Comparison of prohormone-processing activities in islet microsomes and secretory granules: evidence for distinct converting enzymes for separate islet prosomatostatins

In previous work we have examined the nature of converting enzymes for proinsulin, proglucagon, and prosomatostatin-I (PSS-I) in secretory granules isolated from anglerfish islets. The purpose of the present study was to extend the examination of precursor conversion to islet microsomes and to compare prohormone processing, including that of PSS- I and prosomatostatin-II (PSS-II), in islet secretory granules and microsomes. Microsomes (rough endoplasmic reticulum [RER] and Golgi complex) and secretory granules were prepared from anglerfish islets by differential and discontinuous density-gradient centrifugation. Microsomes were further fractionated into Golgi- and RER-enriched subfractions. Lysed secretory granule or microsome preparations were incubated in the presence of a mixture of radioactively labeled islet prohormones. Extracts of products generated were subjected to analysis by gel filtration and high-pressure liquid chromatography. Accuracy of product cleavage was monitored by comparing high-pressure liquid chromatography retention times from the radiolabeled in vitro conversion products with the retention times of labeled products from tissue extracts. All converting activity in microsomes was found to be similar to that in granules in that it had a pH optimum near pH 5 and was inhibited by p-chloromercuribenzoate. No significant differences in the converting activity of Golgi complex- and RER-enriched subfractions of microsomes was observed. The proinsulin, proglucagon, and PSS-II converting-enzymes, which were found in islet secretory granules, were also present and membrane-associated in islet microsomes. However, converting activity for PSS-I was displayed only in secretory granules. This suggests that two or more separate enzymes are involved in processing PSS-I and PSS-II, and that these enzymes have either differential distribution or differential activity in RER/Golgi complex and secretory granules. The demonstration of converting enzyme activity in islet microsomes supports the proposal that these enzymes may be synthesized at the RER and are internalized along with the prohormones.     

Pattern of islet lysosomal enzyme activities and insulin secretory response.

The pattern of lysosomal enzyme activities in isolated pancreatic islets was studied in 3 different strains of mice, NMRI, CBA, and C-57, and related to the in vivo insulin release following injection of the insulin scretagogues glucose and carbachol. It was observed that the relative specific activities among the islet enzymes studied did not show the same pattern in the different strains although beta-gluc-ronidase always displayed the lowest activity. Comparison between the strains revealed that acid phosphatase activity was of the same magnitude in all 3 strains. Islet activities of acid amyloglucosidase, beta-glucuronidase, and N-acetylglucosaminidase, however, were largest in NMRI, intermediate in CBA, and lowest in C-57. This activity pattern roughly correlated with the insulin secretory response to an intravenous injection of glucose, whereas insulin release induced by the cholinergic agonist carbachol was of similar magnitude in all strains.     

Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump

Isolated rat pancreatic islets were pulse-labeled for 5 min with [3H]leucine then chased for 25 min, during which time endogenously labeled [3H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [3H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases (sodium azide, 20 mM, or oligomycin, 10 micrograms/ml) had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer (pH 5.5), proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect. Disrupted granules only converted proinsulin to a limited extent, and neither ATP nor the inhibitors affected conversion. It is therefore suggested that ATP stimulation of proinsulin conversion in isolated, intact, beta-granules is secondary to intragranular acidification by an ATP-dependent proton pump (reflecting the low pH optimum for proinsulin conversion), rather than ATP dependence of converting activity per se.     

Divalent cation inhibition of hormone release from isolated adenohypophysial secretory granules

Divalent cations inhibited in vitro release of growth hormone (GH) and prolactin (PRL) from bovine adenohypophysial secretory granules. Zinc, nickel, and cadmium were most potent, exerting 50% inhibition of protein release near 0.1 mM; relative potency was Ni2+ greater than or equal to Zn2+ greater than Cd2+ much greater than Mn2+ greater than Co2+ greater than Cu2+ much greater than Mg2+ greater than Ca2+. The pH optimum for inhibition, 8.0, was lower than that for stimulation of release by thiols. EDTA augmented release and reversed metal inhibition. Both immunoassay and polyacrylamide gel electrophoresis results indicated that metals inhibited both PRL and GH release in a dose-related fashion, and that PRL was more sensitive to all cations tested. With zinc present, known stimulators of release (reduced glutathione, ATP, and bicarbonate) restored GH release, but only ATP restored PRL release. Bicarbonate potently stimulated GH release, but only affected PRL when Mg2+ and ATP were present. We suggest that divalent cations influence GH and PRL release in a reversible fashion and at multiple sites. Some loci may be common to both lactotrope and somatotrope granules; however, the different sensitivities to metals and differential reversal by stimulators of release indicate that metal-protein interactions may also be specific for either granule, or for the hormones themselves.     

Relative lack of ATP-driven H+ translocase activity in isolated parotid secretory granules

The possible presence of ATP-driven H+ translocase activity in isolated rat parotid secretory granules has been examined by several approaches. First the transmembrane pH difference measured by either [14C] methylamine or [3H]acetate distribution is not substantially affected by ATP in the presence of membrane-permeating anions. Second, despite a low intrinsic H+ permeability of parotid granule membranes, only a small variably detectable inside-positive transmembrane potential is observed (by altered distribution of radioactive ions) when ATP is added in the absence of permeant anions. Third, ATP-induced lysis of parotid granules is minor and appears to be independent of ATP hydrolysis. Finally, ATP-hydrolase activity of the parotid granule fraction is not stimulated by an H+ ionophore, nor is it susceptible to inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole at a concentration which decreases the measured ATPase of purified chromaffin granule membranes by more than 80%. These findings suggest that this exocrine secretory granule type, which is characterized by storage of a heterogeneous mixture of secretory proteins, exhibits H+ pump activity which is at most a small fraction of that observed in biogenic amine storage granules of neural and endocrine tissues.     

Actions of releasing factors on isolated secretory granules mediated by calcium.

Synthetic TRH, crude hypothalamic extract and partially purified prolactin releasing factor stimulated prolactin and growth hormone release from isolated secretory granules. Somatostatin and partially purified prolactin release-inhibiting factor inhibited release of both hormones. Calcium promoted hormone release from granules; its releasing action was potentiated by TRH and ionophore A23187 but reduced by somatostatin.     

Arginine esterase from isolated dog prostate secretory granules is fully active enzymatically

We have isolated secretory granules from dog prostate homogenates and have determined whether a major portion of arginine esterase was localized in this fraction and if it was enzymatically active. Secretory granules were purified by density gradient centrifugation on sucrose, metrizamide, or Percoll. A major proportion of whole prostate homogenate arginine esterase was found in the granule fractions. Furthermore, the specific enzymatic activity in the granules was similar to the one observed in seminal plasma. No evidence could be found for the existence of significant amount of a zymogen inactive form of arginine esterase. These results suggest that arginine esterase could be active within the secretory granules in vivo and that it could hydrolyze protein substrates contained in this organelle.     

Neutrophil granules and secretory vesicles in inflammation

The neutrophil is a major effector cell of innate immunity. Exocytosis of granules and secretory vesicles plays a pivotal role in most neutrophil functions from early activation to the destruction of phagocytosed microorganisms. Neutrophil granules contain a multitude of antimicrobial and potentially cytotoxic substances that are delivered to the phagosome or to the exterior of the cell following degranulation. This review summarises current knowledge of granule biology and highlights the effects of neutrophil degranulation in the acute inflammatory response.     

Development of platelet secretory granules

Platelet granule exocytosis plays a critical role in thrombosis and wound healing. Platelets have three major types of secretory granules that are defined by their unique molecular contents, kinetics of exocytosis and morphologies. Although the ontogeny of platelet granules is poorly understood, a convergence of new insights into megakaryocyte development, the molecular mechanisms of vesicle trafficking and the genetic basis of platelet granule defects, is beginning to define the cellular and molecular pathways responsible for platelet granule ontogeny.     

Presence of sulfated proteoglycans in prolactin secretory granules isolated from the rat pituitary gland.

The composition of the segregated content of rat prolactin granules was investigated taking advantage of the fact that these organelles, isolated as a pure fraction, retain their structural organization after solubilization of their limiting membrane by mild detergent treatment. We found that these membraneless granules contain not only the hormone, but also a number of minor macromolecular components including sulfated glycosaminoglycans, which are labeled when pituitary slices are incubated in vitro with [35S] sulfate. In order to characterize the latter components, the isolated radioactive granules were solubilized (by treatment with either a high ionic strength solution orNaOH) and 35S-labeled acidic glycosaminoglycans precipitated by complexing with cetylpirydinium chloride. A high degree of heterogeneity was observed when the ensuing precipitates were analyzed by cellulose acetate electrophoresis: different components were found to co-migrate with authentic heparin and chondroitin sulfate A and C standards. Another component, which accounts for approx. 50% of the glycosaminoglycan-bound radioactivity, might be heparin sulfate. These acidic glycosaminoglycans are linked to peptide moieties to form proteoglycans.     

Binding of microtubules to pituitary secretory granules and secretory granule membranes

Microtubules assembled in vitro were bound to purified porcine pituitary secretory granules and to isolated granule membranes. The interaction between microtubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with microtubules resulted in pelleting of microtubules which increased as a function of the number of granules added. Binding was quantitated by measurement of the tubulin remaining in the supernate after centrifugation. The interaction of secretory granules and microtubules was inhibited by nucleoside triphosphates and augmented by adenosine 5'-monophosphate and adenosine. When depolymerized protein from microtubules was incubated with secretory granules, the granules did not appear to bind the soluble tubulin dimer present in these preparations. However, the high molecular weight protein associated with microtubules was adsorbed by secretory granules during the binding process. Incubation of isolated secretory granule membranes with microtubules followed by centrifugation to density equilibrium in a discontinuous sucrose density gradient caused pelleting of the membranes, which otherwise banded higher in the gradient. The visible alteration in membrane sedimentation was confirmed by measurements of the membrane-associated magnesium-ATPase activity and by a shift in radioactivity in iodinated membrane preparations. Our data suggest a role for microtubules in the intracellular movement of secretory granules; this movement is perhaps brought about by dynein-like cross bridges which link the tubulin backbone and granule surface.     

Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human B-cells and in islets of type-2 diabetic subjects

Summary Islet amyloid peptide (or diabetes-associated peptide), the major component of pancreatic islet amyloid found in type-2 diabetes, has been identified by electronmicroscopic immunocytochemistry in pancreatic B-cells from five non-diabetic human subjects, and in islets from five type-2 diabetic patients. The greatest density of immunoreactivity for islet amyloid peptide was found in electrondense regions of some lysosomal or lipofuscin bodies. The peptide was also localised by quantification of immunogold in the secretory granules of B-cells, and was present in cytoplasmic lamellar bodies. Acid phosphatase activity was also demonstrated in these organelles. Immunoreactivity for insulin was found in some lysosomes. These results suggest that islet amyloid peptide is a constituent of normal pancreatic B-cells, and accumulates in lipofuscin bodies where it is presumably partially degraded. In islets from type-2 diabetic subjects, amyloid fibrils and lipofuscin bodies in B-cells showed immunoreactivity for the amyloid peptide. Abnormal processing of the peptide within B-cells could lead to the formation of islet amyloid in type-2 diabetes.     

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.     

Lumenal protein multimerization in the distal secretory pathway/secretory granules

Differences in protein solubility appear to play an important role in lumenal protein trafficking through Golgi/post-Golgi compartments. Recent advances indicate that multimeric protein assembly is one of the factors regulating the efficiency of protein storage within secretory granules, by mechanisms that, with slight modification, might be considered to represent the culmination of a process of Golgi cisternal maturation.     

Association of glycoproteins and pepsinogen in the secretory granules of fundic epithelial cells isolated from guinea pig stomach

Epithelial cells were isolated from the fundic portion of the guinea pig stomach. Cells were separated by velocity sedimentation at unit gravity in a Ficoll 70 gradient and pooled in three fractions. By morphological and biochemical criteria, each fraction was characterized as a population highly enriched in one of the three main functional types: oxyntic cell; chief cell and mucus-secreting cell. Measure of the pepsinogen content and specific stainings of the secretory granules for light and electron microscopy led to the definition of two types of mucus-secreting cells in nearly equal quantity; mucous cells with smaller secretory granules entirely glycoproteic in nature and muco-peptic cells containing larger heterogeneous secretory granules. These granules were made of a proteic core containing pepsinogen surrounded by a thin membrane and a voluminous cap, both containing carbohydrates. The cap appeared as if built of orderly packed layers of glycoproteins. Secretory granules of chief cells were also surrounded by a membrane containing glycoproteins and occasionally a small glycoproteic cap. Pepsinogen content was estimated to be three times higher in a single chief cell than in a muco-peptic cell.     

Identification and characterization of calmodulin-binding proteins in islet secretion granules

The binding of 125I-calmodulin to intact secretion granules and protein gel blots of secretion granules from pancreatic islet tissue was examined. Binding of 125I-calmodulin to intact secretion granules was Ca2+-dependent and inhibited by the calmodulin inhibitors trifluoperazine and calmidazolium. Binding was inhibited by excess (200 nM) unlabeled calmodulin, but not by parvalbumin, a Ca2+-binding protein which has little sequence homology to calmodulin. In order to study the binding of calmodulin to specific secretion granule proteins, secretion granules were solubilized, and the solubilized proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose, and incubated with 125I-calmodulin. Autoradiograms of the protein gel blots revealed the presence of three major calmodulin-binding proteins with approximate molecular weights of 73,000, 64,000, and 58,000. These proteins reversibly bound calmodulin in a calcium-dependent manner. Unlabeled calmodulin in the range of 0.1-1.0 nM competed with 125I-calmodulin for binding to these proteins, whereas troponin and parvalbumin were 100 and 1000-fold less effective, respectively. Trifluoperazine blocked binding to the granule proteins in a range of 10(-4) to 10(-5) M, and calmidazolium was effective between 10(-5) and 10(-6) M. Trypsin, at a concentration which did not lyse granules, markedly inhibited calmodulin binding to intact secretion granules. Protein blots from trypsin-treated granules showed that the three major calmodulin-binding proteins were absent. These results indicate that Ca2+-dependent calmodulin-binding proteins are present on the cytoplasmic surface of islet secretion granules and are consistent with the hypothesis that these proteins may play a role in secretion granule exocytosis.