ABSTRACT
The signaling molecule RGS9-2 is a potent modulator of G-protein-coupled receptor function in striatum. Our earlier work revealed a critical role for RGS9-2 in the actions of the µ-opioid receptor (MOR) agonist morphine. In this study, we demonstrate that RGS9-2 may act as a positive or negative modulator of MOR-mediated behavioral responses in mice depending on the agonist administered. Paralleling these findings we use coimmunoprecipitation assays to show that the signaling complexes formed between RGS9-2 and Gα subunits in striatum are determined by the MOR agonist, and we identify RGS9-2 containing complexes associated with analgesic tolerance. In striatum, MOR activation promotes the formation of complexes between RGS9-2 and several Gα subunits, but morphine uniquely promotes an association between RGS9-2 and Gαi3. In contrast, RGS9-2/Gαq complexes assemble after acute application of several MOR agonists but not after morphine application. Repeated morphine administration leads to the formation of distinct complexes, which contain RGS9-2, Gß5, and Gαq. Finally, we use simple pharmacological manipulations to disrupt RGS9-2 complexes formed during repeated MOR activation to delay the development of analgesic tolerance to morphine. Our data provide a better understanding of the brain-region-specific signaling events associated with opiate analgesia and tolerance and point to pharmacological approaches that can be readily tested for improving chronic analgesic responsiveness.
Subject(s)
Analgesics, Opioid/pharmacology , Corpus Striatum/physiology , RGS Proteins/physiology , Animals , Arrestins/genetics , Arrestins/physiology , Blotting, Western , Corpus Striatum/drug effects , Drug Tolerance , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/physiology , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Immunoprecipitation , Mice , Mice, Knockout , Morphine/pharmacology , Phospholipase C beta/metabolism , Phosphorylation , RGS Proteins/drug effects , RGS Proteins/genetics , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , beta-ArrestinsABSTRACT
Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC(50) = 0.1-0.3 µM) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC(50) = 0.08 ± 0.01 µM) and with â¼18-fold lower affinity hGDH1 (IC(50) = 1.67 ± 0.06 µM; p < 0.001). Similarly, 17ß-estradiol showed a â¼18-fold higher affinity for hGDH2 (IC(50) = 1.53 ± 0.24 µM) than for hGDH1 (IC(50) = 26.94 ± 1.07 µM; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.
Subject(s)
Enzyme Inhibitors/chemistry , Estrogens/chemistry , Glutamate Dehydrogenase/chemistry , Amino Acid Substitution , Animals , Astrocytes/enzymology , Brain/enzymology , Cell Line , Enzyme Activation/physiology , Enzyme Inhibitors/metabolism , Estrogens/metabolism , Gene Expression Regulation, Enzymologic/physiology , Glutamate Dehydrogenase/genetics , Glutamate Dehydrogenase/metabolism , Glutamic Acid/chemistry , Glutamic Acid/genetics , Glutamic Acid/metabolism , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Mutation, Missense , Organ Specificity , Spodoptera , Structure-Activity RelationshipABSTRACT
The regulator of G protein signaling 9-2 (RGS9-2) is a constituent of G protein-coupled receptor (GPCR) macromolecular complexes with a major role in regulation of GPCR activity in the central nervous system. Previous in situ hybridization and Western blot studies revealed that RGS9-2 is expressed in the superficial dorsal horn of the spinal cord. In the present study, we monitored tail withdrawal latencies to noxious thermal stimuli and performed in vitro whole-cell patch clamp electrophysiological recordings from neurons in lamina II of the spinal dorsal horn to examine the role of RGS9-2 in the dorsal horn of the spinal cord in nociceptive behaviours and opiate mediated modulation of synaptic transmission. Our findings obtained from RGS9 knockout mice indicate that the lack of RGS9-2 protein decreases sensitivity to thermal stimuli and to the analgesic actions of morphine in the tail immersion paradigm. This modulatory role of RGS9-2 on opiate-mediated responses was further supported by electrophysiological studies showing that hyperpolarization of neurons in lamina II of the spinal dorsal horn evoked by application of DAMGO ([d-Ala2, N-MePhe4, Gly-ol]-enkephalin, a mu opioid receptor agonist) was diminished in RGS9 knockout mice. The results indicate that RGS9-2 enhances the effect of morphine and may play a crucial role in opiate-mediated analgesic mechanisms at the level of the spinal cord.