Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study

Summary The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 m in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled. From these findings, it is suggested that the granules of rat Clara cells consist of two types of granules of distinct origin; one appears to derive from condensing vacuoles of Golgi origin, whereas the other may be formed by membranefusions with small cytoplasmic vesicles of unknown source.     

An immunologic study of the secretory products of rat Clara cells

Lungs of adult rats were lavaged with normal saline containing 0.25 mM phenylmethylsulfonyl fluoride. The surfactant pellet was removed by centrifugation and serum proteins in the lavage were removed by affinity chromatography using rabbit anti-rat whole serum antiserum. The residual proteins, thought to represent products of secretory cells, were used as the immunogen to inject rabbits. The resulting antiserum was absorbed with affinity columns of rat serum and rat liver extract. The gamma globulin fraction of the unbound antiserum was found to react with two proteins in the lavage by immunodiffusion and crossed immunoelectrophoresis. The antiserum specifically stained, by the immunoperoxidase method, a subpopulation of cells consistent in morphology with Clara cells lining the bronchioles and bronchi. The antigens were detectable, by immunohistochemistry, in rat fetus at 19 days of gestation, a progressive increase in the antigen content was noted with increasing gestational age and an adult pattern was noted at 2 weeks of age. In adult animals the intracellular content of the antigens appears to be about twofold greater than their content in the lavage fluid.     

Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat

In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.     

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.     

Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

Chromogranin A: immunocytochemical localization in secretory granules of frog atrial cardiomyocytes

Chromogranin A (CgA) is a member of the granin family of acidic proteins that present in the secretory granules (SGs) of many endocrine, neuroendocrine and neuronal cells. Atrial natriuretic peptide (ANP)-storing SGs in atrial cardiomyocytes of rat heart also contain CgA. Cardiosuppressive effect of CgA-derived peptides (vasostatins) on in vitro isolated and perfused working frog and rat hearts has been shown under both basal conditions and beta-adrenergic stimulation. More recently it has been revealed that rat heart produces and processes CgA-derived vasostatin-containing peptides. Until now nothing has been known about the presence of CgA in an amphibian heart. We have investigated the subcellular localization of CgA in atrial myocytes of adult frog Rana temporaria heart using ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP has shown that out of three morphologically different types (A, B and D) of specific cytoplasmic granules (SCGs) present in myocytes only two (A and B)--large (120-200 nm in diameter) granules with more and with less electron dense core--exhibit immunoreactivity (IR) to these two antigens. The third type (D) of granules (80-100 nm in diameter) are small membrane bound granules characterized by highly electron dense core surrounded with a thin halo. These granules revealed negative reaction on immunostaining for both CgA and ANP. The presence of CgA- and ANP-IR in the same SCGs in frog atrial myocytes is consistent with the endocrine nature of these granules. Taking into account our and literature data we propose that CgA present in frog atrial cardiomyocite SCGs might be a precursor of vasostatin-containing peptides, as it takes place in rat heart. It is possible that these CgA-derived peptides together with ANP exert their regulatory function through the autocrine and/or paracrine mechanisms and play important cardioprotective role in frog heart under stress condition.     

Oxytocin in Leydig cells: an immunocytochemical study of Percoll-purified cells from rat testes

Summary Testicular interstitial cells, from rats aged 35 days, were dispersed with collagenase and separated through Percoll into 5 fractions (I–V); fraction I being the least dense. Measurement of basal testosterone production, histo-enzymological staining for 3-hydroxysteroid dehydrogenase activity and electron microscopy indicated that the majority of Ley dig cells were found in fraction IV (corresponding to a density of 1.076–1.097 g/ml). In addition, cells from this fraction responded to hCG treatment in a dose-dependent manner on day 0 and remained responsive after being cultured for 1 day. Immunostaining for oxytocin indicated that this fraction also contained the majority of the oxytocin-immunoreactive cells. On day 1 of culture, 56% of the cell population from fraction IV were positively stained for the steroidogenic enzyme and 75% immunoreactive for oxytocin. This overlap indicates that the Leydig cells were also the oxytocin immunoreactive cells.     

5-Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma.

Mechanisms of transport of 5-hydroxytryptamine in the pancreatic B-cell were investigated by using cell suspensions and secretory granules prepared from a transplantable rat insulinoma. (1) Cells incubated with 5-hydroxy[G-3H]tryptamine at concentrations ranging from 0.1 microM to 5 mM accumulated the radioisotope principally by a simple diffusion process. The incorporated radioactivity was recovered principally as the parent molecule and was recovered predominantly in soluble protein and secretory-granule fractions prepared from the tissue. (2) Isolated granules incubated in buffered iso-osmotic medium without ATP accumulated the amine to concentrations up to 38-fold that of the medium. This process was insensitive to reserpine and occurred over a wide range of 5-hydroxytryptamine concentrations (0.075 microM-25 mM). Above 5 mM, 5-hydroxytryptamine accumulation decreased in parallel with the breakdown of the delta pH across the granule membrane. Uptake was favoured by alkaline media and was reduced by the addition of (NH4)2SO4. In both cases a close correlation was observed between uptake and the transmembrane delta pH, a finding that suggested that 5-hydroxytryptamine permeated the membrane as the free base and equilibrated across the membrane with the delta pH. Binding of 5-hydroxytryptamine to granule constituents also played a part in this process. ATP caused a further doubling of granule 5-hydroxytryptamine uptake by a process that was sensitive to reserpine (0.5 microM). Inhibitor studies suggested that amine transport in this instance was linked to the activity of the granule membrane proton-translocating ATPase. (3) It was concluded that the uptake of amines driven by proton gradients across the insulin-granule membrane could account for the accumulation in vivo of amines in the B-cell.     

Morphological and functional heterogeneity of Zajdela rat ascites hepatoma cells

This work studies the mechanisms of dysdifferentiation at cell neoplastic transformation based on the example of heterogeneity of the cell populations that form malignant tumors. Two natural fractions of Zajdela rat hepatoma cells are revealed that differ in the type of growth in the primary culture. Cells of one fraction are attached to substrate and are growing in monolayer (S-fraction), whereas cells of the other fraction are floating in the culture medium (F-fraction). Using the method of supravital observation of the primary culture cells (of 1–2 passages) at the limit of resolution of DIC microscopy, it has been established that both fractions contain cells of several types. Some of these cells are specific to one of the fractions and others are present in both fractions, but with different frequencies. Using the same method, it has been shown that, at the long-term separate cultivation of the fractions in vitro (more than 50 passages), both the cell composition and the initial ratio of cells of different types are changed in both of them. According to the data of flow DNA cytometry, cells of both fractions are hypotetraploid and have insignificant differences in the amount of DNA. After adaptation to conditions of cultivation in vitro, S-fraction cells have been found to have elevated proliferative activity compared to the cells of F-fractions; after long cultivation, the fractions already differ significantly (2.3 times) by this criterion. The content of the cell surface laminin, a marker of hepatocellular carcinomas, is higher on cells of the F-fraction than on those of the S-fraction. The interfraction differences are confirmed by immunologic estimations of the resistance of hepatoma cells to lyses of natural killer cells; cells of the S-fraction of the primary culture are 2.4 times more sensitive than cells of the F-fraction, while, after long-term cultivation, cells of the F-fraction become almost resistant to the cytotoxic action of natural killer cells. Based on the obtained data, the most probable pathways of the dysdifferentiation of rat hepatocytes upon the establishment of Zajdela hepatoma and at the long-term cultivation of cells of this tumor in vitro are discussed.     

Morphological and functional heterogeneity of rat ascitic hepatoma Zajdela cells

This work is devoted to the study of molecular and cellular mechanisms of disdifferentiation during neoplastic transformation of cells by investigating the malignant tumor cell heterogeneity. We have revealed two cell fractions of hepatoma Zajdela which differ in patterns of growth in primary culture. The cells of one fraction were attached to the culture plastic and grew in a monolayer (S-fraction), whereas cells of another fraction floated in the culture medium (F-fraction). Using method of lifetime supervision of primary culture cells (1-2 passages) at the limit of the resolving power of DIC-microscopy it has been revealed, that both fractions contain cells of several types. Some of them were specific for one of the fractions, and others were found in both fractions, but their frequencies differed. It has been shown by the same method, that long separate cultivation of these fractions in vitro (more than 50 passage) change both cellular structure and the initial ratio of different types of cells in both fractions. According to DNA flow cytometry, the cells of both fractions were hypotetraploid and had insignificant differences in DNA contents. After adaptation to in vitro conditions, S-fraction cells raised their proliferative activity in comparison with the F-fraction cells, and after long cultivation showed 2.3 times higher DNA content. Greater amount of cell surface laminin, a hepatocellular carcinoma marker, was observed on F-fraction cells than on S-fraction cells. Interfractional distinctions were confirmed also by immunologic assessment of hepatoma cells resistance to natural killer lyses: the sensitivity of S-fraction cells in primary culture was 2.4 times higher than F-fraction cells sensitivity, and, after long cultivation, F-fraction cells became practically resistant to cytotoxic action of natural killers. Based on the data obtained, the most probable paths of cell disdifferentiation during hepatoma Zajdela formation and during long cultivation of this tumor cells in vitro are discussed.     

Topology of chromogranins in secretory granules of endocrine cells.

Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra-granular topology are of special interest with respect to their putative functions. Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities. Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide- and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules.     

Modification of low density lipoproteins by secretory granules of rat serosal mast cells

When low density lipoprotein (LDL) is incubated with granules isolated from rat serosal mast cells, a fraction of LDL is bound to the granule heparin proteoglycan. If incubation is continued at 37 degrees C, the bound LDL, but not the unbound LDL, is degraded by granule neutral proteases. In the early stage of incubation, all the granule-bound LDL can be released by 0.3 M NaCl (the "salt-sensitive" fraction of LDL). With time, an increasing proportion of the granule-bound LDL requires 0.5 M NaCl for release (the "salt-resistant" fraction of LDL). Chemical analysis showed that, on average, 20% of the apolipoprotein B LDL was lost from the salt-sensitive fraction and 60% from the salt-resistant fraction, without any change in the composition of the lipid portion. Electron microscopic analysis disclosed large fused particles of LDL (diameters up to 100 nm) in the highly proteolyzed salt-resistant fraction, but no fused particles could be found in the less proteolyzed salt-sensitive fraction. We conclude that both binding and extensive degradation of LDL by mast cell granules is required for fusion of LDL particles on the granule surface. As compared with native LDL, the mast cell granule-modified LDL particles exhibit (i) increased particle size, (ii) selective loss of protein (apoB), (iii) a decrease in hydrated density, and (iv) stronger ionic interaction between apoB and heparin proteoglycan. The particles resemble the extracellular lipid droplets found in atherosclerotic lesions of both man and animals. Modification of LDL by mast cells may therefore provide a model of how these lipid structures are formed.     

Semi-automatic morphometric analysis of secretory granules in the cells of the rat adenohypophysis

The authors carried out a morphometric analysis--using a semi-automatic system for image analysis--of the secretory granules in the cells of the Wistar rat adenohypophysis. They evaluated the mean diameter (CIRCLE D), maximum diameter (MAX D), the factor of circular form (PE FORM) and the harmonic factor of form (HAR FORM). They also computed the coefficient of correlation between CIRCLE D and PE FORM and the percentual distribution of the values according to mean diameter.     

Formation and exocytosis of secretory granules in the endocrine cells of normal and transplanted pancreas: an electromicroscopic study

The mechanism of secretory granule formation and exocytosis in the endocrine cells of normal and transplanted rat pancreas was studied using electron microscopy. On the one hand, formation of secretory granules starts with the dilatation of the 2 ends or the vesicularization of the middle parts of rough endoplasmatic reticulum (RER). On the other hand, prohormone ribosomes condense into the vesicles of the GOLGI apparatus. This probably indicates that the GOLGI complex is not the only source of formation of secretory granules. Exocytosis occurs with the formation of an electron dense streak between the perigranular membrane and the apical cell membrane. This is followed by the rupture of the streak at this midpoint allowing the granule to extrude into the space between the cell membrane and the parenchymal basal membrane. This fusion-rupture-extrusion mechanism repeats itself at the parenchymal and capillary basal membranes and also at the endothelium until it gets into the capillary lumen, showing that hormones of pancreatic endocrine cells may be actively transported into circulation as intact secretory granules. There is no significant morphological difference between the mechanism of secretory granule formation in normal and transplanted pancreatic tissue.     

Nitric oxide-evoked cGMP production in Purkinje cells in rat cerebellum: an immunocytochemical and pharmacological study

The cerebellar cells that account for glutamate-dependent cyclic GMP (cGMP) production, involving activation of the ionotropic glutamate receptors/nitric oxide synthase/soluble guanylyl cyclase pathway, are not fully established. In the present paper we have searched for the localisation of the cGMP response to the nitric oxide (NO) donor S-nitroso-penicillamine (SNAP 1muM), expected to generate local NO concentrations in the low nanomolar physiological range and evoking a cGMP response dependent on glutamate release and on the consequent activation of ionotropic glutamate NMDA/non-NMDA receptors, in cerebellar slices from adult rat. We have found that low concentration of exogenous NO evoked cGMP accumulation in Purkinje cells in an ionotropic glutamate receptor-dependent and tetrodotoxin-sensitive manner. Such immunocytochemical localisation appears consistent with functional evidence for physiologically relevant glutamate-dependent cGMP production in Purkinje cells in rat cerebellar cortex.     

Morphological and immunocytochemical characterization of snake-like chromatin cells

Snake-like chromatin (SLC) is a nuclear alteration occurring under various pathological conditions and in different tissues. The aim of this study was the morphological and immunocytochemical characterization of SLC-positive conjunctival epithelial cells from keratoconjunctivitis sicca (KCS) patients. Impression cytology specimens from the upper bulbar conjunctiva of 10 controls and 10 KCS patients with a high incidence of SLC cells were assessed, the morphology of SLC nuclei evaluated by light microscopy, and proliferation markers, nucleolar proteins, lamins and cytokeratin filaments detected immunocytochemically. In KCS patients, SLC cells with a normal nuclear shape, with nuclear membrane notching (2.3% of cells) and with binuclear dumb-bell structures (4.4% of cells) were observed. The most striking features of SLC cells were the absence of an A/C lamin signal, the redistribution of fibrillarin into two spots adjacent to SLC structures and cytokeratin 14 positivity in the strangulation belt of the dumb-bell structures. The deficiency of lamin A/C is the probable reason for the disintegration of chromatin from the nuclear lamina in SLC cells. The occurrence of SLC-positive cells, SLC-positive dumb-bell shaped nuclei and SLC-positive binucleated cells, together with the absence of mitotic markers, leads to the conclusion that the SLC phenomenon might be a form of nuclear segregation.     

Morphological and functional heterogeneity in the rat prostatic gland.

Ductal morphogenesis and adult ductal branching patterns were examined in the rat prostate by a microdissection method. The rat prostate consists of paired (right and left) subdivisions which correspond in large part to the classically defined lobes: ventral prostate, lateral prostate, dorsal prostate, and coagulating gland. Of particular interest was the finding that the lateral prostate consists of two different ductal zones: (1) lateral type 1 prostate with 5-7 long main ducts (resembling miniature palm trees) that extend cranially towards both the seminal vesicle and dorsal prostate to arborize near the bladder neck, and (2) lateral type 2 prostate with 5-6 short main ducts that arborize caudal to the bladder neck and give rise to compact bushy glands. Both lateral prostatic groups had a ductal-acinar organization. The adult structure of the other rat prostatic lobes was also examined, and closely resembled their mouse counterparts. The ventral prostate, which had 2-3 pairs of slender main ducts per side, and the coagulating gland, which had 1 main duct per side, was completely ductal in structure. In contrast, the dorsal prostate, which had 5-6 pairs of main ducts per side, had a ductal-acinar structure. Ductal branching morphogenesis occurred at different rates in different lobes and was essentially complete in the prostate at the 30 days. Immunocytochemical studies with an antibody to DP-1, a major secretory protein of the rat dorsal prostate, revealed that secretory function was initiated at approximately 30 days after birth in the coagulating gland, the dorsal prostate, and lateral type 1 prostate. A consistent feature of the lateral type 2 prostate was the absence of DP-1. On Western blots, DP-1 was detected in the secretion of the coagulating gland, lateral type 1 and dorsal prostate, but not in the ventral and lateral type 2 prostate. Polyacrylamide gel electrophoresis confirmed this result and demonstrated that the lateral type 2 prostate expressed several low-molecular weight secretory proteins not found in the other lobes of the prostate. On the whole, the rat prostate exhibited considerable heterogeneity both between and within lobes in developmental processes, ductal patterning, histology, and functional expression.