Degranulation and appearance of vesicular inclusions in canine C cells after administration of antithyroid drug

Summary When stimulated by the feeding of ethylenethiourea for a period of 6 months, dog thyroid glands increased to approximately 30 times their normal size. Not only follicular cells but also C cells had hyperplastic and hypertrophic features. Compared with normal controls there was in the C cells a marked decrease in secretory granules immunoreactive for calcitonin. Furthermore, vesicular inclusions of various sizes and dilated nuclear envelopes, which showed a positive immunoreaction for calcitonin, were observed in the C cells. These findings indicate that the antithyroid drug interferes with synthesis of calcitonin by C cells, probably by inhibiting the conversion of a larger precursor to calcitonin.     

Thiol and aspartyl proteolytic activities in secretory vesicles of bovine pituitary.

Thiol and aspartyl proteolytic activities in isolated secretory vesicles of neural (NL) and intermediate (IL) lobes of bovine pituitary were characterized with heterologous enkephalin and tachykinin precursor substrates, 35S-(Met)-preproenkephalin and 35S-(Met)-beta-preprotachykinin. IL and NL secretory vesicles contained thiol-dependent proteolytic activity that cleaved the enkephalin precursor with a pH optimum of 4.5; this activity resembled a novel "prohormone thiol protease' previously purified and characterized from adrenal medulla chromaffin granules. IL and NL vesicles also demonstrated aspartyl proteolytic activity with acidic pH optimum, as shown by pepstatin A inhibition of tachykinin and enkephalin precursor cleaving activity. This activity may be related to a previously characterized chromaffin granule aspartyl protease (CGAP) related to cathepsin D (2), as indicated by the presence of immunoreactive CGAP in NL secretory vesicles by anti-CGAP immunoblots. These results show that pituitary secretory vesicles, like chromaffin granules, may contain similar thiol-dependent and aspartyl proteolytic activities.     

Study of the protein-peptide composition of secretory granules of bovine lactogenic hormone

A combination of gel chromatography, fluorimetric analysis and polyacrylamide gel electrophoresis with immunochemical identification, the protein-peptide composition of secretory granules of the lactogenic hormone (LTH) isolated from the anterior lobe of bovine hypophysis was investigated. It was found that the peptide content of the granules is less than 3% of that of immunoreactive LTH. Using gel chromatography, the secretory granules were found to contain a hormone monomer and two immunoreactive forms with Mr 42 and 64 kD. With respect to immunoreactivity, the hormone form content was 90, 3 and 7%, respectively. Using polyacrylamide gel electrophoresis and subsequent immunochemical identification, the presence of four immunoreactive forms of LTH were identified in the secretory granules.     

Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000- 15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p- chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.     

Distribution of nerve growth factor in the submandibular gland of the male and female mouse

Summary Nerve growth factor (NGF) was localized in the mouse submandibular gland by means of indirect immunofluorescence applied to 0.5 mthick sections of freeze-dried, plastic-embedded tissue. The antibody to NGF (I_gG-fraction) was raised in rabbits immunized with pure 2.5 S NGF from submandibular glands of adult male mice.In the male gland anti-NGF bound selectively to the secretory granules was present in the cells of the granular ducts. Immunoreactive granules extended from the perinuclear region toward the apical pole. In the female gland immunoreactive cells and granules were considerably less abundant than in males. Immunofluorescence was confined to individual secretory cells located in the wall of the granular striated duct.In the present study no support was found for the hypothesis suggesting that immunoreactive NGF is formed within the secretory granules during their transport from the perinuclear region to the apical pole.     

Properties of plasma membrane-induced amylase release from rat parotid secretory granules: effects of Ca2+ and Mg-ATP.

A secretory granular fraction (SG) and a plasma membrane rich fraction (PM) have been isolated from rat parotid gland by differential and Percoll gradient centrifugation. With these two fractions, a cell-free interaction system has been reconstituted to clarify the exocytotic interaction between the secretory granules and plasma membranes, and the conditions of amylase release from SG have been characterized in vitro. The addition of PM into this assay system induced a rapid and transient release of amylase from SG. Some other membranes such as erythrocyte ghosts also mimicked the effect of PM. This release was increased by Ca2+, but was not completely blocked by EGTA. Simultaneous addition of 1 mM ATP with 1 mM MgCl2 (Mg-ATP) in the presence of Ca2+ reduced this release. However, in spite of the existence of Mg-ATP, the stimulation of PM-induced amylase release was caused by Ca2+ in a concentration-dependent manner (10(-7)-10(-3) M). These results suggest that Ca2+ and Mg-ATP should participate as important regulators in the exocytotic interaction between secretory granules and plasma membranes in this system. Furthermore, the differences between our system and intact cells are also discussed.     

Nonconverted, amino acid analog-modified proinsulin stays in a Golgi- derived clathrin-coated membrane compartment

The secretion of insulin by the pancreatic B-cell involves a passage of the newly synthetized (pro)insulin polypeptides across the Golgi apparatus, at the trans pole of which secretory proteins are released as a population of secretory granules characterized by a clathrinlike coat on segments of their limiting membrane. When the conversion of radiolabeled proinsulin to insulin was inhibited by replacing arginine and lysine with the aminoacid analogs, canavanine and thialysine, the nonconverted radioactive material remained associated with Golgi-derived, coated secretory granules. The coat was characterized as clathrin-containing by immunocytochemistry. Under analog treatment, the noncoated, storage secretory granules did not become markedly labeled during the pulse-chase experiment. These data are compatible with the hypothesis that in normal conditions, the maturation of the coated compartment into noncoated granules is linked to the effective conversion of the prohormone.     

Comparison of the Molecular Forms of the Kex2/Subtilisin-Like Serine Proteases SPC2, SPC3, and Furin in Neuroendocrine Secretory Vesicles Reveals Differences in Carboxyl-Terminus Truncation and Membrane Association

Abstract: The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full-length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full-length 86-kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl-terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane-associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86-kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus-truncated 64-kDa form.     

Purification of mitochondria and secretory granules isolated from pancreatic beta cells using Percoll and Sephacryl S-1000 superfine

Mitochondria and secretory granules were isolated from beta-cell-rich pancreatic islets of ob/ob mice. Crude fractions obtained by differential centrifugation were subjected to centrifugation on isotonic Percoll. The gradient medium was removed from the resulting fractions by gel filtration on Sephacryl S-1000 Superfine. When compared to the crude fractions, the purified mitochondrial fraction exhibited a 4.5-fold increase in citrate synthase activity, a 70% reduction of lysosomal arylsulfatase, and a 40% decrease of contamination with granular insulin. In the purified secretory granule fraction, the insulin content was as high as 60% of the total protein (albumin standard) with arylsulfatase unchanged and no detectable citrate synthase activity.     

鲫鱼后鳃体结构与功能的研究

本文对鲫鱼后鳃体的形态结构及其功能进行了初步研究。结果表明,鲫鱼后鳃体是一种内分泌腺体。腺体由滤泡组成,滤泡上皮包括二种颗粒细胞(亮细胞和暗细胞)和一种支持细胞。对不同性腺期的鲫鱼后鳃体的观察表明,后鳃体滤泡细胞随性腺发育增加高度,滤泡增生,腺体体积增大。而性腺成熟后期,后鳃体滤泡上皮有崩溃现象。揭示后鳃体可能与性腺发育有关。用免疫组织化学的非标记过氧化物酶技术对鲫鱼后鳃体进行激素降钙素的定性定位研究,观察到滤泡上皮的颗粒细胞发生阳性反应,由此证明颗粒细胞可分泌降钙素。     

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.     

Studies on in vitro effect of serotonin on calcitonin secretion by rat thyroid C cells

Thyroid glands of young rats were incubated for 3 h in Eagle's solution supplemented with 5-hydroxy-l-tryptophan (5-HTP) or with serotonin. Following control incubations or incubations with serotonin, no serotonin could be demonstrated in C cells using immunocytochemical techniques. However, serotonin was demonstrated in the secretory granules of all C cells following incubation with 5-HTP. The secretory function of C cells was evaluated by ultrastructural and immunocytochemical studies, and by calcitonin radioimmunoassays of the incubation medium. Following incubation with 5-HTP, the secretory function of the majority of C cells was inhibited, and calcitonin levels in the media were decreased. Incubation with serotonin produced an increased secretory function of C cells and higher calcitonin levels in the media. The results indicate that serotonin and its direct precursor, 5-HTP, affect calcitonin secretion by rat thyroid C cells by distinct mechanisms.     

Morphometric and electron microscopic studies of the secretory granules of neurosecretory cells of the supraoptic and paraventricular nuclei of white rats and mice]

The morphometrical and electron microscopic analysis of secretory granules in the perikaryons of neurosecretory cells of the supraoptic and paraventricular nuclei in male rats and mice has shown than in the cells of these nuclei in both species of animals there occur secretory granules of the same kind and size. Therefore this method fails to determine which of them contain oxytocin and which of them contain vasorpressin. The neurosecretory granules located in the Golgi apparatus zone are of a less size and have more osmiophilic cnetral material than the granules localized on the periphery which mainly have granular central material and are of a greater size. The distinctions in the size and type of secretory granules are associated with certain stages of their "maturation". Granular particles appear to be "swallen", more active forms of storing neurohormones. The presence of larger granular particles in the supraoptic nucleus of mice allows to suggest greater reactivity of this nucleus than in rats which is likely to be associated with a higher ability of mice, as compared with rats, to adaptate to disturbances in water-salt metabolism.     

Studies on in vitro effect of serotonin on calcitonin secretion by rat thyroid C cells

Summary Thyroid glands of young rats were incubated for 3 h in Eagle's solution supplemented with 5-hydroxy-l-tryptophan (5-HTP) or with serotonin. Following control incubations or incubations with serotonin, no serotonin could be demonstrated in C cells using immunocytochemical techniques. However, serotonin was demonstrated in the secretory granules of all C cells following incubation with 5-HTP. The secretory function of C cells was evaluated by ultrastructural and immunocytochemical studies, and by calcitonin radioimmunoassays of the incubation medium. Following incubation with 5-HTP, the secretory function of the majority of C cells was inhibited, and calcitonin levels in the media were decreased. Incubation with serotonin produced an increased secretory function of C cells and higher calcitonin levels in the media. The results indicate that serotonin and its direct precursor, 5-HTP, affect calcitonin secretion by rat thyroid C cells by distinct mechanisms.     

Thyroid medullary carcinoma of the Djungarian hamster Phodopus sungorus. Ultrastructural evidence for the production of normal and atypical intracellular granules

We have carried out an electron microscopic and immunocytochemical study of thyroid medullary carcinoma arising spontaneously in the Djungarian hamster, Phodopus sungorus. At the ultrastructural level the cytoplasm of tumor cells contained numerous round to slightly elongated, dense-cored secretory granules. The number of secretory granules differed from cell to cell in the tumor, being scanty in some cells but more or less abundant in most. Electron microscopic-immunocytochemistry demonstrated that all dense-cored secretory granules in all tumor cells exhibited calcitonin immunoreactivity. In approximately 10% of the tumor cells, unusual star-shaped secretory vesicles were also found in the cytoplasm. These vesicles contained a small, but well-defined, lucent core surrounded by a region of finely granular material of greater electron density. The outer contour of these unusual vesicles was stellate rather than smooth. They appeared to originate not from the Golgi complex, but from the rough endoplasmic reticulum. These atypical stellate vesicles did not show any calcitonin immunoreactivity. Furthermore, in a small number of tumor cells (approximately 1%) a third type of membrane enclosed structure was found. These were conspicuous rods 1-5 micron in length with tapering ends and a crystalline substructure. The presence of both normal and atypical secretory granules in some tumor cells suggests that carcinogenic transformation may interfere with the normal synthesis and assembly of secretory products by the cell.     

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.     

Immunocytochemical localization of serotonin-like immunoreactivities in the ciliated epithelium of the frog palatine mucosa

Immunocytochemical localization of serotonin (5-HT)-like immunoreactivities was studies in the ciliated epithelium of the frog palatine mucosa by the peroxidase anti-peroxidase (PAP) method. 5-HT-like immunoreactivity was found only in the small granular vesicles (100-150 nm in diameter) and not in any mature large secretory granules or in other cell organellae in the goblet cells. No 5-HT-like immunoreactivities were found in any other epithelial and secretory cells in the palatine epithelium. It appears therefore that 5-HT-like immunoreactive granular vesicles have certain physiological effects on the ciliary movement of the ciliated cells or in the goblet cells.     

The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein

The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.     

The pancreatic membrane protein GP-2 localizes specifically to secretory granules and is shed into the pancreatic juice as a protein aggregate

GP-2 is the major secretory granule membrane glycoprotein of the exocrine pancreas and appears in the pancreatic juice in a modified sedimentable form. We have localized GP-2 in the rat pancreas at the electron microscopic level using affinity-purified antibodies and found it to be concentrated in the zymogen granules and in the acinar lumen. Label was also present on the apical and basolateral plasma membranes but prior treatment of the sections with periodate to eliminate the contribution of highly antigenic oligosaccharide moieties reduced substantially the staining of the basolateral surface. Approximately 45% of the GP-2 in the granules was not membrane-associated but appeared instead in the granule lumen. Parallel biochemical characterization of GP-2 in isolated secretory granules demonstrated that 60% fractionated with the membranes after granule lysis while 40% remained in the content fraction. Unlike the membrane-associated form of the protein, which is linked to the membrane via glycosyl-phosphatidylinositol (GPI), GP-2 in the content did not enter the detergent phase upon Triton X-114 extraction; nor was it sedimentable at 200,000g, as is characteristic of the form collected in the pancreatic juice. In addition, GP-2 in the pancreatic juice was recovered in the aqueous phase during Triton X-114 extraction and yet remained sedimentable after detergent extraction, demonstrating that its ability to remain in large aggregates was independent of lipid. These results are consistent with a life cycle for the protein that begins with synthesis of a membrane-associated precursor that can be converted by lipolytic or proteolytic cleavage to a soluble form within the zymogen granule. Further modification to a sedimentable form may then occur in the pancreatic juice.     

Properties of membrane phospholipids and their fatty acyl compositions of secretory granules from rat parotid gland

A secretory granular fraction isolated from rat parotid glands was remarkably different from a microsomal fraction in its phospholipid composition. It had higher levels of lysophospholipids (8%) and phosphatidylethanolamine (31%), while there were lower levels of phosphatidylcholine (40%) and phosphatidylserine (2.1%) than the microsomal fraction. However, fatty acid compositions of individual phospholipid classes from the two subfractions were found to be nearly similar to each other. ESR analysis demonstrated that extracted phospholipids from the secretory granular fraction were more fluid than those from microsomes. The relevance of these observations to physiological function of secretory granules is discussed.