SNAP-25 regulation during adrenal gland development: comparison with differentiation markers and other SNAREs

Abstract

Synaptosomal-associated protein of 25 kDa (SNAP-25) is one of a limited number of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) that play a major role in membrane docking of synaptic vesicles and secretory granules during regulated exocytosis. We have previously shown that SNAP-25 levels differ between noradrenergic and adrenergic chromaffin cell populations of the adult adrenal gland. We examine SNAP-25 expression by immunofluoresence in cells of the sympathoadrenal lineage in the rat during late embryonic and postnatal development. In parallel, tyrosine hydroxylase was used to identify sympathoadrenal cells, phenylethanolamine N-methyltransferase to distinguish adrenergic from noradrenergic chromaffin cells, and chromogranin A to define the presence of secretory granules. In addition, SNAP-25 protein and mRNA levels were followed in adrenal gland extracts by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Protein levels were compared with those of other molecules also implicated in organelle trafficking, including syntaxin 1 and vesicle-associated membrane protein (VAMP-2) and the nonneuronal analogues SNAP-23 and cellubrevin. This study provides evidence that SNAP-25 is expressed early during development in sympathoadrenal neurons and migrating cells. It is detected in intra-adrenal chromoblasts as soon as they enter the adrenal primordium. Its differential expression between catecholamine chromaffin cell phenotypes is already evident from the 17th embryonic day, future noradrenergic cells appearing to express higher levels than adrenergic cells. The granule maturation marker chromogranin A is expressed in chromaffin cells later than SNAP-25. Both SNAP-25 protein and mRNA increased rapidly in the adrenal gland in the perinatal period to peak during the first postnatal week, after which levels dropped dramatically to adult values. In contrast, levels of both syntaxin and SNAP-23 appeared to remain fairly constant throughout adrenal gland development. VAMP-2 expression increased gradually around birth to reach maximal levels during the first two postnatal weeks, and then decreased slightly. Cellubrevin levels also appeared to increase gradually until adult values were attained by the end of the second postnatal week. The threefold increase of SNAP-25 mRNA shortly after birth compared to the low adult levels suggests that during this period SNAP-25 is implicated in additional functions than regulated secretion, possibly associated with cellular growth or maturation.