Amine-producing endocrine-like cells in the epithelium of urethra and prostate of the guinea-pig a chemical,fluorescence histochemical,and electron microscopic study

Summary The urethra and prostate of the guinea-pig contain at least two types of endocrine-like cells in the epithelium. The predominant type is argentaffin and stores 5-hydroxytryptamine. Treatment with reserpine or a dopa decarboxylase inhibitor markedly reduces the 5-hydroxytryptamine content of this cell type. The other less numerous cell type, which is argyrophil but not argentaffin, is devoid of 5-hydroxytryptamine but can be induced to store dopamine if supplied with dopa. Both cell types occur disseminated in the urethral epithelium, whilst only the argyrophyl, non-argentaffin cell type devoid of 5-hydroxytryptamine is found in the prostate. At the ultrastructural level the argentaffin cell type contains numerous electron-dense cytoplasmic 800–1000 Å granules. These granules are argentaffin, suggesting that they are the storage site for 5-hydroxytryptamine. The cells sometimes reach the urethral lumen via a narrow neck, the apex being endowed with microvilli. This arrangement suggests that the cells are capable of responding to stimuli in the urethral lumen. Preliminary attempts to test the effect of depriving or loading guinea-pigs with water failed to induced changes in the 5-hydroxytryptamine content of the urethral endocrine-like cells.     

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections

Summary The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.     

Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of reserpine treated guinea pigs

Summary The individual granules of enterochromaffin cells of normal and reserpine treated guinea pigs have been studied by staining slides of the duodenum first by an argentaffin method and subsequently by an argyrophile method. Some argentaffin cells can be shown to contain not only argentaffin granules, but also granules that are purely argyrophile. The relative number of such argentaffin cells is greatly increased following administration of reserpine, as depletion of their 5-hydroxytryptamine content converts argentaffin granules into purely argyrophile ones. On the basis of this finding it is confirmed that the argyrophile granule is merely an argentaffin granule depleted of its 5-HT content, and that the argyrophile (nonargentaffin) and the argentaffin cells represent different phases of a secretory cycle.This investigation was supported by a grant from the Indian Council of Medical Research. I am grateful to Ms. Ciba of India Ltd. for making available reserpine (Serpasil) and solvent for reserpine. It is a pleasure to thank Mr. Anand Parkash for technical assistance and Mr. M. L. Sharma for the photographs.     

Endocrine cells of the human gastric mucosa

Summary By light and electron microscopy investigation of the human gastric mucosa five types of ultrastructurally different endocrine cells have been detected: 5-hydroxytryptamine storing enterochromaffin (EC) cells, gastrin storing G cells, and functionally undefined ECL, D and D₁ cells. By direct application of Masson's argentaffin reaction as well as of Sevier-Munger's and Grimelius' argyrophil method to electron microscopy specimens, selective deposition of silver grains upon the endocrine granules of such cells was obtained. In particular, only EC cell granules reacted to the argentaffin method, granules of both EC and ECL cells heavily reacted to Sevier-Munger's technique, granules of EC, ECL, G and D₁ cells reacted to Grimelius' technique, while D cell granules failed to react either to argentaffin or argyrophil methods. By the application of the same silver methods to paraffin sections as well as by other selective staining methods for endocrine granules (5-hydroxytryptamine techniques, lead-haematoxylin, HCl-basic dye method), at least four of the above cell types were also identified under light microscope. This opens the way for extensive studies of such cells in conventional histologie specimens.This investigation was supported in part by grant N.70.01022.04 from the Italian Consiglio Nazionale delle Ricerche.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.     

Exocrine pancreas under experimental conditions

Summary At least four types of endocrine-like cells have been detected histochemically in the mucosa of the human colon and rectum, i.e. argentaffin cells storing 5-hydroxytryptamine (5 HT) and non-argentaffin cells reacting with glucagon, somatostatin and bovine pancreatic peptide (BPP) antibodies. Ultrastructurally, four main types and three rare types of endocrine-like cells have been identified. Among the former cells were: (1) argentaffin EC₁ cells, known to store 5 HT and substance P, (2) poorly argyrophil L cells, corresponding to the glucagon-immunoreactive cells storing enteroglucagon or glucagon-like immunoreactivity (GL1), (3) inconstantly argyrophil F-like cells, possibly corresponding to BPP-immunoreactive cells, and (4) fairly argyrophil H cells of unknown function. Rare D cells, corresponding to somatostatin cells, N cells, corresponding to neurotensin cells, and P cells, of unknown function, have been also found.Supported in part by the Italian National Research Council (Grant N. 76.01558.04)     

Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Summary Certain disseminated endocrine-like cells have reviously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (paraxons). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.This work was reported in part at the 29th Congress of the International Union of Physiological Sciences, Sydney, Australia, August 28–September 3, 1983     

Histological, histochemical and ultrastructural studies on the interrenal and chromaffin cells of the fathead minnow, Pimephales promelas Rafmesque

Light and electron microscopic examination of fathead minnow head kidneys revealed that the interrenal and chromaffin cells were intermingled and always closely associated with the cardinal veins and their tributaries. Histochemical tests for lipids in the interrenal cells were positive, and two types of chromaffin cells were indicated by chromaffin reactions. Interrenal cells contained abundant smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Only one interrenal cell type was found. Two types of chromaffin cells were present with differences in cytoplasmic density and in types of granules. In light cells, adrenaline granules were most common, and in dark cells noradrenaline granules predominated.     

Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of the guinea pig

Summary Successive staining of cells of the enterochromaffin system of the guinea pig by an argentaffin and an argyrophile method shows that, as in man and rabbit, they can be divided into (a) those in which all granules are apparently argentaffin, (b) those in which all granules are argyrophile but none are argentaffin, and (c) those in which some granules are argentaffin (as well as argyrophile) while others are purely argyrophile. The presence of the third type makes it evident that no hard and fast line of distinction can be drawn between the so called argentaffin and argyrophile cells.I am grateful to Mr. Mohan Lal Sharma for help with the photographs and to Mr. Anand Parkash for technical assistance.     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland.

The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80-160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150-400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogeneous, some are lysosomes and the other are possibly the granules responsible for the secretion of pineal peptides.     

Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Certain disseminated endocrine-like cells have previously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (" paraxons "). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland

Summary The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80–160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150–400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogenous, some are lysosomes and the others are possibly the granules responsible for the secretion of pineal peptides.Supported in part by a grant from the National Science Council, Republic of ChinaDedicated to Professor Doctor Huoyao Wei on the occasion of his 70th birthday     

Ultrastructural similarity of endocrine-like cells of the human lung and some related cells of the gut

Summary The ultrastructure of endocrine-like cells of the human lung was compared to the ultrastructure of endocrine-like cells of the stomach and pancreas in both adult and foetal material.Three types of endocrine-like cells were found in the human foetal lung. Type 1 or P₁ cells contained very small granules (about 110 nm) of two varieties, cored and vesicular; type 2 or P₂ cells with cored granules measuring about 130 nm; and type 3 cells with cored granules of about 180–190 nm. In the adult lung only one type P_a cells with cored granules could be found.Cells resembling foetal P₁ cells were not found in foetal or adult gastric mucosa, or in the pancreas. In the gastric mucosa cells resembling pulmonary P_a or P₂ cells were moderately represented and often difficult to distinguish from each other. Thus, they were grouped together as gastric P cells. Cells with granules resembling those of pulmonary type 3 cells were found most numerous in the adult oxyntic mucosa. Cells resembling gastric P cells (and pulmonary P₂ cells) were rather numerous in foetal pancreas, but very rare in adult pancreas. Few cells containing granules somewhat resembling those of pulmonary type 3 cells were present in both foetal and adult pancreas.The results were discussed in respect to 1) the similarities between some gastric or pancreatic carcinoids and lung carcinoids, 2) the gastro-pancreatic P cells as a separate cell population, 3) the possible secretion by the lung endocrine-like cells of active substances, either amines or peptides, 4) the similarity between the secretory granules of P_a and P₁ cells and neurosecretory granules of the hypothalamus and between P₂ cells and some endocrine cells of the pituitary.Supported in part by the Italian National Research Council (Grants N. 75.00630.04 and N. 76.01558.04)     

Annual cycle of the chromaffin cells of Triturus cristatus

Morphology of the chromaffin cells of Triturus cristatus during a complete annual cycle has been investigated. General ultrastructural characteristics are similar for all chromaffin cells, including numerous small mitochondria, well-developed Golgi apparatus and rough endoplasmic reticulum with short cisternae. The primary difference among cells is the type of the chromaffin granules they posses. These are of two kinds: adrenalin (A) and noradrenalin granules (NA). Both types are simultaneously present in the chromaffin cells but with different ratios during the year. During December–January and May–August, NA granules largely prevail, while in September–November and February–April, A and NA granules are present in about equal quantities. The total quantity of catecholamine granules, however, is relatively constant throughout the year. These findings suggest that T. cristatus has a single type of chromaffin cell, the granule content of which varies according to different functional states. The catecholamines are apparently discharged by exocytosis.     

Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat

Summary The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80–120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eighth postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed.By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining within the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative.It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.Supported by grants from the Deutsche Forschungsgemeinschaft     

Morphology and histochemistry of the adrenal medulla. I. Various types of primary catecholamine-storing cells in the mouse adrenal medulla.

Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections. Four types of primary catecholamine-storing cells were identified. NA1 cells contain cytoplasmic granules up to 0.3 mum in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA2 cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA3 cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA4 cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence. The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA1 cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA2 cells correspond to this latter population (175 nm). NA3 cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA4 cells being in the dimension of 80 nm. Granules in NA1 and NA2 cells show uniformly high density, whereas those in NA3 and NA4 cells display cores of varying density. Granules with moderately dense cores in NA3 and NA4 cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.     

Docking of chromaffin granules—A necessary step in exocytosis?

Putative docking of secretory vesicles comprising recognition of and attachment to future fusion sites in the plasma membrane has been investigated in chromaffin cells of the bovine adrenal medulla and in rat phaeochromocytoma (PC 12) cells. Upon permeabilization with digitonin, secretion can be stimulated in both cell types by indreasing the free Ca²⁺-concentration to M levels. Secretory activity can be elicited up to 1 hr after starting permeabilization and despite the loss of soluble cytoplasmic components indicating a stable attachment of granules to the plasma membrane awaiting the trigger for fusion. Docked granules can be observed in the electron microscope in permeabilized PC 12 cells which contain a large proportion of their granules aligned underneath the plasma membrane. The population of putatively docked granules in chromaffin cells cannot be as readily discerned due to the dispersal of granules throughout the cytoplasm. Further experiments comparing PC 12 and chromaffin cells suggest that active docking but not transport of granules can still be performed by permeabilized cells in the presence of Ca²⁺: a short (2 min) pulse of Ca²⁺ in PC 12 cells leads to the secretion of almost all releasable hormone over a 15 min observation period whereas, in chromaffin cells, with only a small proportion of granules docked, withdrawal of Ca²⁺ leads to an immediate halt in secretion. Transport of chromaffin granules from the Golgi to the plasma membrane docking sites seems to depend on a mechanism sensitive to permeabilization. This is shown by the difference in the amount of hormone released from the two permeabilized cell types, reflecting the contrast in the proportion of granules docked to the plasma membrane in PC 12 or chromaffin cells. Neither docking nor the docked state are influenced by cytochalasine B or colchicine. The permeabilized cell system is a valuable technique for thein vitro study of interaction between secretory vesicles and their target membrane.     

Chromaffin cells and interrenal tissue in the head kidney of the grouper,Epinephilus tauvina (Teleostei,Serranidae): a morphological (optical and ultrastructural) study

This work analyses the distribution, histology and ultrastructure of chromaffin cells (CCs) and interrenal tissue (It) in the head kidney of Epinephilus tauvina. Histological examination revealed that chromaffin cells are found in small groups under the endothelium of the posterior cardinal vein (PCV) and are mostly closely associated with the interrenal tissue. Ultrastructure examination confirmed the existence of two main chromaffin cell types, distinguished by different types of secretory granules. The first type was characterized by the presence of vesicles with round, strongly electron dense core granules, which were eccentrically located. Such cells were interpreted as being noradrenaline cells. Meanwhile, cells with vesicles that were completely electron lucent or that contained small less dense eccentric granules were identified as adrenaline cells. Nerve endings were invaginated into the chromaffin cells through synaptic junctions. Interrenal tissue consisted of nests, cords, or strands of cells in contact with the posterior cardinal vein (PCV) and interposed with haematopoietic tissue. Ultrastructure analysis revealed only one interrenal cell type, which contained abundant smooth endoplasmic reticulum (sER) and numerous mitochondria with tubulo‐vesicular cristae, characteristics of steroid‐producing cells. The interrenal tissue cells have different cytological aspects that can be linked to a steroidogenic cell cycle allowing a periodical renewal of organelles.     

Fine structure of argyrophil and argentaffin cells in the gastro-intestinal tract of the fowl

Summary The fine structure of granular cells in the proventriculus gizzard and intestine of the fowl has been described. The proventriculus and gizzard have only one type of granular cell, the pure argyrophil cell. In the intestine two cell types are shown. One corresponds to the argyrophil cell seen in the proventriculus and gizzard. The other, more common, is considered to be the argentaffin cell of the intestine. Structural differences between these two cell types confirm the known differences in histochemical reaction. Acknowledgement. I wish to thank Mr. R. N. C. Aitken, Department of Veterinary Histology and Embryology for supplying the birds used in this study.