Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A,B, C,D and E: Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of β-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 2 μM cytochalasin A, C >μM cytochalasin B ? 4–5 μM cytochalasin D, E. While maximal rates of O₂^? release and extents of exocytosis require extracellular calcium (1–2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na⁺, K⁺ or choline inhibited either cytochalasin B- or E-stimulated O₂^? production with IC₅₀ values of 5–10 mM and inhibition occurs whether Cl^?, NO₃^? or SCN^? is the anion added with Na⁺ or K⁺. Release of β-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl (IC₅₀ ≈ 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K⁺ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of β-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O₂^? or β-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

Calcium-Dependent and-Independent Release of Glutamate from Synaptosomes Monitored by Continuous Fluorometry

An enzyme-linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea-pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35-0.39 nmol min-1 mg of protein-1 and is not significantly affected by external Ca2+. KCl depolarization (30 mMKCl) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein-1. The final extent of Ca2+-dependent release amounts to 1.9 nmol mg of protein-1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30 degrees C for 2 h before depolarization leads to a decrease in Ca2+-independent release and an increase in Ca2+-dependent release, consistent with an intrasynaptosomal relocation of the amino acid.     

Nerve-induced secretion of parotid acinar granules in cats

Summary Electron microscopy of cat parotid glands revealed great heterogeneity in the secretory granules of normal unstimulated acinar cells. Electrical stimulation of the parasympathetic nerve to the gland evoked a copious flow of parotid saliva which was accompanied by an extensive depletion of the secretory granules from the acinar cells. Exocytosis was captured as it was occurring by means of perfusion-fixation, and showed that the events occur in a conventional manner. Stimulation of the sympathetic nerve caused only a very small flow of saliva, and no acinar degranulation was detected. It can be concluded that the parasympathetic secretomotor axons provide the main drive for parotid acinar degranulation in the cat. This contrasts with the rat in which sympathetic impulses provide the main stimulus for parotid acinar degranulation. These dissimilarities serve to emphasise how extensively species differences may influence autonomic responses in salivary glands.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Occurrence of a Large Ca2+-Independent Release of Glutamate During Anoxia in Isolated Nerve Terminals (Synaptosomes)

Isolated rat cerebral cortical synaptosomes made anoxic by addition of cyanide developed an inhibition of the Ca2+-dependent release of glutamate 2 min after the addition of the metabolic inhibitor when the intrasynaptosomal ATP/ADP ratio decreased below 1.7. In contrast, cyanide induced a continuous efflux of glutamate through a Ca2+-independent pathway that accounted for the release of 25% of total intrasynaptosomal glutamate in 5 min. The results suggest that a Ca2+-independent release of glutamate could be implicated in the neurotoxic action of this amino acid during anoxia.     

Neurosecretion of the mediodorsal cells of the central nervous system of the snail Helisoma duryi

Summary The neurosecretory mediodorsal cells that produce a putative growth hormone of the snail Helisoma duryi were studied in fast-growing virgin snails and in slow-growing reproducing snails. There are about 60 mediodorsal cells in clusters on each side of the cerebral commissure of the central nervous system, and they contain dense-cored granules which are 100–200 nm in diameter. The cells of virgin snails have dense Golgi bodies, scattered ER cisternae, and few granules, while those of reproducing snails have pale Golgi bodies, stacked ER cisternae, and numerous granules. Thus the mediodorsal cells of the virgin snails appear to be more active synthetically than those of the reproducing snails. The cells near the endocrine dorsal bodies contain many dorsal body precesses in their membrane interdigitations. There are junction-like interactions between some of the interdigitations. Gap junction-like contacts are seen between mediodorsal cells and glial cells. The axon endings of the mediodorsal cells at the neurohemal area in the labial nerve show more release profiles in virgin snails than in reproducing snails. A daily pattern of release has been observed in reproducing snails, and rates of release are higher in the evening than in the morning.     

Ultrastructural visualization of exocytosis in the pig pineal gland

Summary The pinealocytes of the pig contain conspicuous dense bodies, the nature and role of which are not yet fully elucidated. The aim of this study was to demonstrate whether or not these structures are involved in the secretion process. The tannic acid-Ringer incubation (TARI)-method, which allows a clear-cut ultrastructural study of secretory discharge by exocytosis, has been used. The results indicate that pig pinealocytes release the content of the dense bodies with an amorphous inner structure into the extracellular space via exocytosis and that this secretion is quantitatively important. The secreted material is proteinaceous in nature; this indicates that polypeptides are released by the pineal.     

Colocalization of α-lactalbumin and a major casein in secretory vesicles of rat mammary epithelial cells

Summary Whether both casein and noncasein (serum or whey) proteins of milk are contained within the same secretory vesicles of milk secreting mammary epithelial cells was explored. Antibodies to a major casein and to -lactalbumin of rat milk were localized in thin sections with colloidal gold-conjugated second antibodies. Antibodies to the casein component bound to an antigen present within lumina of Golgi apparatus cisternae and within secretory vesicles. This antigen was also recognized in structures within secretory vesicles and within alveolar lumina which were ultrastructurally identified as casein micelles. Antigens recognized by antibodies to -lactalbumin also were present in Golgi apparatus cisternae and within secretory vesicles. Both anti-casein and anti--lactalbumin antibodies recognized antigens within the same secretory vesicles. These observations show that one major noncasein protein of rat's milk is present in casein-containing secretory vesicles.     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA