Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts.

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.     

Comparison of the secretory processes in the parotid and sublingual glands of the mouse. 1. Regulation of the secretory processes.

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.     

Protein processing within the secretory pathway.

Endoproteolytic cleavage of hormone and neuropeptide precursors, as well as many complex proteins, such as coagulation factors and viral glycoproteins, is a key process in the generation of bioactive polypeptides. These cleavages typically occur at the dibasic amino acid residues Lys-Arg or Arg-Arg. The enzymes responsible for the processing belong to a newly discovered family of serine proteases related to the bacterial subtilisins. These include PACE (furin), PC1/PC3, PC2 and PACE4, which have all been characterized functionally and structurally.     

Proprotein-processing endopeptidases of the insulin secretory granule.

Enzymological studies have implicated two Ca2+ dependent endopeptidases in the conversion of proinsulin to insulin: a type 1 activity and a type 2 activity which cleave on the C-terminal side of R31R32 and K64R65 in proinsulin, respectively. These activities were further characterized and their relationship to the mammalian family of subtilisin-like proteases was investigated. PC2 was expressed in neuroendocrine tissues and in insulinoma secretory granule fractions predominantly as a 65kDa protein. On anion-exchange chromatography of solubilized granules, PC1/3 immunoreactivity comigrated with a peak of type 1 activity whereas PC2 immunoreactivity coeluted with the peak of type 2 endopeptidase activity. PC2 antiserum gave a specific immunoprecipitation of type 2 activity from insulin granule extracts. It was concluded that the PC2 gene-product has type 2 endopeptidase activity.     

Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway.

Immunoelectron microscopy of Saccharomyces cerevisiae cells embedded in Lowicryl K4M has been used to localize invertase and plasma membrane (PM) ATPase in secretory organelles. sec mutant cells incubated at 37 degrees C were prepared for electron microscopy, and thin sections were incubated with polyclonal antibodies, followed by decoration with protein A-gold. Specific labeling of invertase was seen in the lumen of the endoplasmic reticulum, Golgi apparatus, and secretory vesicles in mutant cells that exaggerate these organelles. PM ATPase accumulated within the same organelles. Double-immune labeling revealed that invertase and PM ATPase colocalized in secretory vesicles. These results strengthen the view that secretion and plasma membrane assembly are biosynthetically coupled in yeast.     

Biogenesis of secretory granules. Implications arising from the immature secretory granule in the regulated pathway of secretion

In endocrine cells the regulated secretion of hormones, peptides, enzymes and neurotransmitters into the external medium occurs when mature secretory granules fuse with the plasma membrane. Secretory granules form at the trans-Golgi network (TGN) by envelopment of the dense-core aggregate of regulated secretory proteins by a specific membrane. The secretory granules initially formed at the TGN, referred to here as immature secretory granules, are morphologically and biochemically distinct from mature secretory granules. The functional similarities and differences between the immature secretory granule and the mature secretory granule, and the events involved in the maturation of the secretory granules are briefly discussed.     

Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network.

The effects of brefeldin A (BFA) on membrane traffic between the trans-Golgi network (TGN) and the plasma membrane were investigated in intact PC12 cells and in a cell-free system derived from PC12 cells. In intact cells, BFA caused a virtually complete block of constitutive secretion, as indicated by the lack of release from, and accumulation in, the cells of a [35S]sulfate-labeled heparan sulfate proteoglycan (hsPG). Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that this block was due to the inhibition of formation of constitutive secretory vesicles (CSVs) from the TGN. BFA did not block the depolarization-induced release of [35S]sulfate-labeled chromogranin B (CgB) and secretogranin II (SgII) from secretory granules formed prior to the addition of the drug, showing that BFA does not block secretory granule fusion with the plasma membrane. The presence of BFA did, however, prevent the appearance of [35S]sulfate-labeled CgB and SgII in secretory granules, indicating that the drug inhibits the formation of secretory granules from the TGN. Evidence for a direct block of vesicle formation by BFA was obtained using a cell-free system derived from [35S]sulfate-labeled PC12 cells. In this system, low concentrations of BFA (5 micrograms/ml) inhibited the formation of the hsPG-containing CSVs and that of the SgII-containing secretory granules from the TGN to the same extent (50-60%) as, and in a non-additive manner with, the nonhydrolyzable GTP analogue GTP gamma S. Consistent with the inhibitory effects of BFA on vesicle formation from the TGN, BFA treatment of intact PC12 cells led to the hypersialylation of CgB, which presumably was due to the increased residence time of the protein in the TGN. In conclusion, our data are consistent with, and allow the generalization of, the concept that the BFA-induced block of anterograde membrane traffic results from the inhibition of vesicle formation from a donor compartment.     

Myristoylation of proteins in the yeast secretory pathway.

Protein myristoylation was investigated in the yeast secretory pathway. Conditional secretory mutations were used to accumulate inteRmediaries in the pathway between the endoplasmic reticulum and Golgi (sec 18, 20), within the Golgi (sec 7), and between the Golgi and plasma membrane (sec 1, 3, 4, 5, 6, 8, 9). The accumulation of vesicles was paralleled by the enrichment of a defined subset of proteins modified either via ester or amide linkages to myristic acid: Myristoylated proteins of 21, 32, 49, 56, 75, and 136 kDa were enriched between the endoplasmic reticulum and Golgi; proteins of 21, 32, 45, 56, 75, 136 kDa were enriched by blocks within the Golgi; and proteins of 18, 21, 32, 36, 49, 68, and 136 kDa were trapped in a myristoylated form by blocks between the Golgi and plasma membrane. This enrichment of myristoylated proteins was reversed upon returning the cells to the permissive temperature for secretion. The fatty acid was linked to the 21-kDa protein via a hydroxylamine-resistant amide linkage (N-myristoylation) and to the proteins of 24, 32, 49, 56, 68, 136 kDa via hydroxylamine-labile ester linkage (E-myristoylation). In addition, myristoylated proteins of 21, 56, and 136 kDa were glycosylated via amino linkages to asparagine. This suggests they are exposed to the lumen of the secretory pathway. Three proteins (24, 32, and 56) were E-myristoylated in the presence of protein synthesis inhibitors, indicating this modification can occur posttranslationally. After using cycloheximide to clear protein passengers from the secretory pathway the 21-, 32-, and 56-kDa proteins continued to accumulate in a myristoylated form when vesicular transport was blocked between the Golgi and plasma membrane. These data suggest that myristoylation occurs on a component of the secretory machinery rather than on a passenger protein.     

Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules.

Non-hydrolysable analogues of GTP, such as GTP gamma S and GMP-PNP, have previously been shown to inhibit the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [A1F4]-, a compound known to act on trimeric G-proteins. Addition of highly purified G-protein beta gamma subunits stimulated, in a differential manner, the cell-free formation of both CSVs and ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein alpha subunit in the TGN. We conclude that trimeric G-proteins regulate the formation of secretory vesicles from the TGN.     

Atomic force microscopy study of the secretory granule lumen.

We have used an atomic force microscope to study the mechanical properties of the matrix found in the lumen of secretory granules isolated from mast cells. The matrices were insoluble and had an average height of 474 +/- 197 nm. The volume of these matrices increased reversibly about tenfold by decreasing the valency of the bathing external cation (La3+ < Ca2+ < Na+). The elastic (Young's) modulus was found to decrease by about 100-fold (4.3 MPa in La3+ to 37 kPa in Na+) upon a tenfold increase in the matrix volume. A swollen granule matrix had an elastic modulus similar to that of gelatin in water. The elastic modulus was inversely related to the change in the volume of the matrix, following a relationship similar to that predicted for the elasticity of weakly cross-linked polymers. Our results show that the matrix of these secretory granules have the mechanical properties of weak ion exchange resins, lending strong support to an ion exchange mechanism for the storage and release of cationic secretory products.     

Thymic control of secretory antibody responses in the rat.

The effect of neonatal thymectomy on salivary and serum antibody responses was studied in rats. Local immunization of thymectomized rats with a T-dependent antigen (DNPBGG) elicited negligible amounts of IgA anti-DNP antibody in saliva. In contrast, both normal and sham-thymectomized animals demonstrated substantial levels of salivary IgA antibody. All thymectomized rats locally injected with a T-independent antigen (DNP-Lys-Ficoll) exhibited salivary IgA antibody production. Salivary IgG antibodies were somewhat decreased in thymectomized rats injected with either antigen; however, the final effect of T cell deprivation on IgG synthesis was not as pronounced as on IgA synthesis. Serum IgA antibody was induced in control rats injected with DNPBGG, whereas this Ig class of antibody was absent in thymectomized rats. The results suggest that thymus-derived cells exert a regulatory influence on both serum and secretory IgA responses to antigens.