In this work, we have examined the subcellular distribution of cysteine string proteins (Csps) in bovine adrenal medullary chromaffin cells. Csps did not leak from digitonin-permeabilized chromaffin cells, suggesting that there is no cytosolic pool of the protein in these cells. Subcellular fractionation studies confirmed that there was essentially no Csp immunoreactivity in the cytosolic fraction. However, immunoreactivity was detected in the membrane fractions of these cells. Csp immunoreactivity codistributed with dopamine ␤-hydroxylase, a granule marker protein, in sucrose gradient-separated granule fractions. Immunofluorescence studies showed that all chromaffin cells in culture were stained with a punctate appearance consistent with a granular localization. These results were confirmed by immunogold labeling, which demonstrated specific labeling of chromaffin granule membranes. In addition to its presence on synaptic vesicles, cysteine string protein is therefore a bona fide chromaffin granule membrane protein.

No abstract

Conversion of prohormone precursors to smaller active products occurs in secretory granules, which also have the capacity to concentrate biogenic amines. We have examined how processing of the gastrin precursor, progastrin, in rat antral mucosa is influenced by modulation of the biogenic amine content of secretory granules. Newly synthesized progastrin‐derived peptides in rat antral mucosa were labelled in vitro with 35SO42− using a pulse–chase protocol and detected after immunoprecipitation by HPLC with on‐line liquid scintillation counting. Secretory granule morphology was examined by electron microscopy. The effects of experimentally manipulating secretory granule pH and amine content were examined. The dopamine precursor l‐β‐3,4‐dihydroxyphenylalanine (l‐DOPA) inhibited cleavage of 35S‐labelled thirty‐four amino acid amidated gastrin, i.e. [35S]G34, and of [35S]G34 with COOH‐terminal glycine, i.e. [35S]G34–Gly, at a pair of lysine residues, but did not influence cleavage of progastrin at pairs of arginine residues. The effect of l‐DOPA was reversed by reserpine, which inhibits the amine–proton exchangers VMAT1 and VMAT2, and by carbidopa, which inhibits aromatic L‐amino acid decarboxylase. Treatments that raise intragranular pH, e.g. the weak base chloroquine, the ionophore monensin and the vacuolar proton pump inhibitor bafilomycin A1, had similar effects to l‐DOPA. Electron microscopical studies showed that the electron‐dense aggregrates in gastrin cell secretory granules were lost after inhibition of the vacuolar proton pump. Treatment with l‐DOPA produced reserpine‐sensitive dissipation of the electron‐dense aggregates, compatible with the idea that increased amine delivery raised intragranular pH. The data suggest that the processes of amine precursor uptake, decarboxylation and sequestration in secretory granules are associated with selective modulation of progastrin cleavage, possibly by raising intragranular pH and thereby inhibiting pH‐sensitive prohormone convertases.

Transverse sections of the ulnar, saphenous, and sural nerves taken at specific levels in normal, young-adult beagle dogs were examined qualitatively and quantitatively at both the light and electron microscopic levels. The aim of this investigation was to provide baseline information for future studies of peripheral nerve disease in this species. A systematic sampling technique was used for the determination of nerve components (i.e., unmyelinated axons and Schwann cell and fibroblast nuclei). In all nerves sampled, the average size distribution for unmyelinated axons was unimodal, and most of the axons were 0.4-1.1 micron in diameter. Within this range, there were slight individual and nerve-to-nerve variations in the location of the largest diameter peak. The mean densities of Schwann cell nuclei (numbers/mm2) ranged from 841/mm2 in the palmar branch of the ulnar nerve, to 1,223/mm2 in the caudal cutaneous sural nerve, being nearly four times the average density or fibroblast nuclei. In every animal and in almost every nerve, a few abnormalities were found; and these should be kept in mind when assessing peripheral nerves in the dog.

Efferent and reciprocal synapses have been demonstrated in the carotid body of the domestic fowl (Gallus gallus domesticus). Synapses were also found with purely afferent morphology, but were probably components of reciprocal synapses. The general morphology of the endings suggested the presence of two types of axon, afferent axons making reciprocal and perhaps afferent synapses with Type I cells, and efferent axons making efferent synapses with Type I cells. A few axo-dendritic synapses were also found. The dense-cored vesicles associated with the afferent components of reciprocal synapses and with the possible true afferent synapses varied in diameter and core but could belong to one population of pre-synaptic vesicles. These observations are consistent wtih a new theory for the carotid body receptor mechanism. This proposes a spontaneously discharging afferent axon inhibited by an inhibitory transmitter substance released by the Type I cell via the "afferent" component of its reciprocal synapse, the "efferent" component inhibiting this release. Besides this chemoreceptor modulation of its afferent axon, the Type I cell may also have a general secretory function.