Modulation of muscarinic signaling in PC12 cells overexpressing neuronal Ca2+ sensor-1 protein.
It has been suggested that overexpression of neuronal Ca2+ sensor-1 (NCS-1) protein is implicated in the pathophysiology of neurodisorders such as schizophrenia, bipolar disturbance and X-linked mental retardation. The mechanism by which NCS-1 would be involved in the causes and/or consequences of these neurodisorders is still far from elucidation. Independent evidence has pointed NCS-1 as a key regulator of synaptic efficacy by altering the expression and activity of voltage-gated channels, inhibiting internalization of dopaminergic receptors, and altering phosphoinositide metabolism. In this study, we examined the possible participation of NCS-1 protein in signal transmission dependent on muscarinic receptor activation, using PC12 cells stably expressing NCS-1 (PC12-NCS-1). Carbachol (CCH; 300 microM) was able to evoke glutamate release more efficiently from PC12-NCS-1 (15.3+/-1.0nmol/mg of protein) than wild type cells (PC12-wt; 8.3+/-0.9nmol/mg of protein). This increase of glutamate release induced by CCH was independent on extracellular Ca2+ influx. Additionally, a larger increase of cytoplasmic levels of InsP3 (663.0+/-63.0 and 310.0+/-39.0% of fluorescence in A.U.) and [Ca2+]i (766.4+/-40.0 and 687.8+/-37.1nmol/L) was observed after CCH stimulus of PC12-NCS-1 compared with PC12-wt. Clearly distinction between intracellular Ca2+ dynamics was also observed in PC12-NCS-1 and PC12-wt. A larger increase followed by fast decay of [Ca2+]i was observed in PC12-NCS-1. A plateau with a delayed decay of [Ca2+]i was characteristic of PC12-wt [Ca2+]i response. Both enhancement of InsP3 production and glutamate release observed in PC12-NCS-1 were blocked by atropine (10 microM). Together, our data show that overexpression of NCS-1 in PC12 cells induces an enhancement of intracellular second messenger and transmitter release dependent on CCH response, suggesting that muscarinic signaling is "up-regulated" in this cell model.
Guimarães
M M
MM
Faculdade de Medicina da UFMGAv Alfredo Balena Laboratorio de Neurociência, Departamento de Saude Mental, INCT de Medicina Molecular Brazil.
Reis
H J
HJ
Guimarães
L P
LP
Carneiro
D S
DS
Ribeiro
F M
FM
Gomez
M V
MV
Jeromin
A
A
Romano-Silva
M A
MA
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D051580:Neuronal Calcium-Sensor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016716:PC12 Cells; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D002614:Chelating Agents; D018679:Cholinergic Agonists; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; C101464:frequenin calcium sensor proteins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D004533:Egtazic Acid; D002217:Carbachol; D002118:Calcium