Isolation and Characterization of Secretory Granules Storing a Vasoactive Intestinal Polypeptide‐Like Peptide in Torpedo Cholinergic Electromotor Neurones

Abstract

Previous immunocytochemical work showed that the cholinergic electromotor neurones of Torpedo marmorata contain a vasoactive intestinal polypeptide-like immunoreactivity (VIPLI) that is conveyed to the terminals by axonal transport from the cell bodies where it is presumably synthesized. In extension of this work, we have now succeeded in isolating the VIPLI storage granules from both the terminals and the axons of these neurones and characterizing them morphologically and biochemically. They were readily separated from synaptic vesicles but contained several components in common that had previously been regarded as specific for synaptic vesicles. Among these were a heparan sulphate type of proteoglycan, synaptophysin, and a Mg2+-dependent ATPase. The VIPLI concentration in lobe tissue and the amount of tissue available were both insufficient to permit the isolation of granules from the electromotor cell bodies by the same technique but it was possible to establish the presence of such granules by particle-exclusion chromatography, using the stable markers mentioned above. In contrast to the VIPLI-containing granules, axonal synaptic vesicles differed from their terminal counterparts in having a very low acetylcholine content relative to stable vesicle markers: they presumably fill up on reaching the terminal where they are exposed to higher concentrations of cytoplasmic acetylcholine.

Isolation and Characterization of Rapid Transport Vesicle Subtypes from Rabbit Optic Nerve

“…At least some of the regulated vesicles contain terminalbound integral membrane proteins (synaptophysin in particular), suggesting an overlap of constitutive and regulated pathways within peak 3 membranes. These data are in agreement with recently published data on exocytic pathways in endocrine cells (Lowe et al, 1988), and in the electromotor neuron of Torpedo marmorata (Agoston et al, 1989). In contrast, Navone et al (1986Navone et al ( , 1989 have described a restricted distribution of synaptophysin among the secretory vesicle types of rat brain and bovine adrenal medulla and pituitary.…”

Some Currently Neglected Aspects of Cholinergic Function

“…VIP is also found in association with cholinergic neurones in the peripheral nervous system, the electromotor system being no exception (Agoston et al 1989). It is packaged in larger ‘throw away’ vesicles which, unlike synaptic vesicles, cannot be refilled in the periphery and are thus entirely dependent on axonal transport for replenishment.…”

Characterization of gastrin-releasing peptide immunoreactivity in distinct storage particles in guinea pig myenteric and Torpedo electromotor neurones

“…From electrw organ. The collagenous nature of the tissue required a more vigorous techmque and utilized a modification (5,12) of our original method (18). Briefly, the tissue (70-80 g) was frozen in liquid nitrogen, crushed to a coarse powder, and after warming to -10°(2, suspended m 0.935 M NaC1 in a tissue to sahne ratio of 1 8.…”

“…Synaptophysin. This putatxve synaptIc vesicle marker (19), also known as p38 (10), has recently been shown to also be present in neuropeptide-contaming particles from MPLM (1) and Torpedo electric organ (5) It was assayed by a dot-blot technique as described (1,3,5).…”

“…Torpedo electric organ (12,18) in zonal rotors, whereby VIPcontaining storage granules may be separated from ACh-rich synaptic vesicles and storage vesicles containing other neuropeptides (1,2,4,5). From extracts of MPLM, distinct fractions of vesicles storing substance P, somatostatin and VIP have been separated (2), but in the purely cholinerglc Torpedo electromotor system, storage granules containing VIP-11ke lmmunoreactivlty have been up to now the only peptlde-contaIning particles to be isolated (5).…”