Proton NMR detection of acetylcholine status in synaptic vesicles

Abstract

The storage granules of cholinergic nerve terminals, the synaptic vesicles, have a central role in the mechanism of cholinergic transmission, and direct evidence as to whether acetylcholine is stored in them in a free or partially immobilized form is therefore of interest. In some secretory systems it has become evident that low molecular weight secretion products are stored within the granules as complexes with proteins or proteoglycans. The application of high resolution NMR spectroscopy to isolated granules has recently elucidated the internal structure of secretory granules. We report here that proton NMR analysis of isolated cholinergic synaptic vesicles from Torpedo marmorata allows the identification of acetylcholine within the organelle. The spectrum shows that all the acetylcholine is dissolved in an essentially fluid phase within the core. Vesicular and uncompartmentalized acetylcholine can be distinguished from each other and in addition, any hydrolysis to choline and acetate can be monitored. The results open the possibility of studying dynamics of acetylcholine pools and their breakdown in cholinergic tissue by a direct and non-perturbing method.

Identification of a heparan sulphate-containing proteoglycan as a specific core component of cholinergic synaptic vesicles from Torpedo marmorata.

“…Although the highly negatively charged heparan sulphate may provide a matrix to which the positively charged acetylcholine is ionically bound, as suggested for mast cell granules in which histamine is stored together with heparin (Uvnas, 1973), positive evidence for this view is lacking. Proton (Stadler and Fuldner, 1980) and 31P-NMR (Fiildner and analysis carried out on isolated Torpedo vesicles indicates that vesicular acetylcholine and ATP are able to move freely; however, weak interaction with a solid matrix cannot be excluded. Thus, the concept that the proteoglycans found in a variety of secretory granules (for review, see Giannattasio et al, 1979) are involved in the packaging of secretion products as proposed by Palade (1975), may not be applicable in the case of cholinergic synaptic vesicles.…”

“…From the electric organ of Torpedo marmorata cholinergic synaptic vesicles have been isolated in high purity (Tashiro and Stadler, 1978;Ohsawa et al, 1979). These organelles store acetylcholine and ATP in high concentrations (Whittaker et al, 1972;Dowdall et al, 1974;Ohsawa et al, 1979) at an acidic pH (Stadler and Fiuldner, 1981) in soluble form (Stadler and Fuldner, 1980;Fuldner and Stadler, 1982). On stimulation they release their contents into the synaptic cleft (for recent review, see Zimmermann et al, 1981).…”

In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry

“…Vesicles isolated from rested fish are known to contain an average concentration of 0.6 M osmotically active ACh (Breer et al 1978;Morris et al 1965;Stadler and Fuldner 1980). Nerve endings of the Torpedo electric organ contain synaptic vesicles (30-120 nm in diameter) wherein the ACh is stored.…”

A Molecular Description of Nerve Terminal Function

“…Catecholamine uptake into brain synaptic vesicle fractions is stimulated by ATP and inhibited by most of the same drugs that prevent uptake into chromaffin granules (247). Cholinergic vesicles, purified from electric organ, have an internal pH of ~5.5 and contain ~0.15 M ATP in free solution (248)(249)(250)(251). The purified vesicles contain an ATPase that shows the same drug sensitivity as the ATPase in chromaffin granule ghosts, sensitivity to DCCD and resistance to oligomycin or efrapeptin (252,253).…”