosine deaminase. Hypoxic inhibition induced after A(1) receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in the presence of NBTG was completely reversed by the A(3) antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino]-1,2,4-triazolo[1,5-c]quinazoline (MRS 1220), indicating the involvement of A(3) receptors in hypoxic PSP inhibition. This was confirmed by A(3) agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) inhibiting PSPs. The effect of IB-MECA was blocked by the rat A(3) receptor-selective antagonist 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS 1523) and was not observed in the presence of G-protein inhibitor guanosine-5'-O-2-thiodiphosphate (GDP-beta-S). CONCLUSION: We conclude that a high level of endogenous adenosine, which occurs during hypoxia, activates A(3) receptors. Their activation contributes to PSP inhibition by adenosine during hypoxia.

Effect of an adenosine A(1) receptor agonist and a novel pyrimidoindole on membrane properties and neurotransmitter release in rat cortical and hippocampal neurons.

Activation of adenosine A(1) receptors by endogenous adenosine plays a neuroprotective role under various pathophysiological conditions including hypoxia. Intracellular recordings were made in rat pyramidal cells of the somatosensory cortex. Hypoxia (5 min) induced a membrane depolarization and a decrease of input resistance. The A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 100 microM) reversibly inhibited the hypoxic depolarization. The inhibition was also present after blockade of the A(2A), A(2B) and A(3) receptor subtypes by selective antagonists. CPA had no effect on the hypoxic decrease of input resistance. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a selective A(1) receptor antagonist, which did not alter hypoxic depolarization when given alone abolished the inhibitory effect of CPA. Neither CPA nor DPCPX influenced membrane potential or apparent input resistance under normoxic conditions. The novel pyrimidoindole (R)-9-(1-methylbenzyl)-2-(4'-pyridyl)-9H-pyrimido[4,5-b]indole-4-amine (APPPI, 1 and 10 microM) reversibly diminished hypoxic depolarization but had no significant effect on input resistance. The effect of APPPI at a concentration of 1 microM, but not at 10 microM, was blocked by DPCPX (0.1 microM). CPA (100 microM) inhibited [(3)H]-noradrenaline ([(3)H]-NA) release from rat hippocampal brain slices significantly only in the presence of rauwolscine (0.1 microM), an alpha(2)-adrenoceptor antagonist. APPPI (1 and 10 microM) exhibited an inhibitory effect similar to that observed with CPA. The effects of both CPA and APPPI were antagonized by DPCPX (0.1 microM). The present data suggest that mainly presynaptic mechanisms prevent neurons from hypoxic changes by an inhibition of transmitter release. However, in contrast to CPA, APPPI exhibited additional effects, which require further investigation.

Adenosine A(1) and A(3) receptors mediate inhibition of synaptic transmission in rat cortical neurons.

Intracellular recordings were made in rat brain slice preparations containing pyramidal cells of the associative frontal cortex in order to characterize the action of selective adenosine A(1) and A(3) receptor ligands on synaptic neurotransmission. The selective A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) inhibited concentration-dependently the excitatory postsynaptic potentials (PSPs) which were evoked by focal electrical stimulation. The CPA-mediated inhibition was blocked by 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX), a highly A(1) receptor-selective antagonist. The A(3) receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) inhibited concentration-dependently the evoked PSPs while the A(1) receptors were blocked continuously by DPCPX. Under these conditions, the A(3) receptor antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino]-1,2,4-triazolo[1, 5-c]quinazoline (MRS 1220) did not influence the PSPs but inhibited completely the effect of IB-MECA. The inhibitory effect of IB-MECA was unaffected by DPCPX. CPA additionally inhibited the PSPs when applied after IB-MECA. Pharmacological dissociation of the N-methyl-D-aspartate (NMDA) and non-NMDA receptor components of the PSPs showed that CPA as well as IB-MECA reduced both. We conclude that adenosine A(1) and A(3) receptors are present on cortical pyramidal cells and involved in the inhibition of excitatory neurotransmission. Our results indicate no interplay between the two receptor subtypes. The separate inhibition may become particularly evident in situations where there are high levels of endogenously released adenosine, as seen in hypoxia.

Therapy of bronchial asthma with adenosine receptor agonists or antagonists.

Adenosine is an endogenous nucleoside that is released under pathological conditions and interacts with four G-protein-coupled receptor subtypes. These receptors are widely distributed throughout the body. They are involved in many central and peripheral processes, including immunological and inflammatory responses. In inflammatory and asthmatic conditions, the extracellular concentration of adenosine increases in the airway tissue. It enhances mast cell degranulation and bronchoconstriction, but may also inhibit eosinophil or lymphocyte function or modulate reactive oxygen species generation in neutrophils. Despite a large number of studies clearly indicating the effects of adenosine in vitro, many aspects of the mechanisms involved in the adenosine-mediated responses are still unclear, and our knowledge is limited in understanding the complex multifactorial interactions occurring in the whole body. The discovery of adenosine receptor compounds acting with increasing selectivity will bring new approaches to the use of adenosine receptor agonists and antagonists and may clarify some of the current uncertainties. On the basis of our present knowledge, the development of adenosine A(2A)- or (A3)-receptor agonists as antiinflammatory agents or A(2B)-receptor antagonists as inhibitors of mast cell degranulation for the treatment of asthma holds promise.

Neuroprotection by ATP-dependent potassium channels in rat neocortical brain slices during hypoxia.

Morphological changes induced by 30 min of hypoxia (incubation in medium saturated with 95% N2-5% CO2 instead of the normal 95% O2-5% CO2) were investigated in neurons (layers II/III of the parietal cortex) of rat neocortical brain slices. The cells were identified as intact, reversibly or irreversibly injured. As expected, hypoxia decreased the number of intact cells and increased the number of irreversibly injured cells. Pretreatment of slices with diazoxide (300 microM), an agonist of ATP-dependent potassium (KATP) channels completely prevented the morphological damage induced by hypoxia, whereas tolbutamide (300 microM), an antagonist of KATP channels, was ineffective when given alone. However, tolbutamide (300 microM) co-applied with diazoxide (300 microM), partly reversed the neuroprotective effect of this agonist during hypoxia. In conclusion, KATP channels appear to be present on neocortical neurons and their opening counteracts hypoxia-induced cell injury.

Hypoxia and neuronal function under in vitro conditions.

Neurons in the mammalian CNS are highly sensitive to the availability of oxygen. Hypoxia can alter neuronal function and can lead to neuronal injury or death. The underlying changes in the membrane properties of single neurons have been studied in vitro in slice preparations obtained from various brain areas. Hypoxic changes of membrane potential and input resistance correspond to a decrease in ATP concentration and an increase in internal Ca2+ concentration. Functional modifications consisting of substantial membrane depolarization and failure of synaptic transmission can be observed within a few minutes following onset of hypoxia. The hypoxic depolarization accompanied by a hyperexcitability is a trigger signal for induction of neuronal cell death and is mediated mainly by activation of glutamate receptors. The mechanisms of the hypoxic hyperpolarization are more complex. Two types of potassium channels contribute to the hyperpolarization, the Ca(2+)- and the ATP-activated potassium channel. A number of neurotransmitters and neuromodulators is involved in the preservation of normal cell function during hypoxia. Therefore, hypoxia-induced cellular changes are unlikely to have a single, discrete pathway. The complexity of cellular changes implies that several strategies may be useful for neuroprotection and a successful intervention may be dependent upon drug action at more than one target site.

Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels.

In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95%O2-5%CO2 with 95%N2-5%CO2) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K(ATP)) channels, diazoxide (3-300 microM). The effect of 30 but not 300 microM diazoxide was reversed by washout. Tolbutamide (300 microM), an antagonist of K(ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 microM) on the HD. Neither diazoxide (3-300 microM) nor tolbutamide (300 microM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 microM) or tolbutamide (300 microM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between -130 and -50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 microM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri- to diphosphate ratios progressively declined for ATP/ADP and GTP/GDP, but not for UTP/UDP when the duration of hypoxia was increased from 5 to 30 min. Hence, the rapid fall in the ratios of nucleoside tri- to diphosphates without the induction of a potassium current failed to indicate an allosteric regulation of a plasmalemmal K(ATP) channel by purine and pyrimidine nucleotides. Diazoxide had no effect on neocortical pyramidal neurons and was effective only in combination with a hypoxic stimulus; it is suggested that both plasmalemmal and mitochondrial K(ATP) channels are involved under these conditions. The hypoxic depolarization may be due to blockade of K+,Na+-ATPase by limitation of energy supplying substrate.

Inhibition by ethanol of excitatory amino acid receptors in rat locus coeruleus neurons in vitro.

Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). In a first series of experiments, various parameters of spontaneous action potentials were evaluated. It turned out that ethanol (100 mM) does not alter the firing rate, the spike amplitude and the afterhyperpolarization following a spike. In subsequent experiments, the generation of action potentials was prevented by passing continuous hyperpolarizing current via the recording electrode. Under these conditions, ethanol (100 mM) had no effect on the membrane potential or input resistance. Pressure-applied N-methyl-D-aspartate (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and alpha,beta-methylene ATP (alpha,beta-meATP) reproducibly depolarized LC neurons. While ethanol (100 mM) depressed the NMDA- and AMPA-induced depolarization to a similar extent, it did not interact with alpha,beta-meATP. Lower concentrations of ethanol (10 and 30 mM) had no effect on depolarizing responses to NMDA or AMPA. Noradrenaline applied by pressure pulses reproducibly hyperpolarized LC cells. These hyperpolarizations were unchanged by ethanol (100 mM). Biphasic synaptic potentials consisting of early depolarizing (PSP) and late hyperpolarizing (IPSP) components were evoked by electrical stimulation. Ethanol (100 mM) depressed the PSP and increased the IPSP. Glutamatergic PSPs recorded in the combined presence of picrotoxin (100 microM) and suramin (100 microM) were also inhibited by ethanol (100 mM). However, IPSPs recorded under these conditions were insensitive to ethanol (100 mM). In conclusion, ethanol may interfere with the AMPA (or NMDA) receptor-mediated fraction of the PSP and slightly facilitate the alpha2 adrenoceptor-mediated fraction of the IPSP.