RESUMO
This work tested the theory that neuronal calcium sensor-1 (NCS-1) has effects on neurotransmitter release beyond its actions on membrane channels. We used nerve-ending preparations where membrane channels are bypassed through membrane permeabilization made by mechanical disruption or streptolysin-O. Nerve ending NCS-1 and phosphatidylinositol 4-kinase (PI4K) are largely or entirely particulate, so their concentrations in nerve endings remain constant after breaching the membrane. Exogenous, myristoylated NCS-1 stimulated nerve ending phosphatidylinositol 4-phosphate [PI(4)P] synthesis, but non-myristoylated-NCS-1 did not. The N-terminal peptide of NCS-1 interfered with PI(4)P synthesis, and with spontaneous and Ca(2+)-evoked release of both [(3)H]-norepinephrine (NA) and [(14)C]-glutamate (glu) in a concentration-dependent manner. An antibody raised against the N-terminal of NCS-1 inhibited perforated nerve ending PI(4)P synthesis, but the C-terminal antibody had no effects. Antibodies against the N- and C-termini of NCS-1 caused significant increases in mini/spontaneous/stimulation-independent release of [(3)H]-NA from perforated nerve endings, but had no effect on [(14)C]-glu release. These results support the idea that NCS-1 facilitates nerve ending neurotransmitter release and phosphoinositide production via PI4K and localizes these effects to the N-terminal of NCS-1. Combined with previous work on the regulation of channels by NCS-1, the data are consistent with the hypothesis that a NCS-1-PI4K (NP, neuropotentiator) complex may serve as an essential linker between lipid and protein metabolism to regulate membrane traffic and co-ordinate it with ion fluxes and plasticity in the nerve ending.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Exocitose/fisiologia , Terminações Nervosas/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/química , 1-Fosfatidilinositol 4-Quinase/química , 1-Fosfatidilinositol 4-Quinase/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Cálcio/química , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/farmacologia , Córtex Cerebral/química , Relação Dose-Resposta a Droga , Exocitose/efeitos dos fármacos , Feminino , Masculino , Terminações Nervosas/química , Terminações Nervosas/efeitos dos fármacos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/farmacologia , Proteínas Sensoras de Cálcio Neuronal , Neuropeptídeos , Neurotransmissores/química , Neurotransmissores/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 4,5-Difosfato/biossíntese , Fosfatidilinositol 4,5-Difosfato/química , Fosfatos de Fosfatidilinositol/biossíntese , Fosfatos de Fosfatidilinositol/química , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
Inhibition of phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) synthesis by phenylarsine oxide (PAO) inhibits both [3H]-noradrenaline ([3H]-NA) and [14C]-glutamate ([14C]-glu) exocytosis from streptolysin-O (SLO)-perforated synaptosomes. When PI4,5P(2) is blocked by an antibody or chelated by neomycin, neurotransmitter exocytosis again is inhibited. Also, when phosphoinositide (PI) synthesis is indirectly decreased by shunting phosphatidic acid (PA) synthesis into phosphatidylbutanol production, both [14C]-glutamate and [3H]-noradrenaline exocytosis are inhibited. All of these results indicate that PI4,5P(2) is necessary for exocytosis of both synaptic vesicles (SVs) and dense core vesicles (DCVs).
