Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.     

The fine structure of the midgut in the mite Anystis baccarum (L.) (Acari, Actinedida: Anystidae)

The ventriculus and the midgut caeca of the fed females of Anystis baccarum (L.) were investigated by using light and electron microscopy. In addition to the main type of polyfunctional digestive cells, special secretory cells were detected in the anterior region of the ventriculus. The shape and the ultrastructure of the digestive cells vary depending on their physiological state. Intracellular digestion, absorption or excretion processes prevail at different stages of the cell cycle. The secretory cells are characterized by the presence of extensive rough endoplasmic reticulum, filling whole space of the cell. These cells do not contain the apical network of pinocytotic canals, which are typical for the digestive cells. Three types of secretory granules were found in the cytoplasm of the secretory cells that probably correspond to three sequential stages of granulogenesis. The primary secretory granules are formed by the fusion of Golgi vesicles. The primary granules fuse to form complex vesicles with heterogeneous contents. These secondary granules aggregate to form very large inclusions of high electron density (tertiary secretory granules), which probably represent the storage of the secretory product. All types of secretory granules were observed close to the apical plasmalemma.     

Immunocytochemical study of the endocrine pancreas in the grey kangaroo (Macropus fuliginosus)

Summary The endocrine pancreas of the grey kangaroo,Macropus fuliginosus, was investigated by means of immunocytochemistry using the PAP method on the same section at the light- and electron-microscopic levels. Semithin plastic sections were stained individually with primary antibodies for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), and then photographed. Sections were osmicated, re-embedded in BEEM capsules, and ultrathin sections made and examined. The same labelled cells as in the semithin sections were localised in the thin sections, photographs taken and the morphology of secretory granules studied. The insulin cells were pleomorphic; their secretory granules displayed an electron-dense core surrounded by an empty halo. The glucagon cells possessed granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells had larger, less dense secretory granules. The PP cells showed small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborted by correlated immunocytochemical data at the light-and electron-microscopic levels.     

The granin (chromogranin/secretogranin) family

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.     

Isolation of a protein from the plasma membrane of adrenal medulla which binds to secretory vesicles.

Solubilized proteins of the plasma membrane of bovine adrenal medulla were fractionated on the basis of their affinity for secretory vesicles. The isolation procedure included preparation of a highly purified fraction of plasma membranes, its solubilization in detergent, and application to a column prepared from glutaraldehyde-fixed chromaffin granules. Using this technique, one major polypeptide (80% of the material bound) was isolated. This protein has been shown to originate from the plasma membrane and has no affinity for fixed bovine adrenal medullary mitochondria or lysosomes. It is eluted most effectively by low pH (3.0) and can be rebound and re-eluted from fixed secretory granules. In sodium dodecyl sulfate and beta-mercaptoethanol it has an apparent molecular weight of 51,000. In addition, two minor components, comprising about 20% of the material bound were detected having apparent molecular weights in sodium dodecyl sulfate of 14,000 and 62,000. It is suggested that such a molecule could function as a plasma membrane-located receptor for chromaffin granules during the secretory process.     

Regulation of secretory granule formation in chronically hypersecretory melanotrophs in the rat pituitary

The formation of secretory granules in chronically hypersecretory melanotrophs in the rat pituitary was studied. Hypersecretion was induced by treatment with the dopamine antagonist haloperidol (1.5 mg/kg daily for 7 days), which releases the normal neural dopaminergic inhibition of secretion from the melanotroph. Morphometric analysis showed a 100% increase in the volume fraction of granular endoplasmic reticulum after haloperidol treatment, while the volume fractions of electron-dense granules, electron-lucent granules and the Golgi apparatus were unaltered. The mean diameter of the mature secretory granules was increased by 10%, indicating a 30% increase in mean granule volume. A similar increase in diameter was observed in condensing granules within the Golgi area. With earlier results on the effect of chronic inhibition the study shows that a main adaptive response of the melanotroph to altered secretory conditions is a change in the volume of the secretory granules, regulated by a mechanism that operates at an early stage of granule formation.     

Immunocytochemical and ultrastructural characterization of mammosomatotrope-, growth hormone-, and prolactin-cells from the gilthead sea bream (Sparus aurata l., Teleostei): an ontogenic study

Growth hormone (GH), prolactin (PRL), and mammosomatotrope (MS) cells of gilthead sea bream, Sparus aurata, a teleost fish, were studied in specimens from hatching to 15 months (adults) using conventional electron microscopy and an immunogold method using anti-tilapia GH sera and anti-chum salmon PRL serum. MS cells, immunoreactive to both anti-GH sera and anti-PRL sera, had been first identified in fish in a previous study in newly hatched larvae and in older larvae and juvenile specimens of Sparus aurata by light microscopic immunocytochemistry. In the present work, MS cells reacted positively to immunogold label only in older larvae and juveniles and their secretory granules immunoreacted with both GH and PRL antisera or with only one of them. MS cells were ultrastructurally similar to the PRL cells, with which they coincided in time. This is the first report on the ultrastructural characterization of MS cells in fish. In adults, the secretory granules of GH cells (immunoreactive to anti-GH serum) were mainly round, of variable size, and had a homogeneous, highly electron-dense content. Irregularly shaped secretory granules were also present. PRL cells (immunoreactive to anti-PRL serum) were usually observed in a follicular arrangement; they showed few, small, and mainly round secretory granules with a homogeneous and high or medium electron-dense content. Some oval or elongated secretory granules were also observed. GH and PRL cells that showed involutive features were also found. In newly hatched larvae, GH, PRL, and MS cells could not be distinguished either by their ultrastructure or by the immunogold labeling of the secretory granules. In 1-day-old larvae, presumptive GH and PRL cells were observed according to their position in the pituitary gland. In 2-day-old larvae, a few cells showed some of the ultrastructural features described for GH and PRL cells of adults. During development, the number, size, and shape of the secretory granules in both cell types clearly increased and the organelles developed gradually. Some GH cells were found undergoing mitosis.     

Studies of the neurohaemal organ (sinus gland) of the portunid crab Portunus sanguinolentus (Herbst) (Brachyura). 1. Morphology and histology in relation to moulting

The morphological and histological characters of the neurohaemal organ (sinus gland) of Portunus sanguinolentus are described in detail. The sinus gland lies on the dorsal surface of the optic ganglia, opposite the medulla interna. Histological techniques showed the presence of three tinctorially different secretory granules in the sinus gland. The predominant type of secretory material is basophilic and occurs as large granules, while two types of acidophilic material occur near the basement membrane. Cyclic changes in the relative amounts of acidophilic and basophilic material in correlation to moulting are also discussed. Allochthonous cells present in the sinus gland are identified.     

A subset of p23 localized on secretory granules in pancreatic beta-cells.

Proteins on the membrane of secretory granules (SGs) involved in their biogenesis and exocytosis are poorly characterized compared with those of synaptic vesicle in neurons. Thus the secretory granule membrane was prepared from a mouse pancreatic beta-cell line MIN6 by subcellular fractionation, and protein constituents were analyzed by microscale two-dimensional liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Using this proteomics approach, one of the p24 family proteins, p23, was unexpectedly found in the granule fraction, although p24 proteins are generally regarded as functioning in the early secretory pathways between the endoplasmic reticulum and the Golgi apparatus. We further showed that p23 is expressed at high levels in endocrine cells. Furthermore, immunocytochemical analyses of pancreatic beta-cells at the light and electron microscopic levels demonstrated that a significant amount of p23 is localized on the insulin granule membrane, although it is most intensely concentrated at the cis-Golgi compartment as previously shown in non-endocrine cells. These findings suggest that a fraction of p23 enters post-Golgi compartments and may function in the biogenesis and/or quality control of SGs.     

ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS : I. Isolation of a Secretory Granule Fraction

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.     

Localization of glutathione-insulin transhydrogenase (protein-disulfide interchange enzyme) in pancreas using immunocytochemistry, immunodiffusion, and enzymatic activity assay techniques

The localization of the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (GIT), in rat and mouse pancreas was studied by protein A-gold immunocytochemistry, immunodiffusion, and assay of enzymatic activity. Immunocytochemistry on tissue sections using antibody to GIT and protein A-gold complex indicated the presence of GIT in alpha and beta cells in islets as well as acinar cells. The beta cells in obese (ob/ob) hyperinsulinemic mice showed increased GIT immunoreactivity. In both alpha and beta cells, GIT immunoreactive sites were associated predominantly with secretory granules. In pancreas from rats injected with glibenclamide, the degranulated beta cells contained GIT immunoreactive sites on the cisternal surface of the rough endoplasmic reticulum (RER). In acinar cells, the RER, Golgi elements, condensing vacuoles, and zymogen granules possessed GIT immunoreactive sites as did mitochondria. Immunocytochemistry on sections of isolated subcellular fractions showed that GIT was associated with different membranes. The enzymatic activity of GIT was found in the following order: Golgi elements greater than mitochondria greater than microsomes greater than zymogen granules greater than cytosol. In Ouchterlony immunodiffusion tests, each subcellular fraction showed a precipitin band which was continuous with that of purified GIT, a result indicating the presence of immunologically identical GIT in all fractions.     

The cytomorphology of goblet cells of the fetal intestine. Studies of the large intestine of cattle (Bos primigenius taurus)

In the region of the base of the intestinal crypts undifferentiated goblet cells display a configuration and constellation of organelles and membrane structures that are indicative of their importance for function. These images at this stage of development deliver a scenario of the mechanism of secretory granule production: aggregates of protein vesicles from the "transitional elements" (PALADE) of the granular endoplasmic reticulum are, so to speak, rolled up on the trans side of the Golgi apparatus by inversion of peripheral membrane segments of the innermost Golgi lamellae, thereby forming corpuscles. The origin of the capsulated vacuoles, which contain vesicles as single elements or as conglomerates, is well established. Their capsule consists of a trilaminar external and external and internal membrane; between them lies condensed material of the Golgi apparatus. In the opinion of the present author, the development of the ensheathed vacuoles represents a basic, more general mechanism. In contrast, the further steps of synthesis, for the formation of secretory granules, are more heterogeneous. Condensation of the vesicles and the inner capsular membrane results in the formation of a prosecretory granule, which in the basic element in the process of secretory granule production. The prosecretory granules develop singly or by fusion with other granules to give primary secretory granules. The complexity of this mechanism of secretory granule formation, however, becomes evident when considering the apposition of capsulated vacuoles and prosecretory--primary--secondary secretory granules, of prosecretory and primary secretory granules as well as prosecretory granules and secondary secretory granules. Generally, primary granules show a tendency to become secondary secretory granules or to fuse with them. During maturation of the goblet cells the secretory granules fuse to form larger mucous bodies in the theca by fusion of the laminae of the membranes; a final product, there is a homogeneous mucous mass devoid of membranes.     

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: II. Distribution of radioactive peptide hormones and hormone precursors in subcellular fractions after pulse and pulse- chase incubation of islet tissue

Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.     

Association between endocrine pancreatic secretory granules and in- vitro-assembled microtubules is dependent upon microtubule-associated proteins

By use of dark-field light microscopy, secretory granules isolated from the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitro from brain homogenates. Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPs) and not to microtubules assembled from purified tubulin. The addition of a MAP fraction to purified tubulin restored secretory granule binding. The secretory granules were released from MAP-containing microtubules by the addition of Mg-ATP but not by other nucleotides. The number of secretory granules bound to MAP-containing microtubules was increased in the presence of cyclic AMP. In addition to the associations of secretory granules with microtubules, MAP-containing microtubules also associated with each other. These laterally associated microtubules were dispersed by the addition of Mg-ATP. Electron micrographs confirmed that the associations between MAP-containing microtubules and secretory granules as well as the associations of microtubules with one another were mediated by the high molecular weight MAPs known to project from the surface of in-vitro-assembled microtubules.     

Morphogenetic processes in cultured cells from integumentary glands of deer sambar (Cervus unicolor Kerr)

The present paper describes an attempt to maintain the metatarsal and caudal glands of deer sambar (Cervus unicolor Kerr) in diffusion chambers. Another purpose was to provide information on the secretory granule of these glands. Our results show that cultured gland cells have the capacity to reassociate into islet-like organoids in diffusion chambers. Competence in some epithelial cell types may probably be regulated as a function of growth. Scanning electron microscopy of secretory cells showed that the cells in the culture often had numerous short filopodia and blebs. The secretory granules of the caudal gland were spherical and uniform in size. Some of the secretory granules had pseudopodia-like protrusions. By means of SEM, the present study provides evidence of rapid structural changes in the surfaces of epithelial cells in the course of cultivation. It has been suggested that the structural organization in the cell colonies serves to "guide" the function of secretory cells.     

Isolation and characteristics of bovine pituitary secretory granules

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measureable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both growth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10(-5) M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.     

Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases.

Native poly(hydroxybutyrate) (PHB) granules, purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei. The R. rubrum depolymerizing system requires pretreatment of granules with a heat stable 'activator' fraction; the activator can be replaced by mild trypsin treatment. Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide (CTAB) and an anionic detergent, (sodium cholate). Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems; however, some differences were noted. Cholate granules were hydrolyzed in the absence of the R. rubrum activator fraction. Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system. A Triton X-114 extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase. The inhibition was reversed by the activator fraction. Detergent extracts of granules activated with the R. rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules. These data suggest that the activator acts to modify an inhibitor present on native granules.     

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae): acid phosphatase and ATPase activities localization in salivary glands of females during the feeding period

This study investigates the presence and the localization of acid phosphatase and ATPase in the salivary glands of Rhipicephalus (Boophilus) microplus female ticks during feeding. Semi-engorged females showed a larger amount of acid phosphatase compared to those at beginning of feeding, localized mainly in the apical portion of the secretory cells, and in the basal labyrinth of the interstitial cells. Ultrastructural observations also demonstrated its presence in secretion granules and inside some nuclei of secretory cells at beginning of feeding. Acid phosphatase in a free form probably has a hemolymph and/or ribosomal origin and participates in salivary gland secretion control. ATPase was detected in basal membrane of all types of acini and/or in the cytoplasm of the secretory cells at both feeding stages. The enzyme activities found strongly suggests that cell death by apoptosis occurs during the degenerative process.     

Corticotroph cells in primary cultures of rat adenohypophysis: A light and electron microscopic immunocytochemical study

Summary Monolayer cultures of trypsin-dispersed cells of the rat adenohypophysis were grown for 5 to 54 days. ACTH was localized by immunocytochemistry using an antiserum to synthetic ACTH^1–28 prepared in rabbit and sheep anti-goat immunoglobulin coupled with peroxidase. ACTH content of the culture medium was measured by radioimmunoassay.Corticotrophs were found in the cultures and ACTH in the medium at each cultivation time. The corticotrophs retained their essential morphological characteristics. Immunological staining was found in the secretory granules, some tubular or saccular structures, parts of the rough endoplasmic reticulum, and the cytoplasmic matrix. Immature secretory granules in the Golgi apparatus as well as some Golgi elements showed different degrees of immunoreactivity. In agreement with the high ACTH content of the culture medium the number, size and shape of the secretory granules, the active Golgi apparatus, the high amount of extragranular ACTH as well as pictures suggesting granule extrusion claim for a high ACTH synthesis and transport (and low ACTH storage) in the cultured corticotrophs.     

Isolation and characteristics of bovine pituitary secretory granules

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measurable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both groth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10⁻⁵ M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.