Neurotransmitter-stimulated breakdown of endogenous polyphosphoinositides in post mortem human brain.
Membranes from human brain cortex (8-12 h post mortem) were labelled with [3H]inositol, in the presence of CMP, through the back reaction catalysed by PtdIns synthase. The enzyme incorporated [3H]inositol into phosphoinositides at a maximal rate of 419 pmol min-1 mg protein-1. In the absence of CMP, the labelling rate due to the PtdIns headgroup exchanging enzyme was 36 pmol min-1 mg protein-1. Human brain PtdIns synthase showed Kmapp values of 0.49 mM and 18 microM for inositol and CMP, respectively. In the presence of ATP, [3H]polyphosphoinositides formed after [3H]PtdIns were hydrolysed by phospholipase C in a GTP gamma S and neurotransmitter receptor agonist-dependent manner. Production of 3H-inositol phosphates as stimulated by GTP gamma S (350% of basal) was increased by the muscarinic agonists carbachol and oxotremorine-M (600% of basal) and by serotonin (485% of basal). The relative potencies of carbachol and oxotremorine-M were consistent with an action at muscarinic receptors. These results show that coupling between muscarinic and serotonin receptors and phospholipase C is preserved in membranes from post mortem human brain cortex and validate the use of a method involving direct [3H]inositol labelling of a membrane fraction to study the functional state of phospholipase C-coupled receptors in human brain samples.
Sarri
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Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Autónoma de Barcelona, Spain.
Picatoste
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F
Claro
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E
D000368:Aged; D001921:Brain / Q000201:enzymology; D001923:Brain Chemistry / Q000187:drug effects*; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D003568:Cytidine Monophosphate / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007294:Inositol / Q000378:metabolism; D008565:Membrane Proteins; D008566:Membranes / Q000378:metabolism; D008875:Middle Aged; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011180:Postmortem Changes; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology; D011985:Receptors, Serotonin / Q000187:drug effects / Q000502:physiology; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D008565:Membrane Proteins; D018377:Neurotransmitter Agents; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D011985:Receptors, Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D017855:Transferases (Other Substituted Phosphate Groups); C496766:CDIPT protein, human; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D010738:Type C Phospholipases; D003568:Cytidine Monophosphate