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Persistent elevations of cAMP levels are generally accompanied by an inhibition of granulocyte functions. Phosphodiesterases play a critical role in regulating intracellular levels of cAMP. The expression of three isoforms of type 4 cAMP-specific phosphodiesterase (PDE4) in neutrophils suggests diversity of isoform localization and targeting in regulating cell function. The sites of cAMP regulation in granulocytes by the PDE4A isoform were investigated by immunoelectron microscopy. PDE4A was localized uniformly in all granule classes of eosinophils, but was restricted in neutrophils to a subset of myeloperoxidase (MPO)-containing granules that were round or elongated with a central crystalloid core. Granulocytes were stimulated with fMLP to investigate the sites of PDE4A targeting during cell activation. In neutrophils, fMLP induced a rapid (1 min) translocation of granules containing PDE4A to the plasmalemma, where some PDE4A and MPO were exocytosed. In these cells, PDE4A labeling within granules was focal and no longer homogeneous. While immunogold labeling of PDE4A was reduced after fMLP stimulation, staining of MPO-containing granules remained high. Extracellular release of PDE4A was also observed in eosinophils stimulated with fMLP. Morphometry revealed that Au labeling was significantly reduced within 1 min, and that there was a shift in PDE4A localization within eosinophil granules from the crystalline core to the matrix. Fluctuations of cAMP levels and ectoprotein kinase activity with PKA properties occur in blood under normal and pathological conditions. The exclusive localization of PDE4A within granules of neutrophils and eosinophils suggests that PDE4A may function to downregulate cAMP signaling at the cell membrane and/or in the extracellular space at the time of granule release.

Prohormone convertases PC1 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. There is a report that prohormone convertase exists in the rat anterior pituitary gonadotrophs, where it had previously been considered that proprotein processing does not take place. In addition to luteinizing hormone and follicle-stimulating hormone, rat pituitary gonadotrophs contain chromogranin A (CgA) and secretogranin II (SgII), two members of the family of granin proteins, which have proteolytic sites in their molecules. In the present study we examined whether there is a close correlation between subcellular localization of prohormone convertases and granin proteins. Ultrathin sections of rat anterior pituitary were immunolabeled with anti-PC1 or -PC2 antisera and then stained with immunogold. Immunogold particles for PC1 were exclusively found in large, lucent secretory granules, whereas those for PC2 were seen in both large, lucent and small, dense granules. The double-immunolabeling also demonstrated colocalization of PC2 and SgII in small, dense granules and of PC1, PC2, and CgA in large, lucent granules. These immunocytochemical results suggest that PC2 may be involved in the proteolytic processing of SgII and that both PC1 and PC2 may be necessary to process CgA.

Fluorescein and horseradish peroxidase-labeled monoclonal antibodies were used to localize the predominant toxic peptide CSTX-1 in the venom gland of the spider Cupiennius salei. There was no polarity of CSTX-1 expression in repleted glands, whereas the glands of previously milked spiders showed a decreasing immunofluorescent response from the distal to the proximal portion. Detailed investigation revealed a new structure in the venom-secreting epithelium, which is postulated to be an evolutionary adaptation to increasing gland volume. CSTX-1 was found to be synthesized and stored as a fully active toxin within complex units, composed of long interdigitating cells running perpendicular to the muscular sheath and extending into the central lumen of the gland. These venom-producing units were found in all sectors of the gland, including the transitional region between the main gland and the venom duct. The venom is liberated from the venom-producing units into the glandular lumen following the contraction of the surrounding muscle layer. Free nuclei or other cellular fragments, which would have provided evidence for a holocrine secretion process, were not found in the glandular lumen or in the crude venom obtained by electrical stimulation. The fine regulation of the spider's venom injection process is postulated to be the function of the bulbous ampulla, situated in the anterior third of the venom duct.

Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7-8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca2+-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca2+. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca2+ with Clostridium perfringens enterotoxin which induces Ca2+ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca2+-induced granule-granule fusion in anterior pituitary cells.

Pancreatic acinar cells of euthermic, hibernating and arousing individuals of the hazel dormouse Muscardinus avellanarius (Gliridae) have been observed at the electron-microscopic level and analysed by means of ultrastructural morphometry and immunocytochemistry in order to investigate possible fine structural changes of cellular components during periods of strikingly different degrees of metabolic activity. During hibernation, the cisternae of the rough endoplasmic reticulum (RER) flatten assuming a parallel pattern, the Golgi apparatus is extremely reduced and the mitochondria contain many electron-dense particles. The cell nuclei appear irregularly shaped, with deep indentations containing small zymogen granules. They also contain abundant coiled bodies and unusual constituents, such as amorphous bodies and dense granular bodies. Large numbers of zymogen granules occur in all animals. However, the acinar lumina are open and filled with zymogen only in euthermic animals, whereas, in hibernating and arousing individuals, they appear to be closed. Morphometrical analyses indicate that, in pancreatic acinar cells, nuclei and zymogen granules significantly decrease in size from euthermia to hibernation, probably reflecting a drastic decrease of metabolic activities, mainly protein synthesis and processing. In all the studied animals, immunocytochemistry with specific antibodies has revealed an increasing gradient in alpha-amylase content along the RER-Golgi-zymogen granule pathway, reflecting the protein concentration along the secretory pathway. Moreover, during deep hibernation, significantly larger amounts of alpha-amylase accumulate in RER and zymogen granules in comparison to the other seasonal phases analysed. Upon arousal, all cytoplasmic and nuclear constituents restore their euthermic aspect and all morphometrical and immunocytochemical parameters exhibit the euthermic values, thereby indicating a rapid resumption of metabolic activities.

Adrenal chromaffin cells have been characterized by the ability to change the phenotype in response to neurotrophic factor stimulation. The adrenal gland expresses numerous trophic factors endogenously, but there is still a lack of knowledge as to how the adrenal medullary cells respond to these factors. Accordingly, we evaluated nerve fiber outgrowth and cell morphology, and measured catecholamine content in adult rat adrenal medullary tissue transplanted to the anterior chamber of the eye after exposure to neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), or glial cell line-derived neurotrophic factor (GDNF) compared with the effects after exposure to recombinant human nerve growth factor (rhNGF). The results show that rhNGF was the most potent factor in inducing neurite outgrowth from the grafted chromaffin cells. CNTF was also a powerful inducer of nerve fiber formation, while NT-4/5, GDNF, and bFGF were less potent. NT-3 did not produce neurite outgrowth above that seen in vehicle-treated eyes. Combining two neurotrophins, rhNGF and NT-3, reduced nerve fiber formation. Tyrosine hydroxylase (TH) immunohistochemistry revealed good cell survival in all grafts, and no morphological differences were detected with the different treatments. The adrenaline: noradrenaline: dopamine ratio was approximately 49%: 49%: 2%, independent of treatment, and the catecholamine content was equal irrespective of treatment. In conclusion, all neurotrophic factors used, except for NT-3, promoted neurite outgrowth from adult rat chromaffin transplants. Differences in outgrowth induced by the various trophic factors did not, however, change the catecholamine content in grafts when analyzed together with the graft-derived nerve plexus.

Allatostatins are neuropeptides that inhibit the production, by the corpora allata, of a major insect hormone, juvenile hormone. These peptides are produced by cells of the brain and ganglia as well as by midgut endocrine cells. Transport from these sites may contribute to the allatostatin content in the hemolymph (insect blood). Using a monoclonal antibody against Diploptera punctata allatostatin I (A-P-S-G-A-Q-R-L-Y-G-F-G-L-NH2) and in situ hybridization with a digoxigenin-labeled cRNA probe generated from a portion of the allatostatin gene, it is demonstrated that allatostatin is present in and synthesized by granular hemocytes of D. punctata. About 5% of the hemocytes react with anti-allatostatin antibody and a similar number hybridize with a cRNA probe that detects allatostatin-specific mRNA. Electron micrographs showed that allatostatin-immunoreactive material occurs in membrane-bound, uniformly dense granules that frequently fill fusiform-shaped cells. Allatostatin in cell and plasma fractions of hemolymph quantified by enzyme-linked immunosorbent assay and by bioassay for inhibition of juvenile hormone synthesis in vitro indicated that about equal quantities (0.1-0.2 fmol/microl) are present in cell and plasma fractions. The production of allatostatin by hemocytes suggests that allatostatins may function as regulatory peptides in hemolymph activities in addition to their other known functions.

The polymerase chain reaction was used to amplify a cDNA encoding the catalytic region of ras-guanine nucleotide release factor (ras-GRF1) from mouse embryonic stem cell mRNA. Antibodies directed against this protein were prepared and affinity purified. Western immunoblotting of rat tissue lysates revealed a 140-kDa protein in brain as expected but, in addition, a strongly immunoreactive 54-kDa protein, p54, was identified in pancreas. Expression of ras-GRF1 in pancreas was confirmed at the RNA level by reverse-transcriptase-coupled polymerase chain reaction analysis; p54 may therefore correspond to a form of ras-GRF1 or a closely related protein. The cellular and subcellular localization of p54 was investigated by enzyme-linked immunocytochemistry and immunogold electron microscopy. In the pancreas, p54 was expressed primarily in acinar cells, where it was localized along the basolateral and apicolateral plasma membranes. Indirect immunofluorescence microscopy of cultured acini further indicated that the plasma membrane localization of p54 was dependent on the maintenance of the acinar histotype and organized acinar structure. When primary acinar cells were permitted to dissipate into monolayer cultures devoid of zymogen granules, ras-GRF1 staining became cytosolic. Our results suggest that ras-GRF1 is involved in the structure and function of the pancreas.

Electron-microscopic studies of the neural complex (neural gland, dorsal strand, and cerebral ganglion) of an ascidian, Halocynthia roretzi, were performed, paying particular attention to the secretory systems. We found that cells scattered along the dorsal strand and neural cells in the cerebral ganglion contained electron-dense secretory granules of variable size. Immunoelectron-microscopic studies with an antiserum to bullfrog prolactin revealed that the secretory granules (100-250 nm in diameter) of some granulated cells contained a prolactin-like substance. Cells belonging to the neural gland and dorsal strand neither contained electron-dense granules nor showed immunoreactivity. The possibility that cells in the cerebral ganglion and those along the dorsal strand are phylogenetic progenitors of vertebrate adenohypophyseal cells is discussed.

The tissue distribution, preferentially in the human male genital system, and the subcellular localization of the lysosome-associated membrane protein 2 (lamp 2) was studied immunohistochemically using a mouse monoclonal antibody, 2D5. Strong immunoreactivity was present in the tubular system of the kidney, in acinar cells of salivary glands and pancreas, prostate, mammary glands, placenta and in cutaneous sweat glands. Moderate immunoreactivity was observed in cerebral neuronal cells, epidermal cells, testis, epididymis, seminal vesicle and endometrium. Very low immunoreactivity was found in liver. In some of the tissues mentioned, the distribution pattern of immunoreactivity is smooth and homogeneous, while in others it is granular and concentrated in the supra- or perinuclear cytoplasm. The subcellular distribution was studied on ultracryosections and on pre-embedding-processed chopper sections of human prostate. In the latter gland, the protein is not restricted to epithelium, but is also present in stromal cells. Ultrastructurally, the immunoreactivity in secretory cells was localized in electron-translucent vacuoles and granules, including the secretory granules. A close association with cell membranes was not generally the case. Only part of the immunoreactive material was linked to the apical plasma membrane pointing to a biosynthesis independent from an association step with the apical plasma membrane. As shown by immunoelectron microscopy and Western blotting, a high amount of lamp 2 is secreted and is found in so-called prostasomes. The findings indicate that in the human prostate most of the membrane-bound lamp 2 is released from the secretory cells, presumably in an apocrine fashion.

Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n = 82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n = 42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.

The organization of the mucomicrovillar complex of the vomeronasal sensory epithelium of adult rats was examined using confocal laser scanning microscopy. In specimens labeled with the FITC-conjugated isolectin B4 of Bandeiraea simplicifolia, which recognizes terminal alpha-galactose sugar residues of glycoconjugates, we demonstrated that the mucomicrovillar complex was composed of islet-like structures with a high-density alpha-galactose core. The mucomicrovillar complex was further resolved into sensory and mucoid components in double-labeling and dual scanning experiments. The sensory component, which consists of the dendritic terminals of olfactory marker protein-immunoreactive vomeronasal receptor neurons, contained cytosolic glycoconjugates with terminal alpha-galactose sugar residues. The extracellular mucoid component consisted of glycoconjugates containing terminal alpha-galactose derived from the glands associated with the vomeronasal organ. These results demonstrated the complex microchemical organization of the sensory and mucoid components of the mucomicrovillar complex.

Adrenal medullary chromaffin cells secrete catecholamines through exocytosis of their intracellular chromaffin granules. Osmotic granule swelling has been implicated to play a role in the generation of membrane stress associated with the fusion of the granule membrane. However, controversy exists as to whether swelling occurs before or after the actual fusion event. Using morphometric methods we have determined the granule diameter distributions in rapidly frozen, freeze-substituted chromaffin cells. Our measurements show that intracellular chromaffin granules increase in size from an average of 234 nm to 274 nm or 277 nm in cells stimulated to secrete with nicotine or high external K+, respectively. Granule swelling occurs before the formation of membrane contact. Ammonium chloride, an agent which inhibits stimulated catecholamine secretion by approximately 50% by altering the intragranular pH, also inhibits granule swelling. In addition, ammonium chloride-treated secreting cells show more granule-plasma membrane contacts than untreated secreting cells. Sodium propionate induces granule swelling in the absence of secretagogue and has been shown to enhance nicotine- and high K(+)- induced catecholamine release. These results indicate that in adrenal chromaffin cells granule swelling is an essential step in exocytosis before fusion pore formation, and is related to the pH of the granule environment.

Immunohistochemical localization of lutropin beta (LH beta) and follitropin beta (FSH beta) in the pituitary gland of the frog Rana japonica was studied by the peroxidase-anti-peroxidase method and the two-face, double-labeling method with different-sized gold particles at the light- and electron-microscopic levels, respectively, using monoclonal antibodies against bullfrog LH beta and FSH beta. Light-microscopic immunohistochemistry indicated that approximately 66.0% of all the gonadotrophs in the pituitary contained both LH beta and FSH beta, whereas 33.4% of gonadotrophs contained only LH beta, and 0.6% contained only FSH beta. The staining intensity of LH beta and FSH beta varied from cell to cell. The gonadotrophs were classified into four types (Types I-IV) in terms of their ultrastructural and immunolabeling characteristics. Moreover, several secretory granule types were recognized according to differences in their shape and electron density. In all the cell types, both LH beta and FSH beta were often seen in the same secretory granules, but the proportion of granules bearing both hormones ranged from 5.5% in Type I to 32.7% in Type IV. Most secretory granules in Types I and II were immunolabeled with LH beta alone, whereas a small number of granules were immunolabeled with FSH beta alone. More immunolabeled FSH beta granules were present in Types III and IV than in Types I and II.

Immunohistochemical study with antisera to chromogranin A and neuron-specific enolase, a general marker for nerves and endocrine cells, was used to quantify changes in bronchial neuroendocrine cells in guinea-pigs sensitized and challenged with ovalbumin. Actively sensitized animals were killed 2, 6, 24, 48, 72, and more than 144 hours after being challenged by an aerosolized solution of ovalbumin. The number of chromogranin A-immunoreactive cells was significantly greater in sensitized but unchallenged animals and in sensitized animals killed 2 and 6 h after challenge when compared to controls; it decreased significantly in animals killed more than 24 h after challenge when compared to sensitized, unchallenged animals. The number of neuron-specific-enolase-immunoreactive cells did not change. We conclude that the peptide content of bronchial neuroendocrine cells increases during sensitization and in the early phase of a hypersensitivity reaction, and that the cells release their granule contents in the late phase of such a reaction. They may therefore play a role in immunoallergic events in the lung.

Placing the clawed toad Xenopus laevis on a black background stimulates the melanotrope cells in the pars intermedia of the pituitary gland to release proopiomelanocortin (POMC)-derived peptides, including alpha-MSH and N-acetyl-beta-endorphin. In this study three types of secretory granules, electron-dense (approximately 130 nm phi), moderately electron-dense (approximately 160 nm phi) and electron-lucent (approximately 180 nm phi), have been identified in these cells. Apparently, only dark granules are formed by the Golgi apparatus and lucent granules release their contents via exocytosis. Immuno-electron microscopy (immunogold double labelling) of glutaraldehyde-fixed and freeze-substituted material shows that desacetyl-alpha-MSH and N-acetyl-beta-endorphin coexist in all three granule types. Quantification of immunostaining revealed that immunoreactivities to these peptides are lowest in the dark granules and highest in the light ones. It is proposed that intragranular processing of POMC to immunoreactive desacetyl-alpha-MSH and N-acetyl-beta-endorphin involves an increase in granule size and a decrease in granule electron density. Black background-induced activation of the melanotrope cell is reflected by an increase in immunoreactivity of the secretory granules to each of the antisera. This suggests that cell activation stimulates the formation of peptides by intragranular processing of POMC and/or of intermediate POMC-processing products. In addition, cell activation evoked an increase in the percentage of the granule population that reacts with anti-N-acetyl-beta-endorphin, probably by stimulating intragranular acetylation of beta-endorphin. Apparently, this acetylation is a regulated event that occurs in the cytoplasm, independently from the acetylation of desacetyl-alpha-MSH which takes place near the plasmalemma at the time of granule exocytosis.

In the chicken, enkephalin-immunoreactive cells and nerve fibers are distributed in the ultimobranchial glands, which consist of C-cell groups and cyst structures. Ultrastructural features of the enkephalin cells and nerve fibers were examined by immuno-electron microscopy using both the streptavidin-biotin-peroxidase method and the protein A-colloidal gold method. Immunoreactivity for enkephalin was located on the secretory granules of C cells. In 1-day-old chickens, three types of C cells were distinguished on the basis of their granule size. Type-I cells were filled with large secretory granules (200-600 nm in diameter). These elements represented a majority of the C-cell population. Type-II cells contained medium-sized granules (100-280 nm in diameter). Type-III cells displayed small secretory granules (60-200 nm in diameter). The latter cells were elongate or irregular in shape and frequently extended cytoplasmic processes into the connective tissue stroma or contacted other C cells. Enkephalin-immunoactivity was revealed by dense deposits of immunogold particles on the secretory granules of type-II and type-III cells. There were only a few type-I cells showing immunoreactivity for enkephalin. A double immunogold labeling procedure demonstrated that calcitonin and enkephalin were colocalized in the same secretory granules of type-I and type-II cells. Type-III cells were devoid of immunoreactivity for calcitonin. Enkephalin-immunoreactive nerve fibers were characterized by the presence of granular vesicles, 60-160 nm in diameter, and frequently established direct contact with the surface of C cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supra-nuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.

Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIPB-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIPB- and anti-MIPC-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurhormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes.

Our recent finding that the number of immunoreactive alpha-subunit cells was invariably greater than the total number of immunoreactive gonadotropin (GTH) and thyrotropin (TSH) cells in the bullfrog (Rana catesbeiana) pituitary gland raises the possibility that the alpha-subunit also exists in pituitary cells other than GTH and TSH cells. The present study demonstrates that there are a considerable number of immunoreactive prolactin (PRL) cells that are also stained with antibody against the alpha-subunit when adjacent sections are immunocytochemically examined. Neither immunoreactive growth hormone nor adrenocorticotropin cells are stained with the antibody against the alpha-subunit. The specificity of the antibody against the alpha-subunit and of that against PRL was demonstrated by preabsorption test, non-competitive binding test, and immunoblot analysis. Double-immunolabeling with gold particles of different sizes for the alpha-subunit and PRL revealed that most of the immunolabeled PRL-secretory granules are also labeled with the alpha-subunit antibody. The gold particles indicating the presence of the alpha-subunit were mostly found in the peripheral zone of the secretory granules.

The development of the basal lamina (BL), the key structure of the basement membrane (BM), was investigated in three xenografted human carcinomas of the sigmoid colon (CA 1), the lung (L 261), and the hypopharynx (H-Stg 1) following heterotransplantation to athymic mice. The study involved the use of electron microscopy and indirect immunofluorescence techniques employing highly specific antibodies against the intrinsic BL components, heparan sulfate proteoglycan, laminin and type-IV collagen. Following transplantation, the extracellular matrix material of the transplanted tumors decomposed and was phagocytozed by invading macrophages within 1 to 2 days. During this stage, no specific binding of the applied antibodies to BL components could be detected within the xenografts. Following the ingrowth of host-derived connective tissue between days 2 to 6, small fluorescence-positive granules appeared within the cytoplasm and around those tumor cells that were located close to the invaded strands of connective tissue. Ultrastructurally, typical secretory granules were detectable in the cytoplasm of many xenografted carcinoma cells. Thereafter, a tannic acid-positive, patchy material appeared in the extracellular space of CA 1 and L 261 and aggregated to form small fragments of a discontinuous BL. In the H-Stg 1 xenografts, this material assembled to form continuous mono-, bi- and multi-layered structures. Large amounts of excess BL material remained accumulated in the L 261 and H-Stg 1 xenografts until the end of the observation period (day 24). These findings reveal that discontinuities of the BL occur independent of the active invasion processes of tumor cells, since xenografted human carcinomas neither grow invasively nor metastasize in nude mice. Moreover, they confirm that these discontinuities are not caused by a quantitatively insufficient production of BL material, but rather arise from qualitative imbalances of the composition of the synthesized BL material.

The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P less than 0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.