Src family tyrosine kinases differentially modulate exocytosis from rat brain nerve terminals.
We have studied the role of src family tyrosine kinases in regulating synaptic transmitter release from rat brain synaptosomes by using two assays that measure different aspects of synaptic vesicle exocytosis: glutamate release (that directly measures exocytosis of vesicle contents) and release of FM 2-10 styryl dye (that is proportional to the time the synaptic vesicle is fused to the plasma membrane). Depolarisation was induced by KCl (30 mM) or 4-aminopyridine (4AP: 0.3mM) to induce release by full fusion (FF) exocytosis, or by 1 mM 4AP to induce release by both FF and kiss-and-run (KR)-like exocytosis. The src family selective inhibitor, PP1 (10 microM), increased KCl and 0.3 mM 4AP-evoked Ca2+ -dependent release of glutamate, but had little effect upon exocytosis evoked by 1mM 4AP. PP1 did not affect the release of FM 2-10 under any of the depolarisation conditions used. PP1 also had no effect on overall intracellular calcium levels, as measured by FURA2, suggesting that the effects of the inhibitor are downstream of calcium entry. At the same concentration the inactive analogue of this compound, PP3, had no effect on any measure. Immunoblotting with an antibody to phosphotyrosine revealed that phosphorylation of many synaptosomal proteins was reduced by PP1. The immunoreactivity of three protein bands increased upon depolarisation and this increase was blocked by PP1. Phosphorylation of src at tyrosine-416 was reduced by PP1 but changes in its phosphorylation did not correlate with the effects of PP1 on release. These results suggest one or more members of the src family of tyrosine kinases is a negative regulator of the KR mode of exocytosis in synaptosomes, perhaps by tonically inhibiting KR under normal stimulation conditions.
Baldwin
Monique L
ML
School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia.
Cammarota
Martín
M
Sim
Alistair T R
AT
Rostas
John A P
JA
D015761:4-Aminopyridine / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000503:physiopathology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D025461:Feedback, Physiological / Q000187:drug effects / Q000502:physiology; D016257:Fura-2; D018698:Glutamic Acid / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D026902:Potassium Channel Blockers / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011726:Pyridinium Compounds; D011743:Pyrimidines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds; D051381:Rats; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D013572:Synaptic Vesicles / Q000187:drug effects / Q000201:enzymology*; D013574:Synaptosomes; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D004791:Enzyme Inhibitors; C086686:FM2 10; D009419:Nerve Tissue Proteins; D026902:Potassium Channel Blockers; D011720:Pyrazoles; D011726:Pyridinium Compounds; D011743:Pyrimidines; D000644:Quaternary Ammonium Compounds; D018698:Glutamic Acid; D011189:Potassium Chloride; D015761:4-Aminopyridine; D019061:src-Family Kinases; D016257:Fura-2