Ultrastructural demonstration of exocytosis of neural,neuroendocrine and endocrine secretions with an in vitro tannic acid (TARI-) method

Summary The release of neural, neuroendocrine, and endocrine secretory products by exocytosis was ultrastructurally studied by means of tissue incubation in Ringer containing tannic acid (Tannic Acid Ringer Incubation-method; TARI-method), followed by conventional fixation. Tannic acid strongly enhances the electron density of extracellular (secretory) substances. During TARI-treatment of tissues exocytosis proceeds, but the exteriorized contents of the secretory granules are immediately fixed by tannic acid and do not diffuse away into the extracellular space. In this way detection of exocytosis is markedly facilitated since the number of exocytosis phenomena visible at the ultrastructural level considerably increases with progressing incubation time. Studies of the central nervous system of the mollusc Lymnaea stagnalis show that the occurrence of exocytosis during TARI-treatment is calcium-dependent. With the TARI-method exocytosis has been clearly demonstrated in a variety of structures (endocrine cells, neurohaemal axon terminals, synapses) of L. stagnalis, the insect Locusta migratoria, and the rat, including cell types where exocytotic release had not been shown before.     

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.     

Ultrastructural localization of exocytotic release sites in immunocytochemically characterized cell types

Summary Preembedding visualization of exocytosis by tannic acid treatment and postembedding immunocytochemical identification of cell types were combined to demonstrate the release of secretory products by exocytosis of characterized cell types. Treatment with tannic acid was carried out by perfusion with Ringer containing tannic acid, followed by fixation, dehydration and embedding. For electron microscopical immunocytochemistry protein A-gold was used as marker. In this study, exocytotic release was demonstrated for prolactin by cells in the pars distalis, and for oxytocin by axon terminals in the pars nervosa of the pituitary gland of the rat.     

Synaptic and nonsynaptic release of neuromediators in the central nervous system

Different types of release site were studied ultrastructurally with tannic acid and immunohistochemical techniques in the central nervous system (CNS) of the invertebrate pond snail Lymnaea stagnalis and in two neuromediator rich core regions in the CNS of the rat, viz., the median eminence (ME) and the mesencephalic central grey substance (MCG). In the CNS of the snail, release of the contents of the secretory granules could be clearly demonstrated in (1) neurohaemal axonterminals, (2) synapses and (3) in nonsynaptic release sites: neuronal processes without morphological synaptic specializations. In the ME, release of secretory products by exocytosis was found in neurohaemal axonterminals in the external part of the palisade layer and in nonsynaptic release sites in all other layers of the median eminence. It was found that oxytocine and vasopressin were released by exocytosis into the extracellular space from such (preterminal) nonsynaptic release sites. Serial section analysis revealed three types of fibre in the MCG, viz. (1) varicose fibres that made synaptic contacts with MCG dendrites on every varicosity, (2) fibres with two types of varicosity, viz. synapse-bearing varicosities and varicosities without synaptic specializations, and (3) varicose fibres without any synaptic specializations. It has been discussed that the nonsynaptic release sites in the CNS of the snail Lymnaea stagnalis, and the nonsynaptic varicosities in the rat brain are the morphological correlates of nonsynaptic communication in the CNS. The results further indicate that particular peptidergic neuromediators are released from such nonsynaptic varicosities, and may reach via the extracellular space receptors located at some distance.     

Exocytotic release of secretory granules from endocrine cells in the midgut of insects

Summary Exocytotic release of the secretory granules of the endocrine cells in the midgut of a cockroach, Periplaneta americana, was studied by means of fixation with tannic acid in combination with glutaraldehyde and osmium tetroxide. A sequence of images indicative of exocytosis suggests the following steps in this process: (1) A delicate connection appears between the granule-limiting membrane and the plasma membrane. (2) The plasma membrane approaches the granule, forming a concave indentation. (3) The granule-limiting membrane fuses with the plasma membrane and opens to give rise to an omega profile. (4) The granule content is voided into extracellular space. Exocytosis occurs not only at the base of the cell but occasionally at its side facing adjacent cells. (5) The exocytotic invagination after release becomes smaller and narrower; sometimes a coated pit with bristles appears. Multiple exocytosis, and exocytosis in the endocrine cells of the nidus, i.e., the regenerative cell mass, are also described.     

Arrested exocytosis of synaptic vesicles at a glutamatergic synapse,the insect fast excitatory neuromuscular junction

New ultrastructural evidence supporting the vesicular theory of neurotransmitter release has been obtained using a tannic acid incubation technique. In tannic acid-treated muscles of the locust (Schistocerca gregaria), sites of arrested synaptic vesicle fusion are present at synapses of the fast excitatory neuromuscular junction (NMJ). Tannic acid treatment, prior to aldehyde fixation, permits a degree of normal cell function to be maintained so that exocytosis can continue. This results in large numbers of fused vesicles, found merging fully with the presynaptic membrane throughout the synapses. A possible mechanism of membrane retrieval has also been identified, involving large invaginations from the presynaptic membrane.     

Coated vesicles are implicated in the post-fusion retrieval of the membrane of rat atrial secretory granules

Summary Using an in situ tannic acid perfusion technique, this study presents evidence that the removal of membrane components from the rat atrial secretory granule membrane after granule exocytosis is mediated by coated vesicles. When tannic acid is used to arrest the post-fusion stages of granule release, coated pit formation occurs on granule membrane, which, although continuous with the sarcolemma, is easily recognised by the membrane omega profile and the continued presence of the granule core. Tannic acid perfusion, before aldehyde fixation, allows a degree of continued cell function, and granule fusions can persist after tannic acid has reached the cell. This results in an increase in the numbers of fusion profiles and the appearance of coated pits on granule membrane at these sites. The proportion of granules with coats increases with perfusion time, suggesting that endocytotic, as well exocytotic events, may be arrested by the action of tannic acid. Coated vesicles are also involved at earlier stages of the release pathway. In other types of secretory system this is considered to represent recycling of membrane proteins as part of the maturation process of the granule. Although arrested granules exhibiting this clathrin coat could have had the coat prior to fusion, as part of the maturation process, our results show that it is more likely to represent a second stage of membrane protein recycling; the postfusion reclamation of proteins from the sarcolemma. This facet of the tannic acid perfusion procedure suggests a general method for quantifying coated pit formation during secretory granule release.     

Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannic-acid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural characterization of exocytotic release sites in different layers of the median eminence of the rat

Summary The release of neuronal secretory products by exocytosis in different layers of the median eminence of the rat was investigated ultrastructurally after perfusion with Ringer solution containing tannic acid. Exocytotic images were observed in all layers studied. Neurohaemal release sites were found in the pars externa of the palisade layer, where they occurred not only against the basal lamina of the pericapillary space, but also opposite, adjacent to neuronal and glial elements. In the lateral portion of the pars externa of the palisade layer most release sites were separated from the pericapillary space or the pial surface by ependymal or glial processes. In the pars interna of the palisade layer, and in the reticular, fibre and subependymal layers, release was observed in different types of axonal processes without morphological synaptic specializations. We suggest that products released in the pars externa of the palisade layer are destined to reach the capillaries of the primary portal plexus. Although the non-vascular release sites may serve a similar hormonal function, they may alternatively represent the morphological correlate of axoaxonal contacts or of paracrine, non-synaptic release sites.     

Effect of neuromimetics upon the release of atrial natriuretic peptide granules: Are multiple pathways involved in secretion?

The release of atrial natriuretic peptide (ANP) in response to the application of neurohumoral agonists (neuromimetics) is directly demonstrated and quantified at the cellular level, using an ultrastructural assay developed to quantify secretion. The assay uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial secretory granules in the anesthetized rat. The animal is perfused with the neuromimetic, and secretory granules, which retain the capacity to undergo exocytosis throughout the subsequent 30 min tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different agents to be assessed. The actions of three different agents were investigated: isoproterenol, phenylephrine, and acetylcholine. In previously published studies, investigations of the actions of these agents on ANP release has produced unclear and sometimes contradictory results. Using our ultrastructural assay, it was found that during the 30 min perfusion period neither isoprenaline nor phenylephrine caused a significant change in the rate of secretory granule release, whereas acetylcholine significantly decreased the rate of granule release. A new model of secretion is proposed to integrate these findings with previous results and help clarify the complex picture of atrial natriuretic peptide release. © 1996 Wiley-Liss, Inc.     

A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

Stretch and anesthetic dependency of atrial natriuretic peptide release demonstrated by an ultrastructural assay

Using an ultrastructural assay developed to quantify the secretion of atrial natriuretic peptide-containing granules, release of the hormone, in response to different degrees of atrial distension, is directly demonstrated at the cellular level. The ultrastructural assay developed uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial granules in the anesthetized rat. Secretory granules, which retain the capacity to undergo exocytosis throughout a 30-minute tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane, with the core contents retained. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different stimuli to be assessed. By altering the height of the perfusate, perfusion pressure and hence the degree of distension of the right atrium can be increased, and this causes a proportional rise in the release of secretory granules from individual myocytes. An anesthetic regime incorporating fentanyl citrate was found to increase significantly the rate of granule release, and this was further augmented by atrial distension. Quantification of the numbers of cytoplasmic granules under the same conditions did not reveal a reduction in granules. This is thought to be because only a small pool of granules is recruited for exocytosis, and granule production may continue during the perfusion period. Our assay of atrial secretory granule release allows the effect of a variety of stimulatory and inhibitory agents to be assessed directly at the cellular level and provides an independent comparison with previous biochemical data from whole animal and isolated organ studies. © 1993 Wiley-Liss, Inc.     

Photoperiod-dependent changes in exocytotic activity in the hypophyseal pars tuberalis of the Djungarian hamster,Phodopus sungorus

The pars tuberalis of the hypophysis of the Djungarian hamster, Phodopus sungorus, was investigated with regard to secretory activity by applying the tannic acid-Ringer perfusion technique. Two groups were maintained under long photoperiods (16 h light: 8 h dark) or short photoperiods (8 h light: 16 h dark), respectively. Perfusion with tannic acid showed that specific pars tuberalis cells release some of their secretory granules as indicated by typical exocytotic figures. The percentage of cells displaying exocytotic activity was significantly higher in the pars tuberalis of hamsters kept under long photoperiods. The number of exocytotic figures per single cell was not increased. These results provide further evidence for a secretory activity of the pars tuberalis and support the hypothesis of its involvement as a mediator between photoperiodic stimuli and the endocrine system.     

The release of secretory vesicle in encysting Giardia lamblia

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. It secretes an extracellular cyst wall using a poorly understood process. An encystation-specific secretory vesicle (ESV) was previously described containing cyst wall proteins. The process of release of these vesicles has been suggested to occur after fragmentation of large ESV in small secretory vesicles, followed by exocytosis, but it was not demonstrated. The release of the ESV was studied by transmission electron microscopy. It was observed: (1) the moment of vesicle release; (2) that a large vesicle is exocytosed and does not fragment into small vesicles; (3) membrane fusion is distinct from traditional exocytosis since it is incomplete; (4) the occurrence of membrane fragmentation and that those membranes reseal to form ghosts; (5) these membrane ghosts may be endocytosed, adhered to flagellar surface or/and form empty vesicles in the extracellular medium.     

Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte

Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC₅₀) of 0.14 μM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARγ during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity.     

Investigations of the Diurnal Activity of the Endocrine Dorsal bodies in Helix aspersa

Summary

The activity of the endocrine dorsal bodies (DB) of the adult land snail Helix aspersa living in the field shows striking fluctuations during a 24 hr cycle. Quantitative electron microscopical data revealed that the number of secretory granules, the volume of the Golgi apparatus and the number of Golgi saccules containing electron-dense material were maximal at 1 am and minimal at 1 pm. The use of tannic acid indicated the exocytosis of secretory material was intense around 1 pm and only moderate at 1 am. The results suggest that, under natural conditions, the DB have a diurnal rhythm of activity, packaging secretory material into secretory granules mainly during the night and releasing it during the afternoon.     

Effect of infection with Trichobilharzia ocellata and Schistosoma mansoni on the ultrastructure of the albumen gland of their respective hosts, Lymnaea stagnalis and Biomphalaria glabrata

The albumen gland, a female accessory sex gland of pulmonate snails, produces the perivitelline fluid. The ultrastructure of the albumen glands of control and infected specimens of Lymnaea stagnalis and Biomphalaria glabrata was studied. The albumen gland of L. stagnalis contains two types of secretory cells--light (active) and dark (inactive)--and two types of supporting cells--centroacinar and myoepithelial. The secretory cells apparently represent two activity stages of one type of cell. The gland B. glabrata possesses only one secretory cell type, which alternates with one type of supporting cell. The albumen glands of L. stagnalis and B. glabrata infected at a juvenile stage were studied 4 and 14 weeks (L. stagnalis) and 4 and 9 weeks (B. glabrata) after exposure. After four weeks' infection, B. glabrata produced some egg masses, but in subsequent stages egg mass production completely coased. Infected L. stagnalis never produced eggs. B. glabrata was apparently infected at a "physiologically" more mature stage than L. stagnalis. The morphology of the albumen glands four weeks after exposure (the daughter sporocyst stage) is in agreement with this hypothesis. At this interval the secretory cells of L. stagnalis appeared to be much more severely affected (inactive Golgi bodies and rough endoplasmic reticulum, crinophagy of the secretory granules) than the cells of B. glabrata. In the later stages studied (shedding of the cercariae), the glands of both species appeared to be completely inactive (reduced height of the epithelium, inactive organelles, crinophagy, absence of secretory granules). At this stage of infection, daughter sporocysts containing cercaria embryos were seen in the connective tissue of the albumen gland of B. glabrata, but not of L. stagnalis. The results thus indicate that the development and synthetic activity of the albumen gland are seriously affected by infection. These processes are known to be under the endocrine control of the female gonadotrophic hormones. Since it has been established that these hormones are normally present in the haemolymph of infected snails, the findings can be explained by assuming that the parasite interferes in some way or other with the snail's endocrine system.     

Tannic acid in histology: an historical perspective

The development of tannic acid as a reagent in histological methods is traced against a background of widespread use in science and technology from times of antiquity. Numerous light microscopic methods involving tannic acid, particularly in conjunction with iron and silver, have been described for a variety of tissue components. In most applications, tannic acid functions as a mordant. Current use is generally restricted to methods based on its affinity for collagen. The most significant histological use of tannic acid in contemporary times is as an adjunct to conventional glutaraldehyde-osmium-heavy metal fixation and staining for ultrastructural studies of tissue structures not normally clearly demonstrated. Tannic acid reacts with various components by mechanisms which are often not fully understood.     

Some unusual staining properties of tannic acid in plants

Summary Maize root tips were fixed in glutaraldehyde fixatives containing tannic acid and then processed for electron microscopy. Under these conditions, tannic acid selectively stained the contents of the Golgi apparatus secretory vesicles of some outer root cap cells, the cell walls of all cells, and substances in, and adjacent to, intercellular connections of mature primary walls and of secondary walls. Intercellular connections of the young primary walls were not stained. Plasma membranes, and substances associated with the outer leaflets of the plasma membranes, were also stained. Tannic acid-positive material was associated with the cell plate vesicles of forming walls but very little, or none, was associated with the Golgi apparatus vesicles of dividing cells.