Group I mGluRs coupled to G proteins are regulated by tyrosine kinase in dopamine neurons of the rat midbrain.
Metabotropic glutamate receptors (mGluRs) modulate neuronal function via different transduction mechanisms that are either dependent or independent on G-protein function. Here we investigated, using whole cell patch-clamp recordings in combination with fluorimetric measurements of intracellular calcium concentration ([Ca(2+)](i)), the metabolic pathways involved in the responses induced by group I mGluRs in dopamine neurons of the rat midbrain. The inward current and the [Ca(2+)](i) increase caused by the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 100 microM) were permanently activated and subsequently abolished in cells loaded with the nonhydrolizable GTP-analogue GTP-gamma-S (600 microM). In addition, when GDP-beta-S (600 microM) was dialyzed into the cells to produce the blockade of the G proteins, the DHPG-dependent responses were reduced. When the tissue was bathed with the phospholipase C inhibitor 1-[6[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]exyl]-1H-pyrrole-2,5-dione (10 microM), the DHPG-induced calcium transients slightly diminished but the associated inward currents were not affected. Interestingly, a substantial depression of the DHPG-induced inward current and transient increase of [Ca(2+)](i) was caused by the protein tyrosine kinase inhibitors tyrphostin B52 (40 microM) and 4',5,7-trihydroxyisoflavone (genistein; 40 microM), whereas genistein's inactive analogue 4',5,7-trihydroxyisoflavone-7-glucoside (40 microM) was ineffective. The blockade of the Src family of tyrosine kinase by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (20 microM), mitogen-activated protein kinase by 2'-amino-3' methoxyflavone (50 microM), and protein kinase C by staurosporine (1 microM) had no effect on the cellular responses caused by DHPG. The mGluR5-selective antagonist 2-methyl-6-(phenylethynyl)-pyridine (10--100 microM) did not affect the actions of DHPG. Thus our results indicate that the responses, mainly mediated by mGluRs1 in dopamine neurons, are activated by intracellular mechanisms coupled to G proteins and regulated by tyrosine kinases.
Tozzi
A
A
Istituto di Ricovero e Cura a Carattere Scientifico Fondazione S. Lucia, 00179 Rome, Italy.
Guatteo
E
E
Caputi
L
L
Bernardi
G
G
Mercuri
N B
NB
D000818:Animals; D004298:Dopamine / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008636:Mesencephalon / Q000166:cytology; D009474:Neurons / Q000201:enzymology*; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*; D012118:Resorcinols / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D004963:Estrenes; D018691:Excitatory Amino Acid Antagonists; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D018094:Receptors, Metabotropic Glutamate; D012118:Resorcinols; D013873:Thionucleotides; D020032:Tyrphostins; C104077:metabotropic glutamate receptor type 1; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C079215:3,5-dihydroxyphenylglycine; C023427:guanosine 5'-O-(2-thiodiphosphate); D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005998:Glycine; D004298:Dopamine