Stefan Boehm

Medical University of Vienna, Vienna, Vienna, Austria

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Publications (102)432.16 Total impact

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    ABSTRACT: Many membrane proteins incur a folding problem during biosynthesis; only a fraction thereof is exported from the endoplasmic reticulum (ER), because quality control is stringent. This is also true for G protein-coupled receptors. Here, we identify the deubiquitinating enzyme Usp4 as an interaction partner of the A2a adenosine receptor, a Gs-coupled receptor. Usp4 binds to the carboxyl terminus of the A2A receptor and allows for its accumulation as deubiquinated protein. This relaxes ER quality control and enhances cell surface expression of functionally active receptor. The effect of Usp4 on the A2A receptor was specific because 1) it was not seen in C-terminally truncated versions of the receptor; 2) it was not mimicked by Usp14, another member of the ubiquitin-specific protease family; and 3) it was not seen with the metabotropic glutamate receptor-5, another G protein-coupled receptor with a high propensity for intracellular retention. These observations show that deubiquinating enzymes can regulate quality control in the ER.
    Molecular Pharmacology 05/2006; 69(4):1083-94. · 4.41 Impact Factor
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    ABSTRACT: Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.
    The Journal of Cell Biology 02/2006; 172(2):221-31. · 10.82 Impact Factor
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    ABSTRACT: The physiological function of neurotransmitter transporter proteins like the serotonin transporter (SERT) is reuptake of neurotransmitter that terminates synaptic serotoninergic transmission. SERT can operate in reverse direction and be induced by SERT substrates including 5-HT, tyramine and the positively charged methyl-phenylpyridinium (MPP(+)), as well as the amphetamine derivatives para-chloroamphetamine (pCA) and methylene-dioxy-methamphetamine (MDMA). These substrates also induce inwardly directed sodium currents that are predominantly carried by sodium ions. Efflux via SERT depends on this sodium flux that is believed to be a prerequisite for outward transport. However, in recent studies, it has been suggested that substrates may be distinct in their properties to induce efflux. Therefore, the aim of the present study was a pharmacological characterization of different SERT substrates in uptake experiments, their abilities to induce transporter-mediated efflux and currents. In conclusion, the rank order of affinities in uptake and electrophysiological experiments correlate well, while the potencies of the amphetamine derivatives for the induction of efflux are clearly higher than those of the other substrates. These discrepancies can be only explained by mechanisms that can be induced by amphetamines. Therefore, based on our pharmacological observations, we conclude that amphetamines distinctly differ from non-amphetamine SERT substrates.
    Neuropharmacology 12/2005; 49(6):811-9. · 4.11 Impact Factor
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    ABSTRACT: Presynaptic inhibition of transmitter release is commonly mediated by a direct interaction between G protein betagamma subunits and voltage-activated Ca2+ channels. To search for an alternative pathway, the mechanisms by which presynaptic bradykinin receptors mediate an inhibition of noradrenaline release from rat superior cervical ganglion neurons were investigated. The peptide reduced noradrenaline release triggered by K+-depolarization but not that evoked by ATP, with Ca2+ channels being blocked by Cd2+. Bradykinin also reduced Ca2+ current amplitudes measured at neuronal somata, and this effect was pertussis toxin-insensitive, voltage-independent, and developed slowly within 1 min. The inhibition of Ca2+ currents was abolished by a phospholipase C inhibitor, but it was not altered by a phospholipase A2 inhibitor, by the depletion of intracellular Ca2+ stores, or by the inactivation of protein kinase C or Rho proteins. In whole-cell recordings, the reduction of Ca2+ currents was irreversible but became reversible when 4 mM ATP or 0.2 mM dioctanoyl phosphatidylinositol-4,5-bisphosphate was included in the pipette solution. In contrast, the effect of bradykinin was entirely reversible in perforated-patch recordings but became irreversible when the resynthesis of phosphatidylinositol-4,5-bisphosphate was blocked. Thus, the inhibition of Ca2+ currents by bradykinin involved a consumption of phosphatidylinositol-4,5-bisphosphate by phospholipase C but no downstream effectors of this enzyme. The reduction of noradrenaline release by bradykinin was also abolished by the inhibition of phospholipase C or of the resynthesis of phosphatidylinositol-4,5-bisphosphate. These results show that the presynaptic inhibition was mediated by a closure of voltage-gated Ca2+ channels through depletion of membrane phosphatidylinositol bisphosphates via phospholipase C.
    Molecular Pharmacology 12/2005; 68(5):1387-96. · 4.41 Impact Factor
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    ABSTRACT: Bradykinin is known to stimulate neurons in rat sympathetic ganglia and to enhance transmitter release from their axons by interfering with the autoinhibitory feedback, actions that involve protein kinase C. Here, bradykinin caused a transient increase in the release of previously incorporated [3H] noradrenaline from primary cultures of dissociated rat sympathetic neurons. When this effect was abolished by tetrodotoxin, bradykinin caused an inhibition of tritium overflow triggered by depolarizing K+ concentrations. This inhibition was additive to that caused by the alpha2-adrenergic agonist UK 14304, desensitized within 12 min, was insensitive to pertussis toxin, and was enhanced when protein kinase C was inactivated. The effect was half maximal at 4 nm and antagonized competitively by the B2 receptor antagonist Hoe 140. The cyclooxygenase inhibitor indomethacin and the angiotensin converting enzyme inhibitor captopril did not alter the inhibition by bradykinin. The M-type K+ channel opener retigabine attenuated the secretagogue action of bradykinin, but left its inhibitory action unaltered. In whole-cell patch-clamp recordings, bradykinin reduced voltage-activated Ca2+ currents in a pertussis toxin-insensitive manner, and this action was additive to the inhibition by UK 14304. These results demonstrate that bradykinin inhibits noradrenaline release from rat sympathetic neurons via presynaptic B2 receptors. This effect does not involve cyclooxygenase products, M-type K+ channels, or protein kinase C, but rather an inhibition of voltage-gated Ca2+ channels.
    Journal of Neurochemistry 07/2005; 93(5):1110-21. · 3.97 Impact Factor
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    Stefan G Lechner, Stefan Boehm
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    ABSTRACT: Within the last 15 years, at least 8 different G protein-coupled P2Y receptors have been characterized. These mediate slow metabotropic effects of nucleotides in neurons as well as non-neural cells, as opposed to the fast ionotropic effects which are mediated by P2X receptors. One class of effector systems regulated by various G protein-coupled receptors are voltage-gated and ligand-gated ion channels. This review summarizes the current knowledge about the modulation of such neuronal ion channels via P2Y receptors. The regulated proteins include voltage-gated Ca(2+) and K(+) channels, as well as N-methyl-D: -aspartate, vanilloid, and P2X receptors, and the regulating entities include most of the known P2Y receptor subtypes. The functional consequences of the modulation of ion channels by nucleotides acting at pre- or postsynaptic P2Y receptors are changes in the strength of synaptic transmission. Accordingly, ATP and related nucleotides may act not only as fast transmitters (via P2X receptors) in the nervous system, but also as neuromodulators (via P2Y receptors). Hence, nucleotides are as universal transmitters as, for instance, acetylcholine, glutamate, or gamma-aminobutyric acid.
    Purinergic Signalling 01/2005; 1(1):31-41. · 2.64 Impact Factor
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    ABSTRACT: Although feedback inhibition of noradrenaline release by coreleased nucleotides is a well known phenomenon, it remained unclear which P2 receptor subtypes and associated signalling cascades may be involved. In the rat pheochromocytoma cell line PC12, 2-methylthio-ADP reduced noradrenaline release triggered by K+ depolarization more potently than ADP and ATP, whereas UDP or UTP failed to do so. The inhibition by ADP was abolished by pertussis toxin and antagonized by reactive blue 2, 2-methylthio-AMP, and AR-C69931MX, but not by suramin. AR-C69931MX acted as a competitive antagonist with an apparent affinity of 2 nm, but did not alter noradrenaline release, when PC12 cells were continuously superfused. However, when the superfusion was halted during K+ depolarization, release was significantly reduced and this inhibition was attenuated by AR-C69931MX, thus revealing ongoing autoinhibition. Rises in cellular cyclic AMP did not alter depolarization-evoked release nor its reduction by ADP, even though the nucleotide did inhibit cyclic AMP accumulation. ADP and the direct Ca2+ channel blocker Cd2+ inhibited voltage-activated Ca2+ currents, but not ATP-induced currents, and both agents reduced K+-evoked, but not ATP-evoked, release. Hence, if voltage-gated Ca2+ channels do not contribute to stimulation-evoked release, ADP fails to exert its inhibitory action. In primary cultures of rat sympathetic neurons, ADP also reduced Ca2+ currents and K+-evoked noradrenaline release, and AR-C69931MX acted again as competitive antagonist with an apparent affinity of 3 nm. These results show that P2Y12 receptors mediate an autoinhibition of transmitter release from PC12 cells and sympathetic neurons through an inhibition of voltage-gated Ca2+ channels.
    European Journal of Neuroscience 01/2005; 20(11):2917-28. · 3.75 Impact Factor
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    ABSTRACT: The core complex, formed by the SNARE proteins synaptobrevin 2, syntaxin 1 and SNAP-25, is an important component of the synaptic fusion machinery and shows remarkable in vitro stability, as exemplified by its SDS-resistance. In western blots, antibodies against one of these SNARE proteins reveal the existence of not only an SDS-resistant ternary complex but also as many as five bands between 60 and >200 kDa. Structural conformation as well as possible functions of these various complexes remained elusive. In western blots of protein extracts from PC12 cell membranes, an antibody against SNAP-25 detected two heat-sensitive SDS-resistant bands with apparent molecular weights of 100 and 230 kDa. A syntaxin antibody recognized only the 230 kDa band and required heat-treatment of the blotting membrane to detect the 100 kDa band. Various antibodies against synaptobrevin failed to detect SNARE complexes in conventional western blots and detected either the 100 kDa band or the 230 kDa band on heat-treated blotting membranes. When PC12 cells were exposed to various extracellular K(+)-concentrations (to evoke depolarization-induced Ca(2+) influx) or permeabilized in the presence of basal or elevated free Ca(2+), levels of these SNARE complexes were altered differentially: moderate Ca(2+) rises (</=1 microM) caused an increase, whereas Ca(2+) elevations of more than 1 microM led to a decrease in the 230 kDa band. Under both conditions the 100 kDa band was either increased or remained unchanged. Our data show that various SDS-resistant complexes occur in living cells and indicate that they represent SNARE complexes with different structures and diverging functions. The distinct behavior of these complexes under release-promoting conditions indicates that these SNARE structures have different roles in exocytosis.
    Journal of Cell Science 02/2004; 117(Pt 6):955-66. · 5.88 Impact Factor
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    ABSTRACT: Acetylcholine has long been known to excite sympathetic neurons via M1 muscarinic receptors through an inhibition of M-currents. Nevertheless, it remained controversial whether activation of muscarinic receptors is also sufficient to trigger noradrenaline release from sympathetic neurons. In primary cultures of rat superior cervical ganglia, the muscarinic agonist oxotremorine M inhibited M-currents with half-maximal effects at 1 microM and induced the release of previously incorporated [3H]noradrenaline with half-maximal effects at 10 microM. This latter action was not affected by the nicotinic antagonist mecamylamine which, however, abolished currents through nicotinic receptors elicited by high oxotremorine M concentrations. Ablation of the signalling cascades linked to inhibitory G proteins by pertussis toxin potentiated the release stimulating effect of oxotremorine M, and the half-maximal concentration required to stimulate noradrenaline release was decreased to 3 microM. Pirenzepine antagonized the inhibition of M-currents and the induction of release by oxotremorine M with identical apparent affinity, and both effects were abolished by the muscarinic toxin 7. These results indicate that one muscarinic receptor subtype, namely M1, mediates these two effects. Retigabine, which enhances M-currents, abolished the release induced by oxotremorine M, but left electrically induced release unaltered. Moreover, retigabine shifted the voltage-dependent activation of M-currents by about 20 mV to more negative potentials and caused 20 mV hyperpolarisations of the membrane potential. In the absence of retigabine, oxotremorine M depolarised the neurons and elicited action potential discharges in 8 of 23 neurons; in its presence, oxotremorine M still caused equal depolarisations, but always failed to trigger action potentials. Action potential waveforms caused by current injection were not affected by retigabine. These results indicate that the inhibition of M-currents is the basis for the stimulation of transmitter release from sympathetic neurons via M1 muscarinic receptors.
    The Journal of Physiology 01/2004; 553(Pt 3):789-802. · 4.38 Impact Factor
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    ABSTRACT: Activation of P2Y receptors by released nucleotides subserves important autocrine-paracrine functions in various non-neural tissues. To investigate how P2Y receptors are activated in a neuronal environment, we used PC12 cells in which nucleotides were found to elicit increases in inositol phosphates via P2Y2 and decreases in cAMP via P2Y12 receptors. Depolarization of PC12 cells raised inositol phosphates, and blockade of voltage-gated Ca2+ channels by Cd2+ or degradation of extracellular nucleotides by apyrase prevented this effect. In nondepolarized cells, apyrase did not affect inositol phosphates. Depolarization of PC12 cells also reduced the A2A receptor-mediated synthesis of cAMP. This effect was again prevented by Cd2+ or apyrase, but apyrase enhanced the synthesis of cAMP even in nondepolarized cells. Overexpression of rat P2Y2 receptors increased the nucleotide-dependent inositol phosphate accumulation and enhanced the effect of K+ depolarization. Nevertheless, apyrase still failed to alter spontaneous inositol phosphate accumulation. Expression of rat P2Y1 receptors, in contrast, led to huge increases in spontaneous inositol phosphate accumulation, which was reduced by a receptor antagonist or by apyrase. This increased synthesis of inositol phosphates could not be further enhanced by depolarization or receptor agonists, but when endogenous nucleotides were removed by superfusion, recombinant P2Y1 receptors could be activated to mediate an inhibition of M-type K+ channels. These results indicate that nucleoside diphosphate-sensitive (P2Y12 and P2Y1) receptors are activated by spontaneous nucleotide release, whereas triphosphate-sensitive (P2Y2) receptors require an excess of depolarization-evoked release to become activated.
    Journal of Neuroscience 09/2003; 23(20):7479-88. · 6.91 Impact Factor
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    ABSTRACT: The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism.
    Neuropsychopharmacology 08/2003; 28(7):1317-27. · 8.68 Impact Factor
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    Stefan Boehm
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    ABSTRACT: British Journal of Pharmacology (2003) 138, 1–3. doi:10.1038/sj.bjp.0705044
    British Journal of Pharmacology 02/2003; 138(1):1-3. · 5.07 Impact Factor
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    ABSTRACT: 1. In PC12 cells, adenine nucleotides inhibit voltage-activated Ca(2+) currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y(12) receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2-methylthio-AMP (2-MeSAMP) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (AR-C69931MX). 2. ADP reduced A(2A) receptor-dependent cyclic AMP synthesis with half maximal effects at 0.1-0.17 micro M. In the presence of 30 micro M 2-MeSAMP or 100 nM AR-C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA(2) values of 6.1 and 8.5, respectively. 3. The inhibition of Ca(2+) currents by ADP was attenuated by 10-1000 nM AR-C69931MX and by 3-300 micro M 2-MeSAMP. ADP reinhibited Ca(2+) currents after removal of 2-MeSAMP within less than 15 s, but required 2 min to do so after removal of AR-C69931MX. 4. ADP inhibited Ca(2+) currents with half maximal effects at 5-20 micro M. AR-C69931MX (10-100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pK(B) value of 8.7. Similarly, 10-100 micro M 2-MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pK(B) of 5.4. 5. The inhibition of Ca(2+) currents by 2-methylthio-ADP and ADPbetaS was also antagonized by AR-C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPbetaS by a factor of 3.8, indicative of a pA(2) value of 8.1. 6. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca(2+) channels by adenine nucleotides, which suggests that this effect is mediated by P2Y(12) receptors.
    British Journal of Pharmacology 02/2003; 138(2):343-50. · 5.07 Impact Factor
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    ABSTRACT: Bradykinin has long been known to excite sympathetic neurons via B(2) receptors, and this action is believed to be mediated by an inhibition of M-currents via phospholipase C and inositol trisphosphate-dependent increases in intracellular Ca(2+). In primary cultures of rat superior cervical ganglion neurons, bradykinin caused an accumulation of inositol trisphosphate, an inhibition of M-currents, and a stimulation of action potential-mediated transmitter release. Blockade of inositol trisphosphate-dependent signaling cascades failed to affect the bradykinin-induced release of noradrenaline, but prevented the peptide-induced inhibition of M-currents. In contrast, inhibition or downregulation of protein kinase C reduced the stimulation of transmitter release, but not the inhibition of M-currents, by bradykinin. In cultures of superior cervical ganglia, classical (alpha, betaI, betaII), novel (delta, epsilon), and atypical (zeta) protein kinase C isozymes were detected by immunoblotting. Bradykinin induced a translocation of Ca(2+)-independent protein kinase C isoforms (delta and epsilon) from the cytosol to the membrane of the neurons, but left the cellular distribution of other isoforms unchanged. This activation of Ca(2+)-independent protein kinase C enzymes was prevented by a phospholipase C inhibitor. The bradykinin-dependent stimulation of noradrenaline release was reduced by inhibitors of classical and novel protein kinase C isozymes, but not by an inhibitor selective for Ca(2+)-dependent isoforms. These results demonstrate that bradykinin B(2) receptors are linked to phospholipase C to simultaneously activate two signaling pathways: one mediates an inositol trisphosphate- and Ca(2+)-dependent inhibition of M-currents, the other one leads to an excitation of sympathetic neurons independently of changes in M-currents through an activation of Ca(2+)-insensitive protein kinase C.
    Journal of Neuroscience 08/2002; 22(14):5823-32. · 6.91 Impact Factor
  • Stefan Boehm, Helmut Kubista
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    ABSTRACT: The release of transmitters at sympathoeffector junctions is not constant, but subject to modulation by a plethora of different mechanisms. In this respect, presynaptic receptors located on the sympathetic axon terminals are of utmost importance, because they are activated by exogenous agonists and by endogenous neurotransmitters. In the latter case, the transmitters that activate the presynaptic receptors of a nerve terminal may be released either from the very same nerve ending or from a different axon terminal, and the receptors involved are auto- and heteroreceptors, respectively. In terms of their structural and functional features, receptors of sympathetic axon terminals can be categorized as either ionotropic (transmitter-gated ion channels) or metabotropic (most commonly G protein-coupled) receptors. This review summarizes results on more than 30 different metabotropic and four different ionotropic receptors that have been found to control the amount of transmitter being released from sympathetic neurons. Each of these receptors may not only stimulate, facilitate, and reduce sympathetic transmitter release, respectively, but also interact with the functions of other receptors present on the same axonal varicosity. This provides a multitude of mechanisms that regulate the amount of sympathetic transmitter output. Accordingly, a sophisticated cross-talk within and between extra- and intracellular signals is integrated at axon terminals to adapt the strength of sympathoeffector transmission to a given situation. This will not only determine the function of the sympathetic nervous system in health and disease, but also therapeutic and untoward effects of drugs that bind to the presynaptic receptors in sympathetically innervated tissues.
    Pharmacological Reviews 04/2002; 54(1):43-99. · 22.35 Impact Factor
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    ABSTRACT: P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y(12). Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. ADP and ATP (0.1 - 100 microM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A(2A) or recombinant beta(2) receptors. Forskolin-dependent cyclic AMP production was reduced by <or=1 microM and enhanced by 10 - 100 microM ADP; this latter effect was turned into an inhibition when A(2A) receptors were blocked. The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin. The rank order of agonist potencies for the reduction of cyclic AMP was (IC(50) values): 2-methylthio-ADP (0.12 nM)=2-methylthio-ATP (0.13 nM)>ADPbetaS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, or adenosine-3'-phosphate-5'-phosphate, but attenuated by reactive blue 2, ATP(alpha)S, and 2-methylthio-AMP. RT - PCR demonstrated the expression of P2Y(2), P2Y(4), P2Y(6), and P2Y(12), but not P2Y(1), receptors in PC12 cells. In Northern blots, only P2Y(2) and P2Y(12) were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. We conclude that P2Y(12) receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity.
    British Journal of Pharmacology 03/2002; 135(3):673-84. · 5.07 Impact Factor
  • Journal of Neurochemistry - J NEUROCHEM. 01/2002; 63(1):146-154.
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    ABSTRACT: Ca(2+)/calmodulin (Ca(2+)/CaM) and the betagamma subunits of heterotrimeric G-proteins (Gbetagamma) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gbetagamma binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca(2+)/CaM. Mutational analysis of the Ca(2+)/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic alpha-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gbetagamma binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gbetagamma recruitment. In addition, we present evidence that Gbetagamma uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gbetagamma-mediated signaling is abolished in receptors lacking the core CaM binding sequence, alpha subunit activation, as assayed by agonist-dependent GTPgammaS binding, was not affected. This suggests that Ca(2+)/CaM may alter the mode of group III mGluR signaling from mono- (alpha) to bidirectional (alpha and betagamma) activation of downstream effector cascades.
    Journal of Biological Chemistry 09/2001; 276(33):30662-9. · 4.65 Impact Factor
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    ABSTRACT: Ca2+/calmodulin (Ca2+/CaM) and the βγ subunits of heterotrimeric G-proteins (Gβγ) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gβγ binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca2+/CaM. Mutational analysis of the Ca2+/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic α-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gβγ binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gβγ recruitment. In addition, we present evidence that Gβγ uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gβγ-mediated signaling is abolished in receptors lacking the core CaM binding sequence, α subunit activation, as assayed by agonist-dependent GTPγS binding, was not affected. This suggests that Ca2+/CaM may alter the mode of group III mGluR signaling from mono- (α) to bidirectional (α and βγ) activation of downstream effector cascades.
    Journal of Biological Chemistry 08/2001; 276(33):30662-30669. · 4.65 Impact Factor
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    ABSTRACT: The pathway involved in UTP-evoked noradrenaline release was investigated in cultures of rat superior cervical ganglia. Northern blots revealed an age-related increase in levels of mRNA for P2Y6 receptors in cultures obtained at postnatal days 1 and 5, respectively, but no change in transcripts for P2Y1 and P2Y2. Likewise, UTP-evoked overflow of previously incorporated [(3)H]noradrenaline was six-fold higher in neurons obtained at postanatal day 5. Various protein kinase C inhibitors diminished UTP-, but not electrically, induced tritium overflow by > 70%, as did down-regulation of protein kinase C by 24 h exposure to phorbol ester. beta-Phorbol-12,13-dibutyrate and dioctanoylglycerol caused concentration-dependent increases in [(3)H] outflow of up to 6% of total radioactivity, and the secretagogue actions of these agents were reduced in the presence of protein kinase C inhibitors and in neurons pretreated with phorbol ester. Overflow evoked by dioctanoylglycerol was attenuated in the absence of extracellular Ca(2+) and in the presence of tetrodotoxin or Cd(2+). In addition to triggering tritium overflow, UTP reduced currents through muscarinic K(+) channels which, however, were not affected by phorbol esters. This action of UTP was not altered by protein kinase C inhibitors. These results indicate that P2Y6 receptors mediate UTP-evoked noradrenaline release from rat sympathetic neurons via activation of protein kinase C, but not inhibition of K(M) channels.
    Journal of Neurochemistry 06/2001; 77(3):876-85. · 3.97 Impact Factor

Publication Stats

2k Citations
432.16 Total Impact Points

Institutions

  • 2005–2013
    • Medical University of Vienna
      • • Center for Physiology and Pharmacology
      • • Abteilung für Neurophysiologie
      • • Institut für Pharmakologie
      Vienna, Vienna, Austria
  • 2007–2008
    • Medical University of Graz
      • Institut für Experimentelle und Klinische Pharmakologie
      Graz, Styria, Austria
  • 1991–2006
    • University of Vienna
      • • Department of Pharmacology and Toxicology
      • • Institute of Neurophysiology
      Wien, Vienna, Austria
  • 1997–2001
    • Max Planck Institute for Brain Research
      Frankfurt, Hesse, Germany