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The subcellular storage and release of noradrenaline (NA), dopamine-beta-hydroxylase (D beta H), and neuropeptide Y (NPY) was studied in the isolated perfused sheep spleen. Subcellular distribution studies showed a bimodal distribution for NA which was well reflected by D beta H and indicated the occurrence of two types of NA storage vesicles. The most dense, presumably large dense-cored vesicles (LDV), contain both membrane-bound and soluble D beta H; the less dense presumably corresponds to small dense-cored vesicles (SDV) and at least does not contain soluble D beta H. The distribution of NPY is extended but shows a peak only at the position of LDV, indicating that LDV contain NPY. Continuous electrical stimulation of the splenic nerve at 2 Hz, 5 Hz, 10 Hz, and 20 Hz or at 20 hz with bursts induced the release of NA, NPY, and D beta H. The ratio among these components was constant. The fractional release of D beta H and NA was comparable at all frequencies used; that of NPY was 10-20 times lower, suggesting the occurrence of a large nonreleasable NPY pool. The present data argue against a high frequency stimulation or intermittent stimulation-induced preferential release of NPY from adrenergic neurons and question the concept of frequency-dependent chemical coding of sympathetic transmission in general. The simplest interpretation of our data is that NA and NPY are released at all frequencies from a single pool. The present finding might signify that only large dense-cored vesicles are involved in the sympathetic stimulation-evoked secretion of catecholamines from adrenergic nerve terminals of the isolated sheep spleen.

Chromograam A (CGA) has been localized to the large dense cored vesicles (LDV) of sympathetic neurons SDS-PAGE and lmmunoblotlmg of soluble LDV proteins hem ox and dog adrenergic neuronal cell bodies, unonb and nerve terminals, revealed an increasing number of CGAimmunoreactive forms, consistent with proteolyt~e processing dunng axonal transport. Spleme nerve electrical stimulauon (10 Hz, 2 rain) revealed that, apart from CGA these CGA-proeessmg products are released from the sheep spleen. The secretion of CGA-defived fragments from sympathetic neurons might suggest a role in the regulation of synaptic transmission.

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.

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