Characterization of the release of Met-enkephalin from isolated nerve terminals: release kinetics and cation-dependence.
The release of the neuropeptide Met-enkephalin (Met-ENK) from isolated nerve terminals (synaptosomes) of the rat forebrain was characterized with respect to the subcellular distribution, the release upon addition of various stimulatory agents, the release kinetics, the cation-dependence of release and the relationship between Met-ENK release and elevations of the intraterminal free Ca(2+)-concentration ([Ca]i). A highly specific radioimmunoassay was developed for determination of Met-ENK (H-Tyr-Gly-Gly-Phe-Met-OH). Truncated and elongated forms of Met-ENK, Leu-enkephalin, beta-endorphin and dynorphin displayed negligible cross-reactivity. Met-ENK-like immunoreactivity (Met-ENK-LI) is enriched in the purified synaptosomal fraction of rat forebrain homogenates and is released in a strictly Ca(2+)-dependent manner upon chemical depolarization or stimulation with the Ca2+ ionophore, ionomycin. A correlation exists between the release of Met-ENK-LI and the elevations of [Ca]i. Barium ions are able to replace Ca2+ in triggering Met-ENK-LI release. The release of Met-ENK-LI is initiated within 20 s after depolarization and is terminated after 3-5 min, although depolarization and [Ca]i elevation are maintained. At this time, > 90% of the initial Met-ENK-LI is still present inside the synaptosomes. Repolarization and renewed stimulation again evokes Ca(2+)-dependent release of this retained Met-ENK-LI. It is concluded that Met-ENK release from isolated nerve terminals is exocytotic, and that exocytosis is terminated by a regulatory mechanism in synaptosomes after 3-5 min of depolarization, a process which can be reversed by repolarization.(ABSTRACT TRUNCATED AT 250 WORDS)
Verhage
M
M
Rudolf Magnus Institute, Department of Medical Pharmacology, University of Utrecht, The Netherlands.
Ghijsen
W E
WE
Wiegant
V M
VM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002395:Catecholamines / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000378:metabolism*; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009411:Nerve Endings / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012844:Sincalide / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism*
D000596:Amino Acids; D002395:Catecholamines; D015759:Ionomycin; D004744:Enkephalin, Methionine; D012844:Sincalide; D011188:Potassium; D002118:Calcium