Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material

The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.     

The Chemical Nature and Function of the Endoplasmic Granules of the Ciliate Conchophthirus curtus

SYNOPSIS. Conchophthirus curtus is a thigmotrich that occurs on the gills of Unionid clams ( Elliptio complanatus in this study). The anterior third of the endoplasm, unlike the remainder, is relatively firm and without gastrioles; it contains an extensive aggregation of specialized endoplasmic granules and is therefore called granuloplasm. On the surface ventral to the granuloplasm is a thigmotactic area that bears closely set, strongly adherent cilia. There is no evidence that the granules are intracellular microorganisms; they are Feulgen-negative and do not divide, nor do they stain like bacteria. Cytochemical tests show that the granules contain neutral fat, fatty acid, phospholipid, glycogen, and mucin. The evidence indicates that the principal function of the granules and granuloplasm is the production of mucin, which is supplied to the underlying thigmotactic cilia, thereby conferring on them their adhesive properties. Thus, the granules and granuloplasm constitute a mucous organelle, and to the extent that they are osmiophilic and secretory they qualify as Golgi bodies and Golgi material, respectively. Since endoplasmic granule is a general term for any of the granules of protozoan endoplasm, it is recommended that the granules of the present study be called muciferous granules.     

ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS : III. Studies of the Stability of the Isolated Beta Granules

A partially purified secretory granule fraction, isolated from rat islets of Langerhans by differential centrifugation, was used for investigating the stability of the beta granules during incubation in various conditions. Effects of pH, temperature, and time were studied; the granules possessed optimal stability at 4° and pH 6.0, and could be solubilized at pH 4.0 or 8.5, or in the presence of sodium deoxycholate, but not by phospholipase c, ouabain, or alloxan. Incubation with glucose or some of its metabolites, or with tolbutamide, ATP, or cyclic 3',5'-AMP did not alter the stability of the beta granules Exogenous insulin-¹³¹I was not bound by the isolated granules under the conditions used; no specific insulin-degrading activity could be detected in subcellular fractions of the islets. These findings indicate that intracellular solubilization of the granules with subsequent diffusion of the insulin into the extracellular space is not a likely mode of insulin secretion in vivo, and suggest that a crystalline zinc-insulin complex may exist in the matrix of the beta granules.     

The localization of aluminium in the digestive gland of the terrestrial snail Helix aspersa

The terrestrial snail Helix aspersa was exposed to food containing elevated levels of aluminium for up to 33 days and the digestive gland examined by light and electron microscopy and X-ray microanalysis. Four types of cell are found in the digestive gland, (digestive, excretory, calcium and thin) although aluminium was only found in the excretory cells. The aluminium was localised in the 'yellow' or excretory granules that are a characteristic feature of the excretory cells. Aluminium was only found in the granules of snails fed aluminium but there was no difference in the appearance of granules from control or aluminium-fed snails. The granules were large (up to 20 mum in diameter), irregularly shaped and electron-dense. Sulphur, phosphorus and calcium were detected in granules from all snails. The presence of sulphur may indicate protein residues. The amount of aluminium and phosphorus in the granules increased over the experimental period but the number of granules did not change. Levels of aluminium in the granules decreased when the snails were given control food. The role of the excretory granules in the localisation, detoxification and excretion of aluminium is discussed.     

Electron microscopic demonstration of acid phosphatase activity in the developing and mature heterophils of the chicken

Summary As shown by electron microscopic histochemistry using a modified Gomori lead salt technique, acid phosphatase is present in large dense granules and the Golgi apparatus —but not the light granules—in both immature and mature heterophils in the chicken. The large dense granules appear to form by budding from the Golgi cisternae while the light granules appear to be unassociated with the Golgi apparatus. The findings indicate that the large, dense granules are the lysosomes of the heterophils in the chicken.     

The morphometry of the Sudan-III-positive granules in the cytoplasm of the human amniotic epithelium

Seventy-nine human amnions from different gestational ages were studied by new morphometric methods. The Sudan-III-positive granules in the cytoplasm of the amniotic epithelial cells were first observed in the reflected amnion from the second trimester of normal gestation. During the third trimester of normal gestation, the number of cytoplasmic granules increased remarkably with gestational age not only in the reflected amnion but also in the placental amnion. The number of granules was generally related to the increasing rates of anucleated cells and binucleated cells in the amniotic epithelium during the third trimester of normal gestation. During the third trimester of gestation, the number of granules was greater in the distant reflected amnion with its less adequate blood supply than in the placental amnion. A vast accumulation of the granules in the entire amnion was observed in cases of fetal death, in which blood supply had ceased. It is suggested, therefore, that the progressive accumulation of Sudan-III-positive cytoplasmic granules in the human amniotic epithelium during the third trimester of normal gestation is the result of cell degeneration.     

Ultrastructural immunocytochemical localization of vitellogenin in the fat body of the blowfly,Calliphora vicina Rob.-Desv. (erythrocephala Meig.) by use of the unlabeled antibody-enzyme method

Summary The unlabeled antibody-enzyme method was used to demonstrate ultrastructurally the specific localization of vitellogenin in the fat body of Calliphora. In control flies the binding sites to vitellogenin were localized in secretory granules situated in the Golgi complex, and in larger bodies named composite secretory granules. These composite granules appear to be formed when a part of a Golgi complex containing secretory granules and a number of small vesicles become surrounded by a common membrane. Ovariectomized flies, which apparently do not produce secretory granules, exhibited no immunocytochemical staining. Ovariectomized flies in which the administration of ecdysterone induced formation of secretory granules, also revealed specific staining on these granules. This is the first ultrastructural evidence of: (a) the specific localization of vitellogenin in secretory granules of the fat body of an insect; (b) the relationship between the presence of the ovary, and of ecdysterone, and the synthesis of vitellogenin by the fat body.     

Inhibitory effects of dibutyryl cyclic AMP and theophylline on the melanosome transformation in the embryonic chick pigmented retina cultured in vitro.

When the retinal pigment epithelial cells of chick embryo are cultured in monolayer conditions, the pigment granules are lost from the cytoplasm. The first structural change in depigmentation is the transformation of pigment granules into the degradative organelles designated as the dense body and melanosome complex. The cells are grown in medium containing DBcAMP of various doses from 10^?5 to 10^?2M. Cell proliferation is retarded by treatment with DBcAMP (10^?3M). The transformation of pigment granules is almost completely prevented in all 1-day cultured cells. In 5-day cultured cells continuously treated with more than 10^?4M, the transformation is not only prevented, but the synthesis of pigment granules is stimulated. A similar result is obtained by the administration of 10^?3M theophylline. 5′-AMP does not prevent the transformation of pigment granules but seems to stimulate the synthesis of pigment granules. On the other hand, cGMP is ineffective both on prevention of transformation and on synthesis of pigment granules. The mechanisms of the transformation of pigment granules are discussed.     

The ultrastructure of guinea pig heterophil granulocytes and the heterogeneity of the granules

Summary The development of the heterophil granulocytes in the bone marrow of the guinea pig is described. During the maturation of these cells, three types of granule are formed, not only the azurophil and specific granules already described in other mammals but also a third type of granule referred to here as the nucleated granule. During the process of maturation of the cells, these three types of granule are formed successively. On this basis, two steps can be distinguished in the promyelocyte phase in which primary (nucleated and azurophil) granules are formed, i.e. an early and a late stage, nucleated granules being formed in early and azurophil granules in late promyelocytes. Secondary (specific) granules occur first in myelocytes. In mature heterophils of the guinea pig the granule population is composed of about 85% secondary granules, about 10% azurophil granules, and about 5% nucleated granules. The changes in the granule population during the maturation process were quantified. The observations and calculations point to the occurrence of three mitoses: one in the early and one in the late promyelocyte and the third in the myelocyte.     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands

Summary Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B₄ staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins. However, difference was also observed between monoclonal antibody and lectin staining, that is, monoclonal anti-A antibody reacted weakly but consistently with granules from blood group A nonsecretors but DBA (HPA) did not; staining with UEA-I was observed in granules from the secretor individuals of any blood groups whereas monoclonal anti-H antibody reacted with granules from blood group O and some A secretor individuals but not from B and AB secretor individuals; GSAI-B₄ reacted uniformly with granules throughout the cells whereas monoclonal anti-B antibody bound to limited number of granules in the same cells. This was confirmed by the double labeling experiments with the lectin and the antibody. These results suggest that the different types of antigens as to the binding ability for monoclonal antibodies and lectins are expressed on different granules in the same cell.     

Immunocytochemical evidence of a met-enkephalin-like substance in the dense-core granules of mouse Merkel cells

Summary The electron-microscopic immunogold method was applied to Merkel cells of adult mice to demonstrate the subcellular localization of met-enkephalin-like immunoreactivity. Post-embedding incubation with metenkephalin antisera showed that the gold particles were associated with the dense-core granules of the Merkel cells. The majority, but not all, of the dense-core granules were strongly labelled. Osmication caused a significant reduction in the number of gold particles on these granules. The nerve terminal associated with the Merkel cell did not show met-enkephalin-like immunoreactivity. To the best of our knowledge, this is the first report of the ultrastructural localization of a positive met-enkephalin immunoreactivity in the dense-core granules of Merkel cells in mice.     

Retention of amylase in the secretory granules of parotid gland after extensive release of Ca++ by ionophore A-23187

The effect of the ionophore A-23187 was tested on isolated secretory granules of rat parotid gland. The ionophore caused extensive release of calcium from the granules without effecting release of amylase or other secretory proteins. It is therefore concluded that the role of calcium in the granules is probably not that of a stabilizing agent.     

Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane

Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino- N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH- treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP- driven H+ translocation into parafollicular granules isolated from TSH- stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.     

Formation of tRNA granules in the nucleus of heat-induced human cells

The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA(Met) (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA(Met) was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.     

Fine structure of the bone marrow of the chicken and pigeon

The structure of bone marrow from chickens and pigeons was studied with light and electron microscopy. Erythropoiesis occurs in the lumen of the medullary sinuses. Immature erythroid cells appear to adhere to the sinus wall and may thus be prevented from entering the peripheral circulation. The wall of the medullary sinuses is formed by elongated lining cells, lacking a basement membrane, which are continous except at sites where blood cells are passing through them. When viewed with the electron microscope, developing heterophil myelocytes, which occur only in the extravascular spaces, possess two populations of granules; one type is globular in content, the other is fibrillar in content. The globular type predominates during all stages of development and appears to be the specific granule. Specific granules originate from material which is formed in the Golgi complex, pinches off, and accumulates in expanded vesicles. The origin of the material in the fibrillar granules was not determined. Like the globular granules of heterophil leucocytes, granules of eosinophil leucocytes arise from material which is formed in the Golgi complex.     

Targeting of membrane proteins to the regulated secretory pathway in anterior pituitary endocrine cells

Unlike the neuroendocrine cell lines widely used to study trafficking of soluble and membrane proteins to secretory granules, the endocrine cells of the anterior pituitary are highly specialized for the production of mature secretory granules. Therefore, we investigated the trafficking of three membrane proteins in primary anterior pituitary endocrine cells. Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential to the production of many bioactive peptides, is cleaved and enters the regulated secretory pathway even when expressed at levels 40-fold higher than endogenous levels. Myc-TMD/CD, a membrane protein lacking the lumenal, catalytic domains of PAM, is still stored in granules. Secretory granules are not the default pathway for all membrane proteins, because Tac accumulates on the surface of pituitary endocrine cells. Overexpression of PAM is accompanied by a diminution in its endoproteolytic cleavage and in its BaCl(2)-stimulated release from mature granules. Because internalized PAM/PAM-antibody complexes are returned to secretory granules, the endocytic machinery of the pituitary endocrine cells is not saturated. As in corticotrope tumor cells, expression of PAM or Myc-TMD/CD alters the organization of the actin cytoskeleton. PAM-mediated alterations in the cytoskeleton may limit maturation of PAM and storage in mature granules.     

An assessment of the role of protein kinase and zymogen granule phosphorylation during secretion by the rat exocrine pancreas.

1. The levels of protein kinase activity and zymogen granule phosphorylation were studied in the adult rat during stimulus-coupled secretion in vitro. 2. The specific activity of protein kinase associated with intact zymogen granules was 11 pmol [32P]phosphate transferred to histone per min per mg protein. Most of this activity was recovered in purified granule membranes. 2. The addition of 10(-6) M cyclic AMP to a mixture of zymogen granules and the postmicrosomal supernatant resulted in a 5-fold increase in protein kinase activity associated with zymogen granules. The adsorbed activity was eluted from granules by 0.15 M NaCl. Cyclic GMP did not promote protein kinase binding to isolated granules. 4. Incubation of tissues with carbachol (10(-5) M), pancreozymin (0.1 unit/ml), caerulein (10(-8) M) or dibutyryl cyclic AMP (2.10(-4) M) between 2.5 and 60 min did not increase the levels of protein kinase activity in isolated zymogen granules above control values. 5. Protein phosphorylation of zymogen granule membranes and granule content was not detectable in tissues incubated with carbachol, pancreozymin-C-octapeptide, or caerulein. 6. These results suggest that neither the phosphorylation of zymogen granule membrane protein nor the adsorption of protein kinase activity to zymogen granules is an obligatory step in secretion.     

Ultrastructure of the acinar cells in the submandibular gland of the nine-banded armadillo

There are two discrete lobes comprising the armadillo subman-dibular gland. These two lobes can be defined grossly, histochemically and morphologically with the light and electron microscope. The minor lobe stains more intensely with PAS and AB. When viewed in the electron microscope, the secretory granules of the acinar cells within this lobe appear mucous-like. The granules of the demilune cells are slightly different in appearance. The secretory granules of the acinar cells in the major lobe contain many dense foci embedded in a fibrillar matrix, a substructure not described previously. The demilune cells of this lobe contain secretory granules with a mucous-like structure which is consistent throughout the entire lobe. As in the minor lobe, these demilune cells stain very intensely with PAS and AB.     

P granules extend the nuclear pore complex environment in the C. elegans germ line

The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)-like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.     

Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and [14C]methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca2+ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.