Src family tyrosine kinases differentially modulate exocytosis from rat brain nerve terminals

• Published in: Neurochemistry international 2006-07, Vol.49 (1), p.80-86
• Main Authors: Baldwin, Monique L., Cammarota, Martín, Sim, Alistair T.R., Rostas, John A.P.
• Format: Article
• Language: English
• Subjects: 4-Aminopyridine - pharmacology; Animals; Biological and medical sciences; Brain - enzymology; Brain - physiopathology; Calcium Signaling - drug effects; Calcium Signaling - physiology; Depolarisation; Down-Regulation - drug effects; Down-Regulation - physiology; Enzyme Inhibitors - pharmacology; Exocytosis; Exocytosis - drug effects; Exocytosis - physiology; Feedback, Physiological - drug effects; Feedback, Physiological - physiology; Fundamental and applied biological sciences. Psychology; Fura-2; Glutamic Acid - metabolism; Membrane Fusion - drug effects; Membrane Fusion - physiology; Nerve Tissue Proteins - drug effects; Nerve Tissue Proteins - metabolism; Phosphorylation - drug effects; Potassium Channel Blockers - pharmacology; Potassium Chloride - pharmacology; Presynaptic Terminals - drug effects; Presynaptic Terminals - enzymology; Presynaptic Terminals - secretion; Pyrazoles - pharmacology; Pyridinium Compounds; Pyrimidines - pharmacology; Quaternary Ammonium Compounds; Rats; Src; src-Family Kinases - antagonists & inhibitors; src-Family Kinases - metabolism; Synaptic Transmission - drug effects; Synaptic Transmission - physiology; Synaptic Vesicles - drug effects; Synaptic Vesicles - enzymology; Synaptosomes; Vertebrates: nervous system and sense organs
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/j.neuint.2006.01.002

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: 0.3 mM) 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 μM), increased KCl and 0.3 mM 4AP-evoked Ca 2+-dependent release of glutamate, but had little effect upon exocytosis evoked by 1 mM 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.