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ABSTRACTIntroductionAstrocytes have previously been considered as cells supporting neuronal functions, but they are now recognized as active players in maintaining central nervous system (CNS) homeostasis. Astrocytes can communicate with other CNS cells, i.e. through the gliotransmitter ATP and P2X7 receptors (Rs).Areas coveredIn this review, we will discuss how the P2X7R initiates the release of gliotransmitters and proinflammatory cytokines/chemokines, thereby establishing a dialog between astrocytes and neurons and, in addition, causing neuroinflammation. In astrocytes, dysregulation of P2X7Rs has been associated with neurodegenerative illnesses such as Alzheimer’s disease (AD), as well as the consequences of cerebral ischemic injury and status epilepticus (SE).Expert opinionAlthough all CNS cells are possible sources of ATP release, the targets of this ATP are primarily at microglial cells. However, astrocytes also contain ATP-sensitive P2X7Rs and have in addition the peculiar property over microglia to continuously interact with neurons via not only inflammatory mediators but also gliotransmitters, such as adenosine 5’-triphosphate (ATP), glutamate, γ-amino butyric acid (GABA), and D-serine. Cellular damage arising during AD, cerebral ischemia, and SE via P2X7R activation is superimposed upon the original disease, and their prevention by blood–brain barrier permeable pharmacological antagonists is a valid therapeutic option.

ObjectiveThe extent to which tryptophan (Trp) metabolism alterations explain or influence the outcome of inflammatory bowel diseases (IBDs) is still unclear. However, several Trp metabolism end-products are essential to intestinal homeostasis. Here, we investigated the role of metabolites from the kynurenine pathway.DesignTargeted quantitative metabolomics was performed in two large human IBD cohorts (1069 patients with IBD). Dextran sodium sulphate-induced colitis experiments in mice were used to evaluate effects of identified metabolites. In vitro, ex vivo and in vivo experiments were used to decipher mechanisms involved. Effects on energy metabolism were evaluated by different methods including Single Cell mEtabolism by profiling Translation inHibition.ResultsIn mice and humans, intestinal inflammation severity negatively correlates with the amount of xanthurenic (XANA) and kynurenic (KYNA) acids. Supplementation with XANA or KYNA decreases colitis severity through effects on intestinal epithelial cells and T cells, involving Aryl hydrocarbon Receptor (AhR) activation and the rewiring of cellular energy metabolism. Furthermore, direct modulation of the endogenous tryptophan metabolism, using the recombinant enzyme aminoadipate aminotransferase (AADAT), responsible for the generation of XANA and KYNA, was protective in rodent colitis models.ConclusionOur study identified a new mechanism linking Trp metabolism to intestinal inflammation and IBD. Bringing back XANA and KYNA has protective effects involving AhR and the rewiring of the energy metabolism in intestinal epithelial cells and CD4+T cells. This study paves the way for new therapeutic strategies aiming at pharmacologically correcting its alterations in IBD by manipulating the endogenous metabolic pathway with AADAT.

ABSTRACTIntroduction: Alzheimer’s disease (AD) is a neurodegenerative illness with genetic risk as an etiological factor in a subset of cases. In AD with autosomal dominant inheritance, the extracellular β-amyloid (Aβ) aggregates and intracellular neurofibrillary tangles which consist of hyperphosphorylated tau, appear to be involved in the neuronal damage; however, other forms of AD may have a polygenetic causality. Microglial cells orchestrate pathophysiological events responsible for neuronal damage in AD. They surround Aβ aggregates and the stimulation of microglial P2X7 receptors (P2X7Rs) by high local concentrations of ATP which originates from damaged CNS cells, results in degeneration of nearby neurons.Areas covered: We discuss the pathogenesis of Alzheimer’s disease, the role of P2X7 receptors and their potential as therapeutic targets. We also address the fundamental hurdles in the development of new therapeutic strategies for Alzheimer’s disease.Expert opinion: There are many difficulties associated with the development of efficient pharmacological strategies for AD; the lack of good animal and cellular models for this illness is a key obstacle. None of the pharmacological strategies developed so far have led to an improvement of the treatment of AD. Hence, the consideration of blood-brain barrier-permeable P2X7R antagonists as possible therapeutic agents in AD is a must.

ABSTRACTAryl hydrocarbon receptor (AhR) is a critical player in the crosstalk between the gut microbiota and its host. However, factors regulating AhR within the gut, which is a complex metabolomic environment, are poorly understood. This study investigates the effect of a combination of metabolites on the activation mechanism of AhR. AhR activity was evaluated using both a luciferase reporter system and mRNA levels of AhR target genes on human cell lines and human colonic explants. AhR activation was studied by radioligand-binding assay, nuclear translocation of AhR by immuofluorescence and protein co-immunoprecipitation of AhR with ARNT. Indirect activation of AhR was evaluated using several tests and inhibitors. The promoter of the target gene CYP1A1was studied both by chromatin immunoprecipitation and by using an histone deacetylase HDAC inhibitor (iHDAC). Short-chain fatty acids, and butyrate in particular, enhance AhR activity mediated by endogenous tryptophan metabolites without binding to the receptor. This effect was confirmed in human intestinal explants and did not rely on activation of receptors targeted by SCFAs, inhibition of AhR degradation or clearance of its ligands. Butyrate acted directly on AhR target gene promoter to reshape chromatin through iHDAC activity. Our findings revealed that butyrate is not an AhR ligand but acts as iHDAC leading to an increase recruitment of AhR to the target gene promoter in the presence of tryptophan-derived AhR agonists. These data contribute to a novel understanding of the complex regulation of AhR activation by gut microbiota-derived metabolites.

Background: We carried out a test whether or not transplant drugs such as cyclosporine A, Rapamycin (Sirolimus), Tacrolimus, Everolimus and Mycophenolate mofetil affects the expression of phase II enzymes comprising of UDP-glucuronosyltransferases (UGTs) and glutathione-S-transferases (GSTs), and antioxidant enzymes that consist of glutathione reductase (GSR), glutathione peroxidase 1 (GPX1) and heme-oxygenase 1 (HMOX1). Methods: Experiments were performed in primary cultures of human hepatocytes and in human hepatocarcinoma HepG2 cells, the models of metabolically competent and incompetent cells, respectively. We used quantitative real-time PCR. Results: We found that none of the tested compounds affected the expression of investigated genes in human hepatocytes. On the other hand, Mycophenolate mofetil induced GPX1 mRNA, although it suppressed mRNA level of UGT1A4/1A9/2B7/2B10, GSTA1/O1/T1, GSR and HMOX1 in HepG2 cells. Conclusion: We showed that the tested transplant drugs have no effect on the expression of selected phase II and antioxidant enzymes in human hepatocytes. Nevertheless, the experiments carried out in two common and frequently used in vitrocellular models, we emphasize that finding based solely on carcinoma cells must be taken with caution when transposing to in vivosituations.

The article focuses on the aspects of the use of cardiovascular implantable electronic devices as therapeutic devices which includes pacemakers, loop recorders, and implantable cardiac devices. Topics include the aetiology of cardiac-related diseases such as bradyarrhythmias, tachyarrhythmias, and arrhythmias, the aspects of cardiac resynchronisation therapy, and the management and follow-up monitoring of the devices.

In this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed ATP binding site of the human P2X3 receptor on the agonistic effect of nucleotide analogs. The wild-type and mutant receptors were expressed in HEK293 cells, and the nucleotide effects were measured by means of the whole-cell patch-clamp method. Modifications in the receptor binding site changed the concentration-response relationship, the current kinetics, and the recovery from desensitization during fast, pulsed, local agonist applications. On the basis of this fact, we were able to distinguish binding from other effects, such as gating/desensitization, by using a hidden Markov model that describes the complete channel behavior with a matrix of rate constants. The binding energies of the nucleotide analogs were calculated and compared with the binding energies of ATP at both the wild-type receptor and its alanine mutants. Changes in the binding energies caused by alterations in the receptor and/or agonist structures were concluded to be attributable to the preferential binding of certain structural constituents of ATP to certain amino acid moieties of the receptor. The results were also checked for consistency with a P2X3 homology model that we developed from the known zebrafish P2X4 crystal structure in the closed state. The functional data correlated well with the predictions of the agonist dockings to the structural model.

P2X2 receptors are members of the ATP-gated P2X family of cation channels, and they participate in neurotransmission in sympathetic ganglia and interneurons. Here, we identified 7,7'-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methyl-phenylene)carbonylimino))bis(1-methoxy-naphthalene-3,6-disulfonic acid) tetrasodium salt (NF770) as a nanomolar-potent competitive P2X2 receptor antagonist within a series of 139 suramin derivatives. Three structural determinants contributed to the inhibition of P2X2 receptors by NF770: 1) a "large urea" structure with two symmetric phenylenecarbonylimino groups; 2) attachment of the naphthalene moiety in position 7,7'; and 3) the specific position of two sulfonic acid groups (3,3'; 6,6') and of one methoxy group (1,1') at the naphthalene moiety. This structure-activity relationship was interpreted using a rat P2X2 homology model based on the crystal structure of the closed zebrafish P2X4 receptor. Docking of the suramin derivatives into the modeled ATP-binding pocket provides a uniform explanation for the observed differences in inhibitory potencies. Changes in the chemical structure that increase the inhibitory potency of the suramin derivatives improved the spatial orientation within the ATP-binding pocket to allow for stronger polar interactions of functional groups with Gly72, Glu167, or Arg290. Gly72 is responsible for the orientation of the methoxy group close to Arg290 or Glu167. Combined mutational and functional analysis confirmed that residues Gly72 and Glu167 are as important for ATP binding as Arg290, the ATP-binding role of which has been shown in previous studies. The in silico prediction of Gly72 and Glu167 as ATP-binding residues strongly supports the validity of our homology docking.

This review deals with the molecular modelling of a subtype of the membrane-embedded purinoreceptor P2X family, which belongs to the large class of membrane-embedded glycoproteins. The P2X family has two transmembrane domains and a core of five extracellularly occurring disulfide bonds. At present, seven different P2X receptor subtypes (P2X1 – X7) have been cloned. The human purinoreceptor P2X3 (h-P2X3) is a putative drug target for the development of inhibitors against chronic inflammatory, neuropathic and mixed-pain conditions. No details on P2X receptor architecture are known at the atomic resolution level by X-ray or NMR analyses. An attempt was made to predict the conformation of h-P2X3 using homology based comparative modelling and threading, but the modelling could not be carried out due to missing template proteins. State-of-the-art abinitioprotein structure prediction methods also failed. A novel approach has been applied and exemplified on the h-P2X3 receptor. The coordinates of the secondary structure of h-P2X3 were determined by a profile-based neural network prediction. The conformation was geometry optimised using the quantum chemistry RHF/3-21G minimal basic set and all-atom molecular mechanics AMBER force field. A dielectric constant of ε = 3.5 was used to simulate the lipophilic environment of the membrane-bound protein. The h-P2X3 protein has a number of interacting peptide modules. An example is an extracellularly occurring triad of sterically interacting domains, which consists of a nucleotide binding domain (amino acids in positions 62 – 66), a PKC phosphorylation site (196 – 198) and a N-glycosylation attachment site (194 – 197). The discovery of this peptide module, and of other interacting modules, raises the possibility of exploiting structure-based strategies to design P2X3 inhibitors. Nevertheless, it should be noted that the predicted structures are defined in a probabilistic sense. Only biological and chemical knowledge can determine whether or not these predictions are meaningful. Thus, the results from the computational tools are probabilistic predictions and subject to further experimental verification.

We have previously identified the suramin analog 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) as a low nanomolar potency antagonist of recombinant P2X1receptors. Here, we characterize, by two-electrode voltage-clamp electrophysiology, three isomeric suramin analogs designated para-4,4',4'',4''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetrakis-benzenesulfonic acid (NF110), meta-(3,3',3'',3''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF448), and ortho-(2,2',2'',2''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (MK3) with respect to their potency in antagonizing rat P2X receptor-mediated inward currents in Xenopus laevis oocytes. Meta, para, and ortho refer to the position of the single sulfonic acid group relative to the amide bond linking the four symmetrically oriented benzenesulfonic acid moieties to the central, invariant suramin core. NF448, NF110, and MK3 were >200-fold less potent in blocking P2X1receptors than NF449, from which they differ structurally only by having one instead of two sulfonic acid residues per benzene ring. Although the meta- and ortho-isomers retained P2X1receptor selectivity, the para-isomer NF110 exhibited a significantly increased activity at P2X3receptors (Ki∼ 36 nM) and displayed the following unique selectivity profile among suramin derivatives: P2X2+3= P2X3> P2X1> P2X2>> P2X4> P2X7. The usefulness of NF110 as a P2X3receptor antagonist in native tissues could be demonstrated by showing that NF110 blocks αβ-methylene-ATP-induced currents in rat dorsal root ganglia neurons with similar potency as recombinant rat P2X3receptors. Together, these data highlight the importance of both the number and exact location of negatively charged groups for P2X subtype potency and selectivity.

A novel method of structure prediction for membrane-bound proteins is reviewed. The approach is based on a sequence-function analysis, secondary structure prediction and subsequent geometry optimization. The prediction of the structure of ligand-gated P2X4 receptor subunit, which is a membrane-embedded cation channelforming protein with extracellularly occuring ATP binding sites, is shown as an example. The potential Nglycosylation sites of the glycoprotein, location of the five disulfide bridges, and phospholipid-dependent protein kinase C (PKC) phosphorylation attachment sites were determined by sequence-function analysis. Subsequently an attempt was made to predict its conformation using homology-based comparative modeling and threading; however, the modeling could not be accomplished. Because of this, secondary structure prediction of the protein was carried out. The input coordinates of the spatial structure were obtained by a profile-based neural network prediction method. The resulting secondary structure was converted into a three-dimensional geometry. The secondary and tertiary structures were optimized by the quantum chemistry RHF/3-21G minimal basic set and all-atom molecular mechanics AMBER96 force field. The predicted shape is similar to the shape of the experimentally obtained monomeric structure of the classical ion channel, the K+ion channel from Streptomyces lividans (KcsA channel), and agrees with the P2X shape proposed by biological experimenters. The geometry optimized structure of the P2X4 receptor is freely available (Protein Data Bank format) from the authors on e-mail request (magp@medizin.unileipzig. de).

Whole-cell patch-clamp recordings were performed on HEK293 cells transiently transfected with the rat (r) wild-type transient receptor potential vanilloid 1 (TRPV1) (rTRPV1) receptor or with a mutant that lacks the potential N-glycosylation site at position N604 (rTRPV1-N604T). Replacement of Asn by Thr (N604T) depressed the maximum of the concentration–response curve for capsaicin and decreased the EC50value of this agonist. Further, such a manipulation modified the sensitivity to the TRPV1 receptor-antagonist capsazepine and altered the dependence of the capsaicin effect on extracellular pH. Hence, glycosylation may affect the basic functional characteristics of the rTRPV1 receptor channel in accordance with the knowledge that N-glycosylation may regulate ligand binding or gating properties of ionotropic neurotransmitter receptors.

The membrane-embedded, ligand-gated P2X glycoprotein receptor is a monovalent-bivalent cation channel that is activated by physiological concentrations of extracellular ATP. A quantitative structure-activity relationship (QSAR) analysis was developed to model the cation permeability of the P2X2 channel and its mutants. As chemical properties, the helix-coil equilibrium constants and the distribution coefficients of the system octanol/water at pH 7.4 were applied and modified (sliding windows) according to Eroshkin et al. (Comput. Appl. Biosci., 1995, 11, 49-44). The results were visualized by a dimeric P2X2 channel construct. The results support the hypothesis that residues which put into the cavity and contribute to hydrogen bonding forces are involved to a control of the transport of hydrated cations through the P2X2 channel. The model may be useful to develop P2X2 receptor antagonists.

Motivation: No details on P2X receptor architecture had been known at the atomic resolution level. Using comparative homology-based molecular modelling and threading, it was attempted to predict the three-dimensional structure of P2X receptors. This prediction could not be carried out, however, because important properties of the P2X family differ considerably from that of the potential template proteins. This paper reviews an alternative approach consisting of three research fields: bioinformatics, structural modelling, and a variety of the results of biological experiments. Model: Starting point is the amino acid sequence. Using the sequential data, the first step is a secondary structure prediction. The resulting secondary structure is converted into a three-dimensional geometry. Then, the secondary and tertiary structures are optimized by using the quantum chemistry RHF / 3-21G minimal basic set and the allatom molecular mechanics AMBER96 force field. The fold of the membrane-embedded protein is simulated by a suitable dielectricum. The structure is refined using a conjugate gradient minimizer (Fletcher-Reeves modification of the Polak-Ribiere method). The results of the geometry optimization were checked by a Ramanchandran plot, rotamer analysis, all-atom contact dots, and the C() deviation. As additional tools for the model building, multiple alignment analysis and comparative sequence-function analysis were used. The approach is exemplified on the membrane-embedded, ligand-gated P2X3 receptor subunit, a monovalent-bivalent cation channel-forming glycoprotein that is activated by extracellular adenosine 5-triphosphate. From these results, a topology of the pore-forming motif of the P2X3 receptor subunit was proposed. It is believed that a fully functional P2X channel requires a precise coupling between (i) two distinct peptide modules, an extracellularly occuring ATP-binding module and a pore module that includes a long transmembrane and short intracellular part, (ii) an interaction surface with membranes, and (iii) hydrogen bonding forces of the residues and hydrated cations. Furthermore, this paper demonstrates the role of quantitative structure-activity relationships (QSARs) in P2X research (calcium ion permeability of the wild-type and after site-directed mutagenesis of the rat P2X2 receptor protein, KN-62 analogs as competitive antagonists of the human P2X7 receptor). Experimental proofs: The predictions are experimentally testable and may provide an additional interpretation of experimental observations published in literature. In particular, there is the good agreement of the geometry optimized P2X3 structure with experimentally proposed P2X receptor models obtained by neurophysiological, biochemical, pharmacological, and mutation experiments. Although the rat P2X3 receptor subunit is more complex (397 amino acids) than the KcsA protein (160 amino acids), the overall folds of the peptide backbone atoms are similar. Limitations: To avoid semantic confusion, it should be noted that “prediction” is defined in a probabilistic sense. Matches to generic rules do not mean “this is true“ but rather ”this might be true”. Only biological and chemical knowledge can determine whether or not these predictions are meaningful. Thus, the results from the computational tools are probabilistic predictions and subject to further experimental verification. Availability: The geometry optimized P2X3 receptor subunit is freely available for academic researchers on e-mail request (PDB format).

Neurons of the central nervous system (CNS) are endowed with ATP-sensitive receptors belonging to the P2X (multimeric ligand-gated cationic channels) and P2Y (G protein-coupled 7TM receptors) types. To date seven P2X and eight P2Y receptors of human origin have been molecularly identified and functionally characterized. P2X subunits may occur as homooligomers or as heterooligomeric assemblies of more than one subunit. P2X7 subunits do not form heterooligomeric assemblies and are uniqe in mediating apoptosis and necrosis of glial cells and possibly also of neurons. The P2X2, P2X4, P2X4 / P2X6 and P2Y1 receptors appear to be the predominant neuronal types. Whereas a number of P2X receptors mediate fast synaptic responses to the transmitter ATP, P2Y receptors mediate either slow changes of the membrane potential in response to non-synaptically released ATP or the interaction with receptors for other transmitters. The localisation of these receptors may be at the terminal axons (presynaptic) or at the somato-dendritic region (postsynaptic). Whereas presynaptic P2 receptors may be either excitatory (P2X) or inhibitory (P2Y), postsynaptic P2 receptors appear to be without exception excitatory. Finally, the enzymatic degradation of ATP may lead to the local generation of adenosine which can modulate ATP-related neurotransmission via activation of A1 or A2A receptors.

ATP and its metabolite adenosine activate membrane receptors thought to be functionally relevant in the mediation of reward and reinforcement (e.g., feeding behavior). In this study, the effects of restricted feeding on the purinergic transmission at the level of the adenosine A2Aand ADP/ATP‐sensitive P2Y1receptor mRNA were investigated in the rat nucleus accumbens after 3 days of acute food deprivation and after 10 days when adaptive changes can be expected to occur. Furthermore, plasma leptin and the expression of its receptor mRNA (long form) to characterize the status of satiety were examined as well as the expression of the cocaine‐ and amphetamine‐related transcript (CART) mRNA as a possible marker for changes in the sensitiveness of the reward system. Plasma levels of the anorectic peptide leptin were reduced within 3 days of feeding restriction; the mRNA of the leptin receptor was elevated at day 10. The P2Y1receptor mRNA expression showed the same time course, whereas the A2Areceptor mRNA reached the control level on day 10 after a downregulation found on day 3. CART mRNA expression was decreased in the acute situation and upregulated after 10 days. The data indicate that the accumbal gene expression during food restriction is triggered by peripheral signals like plasma leptin concentration and by neuronal signals in the CNS, including extracellular adenosine and ADP/ATP, and that the investigated receptor mRNAs are regulated to habituate to the reduced nutrition. In conclusion, chronic food restriction increases the mesolimbic sensitivity directed to achieve satisfaction and reward via regulation of mRNA expression involving that of P2Y1and A2Areceptors in a functionally antagonistic manner. Drug Dev. Res. 59:95–103, 2003. © 2003 Wiley‐Liss, Inc.

Changes in glial fibrillary acidic protein (GFAP) are believed to be a sensitive and specific marker for astroglial responses to various kinds of injury. Neurotrophic factors and their receptors involved in the physiological and pathological responses of astrocytes are of special interest. For example, ATP, released from damaged or dying cells, may represent one of the endogenous factors mediating the activation of astrocytes. In the present study, the influence of different ATP analogs on the GFAP‐immunoreactivity (IR) and on the expression of P2 receptors on GFAP‐labeled cells were studied. Microinjection of the selective P2 receptor agonists α,β‐methylene ATP (α,β‐meATP; P2X1,3), adenosine 5′‐O‐(2‐thiodiphosphate) (ADP‐β‐S; P2Y1,12), and uridine 5′‐O‐(3‐thiotriphosphate) (UTP‐γ‐S; P2Y2,4,6) in the nucleus accumbens (NAc) of rats resulted in an increase of GFAP‐IR in comparison to the treatment with artificial cerebrospinal fluid. The agonist‐induced morphogenic changes documented a strong potency of α,β‐meATP and ADP‐β‐S. The effect of UTP‐γ‐S was small. Blockade of P2 receptors with the nonselective antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) decreased the injury‐induced elevation of GFAP‐IR when given alone and in addition inhibited the agonist effects. Double immunofluorescence measurements confined the localization of P2X2–4and P2Y1receptor subtypes to GFAP‐labeled astrocytes of untreated rats. The receptor subtypes P2X1,5,7were not present in the NAc of native rats, but became expressed after mechanical damage. P2X6receptor‐labeling was absent both with and without mechanical damage. After microinjection of α,β‐meATP and ADP‐β‐S into the NAc, the P2X1,3‐ and P2Y1‐IR, respectively, appeared to increase. These results show that the stimulation of P2 receptors influences the expression of GFAP‐IR in vivo. GFAP‐positive astrocytes possess P2X as well as P2Y receptor subtypes and change the expression of these receptors after injury and subsequent long‐term activation by ATP. Drug Dev. Res. 59:175–189, 2003. © 2003 Wiley‐Liss, Inc.

The present review summarizes data about the properties, distribution, and effects of neuronal P2Y receptors. Extracellular nucleotide receptors can be classified into two types belonging either to the P2X (ligand‐gated cationic channels) or the P2Y type (G protein‐coupled receptors). Neuronal P2Y receptors comprise five cloned and functionally defined classes of which P2Y1, P2Y6, P2Y11, P2Y12, and P2Y13occur in the central nervous system. Various types of P2Y receptors stimulate inwardly rectifying potassium channels, positively or negatively modulate the high‐voltage activated Ca2+current and increase the intracellular free Ca2+concentration in neuronal preparations. Moreover, in a subpopulation of pyramidal cells of the rat prefrontal cortex (PFC), the P2Y2receptor potentiates the function of a ligand‐gated cationic channel, the N‐methyl‐D‐aspartate (NMDA)‐type excitatory amino acid receptor. This effect is G protein‐mediated and utilizes the phospholipase C/inositol 1,4,5‐trisphosphate/Ca2+/calmodulin kinase II pathway. It appears that P2Y2receptors situated at astrocytes release glutamate, which in turn modulates NMDA receptors of neighbouring neurons via stimulation of group I metabotropic glutamate receptors. In contrast, P2Y1receptors inhibit the conductance of NMDA receptor‐channels in all PFC pyramidal cells investigated. This effect is fast in onset and does not depend on G protein activation. It is suggested that P2Y1receptors alter the function of NMDA receptors by a direct protein–protein coupling in the membrane. Because ATP and dopamine are supposed to be coreleased from dopaminergic fibers onto layer V pyramidal cells, both neurotransmitters may interact with the neuronal NMDA receptor. Hence, D1dopamine receptors and P2Y1/P2Y2receptors may be involved in the fine tuning of higher order cognitive functions including learning and memory. Drug Dev. Res. 59:49–55, 2003. © 2003 Wiley‐Liss, Inc.

Changes in glial fibrillary acidic protein (GFAP) are believed to be a sensitive and specific marker for astroglial responses to various kinds of injury. Neurotrophic factors and their receptors involved in the physiological and pathological responses of astrocytes are of special interest. For example, ATP, released from damaged or dying cells, may represent one of the endogenous factors mediating the activation of astrocytes. In the present study, the influence of different ATP analogs on the GFAP-immunoreactivity (IR) and on the expression of P2 receptors on GFAP-labeled cells were studied. Microinjection of the selective P2 receptor agonists α,β-methylene ATP (α,β-meATP; P2X1,3), adenosine 5'-O-(2-thiodiphosphate) (ADP-β-S; P2Y1,12), and uridine 5'-O-(3-thiotriphosphate) (UTP-γ-S; P2Y2,4,6) in the nucleus accumbens (NAc) of rats resulted in an increase of GFAP-IR in comparison to the treatment with artificial cerebrospinal fluid. The agonist-induced morphogenic changes documented a strong potency of α,β-meATP and ADP-β-S. The effect of UTP-γ-S was small. Blockade of P2 receptors with the nonselective antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) decreased the injury-induced elevation of GFAP-IR when given alone and in addition inhibited the agonist effects. Double immunofluorescence measurements confined the localization of P2X2–4 and P2Y1 receptor subtypes to GFAP-labeled astrocytes of untreated rats. The receptor subtypes P2X1,5,7 were not present in the NAc of native rats, but became expressed after mechanical damage. P2X6 receptor-labeling was absent both with and without mechanical damage. After microinjection of α,β-meATP and ADP-β-S into the NAc, the P2X1,3- and P2Y1-IR, respectively, appeared to increase. These results show that the stimulation of P2 receptors influences the expression of GFAP-IR in vivo. GFAP-positive astrocytes possess P2X as well as P2Y receptor subtypes and change the expression of these receptors after injury and subsequent long-term activation by ATP. Drug Dev. Res. 59:175–189, 2003. © 2003 Wiley-Liss, Inc.

ATP and its metabolite adenosine activate membrane receptors thought to be functionally relevant in the mediation of reward and reinforcement (e.g., feeding behavior). In this study, the effects of restricted feeding on the purinergic transmission at the level of the adenosine A2A and ADP/ATP-sensitive P2Y1 receptor mRNA were investigated in the rat nucleus accumbens after 3 days of acute food deprivation and after 10 days when adaptive changes can be expected to occur. Furthermore, plasma leptin and the expression of its receptor mRNA (long form) to characterize the status of satiety were examined as well as the expression of the cocaine- and amphetamine-related transcript (CART) mRNA as a possible marker for changes in the sensitiveness of the reward system. Plasma levels of the anorectic peptide leptin were reduced within 3 days of feeding restriction; the mRNA of the leptin receptor was elevated at day 10. The P2Y1 receptor mRNA expression showed the same time course, whereas the A2A receptor mRNA reached the control level on day 10 after a downregulation found on day 3. CART mRNA expression was decreased in the acute situation and upregulated after 10 days. The data indicate that the accumbal gene expression during food restriction is triggered by peripheral signals like plasma leptin concentration and by neuronal signals in the CNS, including extracellular adenosine and ADP/ATP, and that the investigated receptor mRNAs are regulated to habituate to the reduced nutrition. In conclusion, chronic food restriction increases the mesolimbic sensitivity directed to achieve satisfaction and reward via regulation of mRNA expression involving that of P2Y1 and A2A receptors in a functionally antagonistic manner. Drug Dev. Res. 59:95–103, 2003. © 2003 Wiley-Liss, Inc.

The present review summarizes data about the properties, distribution, and effects of neuronal P2Y receptors. Extracellular nucleotide receptors can be classified into two types belonging either to the P2X (ligand-gated cationic channels) or the P2Y type (G protein-coupled receptors). Neuronal P2Y receptors comprise five cloned and functionally defined classes of which P2Y1, P2Y6, P2Y11, P2Y12, and P2Y13 occur in the central nervous system. Various types of P2Y receptors stimulate inwardly rectifying potassium channels, positively or negatively modulate the high-voltage activated Ca²⁺ current and increase the intracellular free Ca²⁺ concentration in neuronal preparations. Moreover, in a subpopulation of pyramidal cells of the rat prefrontal cortex (PFC), the P2Y2 receptor potentiates the function of a ligand-gated cationic channel, the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. This effect is G protein-mediated and utilizes the phospholipase C/inositol 1,4,5-trisphosphate/Ca²⁺/calmodulin kinase II pathway. It appears that P2Y2 receptors situated at astrocytes release glutamate, which in turn modulates NMDA receptors of neighbouring neurons via stimulation of group I metabotropic glutamate receptors. In contrast, P2Y1 receptors inhibit the conductance of NMDA receptor-channels in all PFC pyramidal cells investigated. This effect is fast in onset and does not depend on G protein activation. It is suggested that P2Y1 receptors alter the function of NMDA receptors by a direct protein–protein coupling in the membrane. Because ATP and dopamine are supposed to be coreleased from dopaminergic fibers onto layer V pyramidal cells, both neurotransmitters may interact with the neuronal NMDA receptor. Hence, D1 dopamine receptors and P2Y1/P2Y2 receptors may be involved in the fine tuning of higher order cognitive functions including learning and memory. Drug Dev. Res. 59:49–55, 2003. © 2003 Wiley-Liss, Inc.

The second coding exon of the adenosine A1receptor gene was eliminated by homologous recombination. The phenotype of mice (mixed C57B6/129OlaHsd background) was studied, using siblings from matings of heterozygous mice. Among the offspring the ratio between+/+,+/−and−/−animals was 1:2:1. Over the first half‐year—at least—growth and viability were the same in all genotypes. Binding of A1ligands was eliminated in−/−mice and halved in+/−mice. Blood pressure was increased in−/−mice and this was paralleled by an increase in plasma renin. Heart rate was unaffected, as was contractility. Furthermore, the response of the perfused heart to ischemia was similar in+/+and −/−hearts. However, remote preconditioning was eliminated in−/−mouse hearts. Tubuloglomerular feedback in the kidney was also lost in−/−mice. The analgesic response to a non‐selective adenosing receptor agonist was lost in−/−mice, which also showed hyperalgesia in the tail‐flick test. There was a slight hypoactivity in−/−mice, but responses to caffeine were essentially normal. The inhibition of excitatory neurotransmission in hippocampus by adenosine was lost in−/−mice and reduced in+/−mice. Responses to ATP were affected similarly. Hypoxic depression of synaptic transmission was essentially eliminated in hippocampus and hypoxic decrease in spinal respiratory neuron firing was markedly reduced. These results show that adenosine A1receptors play a physiologically important role in the kidney, spinal cord, and hippocampus and that they are critically important in the adaptive responses to hypoxia. Drug Dev. Res. 58:350–353, 2003. © 2003 Wiley‐Liss, Inc.

The second coding exon of the adenosine A1 receptor gene was eliminated by homologous recombination. The phenotype of mice (mixed C57B6/129OlaHsd background) was studied, using siblings from matings of heterozygous mice. Among the offspring the ratio between+/+,+/−and−/−animals was 1:2:1. Over the first half-year—at least—growth and viability were the same in all genotypes. Binding of A1 ligands was eliminated in−/−mice and halved in+/−mice. Blood pressure was increased in−/−mice and this was paralleled by an increase in plasma renin. Heart rate was unaffected, as was contractility. Furthermore, the response of the perfused heart to ischemia was similar in+/+and −/−hearts. However, remote preconditioning was eliminated in−/−mouse hearts. Tubuloglomerular feedback in the kidney was also lost in−/−mice. The analgesic response to a non-selective adenosing receptor agonist was lost in−/−mice, which also showed hyperalgesia in the tail-flick test. There was a slight hypoactivity in−/−mice, but responses to caffeine were essentially normal. The inhibition of excitatory neurotransmission in hippocampus by adenosine was lost in−/−mice and reduced in+/−mice. Responses to ATP were affected similarly. Hypoxic depression of synaptic transmission was essentially eliminated in hippocampus and hypoxic decrease in spinal respiratory neuron firing was markedly reduced. These results show that adenosine A1 receptors play a physiologically important role in the kidney, spinal cord, and hippocampus and that they are critically important in the adaptive responses to hypoxia. Drug Dev. Res. 58:350–353, 2003. © 2003 Wiley-Liss, Inc.

ATP and its metabolite adenosine activate membrane receptors, termed P2 and P1, respectively. In the present study, the modulation of the mesolimbic neuronal circuit by ATPergic and adenosinergic mechanisms was investigated by microdialysis in the nucleus accumbens (NAc) and by telemetrically recorded EEG from both the NAc and the ventral tegmental area (VTA) of freely moving rats. The basal extracellular dopamine concentration was enhanced after accumbal perfusion with the ATP analog 2‐methylthio ATP (2‐MeSATP; 100 μM); by contrast, adenosine (100 μM) caused a reduction of extracellular dopamine. When given alone, the P2 receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS; 20 μM) decreased the concentration of dopamine, whereas the P1 receptor antagonist 8‐(p‐sulfophenyl)theophylline (8‐SPT; 100 μM) increased it. In the same animals, P2 receptor stimulation by 2‐MeSATP caused neuronal activation, indicated by an elevation of the absolute power in the EEG of the NAc mainly by enhancement of the relative power in the alpha band (8–13 Hz) of the EEG spectrum. By contrast, adenosine led to a depression of the absolute power in the VTA accompanied by an elevation of the delta‐band power (0.4–6 Hz) in the NAc corresponding to a slowing of neuronal activity. When given alone, PPADS reduced the absolute EEG power in the NAc accompanied by a decrease in the high‐frequency power, but had no effects on the VTA. 8‐SPT on its own enhanced the total power in both the NAc and the VTA, reflected by an enhancement in the slow and the high‐frequency bands. Whereas the 8‐SPT‐evoked changes of EEG pattern as well as of dopamine concentration in the NAc were abolished by the co‐application of PPADS, the 8‐SPT‐induced EEG changes in the VTA persisted under these conditions. In conclusion, the accumbal neuronal output, reflected by both dopamine release and neuronal electrical activity, is modulated in a functionally antagonistic manner by P2 and P1 receptor stimulation. It is suggested that an inhibitory GABAergic feedback projection to the VTA is stimulated by adenosine, either directly or indirectly via glutamate release. Synapse 47:134–142, 2003. © 2002 Wiley‐Liss, Inc.

In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3(α,β‐methylene adenosine 5′‐triphosphate; α,β‐meATP), P2Y1,12(adenosine 5′‐O‐(2‐thiodiphosphate; ADP‐β‐S) and P2Y2,4,6receptors (uridine 5′‐O‐(3‐thiotriphosphate; UTP‐γ‐S). Pyridoxalphosphate‐6‐azophenyl‐2,4‐disulphonic acid (PPADS) was used as a non‐selective antagonist. The astroglial reaction was studied by means of immunocytochemical double‐labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5‐bromo‐2′‐deoxyuridine (BrdU).The agonist‐induced changes in comparison to the artificial cerebrospinal fluid (aCSF)‐treated control side reveal a strong mitogenic potency of ADP‐β‐S and α,β‐meATP, whereas UTP‐γ‐S was ineffective. The P2 receptor antagonist PPADS decreased the injury‐induced proliferation when given alone and in addition inhibited all agonist effects.The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up‐regulation of GFAP. A significant increase of both GFAP‐immunoreactivity (IR) and GFA‐protein content (by using Western blotting) was found after microinfusion of α,β‐meATP or ADP‐β‐S. In contrast, UTP‐γ‐S failed to increase the GFAP‐IR. The morphogenic effects were also inhibited by pre‐treatment with PPADS.A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3and P2Y1receptors on the GFAP‐labelled astrocytes.In conclusion, the data suggest that P2Y (P2Y1or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.

The temperature-dependence of ATP release and contraction response evoked by different agonists were investigated in superfused guinea-pig vas deferens. α-Adrenoceptor agonists, i.e. noradrenaline (300 μM), and α-methyl-noradrenaline (300 μM), increased the basal ATP outflow, measured by the luciferin-luciferase assay, and induced biphasic contractile response. Cooling the bath temperature to 12°C almost completely inhibited ATP release and twitch contraction evoked by α-adrenoceptor agonists, whereas the phasic contraction remained unaffected. In contrast, twitch contraction and subsequent ATP release induced by β,γ-methylene-ATP, a selective P2 receptor agonist (100 μM), was not reduced by low temperature. The ectoATPase activity, measured by HPLC technique was not significantly different at 37°C and 12°C. Nifedipine (1 μM), the voltage sensitive Ca2+channel blocker eliminated β,γ-methylene-ATP evoked twitch contraction but not ATP release. In conclusion, α-adrenoceptor and P2 receptor agonists utilize distinct mechanisms to elicit ATP release and contraction: α-adrenoceptor-mediated ATP release and contraction is temperature-dependent, indicating the involvement of a carrier-mediated process in it, whereas P2x purinoceptor evoked ATP release and twitch is mediated by a different mechanism.

The present review summarizes data about the expression of P2 receptors in two types of macroglial cells, i.e., in astrocytes of the nucleus accumbens (NAc) and Müller glial cells of the retina. Changes in the expression of these receptors during pathological conditions may indicate an involvement in the induction and/or maintenance of reactive astrogliosis. In fact, astrocytic proliferation was induced in the NAc by the application of different P2X and P2Y receptor agonists, such as 2‐methylthio ATP (2‐MeSATP), α,β‐methylene adenosine 5′‐triphosphate (α,β‐meATP) and adenosine 5′‐O‐(2‐thiodiphosphate (ADP‐β‐S). An increased content of glial fibrillary acidic protein, astrocytic hypertrophy, and proliferation as indicated by enhanced incorporation of 5‐bromo‐2′‐deoxyuridine (BrdU) was found under these conditions; an enhanced BrdU incorporation signalizes a higher rate of DNA synthesis. Müller glial cells of the human retina express both metabotropic P2Y and ionotropic P2X7receptors. Application of the P2X7receptor agonist benzoyl‐benzoyl ATP (BzATP) evoked a cationic conductance increase that led to the depolarization of cells and to Ca2+entry from the extracellular space. Long‐lasting application of BzATP, however, did not cause the formation of pores permeable for large molecules, excluding the presence of classic P2X7receptors. Müller glial cells of the human retina show increased P2X7receptor‐mediated cationic currents during proliferative gliosis. A role of P2X7receptors in glial proliferation is also indicated by the observation that the DNA synthesis rate of cultured Müller cells is enhanced in the presence of BzATP, as shown by the measurement of BrdU incorporation. The data indicate that activation of P2 receptors may exert both short‐ and long‐term effects in macroglial cells, and that these receptors may be involved in glial responses to pathological conditions. Drug Dev. Res. 53:140–147, 2001. © 2001 Wiley‐Liss, Inc.

The present review summarizes data about the expression of P2 receptors in two types of macroglial cells, i.e., in astrocytes of the nucleus accumbens (NAc) and Müller glial cells of the retina. Changes in the expression of these receptors during pathological conditions may indicate an involvement in the induction and/or maintenance of reactive astrogliosis. In fact, astrocytic proliferation was induced in the NAc by the application of different P2X and P2Y receptor agonists, such as 2-methylthio ATP (2-MeSATP), α,β-methylene adenosine 5'-triphosphate (α,β-meATP) and adenosine 5'-O-(2-thiodiphosphate (ADP-β-S). An increased content of glial fibrillary acidic protein, astrocytic hypertrophy, and proliferation as indicated by enhanced incorporation of 5-bromo-2'-deoxyuridine (BrdU) was found under these conditions; an enhanced BrdU incorporation signalizes a higher rate of DNA synthesis. Müller glial cells of the human retina express both metabotropic P2Y and ionotropic P2X7 receptors. Application of the P2X7 receptor agonist benzoyl-benzoyl ATP (BzATP) evoked a cationic conductance increase that led to the depolarization of cells and to Ca²⁺ entry from the extracellular space. Long-lasting application of BzATP, however, did not cause the formation of pores permeable for large molecules, excluding the presence of classic P2X7 receptors. Müller glial cells of the human retina show increased P2X7 receptor-mediated cationic currents during proliferative gliosis. A role of P2X7 receptors in glial proliferation is also indicated by the observation that the DNA synthesis rate of cultured Müller cells is enhanced in the presence of BzATP, as shown by the measurement of BrdU incorporation. The data indicate that activation of P2 receptors may exert both short- and long-term effects in macroglial cells, and that these receptors may be involved in glial responses to pathological conditions. Drug Dev. Res. 53:140–147, 2001. © 2001 Wiley-Liss, Inc.

The endogenous mechanisms modulating ATP‐induced dopamine release in the nucleus accumbens (NAc) were studied by microdialysis in freely moving rats. The ATP analog 2‐Methylthio ATP (2‐MeSATP) facilitated the release of dopamine in a manner sensitive to pertussis toxin and tetrodotoxin. It is suggested that G‐protein‐coupled P2Y receptors and voltage‐gated sodium channels are involved in this process. N‐methyl‐D‐aspartate (NMDA) applied in a concentration of 100 μM decreased the extracellular dopamine level, whereas 1 and 10 mM NMDA enhanced it. The endogenous agonist glutamate (10 μM) inhibited the basal and facilitated release of dopamine. Infusion with a combination of the ionotropic glutamate receptor antagonists (±)‐3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), as well as with the metabotropic glutamate receptor antagonist (±)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) increased the basal level of dopamine and potentiated the 2‐MeSATP‐facilitated dopamine release, suggesting an ATP‐mediated glutamate release. The GABAAreceptor antagonist bicuculline infused into the NAc also enhanced the basal level of dopamine; however, the application of 2‐MeSATP in the presence of bicuculline caused an early decrease and a subsequent increase of dopamine release. The facilitatory phase of the 2‐MeSATP effect was comparable with that measured in the absence of bicuculline. By contrast, when bicuculline was infused into the ventral tegmental area (VTA) it elevated the accumbal basal dopamine level and in addition facilitated the 2‐MeSATP‐ and the glutamate‐induced dopamine release above that measured in the absence of bicuculline. These results suggest that ATP in the NAc has a physiologically relevant function in modulating dopaminergic transmission depending on the mesolimbic neuronal activity. The first component of the ATP effect involves a direct stimulation of the terminals of VTA neurons, while the second inhibitory component involves a sequential activation of glutamate and, finally, via ionotropic and metabotropic glutamate receptors, of GABA neurons projecting to the VTA. Synapse 39:222–232, 2001. © 2001 Wiley‐Liss, Inc.

Although it has been demonstrated repeatedly that ATP is a fast excitatory transmitter in the central nervous system (CNS), there are only limited data to indicate a corelease of ATP with other transmitters such as γ–aminobutyric acid or noradrenaline. Somewhat surprisingly, there is no evidence hitherto for glutamate-ATP cotransmission in spite of the widespread distribution of glutamatergic neurons in the brain and spinal cord. However, ATPergic and glutamatergic neurons may interact both at the pre- and postsynaptic level. Presynaptic P2X receptors facilitate glutamate release both from the terminals of mesencephalic proprioceptive trigeminal neurons projecting to the motor trigeminal nucleus in the brainstem and from primary afferent fibers onto dorsal horn neurons of the spinal cord. The inhibitory effect of ATP via presynaptic P2Y receptors has been demonstrated convincingly at noradrenergic but not glutamatergic nerve terminals. However, in addition to the direct effects of ATP, adenosine formed by the enzymatic degradation of ATP can inhibit the release of glutamate from the Schaffer collateral-commissural pathway onto CA1 pyramidal cells of the hippocampus. Similarly, ATP degraded to adenosine may inhibit the N-methyl-D-aspartate (NMDA)-induced current in the striatopallidal subpopulation of medium spiny neurons via A2A receptor activation. Finally, ATP may potentiate the NMDA receptor–mediated depolarization or the underlying inward current at layer V pyramidal neurons of the prefrontal cortex, and P2Y receptors have been suggested to mediate this interaction. Thus, although as a transmitter ATP alters the membrane potential of a limited subset of CNS neurons, it may have widespread and pronounced effects by modulating glutamatergic mechanisms. Drug Dev. Res. 52:76–82, 2001. © 2001 Wiley-Liss, Inc.

Although it has been demonstrated repeatedly that ATP is a fast excitatory transmitter in the central nervous system (CNS), there are only limited data to indicate a corelease of ATP with other transmitters such as γ–aminobutyric acid or noradrenaline. Somewhat surprisingly, there is no evidence hitherto for glutamate‐ATP cotransmission in spite of the widespread distribution of glutamatergic neurons in the brain and spinal cord. However, ATPergic and glutamatergic neurons may interact both at the pre‐ and postsynaptic level. Presynaptic P2X receptors facilitate glutamate release both from the terminals of mesencephalic proprioceptive trigeminal neurons projecting to the motor trigeminal nucleus in the brainstem and from primary afferent fibers onto dorsal horn neurons of the spinal cord. The inhibitory effect of ATP via presynaptic P2Y receptors has been demonstrated convincingly at noradrenergic but not glutamatergic nerve terminals. However, in addition to the direct effects of ATP, adenosine formed by the enzymatic degradation of ATP can inhibit the release of glutamate from the Schaffer collateral‐commissural pathway onto CA1 pyramidal cells of the hippocampus. Similarly, ATP degraded to adenosine may inhibit the N‐methyl‐D‐aspartate (NMDA)‐induced current in the striatopallidal subpopulation of medium spiny neurons via A2Areceptor activation. Finally, ATP may potentiate the NMDA receptor–mediated depolarization or the underlying inward current at layer V pyramidal neurons of the prefrontal cortex, and P2Y receptors have been suggested to mediate this interaction. Thus, although as a transmitter ATP alters the membrane potential of a limited subset of CNS neurons, it may have widespread and pronounced effects by modulating glutamatergic mechanisms. Drug Dev. Res. 52:76–82, 2001. © 2001 Wiley‐Liss, Inc.

Whole‐cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A2Areceptors in modulating N‐methyl‐D‐aspartate (NMDA)‐induced currents in rat striatal brain slices. The A2Areceptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) current in a subset of striatal neurons.Lucifer yellow‐filled pipettes in combination with immunostaining of A2Areceptors were used to identify CGS 21680‐sensitive cells as typical medium spiny striatal neurons.Dibutyryl cyclic AMP and the protein kinase A activator Sp‐cyclic AMPs, but not the protein kinase A inhibitors Rp‐cyclic AMPS or PKI(14–24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U‐73122, but not the inactive structural analogue U‐73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12‐myristate 13‐acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1,4,5‐trisphosphate (InsP3) and a more efficient buffering of intracellular Ca2+by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680‐induced inhibition.The calmodulin antagonist W‐7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281–309) and KN‐93 but not the inactive structural analogue KN‐92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680.It is suggested that the transduction mechanism of A2Areceptors to inhibit NMDA receptor channels is the phospholipase C/InsP3/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.

Abstract: Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 µM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 µM) and cibacron blue 3GA (3 µM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 µM; by 47%), by the N-type calcium channel blocker ω-conotoxin GVIA (100 nM; by 35%), and by the α2-adrenoceptor agonist UK-14,304 (1 µM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by ω-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 µM) caused a marked increase in intraaxonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and ω-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 µM) induced a large depolarisation and action potential firing. UDP (100 µM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and – probably – the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by α2-adrenocep-tors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to α2-adrenoceptor modulation.

Actin filament (F‐actin) depolymerization leads to the use‐dependent rundown of N‐methyl‐D‐aspartate (NMDA) receptor activity in rat hippocampal neurones. Depolymerization is promoted by Ca2+which enters the cells viaNMDA receptor channels. The rashomologue (Rho) GTPases (RhoA, Rac1 and Cdc42) promote actin polymerization and thus control the actin cytoskeleton. We have investigated, by means of the whole‐cell patch clamp technique, whether the actin fibres which interact with NMDA receptors are controlled by Rho GTPases.In the presence of intracellular ATP which attenuates rundown, the C3 toxin from Clostridium (C.) botulinumwas used to inactivate RhoA. Indeed, it enhanced the use‐dependent rundown of NMDA‐evoked inward currents to a level similar to that obtained in the absence of ATP.Lethal toxin from Clostridium sordelliiwhich inactivates Rac1 and Cdc42 lacked this effect.We suggest that the function of somatodendritic NMDA receptor channels in rat hippocampal neurones can be modulated by RhoA viaits action on F‐actin.

Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 µmol/l, SKF 38393 10 µmol/l, dopamine 10 µol/l and clonidine 0.1 µmol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 µmol/l, domperidone 1 µmol/l, SCH 23390 1 µmol/l and rauwolscine 1 µmol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 µmol/l and rauwolscine 1 µmol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 µol/l had the largest effect. S- and R-sulpiride, 10 µmol/l, as well as domperidone 1 µol/l antagonized the action of LY 171555 1 µmol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 µmol/l and rauwolscine 1 µmol/l blunted the effect of SKF 38393 10 µmol/l. Rauwolscine 1 µmol/l slightly reduced the inhibition by dopamine 10 µmol/l; S-sulpiride 10 µmol/l was antagonistic only in the presence of rauwolscine 1 µmol/l. When rauwolscine 1 µmol/l, prazosin 0.1 µmol/l, propranolol 1 µmol/l and cocaine 10 µmol/l was added to the medium, dopamine 10 µmol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 µmol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 µmol/l prevented the effect of clonidine 0.1 µmol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA2- and postsynaptic DA1-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to a2-adrenoceptor agonist properties; its DA2-effect is unmasked only after a2-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine.

Without Abstract:

The effects of neuropeptide Y (NPY; 1–36) and NPY fragment (16–36) on nicotinic currents (IACh) and voltage-dependent calcium currents (ICa) were studied in bovine chromaffin cells using the whole-cell patchclamp technique. The peak amplitude of inward nicotinic currents was markedly depressed by both NPY(1–36) and NPY (16–36). In contrast, ICa was unaffected by either NPY(1–36) or NPY(16–36). Both pertussis toxin pretreatment and including GDP[ß-S] in the patch pipette solution completely abolished the inhibitory effect of NPY on IACh It is concluded that inhibition of IACh probably represents the mechanism by which NPY decreases catecholamine release from adrenal medulla. This effect appears to be mediated by a G-protein-coupled Y2 receptor.

a2-Adrenoceptor agonists inhibit the firing of locus coeruleus (LC) neurons. It was recently observed that the a2-adrenoceptor agonists clonidine, rilmenidine and cirazoline, when injected intravenously in anaestbetized rats pretreated with the irreversible a2-adrenoceptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), excite the LC. The effect was attributed to activation of 11 imidazoline receptors. The aim of the present experiments was to characterize the direct effect of a2-adrenoceptor and I1 imidazoline receptor agonists on LC neurons.

1Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). The pressure application of α,β‐methylene ATP (α,β‐meATP) caused reproducible depolarizations which were depressed by suramin (30 μM) and abolished by suramin (100 μM). Pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS; 10, 30 μM) also concentration‐dependently inhibited the α,β‐meATP‐induced depolarization, although with a much slower time‐course than suramin. Almost complete inhibition developed with 30 μMPPADS. Reactive blue 2 (30 μM) did not alter the effect of α,β‐meATP, while reactive blue 2 (100 μM) slightly depressed it.2Pressure‐applied (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) also depolarized LC neurones. Kynurenic acid (500 μM) depressed and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 50 μM) abolished the response to AMPA. Suramin (100 μM) potentiated the AMPA effect.3Pressure‐applied noradrenaline hyperpolarized LC neurones. Suramin (100 μM) did not alter the effect of noradrenaline.4Focal electrical stimulation evoked biphasic synaptic potentials consisting of a fast depolarization (p.s.p.) followed by a slow hyperpolarization (i.p.s.p.). A mixture of D(−)‐2‐amino‐5‐phosphonopentanoic acid (AP‐5; 50 μM), CNQX (50 μM) and picrotoxin (100 μM) depressed both the p.s.p. and the i.p.s.p. Under these conditions suramin (100 μM) markedly inhibited the p.s.p., but did not alter the i.p.s.p. In the combined presence of AP‐5 (50 μM), CNQX (50 μM), picrotoxin (100 μM), strychnine (0.1 μM), tropisetron (0.5 μM) and hexamethonium (100 μM), a high concentration of suramin (300 μM) almost abolished the p.s.p. without changing the i.p.s.p.5In the presence of kynurenic acid (500 μM) and picrotoxin (100 μM), PPADS (30 μM) depressed the p.s.p. Moreover, the application of suramin (100 μM) to the PPADS (30 μM)‐containing medium failed to cause any further inhibition. Neither PPADS (30 μM) nor suramin (100 μM) altered the i.p.s.p.6It was concluded that the cell somata of LC neurones are endowed with excitatory P2‐purinoceptors. ATP may be released either as the sole transmitter from purinergic neurones terminating at the LC or as a co‐transmitter of noradrenaline from recurrent axon collaterals or dendrites of the LC neurones themselves.