Megumi Taketo

Muscarinic Receptor-mediated Dual Regulation of ADP-ribosyl Cyclase in NG108-15 Neuronal Cell Membranes

Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma × glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30–52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation.

Nicotinamide-adenine dinucleotide regulates muscarinic receptor-coupled K+ (M) channels in rodent NG108-15 cells.

1. The possible role of nicotinamide‐adenine dinucleotide (NAD+) and cyclic adenosine diphosphate ribose (cADPR) as regulators of M‐type K+ currents (IK(M)) has been studied in whole‐cell patch‐clamped NG108‐15 mouse neuroblastoma x rat glioma cells that had been transformed to express m1 muscarinic acetylcholine receptors (mAChRs). 2. Pre‐incubation of NG108‐15 cells for 6‐8 h with streptozotocin (2‐5 mM) reduced NAD+ levels by 40‐50%. Nicotinamide (2‐5 mM) increased NAD+ levels and prevented depletion by streptozotocin. 3. Streptozotocin pretreatment reduced the inhibition of IK(M) produced by 100 microM acetylcholine (ACh) from 51.6 +/‐ 7.0 to 29.1 +/‐ 7.5%. This was prevented by simultaneous pre‐incubation with 2 mM nicotinamide or by adding 2 mM NAD+ to the pipette solution. Neither procedure significantly affected the initial amplitude of IK(M). 4. Inclusion of 2 microM cADPR in the pipette solution induced a slow loss of IK(M) with a time constant of about 20 min. 5. It is concluded that mAChR‐induced inhibition of IK(M) requires intracellular NAD+. This might be needed for the formation of cADPR as a regulator or messenger for IK(M) inhibition.

Signal Transduction from Bradykinin, Angiotensin, Adrenergic and Muscarinic Receptors to Effector Enzymes, Including ADP-Ribosyl Cyclase

Abstract Muscarinic acetylcholine receptors in NG108-15 neuroblastoma x glioma cells, and ?-adrenergic or angiotensin II receptors in cortical astrocytes and/or ventricular myocytes, utilize the direct signaling pathway to ADP-ribosyl cyclase within cell membranes to produce cyclic ADP-ribose (cADPR) from ?-NAD+. This signal cascade is analogous to the previously established transduction pathways from bradykinin receptors to phospholipase C? and ?-adrenoceptors to adenylyl cyclase via G proteins. Upon receptor stimulation, the newlyformed cADPR may coordinately function to upregulate the release of Ca2+ from the type II ryanodine receptors as well as to facilitate Ca2+ influx through voltage-dependent Ca2+ channels. cADPR interacts with FK506, an immunosuppressant, at FKBP12.6, FK506-bindingprotein, and calcineurin, or ryanodine receptors. cADPR also functions through activating calcineurin released from Akinase anchoring protein (AKAP79). Thus, some Gq/11coupled receptors can control cADPR-dependent modulation in Ca2+ signaling.

INOSITOL TRISPHOSPHATE/CA2+ AS MESSENGERS OF BRADYKININ B2 AND MUSCARINIC ACETYLCHOLINE M1-M4 RECEPTORS IN NEUROBLASTOMA-DERIVED HYBRID CELLS

Neuroblastoma x glioma hybrid NG 108-15 and neuroblastoma x fibroblast hybrid NL308 cells possess endogenous bradykinin B2 receptors and m4 muscarinic acetylcholine receptors (mAChRs), which couple to phospholipase C and adenylate cyclase, respectively. Four genetic subtypes of mAChRs differed in their effects when stimulated in NG108-15 and NL308 cells overexpressing mAChRs. Broadly speaking, the principal effects fell into two categories: the odd-numbered receptors (m1 and m3) activated phospholipase C and increased inositol trisphosphate/Ca2+, as bradykinin did, whereas the even-numbered receptors (m2 and m4) inhibited adenylate cyclase via a pertussis toxin (PTx)-sensitive G-protein in NG108-15 cells. But all four types of NL308 cells overexpressing each m1, m2, m3 and m4 receptor activated phospholipase C, while keeping the PTx-sensitivity in m2/m4, but not in m1/m3 receptors. Coupling to ion channel effectors showed a comparable dichotomy in NG108-15 cells, while cross-activation occurred in NL308 cells.

Ca2+ release and Ca2+ influx in Chinese hamster ovary cells expressing the cloned mouse B2 bradykinin receptor: tyrosine kinase inhibitor-sensitive and- insensitive processes.

A cDNA encoding a mouse B2 bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B2 BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). The initial elevation in [Ca2+]i was abolished by thapsigargin pretreatment in Ca(2+)-free medium. The second phase was dependent on external Ca2+. The BK/inositol trisphosphate and thapsigargin-sensitive Ca2+ stores required extracellular Ca2+ for refilling. Ca2+ influx induced by BK and thapsigargin was confirmed by Mn2+ entry through Ca2+ influx pathways producing Mn2+ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca2+]i increase during the sustained phase and the rate of Mn2+ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B2 BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca2+]i increases, by mobilization of intracellular Ca2+ and store-depletion-mediated Ca2+ influx, the latter of which is tyrosine phosphorylation dependent.

Chapter 13. Bradykinin B2 receptors and signal transduction analyzed in NG108-15 neuroblastoma X glioma hybrid cells, B2 receptor-transformed CHO cells and ras-transformed NIH/3T3 fibroblasts

Publisher Summary This chapter discusses the molecular structure of mouse B2 BK receptors based on its cDNA and functional characterization, describes B2 receptors at the genetic and molecular level, and summarizes the results obtained for B2 receptors expressed in xenopus oocytes and Chinese hamster ovary (CHO) cells. The chapter also describes signal transduction pathways in the downstream of B2 BK receptors in NG108-15 neuroblastoma X glioma hybrid cells, which provide one of the basic mechanisms underlying nociception and neuronal activity modulation by BK. The chapter mentions B2 BK receptor-mediated interaction between tyrosine kinase and phospholipase C signal pathways in ras-transformed NIH/3T3 fibroblast (DT) cells, where BK functions as a mitogen. The chapter also discusses various B2 receptor-mediated inositol phospholipid metabolism. The activation of a mitogen-activated protein (MAP) kinase pathway may result in cell proliferation. In relation to nociception, and pain neurotransmission or neuromodulaion, BK serves as a transmitter or modulator. To do this, B2 receptors induce changes in ion channel conductances, as a consequence of formation of second messengers or of protein phosphorylation.

Potential mechanism for bradykinin-activated and inositol tetrakisphosphate-dependent Ca2+ influx by Ras and GAP1 in fibroblast cells.

Here we propose a molecular model for bradykinin receptor-operated and second messenger (inositol-1,3,4,5-tetrakisphosphate)-evoked Ca2+ influx and its potentiation by oncogenic Ras, which is not store-depletion-induced, so-called capacitative, Ca2+ influx. The principal idea for this hypothesis stems from observation that two bradykinin B2 receptor-activated signal pathways, protein tyrosine phosphorylation and formation of inositol tetrakisphosphate, merge during the Ca2+ influx process and that GTPase activating-protein 1 (GAP 1) is inositol tetrakisphosphate binding protein.

Slow Synaptic Responses in Neuronal Tumor Cells: Dual Regulation of ADP-Ribosyl Cyclase and Inhibition of M-Current by Muscarinic Receptor Stimulation

Muscarinic acetylcholine receptors (mAChRs) utilize the direct signaling pathway to ADP-ribosyl cyclase via G proteins within cell membranes to produce cyclic ADP-ribose (cADPR) from s-NAD+. This signal cascade is analogous to the previously established transduction pathways from mAChRs to adenylyl cyclase and phospholipase Cs. Together with cytosolic Ca2+, cADPR functions to release Ca2+ through ryanodine receptors. This cADPR-dependent and mAChR-controlled increase in cytosolic Ca2+ concentrations may induce various cellular responses.

Developmental change of GABAA receptor in hippocampus

Dept. of Molecular Neurobiology. Advanced Research Institute for Science and Engineering Waseda University Shinjukuku, Tokyo 169-8555 GABA, a main inhibitory neurotransmitter in adult central nervous system, has been suggested to play an important role in neuronal development. Developmental studies on transcripts of the GABA receptor gene showed growth-dependent changes of expression of the subunits. but it has not yet been decided which kind of GABAA receptor complex really functions. In this study, developmental change of GABAA receptor was examined efectrophysiologically in rat CA3 hippocampal neurons. Decay-time course of bicuculline sensitive spontaneous IPSC was found to be faster in young adult than in neonate. GABAergic IPSC evoked by focal electrical stimulation to pyramidal layer and by extrasynaptically applied GABA were also examined. In young adult, both responses were potentiated by a specific agonist of type I GABAA receptor but the potentiation was only marginal in neonate. These results indicate that, during early postnatal period, alteration of subunit assembly occurs in GABAA receptor, leading to functional change of GABAA current.

S2-3 Bradykinin B2 receptor-activated Ca influx; interaction of IP4, ras-GAP IP4-binding protein, and protein tyrosine phosphorylation

We have reported that application of bradykinin (BK), a neuropeptide, increases in [Ca2”)‘, which is extracellular Ca2’-dependent in Ki-ras-transformed NIH/3T3 (DT) fibroblasts. Activation of BK 82 receptors resulted in a decrease of cellular fluorescence at the excitation wavelength of 360 nm after MnC2 application in DT cells. This Mn2+ entry increased with membrane hyperpolarization. Internal application of Ins(l,3,4,5)P4 mimicked the membrane potential-dependent Mn2’ entry. BKand InsP4-induced Ca2’ influx was blocked by a tyrosine kinase inhibitor, genistein. Bradykinin receptor activation induced tyrosine phosphorylation of MAP kinase, which was inhibited by genistein. These results suggest that two 82 receptor-activated signal pathways, protein tyrosine phosphorylation and InsP4, merge at the hyperpolarization-activated Ca2’ influx process in ras-transformed NIH/3T3 fibroblasts.