ABSTRACT
"Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS, a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation, we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine beta-hydroxylase and cytochrome b561. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [3H]Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin; however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of approximately 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine beta-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b561 was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.
