Richard L. Klein

Exocytosis from large dense cored vesicles outside the active synaptic zones of terminals within the trigeminal subnucleus caudalis: A possible mechanism for neuropeptide release

It has been hypothesized that chemical interactions between neurons in the central nervous system can occur in the absence of well defined synaptic complexes, but morphological correlates have been difficult to find. The present study demonstrates exocytotic release from large (70-130 nm) dense cored vesicles at structurally nonspecialized areas along the plasmalemma of structurally different categories of terminals and occasionally from dendrites and axons within the neuropil of the trigeminal subnucleus caudalis. In rats, the marginal (lamina I) and substantia gelatinosa (lamina II) layers contain the central terminals of primary afferent fibers from the infraorbital nerve that supply the skin and whiskers (vibrissae). Different types of interneurons are also present and may modify the input being relayed to higher centers. While exocytotic profiles were present in control animals, they increased significantly (P less than 0.01) on the ipsilateral side 1-24 h after a unilateral skin lesion in the vibrissae area. A second increase (P less than 0.001) occurred 14-15 days after the lesion. Virtually all examples of large vesicle exocytosis were observed at structurally nonspecialized sites while those at the active synaptic zones involved small clear vesicles. Substance P-like immunofluorescence, present in controls and on the ipsilateral side during the first 6 days, subsequently declined until 4 weeks after surgery when some recovery was noted. The increase in large vesicle exocytosis and the decrease in substance P are interpreted to reflect functional adjustments of different neurons in response to the lesion. The exocytosis involving large dense cored vesicles may serve to deliver transmitters and/or neuropeptide modulators to appropriate receptors in a wider area than release into a specialized synaptic cleft would allow.

Exocytosis from Neuronal Large Dense-Cored Vesicles

Publisher Summary This chapter provides an overview of exocytosis from neuronal large dense-cored vesicles. Regulated exocytosis operates in cell types ranging from primitive protozoa to highly differentiated endocrine cells and neurons. Membrane-bounded vesicles, predestined for exocytosis, provide protective storage for substances that targets the intracellular metabolizing enzymes. In neurons, the secretory organelles that undergo exocytosis correspond to a variety of storage vesicles based on content. They fall into two general categories based on size: the large dense-cored vesicles (LDVs) measuring > 70 nm in diameter and the small vesicles in the range of 45–55 nm. The latter category, called “synaptic vesicles,” is familiar to cell biologists because of its role in the release of classical transmitter and amino acid transmitters. The aspects of the structure and contents of large and small vesicles are compared as they pertain to exocytosis. The differential recruitment of LDVs and small synaptic vesicles for exocytotic release is also emphasized when supported by ultrastructural correlates. Protein phosphorylation is important for LDV transport to and fusion with the plasmalemma.

Opioid peptides and noradrenaline co-exist in large dense-cored vesicles from sympathetic nerve.

The possibility that opioid peptides and noradrenaline co-exist not only in the desheathed bundle of bovine splenic nerve which contains approximately 98% sympathetic C-fibers, but also in the population of large dense-cored noradrenergic vesicles from these fibers, has been investigated. The primary fraction of large dense-cored vesicles which can be prepared at about 85% purity has been further subjected to density gradient and fractional centrifugation procedures, including D2O-loading and unloading on modified second gradients, in an attempt to separate any minor population of particles which potentially could contain opioid peptides and contaminate the large dense-cored vesicle fraction. Measurement of opioid peptides, noradrenaline, dopamine and dopamine beta-hydroxylase activity supports the conclusion that opioid peptides are stored in the primary population of large dense-cord vesicles per se, rather than in a minor population of contaminating particles from cells other than sympathetic C-fibers. This conclusion has implications for exocytotic release and the physiological role of the opioid peptides intra- and extra-neuronally. Nerve vesicle opioid peptides have a size less than 5000 daltons, in contrast to the high proportion of large peptides containing enkephalin sequences in the bovine adrenal medulla.

Differential distribution of neuropeptides and serotonin in pig adrenal glands

A differential distribution of vasoactive neuropeptides and serotonin in chromaffin cells and nerve fibers within the adrenal glands of the pig (Sus scrofa) was found using immunohistochemical methods. Met- and leu-enkephalins, present at high levels in the medulla (measured by radioimmunoassay), occurred in adrenaline storing cells, some of which contained calcitonin gene-related peptide. Islets of chromaffin cells beneath the capsule also contained enkephalins and calcitonin gene-related peptide. Nerve fibers with enkephalin-like immunoreactivity were sparse, but many varicose fibers in the inner cortex and medulla showed calcitonin gene-related peptide immunofluorescence in a pattern similar to vasoactive intestinal polypeptide. Neuropeptide Y was mainly associated with perivascular fibers and neither neuropeptide Y nor vasoactive intestinal polypeptide immunoreactive chromaffin cells were detected. In contrast to the neuropeptides, most serotonin-like immunoreactivity coincided with noradrenaline histofluorescence. It is concluded that the distribution of nerve fibers with calcitonin gene-related peptide and vasoactive intestinal polypeptide would allow interactions between chromaffin and inner cortical cells. Stimuli activating noradrenaline chromaffin cells could release serotonin while stimulation of adrenaline storage cells would release enkephalin and, to a lesser extent, calcitonin gene-related peptide. Met-enkephalin, which occurs 3 4:1 over leu-enkephalin, is the most likely of the co-released peptides to reach distant receptors via the venous outflow.

Highly purified splenic nerve vesicles: Early post-mortem effects on norepinephrine content and pools

SummaryIsolated large dense-core vesicles from bovine splenic nerve can be prepared at a purity comparable to that of adrenomedullary vesicles. Improved methods coupled with a short post-mortem delay of ∼10 min, from the moment of sacrifice at the slaughterhouse to chilling the nerves, yield vesicles containing 11–12 μg norepinephrine (NE)/mg protein (range 5–18) at 80–90% purity. In the upper range, therefore, the vesicles contain 100 nmol NE/mg protein uncorrected for purity, which is more than an order of magnitude higher than published values for splenic nerves from other laboratories. The extrapolated estimate for contentin vivo is 200 nmol/mg protein.The shorter post-mortem delay has revealed a second more labile pool of NE accounting for 20% of the vesicle NE content, and 50% if extrapolated to zero post-mortem delay. It has a half-life of 4–5 min for net NE loss at 30 °C and is relatively insensitive to the presence of ATP in a medium containing 0.5–1.0 μg 1-NE/ml. This rapid single exponential component is in addition to the well-known slower ATP-sensitive component, which was the only one found in earlier preparations with a 20–30 min post-mortem delay. Characteristics of the latter can be reproduced in the present preparation after an unavoidable slow slaughter, and by several other procedures designed to mimic the additional post-mortem delay.

Quinidine and Unidirectional Cation Fluxes in Atria

The effect of quinidine on unidirectional fluxes of Na and K in isolated rabbit atria has been investigated and correlated with known effects of the drug on the electrical and mechanical properties of the heart. Quinidine caused a rapid inhibition of Na entry, which secondarily depressed K efflux. In addition, the drug depressed membrane permeability to K and caused a marked inhibition of active transport, both of which were relatively slow in onset.

Evidence that most of the dopamine β-hydroxylase is not membrane bound in purified large dense cored noradrenergic vesicles

Abstract The compartmentalization of dopamine β-hydroxylase was studied in a purified fraction of large, dense-cored noradrenergic vesicles from bovine splenic nerve. A correlative biochemical and morphological approach was used to evaluate physical methods designed to produce more efficient vesicle lysis than has been accomplished previously with freeze-thawing, hypo-osmotic shocks or incubation at 37°C, all of which are relatively ineffective. Of the seven purely physical techniques tested, only those which subjected the vesicles to high pressures followed by rapid decompression produced effective lysis, unmasking of latent enzyme activity, and release of enzyme into the soluble phase. Based on results with the French Press, the best estimate for the percentage of dopamine β-hydroxylase which can be released into the soluble phase is 55–70% of the total enzyme activity produced by the addition of 0.05% v/v Triton X-100. This includes two-thirds of the enzyme which was originally in a latent form. The enzyme is considered to occur primarily, if not entirely, in a granular complex in the vesicle matrix. About one-third is thought to be oriented specifically at the inner surface of the vesicle membrane and is active enzymatically. Physical rupture of the vesicles causes redistribution of partially depleted vesicles and free matrix granules containing dopamine β-hydroxylase activity into less dense zones of a sucrose-D 2 O density gradient. After French Press treatment, 30% of the enzyme lost from the heavy vesicle peak is found in the region of the light vesicle peak which is thought to be the equilibrium density of the smaller vesicles characteristic of nerve terminals. The data indicate that much more dopamine β-hydroxylase is potentially available for release from the large, dense-cored vesicles upon nerve stimulation than previously has been believed. This will affect interpretations of exocytotic release from the two vesicle populations in noradrenergic varicosities and will have a bearing on the origin of circulating dopamine β-hydroxylase in response to physiological stress and pharmacological intervention, as well as in neurological diseases.

Ultrastructure of highly purified sympathetic nerve vesicles: Correlation between matrix density and norepinephrine content

Initial glutaraldehyde fixation followed by OsO4 or combined with potassium dichromate or picric acid preserves essentially all 3H-1-norepinephrine (NE) label in isolated sympathetic nerve vesicles (750 ) throughout preparation for electron microscopy. Cacodylate and s-collidine buffers increase the tendency to form contracted dense cores surrounded by a more translucid zone in vesicles, when compared to potassium phosphate buffer which maintains a homogeneous, dense, fine granular vesicle matrix. The typical contracted dense core appearance in nerve vesicles is considered to be an artifact. The loss of sedimentable NE content from vesicles under depleting conditions is roughly paralleled by a concurrent loss of dense staining matrix material. This process is at least partly reversible. The matrix density in vesicles is judged to approximate semi-quantitatively the NE content under the conditions tested, but all staining reactions appear to be indicative of the amount of complexing substance present, rather than of NE per se.

Highly purified splenic nerve vesicles: Early post-mortem effects on ultrastructure

SummaryElectron microscopic analyses of whole pellet depths confirm the claim that large dense-core vesicles (750Å) can be isolated from bovine splenic nerve at a routine purity of 80–90%. After a minimal 10 min post-mortem delay at the slaughterhouse, essentially all vesicles in cold control preparations possess a homogeneous, finely granular, electron-dense matrix. This appearance is maintained after brief incubation with ATP at 30 °C, even though a newly discovered ATP-insensitive norepinephrine (NE) pool (20%) is rapidly and completely lost. Subsequent depletion of the remaining NE (80%) in the slower ATP-sensitive pool is paralleled by proportional decrements in vesicle matrix density.In contrast, cold control vesicles in our earlier preparation with a 20–30 min post-mortem delay appear relatively depleted, but gain electron density after brief incubation with ATP at 30 °C. This effect can be duplicated in the present preparation by several procedures designed to mimic the additional post-mortem delay.Ultrastructural events associated with NE depletion and vesicle degeneration begin with random dense granulation of the original finely granular matrix, followed by aggregation to form very electron-dense 200Å granules. The latter occur intravesicularly at first, but are released in increasing numbers as vesicles swell and rupture. Clusters of the 200Å granules give the appearance of dense cores both intra- and extravesicularly, particularly after certain fixation and staining procedures.

Studies on nuclear amino acid transport and cation content in embryonic myocardium of the chick

Nuclei isolated from 12-day embryonic chick heart are capable of transporting a number of amino acids including arginine, alanine and serine. Dicarboxylic amino acids are not transported. Arginine is concentrated in an intranuclear diffusible pool 17 times that expected from a purely passive equilibrium distribution. There is no competition between stereoisomers nor between unlike amino acids, whether or not they are transported. The nuclei bind Ca ++ (8 mM) and Na + (28 mM) in the absence of added cations. Mg ++ and K + are not tightly bound. Nuclear Na + content reflects that bound plus that in the extranuclear environment; nuclear K + can be concentrated in free form above that in the extranuclear environment. Nuclear cation content is not appreciably affected by the presence of adenosine triphosphate (ATP) and arginine, except that the Mg ++ -ATP chelate can not readily permeate the nuclear membrane. Subcellular distribution of Na + and Ca ++ plus Mg ++ , as revealed by histochemical means, correlates well with direct analytical data. The high nuclear Na + content can account for only about 50 percent of the total bound Na + in the myocardium at 12 days of embryonic age.