Article

Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells

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Abstract

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-(3)H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.

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... At that time isoproterenol (a powerful sympathomimetic stimulant of parotid secretion [10,[15][16][17]) was added to a final concentration of 1 µM, and the incubation was continued for 4 hr . Percentage release of protein radioactivity and amylase into the medium was determined, using discharge assays previously reported (18) . Separate doseresponse experiments had shown that the optimal isoproterenol concentration for our system was I AM. ...
... Lobule Proteins during In Vitro Incubation When parotid lobules were incubated at 37°C in gassed F 12 supplemented with 5 µCi/ml L-leucine-3 H, label was incorporated into protein at a constant rate (very similar to that observed with pancreatic slices [18]) for at least 8 hr (Fig . 3) . ...
... 8 in reference 18) . corporation studies on guinea pig pancreatic slices(5,6,18) . ...
Article
Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2–2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-³H, followed by a chase incubation, showed that the label is initially located (chase: 1–6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16–36 min), condensing vacuoles (chase: 36–56 min), immature granules (chase: 56–116 min), and finally mature storage granules (chase: 116–356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.
... Palade and his collaborators (9,(19)(20)(21)(22)(23)46) showed that, in pancreatic exocrine cells of the guinea pig, zymogen granules arise as irregular "condensing vacuoles" in the Golgi zone. Within the condensing vacuoles the dilute secretory proteins become progressively concentrated. ...
... It was therefore concluded that the transport of newly synthesized proteins to the condensing vacuoles did not involve the Golgi "stacks." Instead, small vesicles were thought to convey these proteins to the condensing vacuoles, effectively bypassing the Golgi stacks (23). Jamieson and Palade (23) subsequently reported that when tissue slices of fasted guinea pig pancreas were stimulated by the secretogogue, carbamylcholine, for 3 h, ARG grains were found over the flattened sacs of the Golgi stack. ...
... Instead, small vesicles were thought to convey these proteins to the condensing vacuoles, effectively bypassing the Golgi stacks (23). Jamieson and Palade (23) subsequently reported that when tissue slices of fasted guinea pig pancreas were stimulated by the secretogogue, carbamylcholine, for 3 h, ARG grains were found over the flattened sacs of the Golgi stack. Thus, they proposed that in the stimulated gland the direct pathway to the condensing vacuoles was replaced by one that involved the Golgi apparatus. ...
Article
Phosphatase cytochemistry was used to distinguish between the Golgi apparatus and GERL (considered as a specialized region of endoplasmic reticulum [ER] at the inner [trans] aspect of the Golgi stack) in pancreatic exocrine cells of guinea pig, rat, rabbit, and hamster. The trans element of the Golgi stack exhibits thiamine pyrophosphatase (TPPase) but no acid phosphatase (AcPase) activity. In contrast, GERL shows AcPase but no TPPase activity. The nascent secretory granules, or condensing vacuoles, are expanded cisternal portions of GERL. Continuities of condensing vacuoles with rough ER are suggested, and it is proposed that some secretory components may have direct access to the condensing vacuoles from ER. Connections of Golgi apparatus with GERL were not seen.
... IntraceUular aspects of the process of protein secretion have been unraveled in the adult pancreatic exocrine acinar cell by the classical autoradiographic studies of Caro and Palade (4) and Jamieson and Palade (20)(21)(22). These studies have identified six successive steps in the secretory process namely, synthesis, segregation, intraceUular transport via the Golgi complex, concentration, intracellular storage of discharge (for detailed discussion of this process see references 18 and 27). ...
... (b) The newly synthesized secretory proteins in these GDF cells bypass the typical storage step and are rapidly discharged due to a functional defect. It is pertinent to note that Jamieson and Palade (22) demonstrated that in secretagogue-stimulated normal pancreatic acinar cells the secretory proteins are concentrated and packaged in numerous small storage vesicles in a normal fashion, but they are rapidly discharged. Preferential release of newly synthesized prolactin in mammotrophs has been described (43). ...
Article
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The pancreatic acinar carcinoma established in rat by Reddy and Rao (1977, Science 198:78-80) demonstrates heterogeneity of cytodifferentiation ranging from cells containing abundant well-developed secretory granules to those with virtually none. We examined the synthesis intracellular transport and storage of secretory proteins in secretory granule-enriched (GEF) and secretory granule-deficient (GDF) subpopulations of neoplastic acinar cells separable by Percoll gradient centrifugation, to determine the secretory process in cells with distinctly different cytodifferentiation. The cells pulse-labeled with [3H]leucine for 3 min and chase incubated for up to 4 h were analyzed by quantitative electron microscope autoradiography. In GEF neoplastic cells, the results of grain counts and relative grain density estimates establish that the label moves successively from rough endoplasmic reticulum (RER) leads to the Golgi apparatus leads to post-Golgi vesicles (vacuoles or immature granules) leads to mature secretory granules, in a manner reminiscent of the secretory process in normal pancreatic acinar cells. The presence of approximately 40% of the label in association with secretory granules at 4 h postpulse indicates that GEF neoplastic cells retain (acquire) the essential regulatory controls of the secretory process. In GDF neoplastic acinar cells the drainage of label from RER is slower, but the peak label of approximately 20% in the Golgi apparatus is reached relatively rapidly (10 min postpulse). The movement of label from the Golgi to the post-Golgi vesicles is evident; further delineation of the secretory process in GDF neoplastic cells, however, was not possible due to lack of secretory granule differentiation. The movement of label from RER leads to the Golgi apparatus leads to the post-Golgi vesicles suggests that GDF neoplastic cells also synthesize secretory proteins, but to a lesser extent than the GEF cells. The reason(s) for the inability of GDF cells to concentrate and store exportable proteins remain to be elucidated.
... Work in recent years has started to elucidate how the cell manages to perform this considerable sorting task. Thirty years after Porter et al. (1945) discovered the endoplasmic reticulum (ER)-the main avenue for the transport of proteins and lipids in eukaryotic cells (Jamieson and Palade, 1971;Palade, 1975)-the signal hypothesis (Blobel and Sabatini, 1971;Blobel and Dobberstein, 1975a,b) provided the first molecular explanation for the sorting of secretory proteins into the ER lumen. The rate of progress in the last 8 years has been exponential. ...
... Originally described in glandular epithelial cells, it is found, although modified, in every eukaryotic cell type (Palade, 19751, including yeast. Secretory proteins undergo the following biosynthetic and processing steps (Jamieson andPalade, 1971, 1977;Palade, 1975): (1) synthesis of a precursor (or preprotein) by polysomes attached to the outer surface of the rough ER; (2) segregation into the ER cistemae, mediated by a signal sequence; (3) intracellular transport via transitional smooth ER vesicles to the Golgi complex; (4) concentration and storage in secretory granules in cells with "regulated" secretion; this step is absent in cells with continuous or "nonregulated" secretion; and (5) exocytosis. ...
Article
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... The higher magnification view of fusion between the Golgi secretory vacuoles and the plasmalemma represent the selective diffusion of the insulin receptor (IBP) along the plane of the fluid (28,29) fused membranes onto its position in the plasmalemma . To maintain specificity of the Golgi membranes and the plasmalemma (4,23,30,31) the secretory vacuole membrane must be internalized within the cell (32,33,34) . present on either side of the membrane, are available for binding with the [ 115 I]insulin. ...
... The exocytic dynamics of this system is exemplified, in animals, by the transport of secretory proteins in pancreatic exocrine cells, which takes approximately 2 hrs. 29 On the other hand, in carrot phloem parenchyma, the secretion of hydroxyproline-rich glycoproteins into the extracellular matrix takes as little as 30 minutes. 30 Comparable dynamics are reported for endocytosis in protoplasts (Fig. 5A) isolated from soybean (Glycine max) plants that were exposed to cationized ferritin. ...
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Size is a key feature of any organism since it influences the rate at which resources are consumed and thus affects metabolic rates. In the 1930s, size-dependent relationships were codified as "allometry" and it was shown that most of these could be quantified using the slopes of log-log plots of any two variables of interest. During the decades that followed, physiologists explored how animal respiration rates varied as a function of body size across taxa. The expectation was that rates would scale as the 2/3 power of body size as a reflection of the Euclidean relationship between surface area and volume. However, the work of Max Kleiber (1893-1976) and others revealed that animal respiration rates apparently scale more closely as the 3/4 power of body size. This phenomenology, which is called "Kleiber's Law", has been described for a broad range of organisms, including some algae and plants. It has also been severely criticized on theoretical and empirical grounds. Here, we review the history of the analysis of metabolism, which originated with the works of Antoine L. Lavoisier (1743-1794) and Julius Sachs (1832-1897), and culminated in Kleiber's book The Fire of Life (1961; 2. ed. 1975). We then evaluate some of the criticisms that have been leveled against Kleiber's Law and some examples of the theories that have tried to explain it. We revive the speculation that intracellular exo- and endocytotic processes are resource delivery-systems, analogous to the supercellular systems in multicellular organisms. Finally, we present data that casts doubt on the existence of a single scaling relationship between growth and body size in plants.
... 80 % confluence, they were washed with PBS buffer, scraped with a rubber policeman, sedimented and disrupted with a Potter-Elvejhem homogenizer in a buffer containing 50 mM Tris\HCl, 10 mM EDTA and 0.25 M sucrose (pH 7.4). Subcellular fractionation was performed by serial centrifugations inspired from Jamieson and Palade [17]. The homogenate was cleared by centrifugation at 2000 g for 10 min at 4 mC. ...
Article
In this article, we report the nucleotide sequence of the cDNA encoding an isoform of bile-salt-dependent lipase (BSDL) expressed by human hepatoma cells. The BSDL is a 100-kDa glycoprotein normally expressed by the human pancreas. Using a polyclonal antibody raised against an internal peptide located between Ile327 and Glu350 of the human pancreatic BSDL, we have immunodetected an isoform of human pancreatic BSDL, with an apparent molecular mass of about 62 kDa. This isoform of BSDL was mainly associated with the cytosol of a human hepatoma cell line (HepG2), the remaining protein being found in the microsome fraction. In addition, esterolytic activity on p-nitrophenyl hexanoate measured in microsomes and cytosol appeared very low and was weakly stimulated by bile salts, such as taurocholate. In contrast to human pancreatic BSDL, which is secreted as a component of pancreatic juice, this isoform appeared to be retained in the HepG2 cells. Reverse transcription, followed by PCR and amplification, performed on RNA extracted from HepG2 cells using specific primers hybridizing to the sequence coding for the entire normal human pancreatic BSDL, allowed us to amplify a 1.7-kb transcript that appeared to be 0.5 kb shorter than the transcript of the pancreatic enzyme (2.2 kb). From the sequence of the transcript thus obtained, a protein with a molecular mass of 62 kDa might be predicted, which is in good agreement with the size of the isoform of BSDL immunodetected in HepG2 cells. The N-terminal amino-acid sequence, deduced from the 1.7-kb transcript sequence, matched that of the pancreatic BSDL. However, the C-terminal domain appeared truncated, bearing only a single mucin-like sequence compared with sixteen for the human pancreatic BSDL. The actual intracellular function of this human BSDL hepatoma isoform remains to be elucidated.
... To compare the secretory responses induced by stimulation with these 3 secretagogues, we measured the amount of amylase release 30 min after the stimulation in the following experiments. Previous studies demonstrated that synthesis of enzymes began 20-30 min after the start of incubation with secretagogues in the rat (1) and rabbit (7) parotid and the guinea pig (11) and rat (20) pancreatic preparation, therefore a longer incubation period was not suitable for this study since both release and synthesis of amylase would take place. ...
Article
Developments of immunology and immunohistochemistry have shown that vasoactive intestinal polypeptide (VIP) is contained in a wide variety of cells including the peripheral nerve innervating the salivary gland of cat, rat and man. Physiological studies of VIP in the salivary gland have been mainly focused on either its action to induce fluid and electrolyte secretion in the submandibular gland or its potent action as a vasodilator. In general, in the salivary gland, VIP potentiates acetylcholine-induced salivation but induces per se little, if any, salivation. The present study provides evidence that VIP acts on the parotid lobules of the rat to induce amylase release. This effect of VIP was not inhibited by pretreatment with atropine, α- or β-blocker, and Ca removal from the extracellular environment. A dose-response relation was obtained for the VIP-induced amylase release. ED 50 was calculated to be 3.6x10 -9M, which was much smaller than that of adrenaline or of carbachol. This favors the view that VIP could be a transmitter candidate.
... For most G-protein coupled receptors, including the D2R, the magnitude of the receptor-mediated signal can be modulated by moving receptors to and from the cellular membrane, a mechanism known as trafficking. Trafficking of clathrin-coated vesicles forming at the cellular membrane (endocytosis) or in intracellular organelles (exocytosis) is one of the major mechanisms used by cells to shuffle proteins at the cellular membrane (Jamieson and Palade, 1971;Pearse, 1976). Among others, β-arrestin and adaptor proteins (AP) are essential proteins for facilitating vesicle budding. ...
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The dopamine 2 receptors (D2R) are G-protein coupled receptors expressed both in pre- and post-synaptic terminals that play an important role in mediating the physiological and behavioral effects of amphetamine (Amph). Previous studies have indicated that the effects of Amph at the D2R mainly rely on the ability of Amph to robustly increase extracellular dopamine through the dopamine transporter (DAT). This implies that the effects of Amph on D2R require the neurotransmitter dopamine. However, because of its lipophilic nature, Amph can cross the cellular membrane and thus potentially affect D2R expression independently of dopamine and DAT, e.g., in post-synaptic terminals. Here we used an in vitro system to study whether Amph affects total expression, cellular distribution, and function of the human D2R (hD2R), endogenously expressed in HEK293 cells. By performing Western blot experiments, we found that prolonged treatments with 1 or 50 μM Amph cause a significant decrease of the endogenous hD2R in cells transfected with human DAT (hDAT). On the other hand, in cells lacking expression of DAT, quantification of the hD2R-mediated changes in cAMP, biotinylation assays, Western blots and imaging experiments demonstrated an increase of hD2R at the cellular membrane after 15-h treatments with Amph. Moreover, imaging data suggested that barbadin, a specific inhibitor of the βarrestin-βadaptin interaction, blocked the Amph-induced increase of hD2R. Taken together our data suggest that prolonged exposures to Amph decrease or increase the endogenous hD2R at the cellular membrane in HEK293 cells expressing or lacking hDAT, respectively. Considering that this drug is often consumed for prolonged periods, during which tolerance develops, our data suggest that even in absence of DAT or dopamine, Amph can still alter D2R distribution and function.
... In summary, mononuclear phagocytes may well be unique in producing two varieties of primary lysosomes-one which is recognizable as a large granule, and another which is a vesicle distinguishable as a lysosome only by cytochemical techniques . The fact that the cell can shift from one mode to another in the packaging of the secretory product resembles the situation described in the pancreatic exocrine cell in which a partially similar shift occurs in vitro upon continuous stimulation (48) . ...
Article
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... (27). Between 1967Between -1975, various researchers conducted numerous studies on the role of the GA in the secretory pathway and vesicular transport (28)(29)(30). In the 1980s, studies were carried out emphasizing the importance of mannose-6 phosphate in the exiting of lysosomal enzymes from the GA and in targeting them (31)(32)(33)(34) In parallel with the technological developments, it was possible to reach detailed information about the location of the GA, its morphological, functional and pathological features. ...
Article
Scientists were introduced to the Golgi apparatus (GA) in 1898, when it was discovered by Camillo Golgi in 1898 as a “cytoplasmic reticular network”. Researchers heard Camillo Golgi’s name not only because of the GA, but also because of many definitions such as Golgi silver impregnation techniques, Golgi type I and II cells, Golgi cells of the cerebellum, and Golgi tendon organ. In fact, although the GA beared the name of this scientist, many scientists did numerous studies on the morphological and functional properties of this unique organelle before him, simultaneously with him or after him. Despite the simple technical possibilities of the old times, the scientists, whom we gratefully commemorated, obtained magnificent findings about the GA and presented them to the world of science. In this short article, which was a review, the following historical developments, starting from the discovery of the GA, were summarized.
... Soon after publishing, these paths were questioned and pointed that proteins could be in the cytoplasm and outside the Golgi cisternae. It took several years and additional refinement of the described autoradiography technique (Jamieson & Palade 1967a;Jamieson & Palade 1967b;Jamieson & Palade 1971), to prove the protein flow from ER to Golgi, from Golgi to vesicles and finally from vesicles to plasma membrane. At the end, evidence of a simple and good model for a basic secretory pathway was sound enough to be accepted. ...
Thesis
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Plant Cell Biology is a semester long course for undergraduates and graduate students which integrates mathematics and physics, two years of chemistry, genetics, biochemistry and evolution disciplines. Having taught this course for over ten years, the author uses his expertise to relate the background established in plant anatomy, plant physiology, plant growth and development, plant taxonomy, plant biochemistry, and plant molecular biology courses to plant cell biology. This integration attempts to break down the barrier so plant cell biology is seen as an entrée into higher science. Distinguishing this book from papers that are often used for teaching the subject which use a single plant to demonstrate the techniques of molecular biology, this book covers all aspects of plant cell biology without emphasizing any one plant, organelle, molecule, or technique. Although most examples are biased towards plants, basic similarities between all living eukaryotic cells (animal and plant) are recognized and used to best illustrate for students cell processes. Thoroughly explains the physiological underpinnings of biological processes to bring original insight related to plants Includes examples throughout from physics, chemistry, geology, and biology to bring understanding to plant cell development, growth, chemistry and diseases Provides the essential tools for students to be able to evaluate and assess the mechanisms involved in cell growth, chromosome motion, membrane trafficking, and energy exchange Companion Web site provides support for all plant cell biology courses.
Chapter
Glycoproteins are a large and heterogeneous group of macromolecules and serve a variety of different functions. This chapter reviews the structure and biosynthesis of these membrane-bound glycoproteins. The carbohydrate of the cell surface membrane is always oriented toward the external environment; this fact and the analogous carbohydrate asymmetry across intracellular membranes are essential in understanding membrane glycoprotein biosynthesis. The chapter discusses protein–lipid interactions through model membrane systems. The use of model membranes in which well-characterized isolated proteins are incorporated into lipid vesicles made from well-defined lipids provides an experimental model to study these protein–lipid interactions. These studies provide information useful for understanding the incorporation of glycoproteins into membranes in vivo. The synthesis of nucleotide sugars and lipid intermediates and with the utilization of these glycose donors by glycosyltransferases is also discussed in the chapter. It illustrates a model for membrane glycoprotein biogenesis. Data on the subcellular localization of various glycosylation reactions support this scheme and some of the evidence is reviewed in the chapter.
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This chapter examines the structural basis of secretory protein transport and packaging by the Golgi apparatus. Recent evidence points to distinct structural, chemical, and functional specialization within the Golgi region, which may be altered or modulated by changes in cellular activity. Insofar as they bear on the processes of protein transport and packaging, aspects of membrane dynamics and the segregation and sorting of various synthetic products are discussed in the chapter. The Golgi apparatus plays a central role in secretory cells. The heterogeneity of the Golgi apparatus—demonstrated in structural, cytochemical, and biochemical studies—suggests that specialized regions of the Golgi apparatus may be responsible for specific functions. The identification of Golgi–Endoplasmic Reticulum Lysosome (GERL) at the trans face of the Golgi apparatus and its apparent role in secretory granule formation add additional complexity to the understanding of the functioning of the Golgi apparatus.
Article
It is becoming clear that all eukaryotic cells synthesize, in association with their rough endoplasmic reticulum (RER), three major classes of proteins utilizing a common set of mechanisms. These proteins can be classified as bona fide secretory proteins which are discharged from the cell for use in the extracellular space; lysosomal hydrolases which are segregated into a set of intracellular compartments for use in intracellular digestion processes; and membrane proteins destined for use in the biogenesis of several intracellular membrane-bounded compartments and of specialized functional domains of the plasmalemma.
Thesis
Le mauvais pronostic du cancer du pancréas (CP) peut s'expliquer par un diagnostic tardif et le manque de traitements efficaces. Une meilleure compréhension des mécanismes moléculaires qui régissent l'initiation, la progression et la résistance aux chimiothérapies est indispensable pour améliorer la prise en charge des patients. TRIP12 (Thyroid hormone Receptor Interacting Protein 12) est une E3 ubiquitine ligase de la famille HECT (Homologous to the E6-AP Carboxyl Terminus). TRIP12 régule le cycle cellulaire et la stabilité chromosomique, elle intervient dans le remodelage de la chromatine, dans la réponse aux dommages à l'ADN et dans l'activation de la voie P53. L'ARNm de TRIP12 est surexprimé dans le CP. De plus, TRIP12 est impliquée dans la dégradation de PTF1a (Pancreas Transcription Factor 1a), facteur de transcription essentiel du développement et de l'homéostasie des cellules acinaires du pancréas, suppresseur de tumeur dont l'expression est perdue au cours de la carcinogénèse pancréatique. Ces données suggèrent un rôle de TRIP12 dans le CP. Mon premier objectif de thèse était de déterminer pourquoi l'expression de TRIP12 augmente dans le CP et quelles en sont les conséquences. J'ai également émis l'hypothèse que TRIP12 joue un rôle dès l'initiation du CP. Mon second objectif de thèse était de déterminer si et comment TRIP12 contribue à la reprogrammation des cellules acinaires en cellules canalaires, première étape de la formation de lésions prénéoplasiques. J'ai découvert que TRIP12 est nécessaire à la croissance des tumeurs pancréatiques et que son expression est variable dans les tumeurs et les lignées cellulaires dérivées de CP. J'ai démontré que l'hétérogénéité d'expression de TRIP12 dans ces lignées cellulaires s'explique 1)- par des niveaux d'ARNm différents liés à l'activité du promoteur du gène Trip12 et 2)- par une régulation différente de la protéine TRIP12 au cours du cycle cellulaire mettant en jeu la déubiquitinase USP7 et un défaut de l'export nucléaire de TRIP12. Les lignées pancréatiques ayant un taux élevé de TRIP12 répondent mieux aux agents anticancéreux mettant en évidence le rôle de TRIP12 dans la chimiosensibilité des cellules cancéreuses pancréatiques. Par ailleurs, les résultats obtenus à partir d'un nouveau modèle murin montrent que l'invalidation conditionnelle de TRIP12 dans le pancréas au cours de l'embryogenèse n'altère pas le développement et la fonction du pancréas à l'âge adulte. Cependant, TRIP12 est capitale pour l'initiation du CP. En effet, la perte de TRIP12 dans les cellules acinaires empêche la métaplasie acino-canalaire. TRIP12 est aussi importante pour la progression du CP car son absence ralentit le développement des tumeurs engendrées par la mutation du gène Kras et la perte du gène P53. L'ensemble de mes travaux de thèse montre que TRIP12 est un acteur décisif du CP et explique l'hétérogénéité d'expression de TRIP12 observée dans les tumeurs pancréatiques. Ces nouvelles connaissances pourraient offrir de nouvelles options thérapeutiques pour le CP où le besoin est énorme. TRIP12 est une protéine candidate de haute importance qui peut contribuer aux stratégies de thérapie par des médicaments innovants.
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The development of new techniques in molecular biology has greatly advanced our understanding of the evolutionary and systematic biology of mammals. Before 1965 our comprehension of mammalian evolution and our approach to systematics rested upon our ability—sometimes strictly intuitive—to interpret the significance of interspecific differences in gross anatomic features. Since then, we have been increasingly able to study proteins, chromosomal structure and organization, comparisons of individual genes, gene loci, and nuclear and mitochondrial DNA. Evolutionary and systematic investigations in mammalogy now routinely include biochemistry of proteins as well as gross anatomy, but often neglect everything in between.
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It is now clear that most, if not all, eukaryotic cells produce a variety of macromolecules (polysaccharides, polypeptides, and covalent derivatives of the former such as glycoproteins and peptidoglycans) which are destined for export. In the context of this chapter, the term “export” refers to the disposition and utilization of these products in a compartment or compartments separated from the cytosol, which includes the extracellular space (in the case of secretory cells) and intracellular compartments which may or may not be functionally connected to the extracellular space (as exemplified by the lysosomal or intracellular digestive system of the cell).
Article
The precise localization of secretory proteins in exocrine pancreatic cells has not yet been elucidated. Especially the role of the Golgi complex in protein processing remains a matter of controversy. Based on morphological and autoradiographical work with the frog pancreas, we described proteins moving from the peripheral elements at the cis (immature) side of the Golgi complex across the stack of Golgi cisternae towards the condensing vacuoles at the trans (mature) side of the Golgi complex. Jamieson and Palade described a similar pathway in the stimulated Guinae pig pancreas, but suggested that in unstimulated cells the Golgi cisternae are bypassed and that vesicles carry proteins directly from the RER to the condensing vacuoles. Novikoff et al. postulate from their study on pancreatic cells of 4 mammalian species including Guinea pig and rat that condensing vacuoles can originate directly from specialized regions of the ER (so-called GERL) at the trans side of the Golgi complex. This makes the role of the peripheral elements and the Golgi cisternae in protein processing even more doubtful.
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This chapter focuses on the molecular mechanisms by which nascent proteins and larger cargos are sequestered, packaged into vesicle carriers, and targeted to specific hepatocellular destinations during the secretory process. The secretory pathway begins with synthesis of proteins at the endoplasmic reticulum. The chapter describes some of the molecular components required to direct nascent proteins and more complex lipoprotein particles from the endoplasmic reticulum (ER) to the Golgi apparatus and then to their final destination. Coat protomer I coated vesicles are required for intra‐Golgi trafficking and for recycling from the Golgi apparatus back to the ER. The first cis‐Golgi element and the trans‐Golgi network are tubular membrane meshworks at the entry and exit sides of the Golgi apparatus, respectively. Transport in the anterograde direction from the ER to the Golgi apparatus is mediated by coat protomer II coated vesicles.
Article
Full-text available
Pancreatic acinar cell vacuolation is spontaneously observed in mice; however, the lesion is rare and has not been well documented. Herein, we present a detailed pathological examination of this lesion. Vacuoles in pancreatic acinar cells were present in 2/15 X gene knockout mice with a C57BL/6J mouse background, 4/298 ICR(CD-1) mice, 1/110 B6C3F1 mice, and 3/399 CByB6F1-Tg(HRAS)2Jic mice. The vacuoles were usually observed in a unit of the acinus, and the lesions were spread throughout the pancreas. These vacuoles contained weakly basophilic material that was positive for the periodic acid–Schiff reaction. Immunohistochemically, the vacuoles were positive for calreticulin antibody. Electron microscopy revealed globular dilatation of the rough endoplasmic reticulum (rER). According to these findings, vacuolation of pancreatic acinar cells is caused by the accumulation of misfolded proteins and enlargement of the rER.
Article
The European Pancreatic Club Lifetime Achievement award is a distinction awarded for research on the pancreas. It comes with the obligation to submit a review article to the society's journal, Pancreatology. Since the research topics of my group have recently been covered in reviews and book chapters I want to use this opportunity to appraise the stations of my clinical and research education, the projects that I pursued and abandoned, the lessons I have learned from them, and the women and men who influenced my training and development as a physician scientist. Some crossed my path, some become collaborators and friends, and some turned into role models and had a lasting impact on my life.
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Gastrointestinal hormones and their receptors play important roles in the regulation and integration of digestive function. An understanding of the organization of this endocrine system, where the hormones are produced, what stimulates their secretion, their targets, and the characterization of their responses provide important insights to understand normal physiology and pathophysiologic presentations. This is also useful in developing diagnostics and therapeutic approaches to disease.
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The general construction of the endoplasmic reticulum (ER) is that of a network of parallel membranes making up lamellar, tubular, cisternal and vesicular structures, somewhat irregular in diameter and form, and enclosing a system of channels separated from the cytoplasm. The ER represents a continuous system in which the different types of membrane are connected with one another to form a continuum1, including the nuclear envelope. There are two morphologically different sections of the ER. One part is studded with ribosomes on the outer surface of the membranes and has been designated the rough ER. The other part of the interconnected system is lacking such attached ribosomes and is therefore called the agranular or smooth ER.
Article
The secretory pathway along which newly synthesized secretory and membrane proteins traffic through the cell was revealed in two articles published 50 years ago. This discovery was the culmination of decades of effort to unite the power of biochemical and morphological methodologies in order to elucidate the dynamic nature of the cell's biosynthetic machinery. The secretory pathway remains a central paradigm of modern cell biology. Its elucidation 50 years ago inspired tremendous multidisciplinary and on-going efforts to understand the machinery that makes it run, the adaptations that permit it to serve the needs of specialized cell types, and the pathological consequences that arise when it is perturbed.
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The general construction of the endoplasmic reticulum (ER) is that of a network of parallel membranes making up lamellar, tubular, cisternal and vesicular structures, somewhat irregular in diameter and form, and enclosing a system of channels separated from the cytoplasm. The ER represents a continuous system in which the different types of membrane are connected with one another to form a continuum1, including the nuclear envelope. There are two morphologically different sections of the ER. One part is studded with ribosomes on the outer surface of the membranes and has been designated the rough ER. The other part of the interconnected system is lacking such attached ribosomes and is therefore called the agranular or smooth ER.
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This chapter discusses the process of cortical exocytosis in sea urchin egg. There is now ample evidence that an increase in cytosolic free calcium precedes and is the signal for exocytosis. The calcium signal is therefore described in the chapter, including the regulatory role of the guanyl nucleotide binding proteins. The use of subcellular fractions of sea urchin eggs has been analyzed with their retained exocytotic apparatus for in vitro studies of the calcium trigger. The fundamental mechanism of Ca2+ signal generation in the sea urchin egg and in mammalian cells appears to be virtually identical: receptor occupation results in the sequential activation of a G protein and P1-specific phospholipase C. Two areas examined are (1) the mechanism of generation of the intracellular calcium signal and (2) the use of egg cortex as an in vitro model for the investigation of the molecular mechanism of the terminal stages of exocytosis. The egg cortex constitutes a particularly advantageous model system for studying exocytotic membrane fusion. The available data suggest that cortical vesicle discharge in the cell-surface complex and cortical-lawn preparations is the in vitro equivalent of exocytosis in the egg; however, ultimate validation of this in vitro approach will come only through the identification of proteins that are required for exocytosis, both in vitro and in vivo.
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Transporting epithelial cells are present in many organs in vivo. These epithelial cells play an important role in regulating the passage of solutes from one tissue compartment to another. In order to perform such functions, transporting epithelial cells must possess a remarkable structural and functional polarity. Their plasma membrane is characterized by two distinct domains, which are separated by tight junctions: an apical (or mucosal) domain with microvilli, which often faces a lumenal compartment, and a basolateral (or serosal) domain, which is associated with another tissue compartment (Fig. 1). These two plasma membrane domains possess different transport systems, membrane receptors, and enzymes. For example, distal tubule cells of the kidney readsorb salt and water from the lumen of the tubule back into the blood. This vectorial transport is facilitated by the polarized structure of the distal tubule cells (Fig. 2). The apical surface (which faces the lumen of the tubule) possesses an amiloride-sensitive Na+ channel, as well as proteolytic enzymes (leucine aminopeptidase and alkaline Phosphatase). In contrast, the basolateral surface possesses high Na+ /K+ ATPase activity. Hormone-sensitive transepithelial solute transport is facilitated by the presence in the basolateral surface of arginine Vasopressin receptors coupled to adenylate cyclase. So, the polarized structure of the epithelial cell is essential to the appropriate expression of its differentiated transport functions.
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Recent advances in quantitative electron-microscopic autoradiography, cytochemistry, and immunoassay techniques have provided more precise information on the location and kinetics of the intracellular processing of adenohypophyseal hormones than has been possible previously using other systems. These studies have elucidated many of the biochemical and morphological pathways in the synthesis, intracellular transport, release, and endocrinophagy of adenohypophyseal hormones. The growth-hormone (GH) cell or somatotroph has served as the model for our laboratory investigations on the mechanism(s) by which secretagogues stimulate hormone synthesis and release from the adenohypophysis.
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In the almost 20 years since Roth and Porter (1964) proposed that coated vesicles mediate the specific transport of proteins, researchers from various fields have joined in investigating both the structure and function of these organelles. Interest in the function of coated vesicles has intensified since it became apparent that coated pits and coated vesicles are the organelles responsible for the initial steps in the receptor-mediated endocytosis of a host of polypeptides, hormones, growth factors and viruses. There is ample evidence also that they function in other intracellular pathways, including transport of proteins from one cell surface to another, from the endoplasmic reticulum to the Golgi and from the Golgi to the cell surface.
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An important range of functions has been ascribed to the exocrine pancreas (Table 1) but very little is known about the quantitative nature of these functions in man. The analysis and description of disturbances of pancreatic function is therefore still very crude.
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Die exokrine Zelle des Pankreas ist ein Modellfall für Zellen mit hoher Eiweißsynthese und Sekretion. Durch ihre polare Struktur mit einem basolateralen und einem luminalen Anteil eignet sie sich ideal zur Erforschung grundlegender Sekretionsmechanismen. Diese Einschätzung stammt von George Palade, der für die Klärung der intrazellulären Transport- und Sekretionsmechanismen den Nobel-Preis verliehen bekam [1]. Im folgenden sollen die einzelnen Transportschritte in der Azinus-zelle von der Synthese bis zur Exozytose erläutert werden. Daneben werden die hierfür erforderlichen Schritte der Signaltransduktion zusammengefaßt.
Chapter
In the exocrine pancreas, intracellular transport of proteins and enzyme secretion is stimulated in response to a variety of hormones and neurotransmitters such as cholecystokinin (CCK), vasoactive intestinal peptide (VIP) and secretin (3,23). Receptor-mediated activation of phospholipase C (PLC) by cholecystokinin-octapeptide (CCK-OP) and direct activation of PLC-coupling G-proteins by weakly hydrolyzable analogues of GTP, such as guanosine 5′-0-(3-thiotriphosphate) (GTP7S), lead to phosphatidylinositol 4,5-biphosphate breakdown and formation of inositol 1,4,5-trisphosphate (IP3; see Ref. 24) and diacylglycerol (DG; see Ref. 19) in both intact and permeabilized pancreatic acinar cells. Whereas IP3 releases Ca2+ from intracellular Ca2+ stores (26), DG or its synthetic analogue 12-0-tetradecanoylphorbol 13-acetate (TPA) activates protein kinase (PK) C (7) and subsequently stimulates enzyme secretion (15). Both secretin and VIP stimulate enzyme secretion (11) by an increase in intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) levels, which leads to activation of PKA (5).
Article
Full-text available
A molecular model of pancreatic zymogen granule (ZG) is critical for understanding its functions. We have extensively characterized the composition and membrane topology of rat ZG proteins. In this study, we report the development of targeted proteomics approaches to quantify representative mouse and human ZG proteins using LC-SRM and heavy isotope-labeled synthetic peptides. The absolute quantities of mouse Rab3D and VAMP8 were determined as 1242 ± 218 and 2039 ± 151 (mean ± SEM) copies per ZG. The size distribution and the averaged diameter of ZGs 750 ± 23 nm (mean ± SEM) were determined by atomic force microscopy. The absolute quantification of Rab3D was then validated using semi-quantitative Western blotting with purified GST-Rab3D proteins as an internal standard. To extend our proteomics analysis to human pancreas, ZGs were purified using human acini obtained from pancreatic islet transplantation center. One hundred and eighty human ZG proteins were identified for the first time including both the membrane and the content proteins. Furthermore, the copy number per ZG of human Rab3D and VAMP8 were determined to be 1182 ± 45 and 485 ± 15 (mean ± SEM). The comprehensive proteomic analyses of mouse and human pancreatic ZGs have the potential to identify species-specific ZG proteins. The determination of protein copy numbers on pancreatic ZGs represents a significant advance towards building a quantitative molecular model of a prototypical secretory vesicle using targeted proteomics approaches. The identification of human ZG proteins lays a foundation for subsequent studies of altered ZG compositions and secretion in pancreatic diseases. Electronic supplementary material The online version of this article (10.1007/s41048-018-0055-1) contains supplementary material, which is available to authorized users.
Chapter
No specific features are seen by gross examination. The inclusions are identified by microscopic examination of tissue sections stained by routine procedures.
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Prior to the advent of electron microscopy, studies of secretory processes were limited primarily to identifying secretory cells and the materials they produce, determining the gross mechanisms of secretion, and delineating the stimuli which cause the release of material to the extracellular environment. The development and refinement of ultrastructural techniques provided an entirely new dimension to the understanding of secretory processes as investigations were made possible into the assembly, intracellular transport, and release of secretory material. Subsequently, from the examination of a wide variety of secretory cells, certain common principles evolved regarding the mechanisms of production and release of secretory products. The importance of cellular membranes in the synthesis, packaging, and intracellular transport of secretory material was recognized, and defects in secretory function were traced to abnormalities in cellular mechanisms. In the ensuing discussion, an attempt will be made to outline the common pathways shared by many different types of secretory cells for producing and releasing their products. Several representative examples of secretory cells and the cellular organelles involved in the manufacture of precursor material will be analyzed in depth. Finally, an effort will be made to describe some of the current controversies, theories, and supporting experimental evidence regarding certain aspects of the intracellular mechanism of secretion.
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The following chapter covers the saga of the Golgi apparatus (GA) from its discovery to the beginning of the 1980s when new tools for research, such as antibodies and molecular probes, became available. Emphasis is given to early insights and to those developments which laid the foundations for new developments covered in this monograph. This historical review, however, cannot include detailed descriptions nor can all significant contributions be mentioned. I apologize to all those whose work may not have found its due consideration. The historical development of our current knowledge of the GA is paradigmatic for the evolution of science in many ways. It exemplifies the importance of new approaches for progress to be made since contacts with apparently unrelated areas of research very often lead to surprising breakthroughs. Camillo Golgi discovered the “apparato reticolare interno”by using his difficult “black reaction”which brought him fame for the first demonstration of neuronal networks. During the following fifty years, the GA remained an intangible and often spurious concept for one part of the scientific community while the other was convinced of its reality, ubiquity and functional association with secretory processes. The end of this “Golgi controversy”was marked by the identification of the GA as a morphological entity by electron microscopy at the beginning of the 1950s. This advance, followed by the general pace of the postwar development of the life sciences, led to a singular explosion of interest in this organelle during the 1960s as reflected by a citation analysis. Major contributions were the observations on cytochemical differentiation of the cisternal stack of the GA, its role in secretion, and post-translational modifications as shown by autoradiography and biochemically by fractionation. At the end of the 1970s, the importance of the GA within the secretory pathway and its contribution to structural changes of secretory products was solidly established. The development of immunochemical and molecular probes for Golgi-specific marker proteins was the beginning of the current era. These tools now permit studies on the biogenesis of the GA, molecular mechanisms of intracellular transport and sorting, assembly and disassembly during the cell cycle, the fine architecture of this organelle, and have shown its ubiquity among eukaryotic cells including yeasts. All these aspects are at the center of current interests and are comprehensively dealt with in this volume.
Article
Synchronization of the secretory cycle in vivo was obtained by injecting isoprenaline as an inducer of secretion. A quantitative correlation between enzyme release, its subsequent reaccumulation, and the sequence of ultrastructural changes was found. At the ultrastructural level secretion was paralleled by depletion of zymogen granules through fusion of the granule membrane with the lumen membrane and discharge of the content. Each zymogen granule membrane, once connected with the lumen, acted as a lumen membrane. Fusion was thus sequential and resulted in a dramatic enlargement of the lumen space. During the entire process the passage between the lumen and the intercellular space remained blocked by the tight junctions, as shown by their impenetrability to ferritin. Reduction of the lumen size following enzyme discharge seemed to be achieved by withdrawal of lumen membrane in the form of small smooth vesicles which appeared mostly in the apical part of the cell. At the same time, the cell retracted towards the lumen, the whole process being completed within 2 hr from onset of secretion. Disappearance of the smooth vesicle followed, concomitant with formation of many condensing vacuoles and appearance of mature zymogen granules. The fate of the zymogen granule membrane, including its fusion with the lumen membrane, resorption in the form of small smooth vesicles, and its eventual reutilization mediated by the Golgi system, is discussed.
Ribonucleoprotein (RNP)1 particles isolated by DOC treatment from pancreatic microsomes have a RNA content of 35 to 45 per cent of their dry weight. In the analytical ultracentrifuge about 85 per cent of the material has a sedimentation coefficient of ∼85 S. These particles contain amylase, RNase, and trypsin-activatable proteolytic activities which cannot be washed off or detached by incubation in 0.44 M sucrose. The enzymes are released, however, by incubation in the presence of low concentrations of ATP, PP, or EDTA, and high concentrations of IP and AMP. At the same time, and at the same concentrations, ∼80 per cent of the RNA and ∼25 per cent of the protein of the particles becomes also non-sedimentable. The simultaneous addition of Mg++ to the incubation medium prevents these losses. This finding, together with the observation that all the Mg++ of the particles is released by the same agents, makes it likely that Mg++ holds the particles together, and that its removal by the chelators used causes the particles to disintegrate. These findings are discussed in relation to the molecular structure of the RNP particles.
Article
The secretion of protein by the acinar cells of the pancreas occurs in two steps: the synthesis of the protein from amino acids and its migration through the cell to the acinar lumen. Since very soon (one minute) after the injection of leucine-H3 the labeled protein is found mainly in the ergastoplasm of the basal half of the cell, protein synthesis is believed to occur in this location. At later intervals the labeled protein is found in the Golgi complex (10 and 30 minutes), and then in the zymogen granules of the apical half of the cell (60 minutes). The newly synthesized protein thus migrates from the ergastoplasm to the Golgi complex, and from there as zymogen granules through the apical half of the cell prior to being released in the acinar lumen.
Article
1.1. A procedure was developed for the isolation of zymogen granules from the parotis gland of the rat.2.2. The distribution of amylase in glands from fasted animals was investigated. 55% of total enzyme was located in the zymogen granules, while 35% remained in the soluble fraction. The microsomes contained only traces of activity.3.3. The isolated zymogen granules were shown to be osmotically sensitive spheres bounded by a membrane 60–70 Å thick. In hypotonic solutions the granules ruptured, releasing 95% of the amylase into the soluble phase. The residue, consisting of water insoluble membranes, retained about one third of the protein of the granules. The membranes also contained phospholipid, but little if any saccharide.4.4. Rat-parotis amylase, purified about four-fold over the homogenate showed a specific activity somewhat higher than that of known pure alpha amylases.5.5. The implications of the findings are discussed with respect to secretory enzyme accumulation.
Article
This chapter focuses on the dynamic aspects of phospholipids in exocrine cells which secrete organic molecules, in particular the exocrine pancreas, which secretes protein. Some interpretations of the possible physiological role of the phospholipid effect in the overall process of protein secretion are also presented. This role seems to center on the mechanism of membrane relocation, which is part and parcel of the process of membrane circulation which appears to occur during protein secretion. The prominent constituents of all cell membranes, such as phospholipids, serve more than one role in membrane function. Phosphatides also play a dynamic role in nervous tissue, and the available evidence suggests that this function is closely related to that in glands which secrete organic molecules. Dynamic functions discussed in the chapter involve changes in metabolism as detected by isotopic tracers. There are no doubt functions of phospholipids which do not involve metabolic changes.
Article
This chapter discusses the structure and function of the Golgi apparatus. The basic ultrastructure of the Golgi apparatus is relatively consistent throughout both the animal and plant kingdoms. The Golgi apparatus is readily observed in the living cell under the phase contrast microscope, where it generally appears comparable to that seen following the metallic impregnation methods. Metallic impregnation shows the Golgi apparatus as a dense, somewhat pleomorphic and ill-defined morphological structure. Early studies revealed its form to vary greatly in different types of cells and in the same cells depending on their state of activity. It may exist as a compact mass or be dispersed through the cytoplasm as isolated fragments. It has been described as a filamentous or platelike reticulum, a network of varying degrees of complexity, an arrangement of clear canals, vacuoles of varying size and crescent, cup or diskshaped bodies (dictyosomes) composed of an outer rim of osmiophilic substance (“Golgi externum”) and an inner central portion of osmiophobic substance (“Golgi internum”). Electron microscope studies served to confirm and strengthen the classical concept of the Golgi apparatus and to clearly show its complex nature. Golgi complex might be involved in the synthesis of this cell product or to what extent it might be dealing with the secretory product in other ways. In some cells, it appears that the Golgi complex may collaborate with other cell organelles in the production of complex or heterogeneous secretory product. Other complex functions are also discussed in the chapter.
Article
During their differentiation in the bone marrow, eosinophilic leukocytes synthesize a number of enzymes and package them into secretory granules. The pathway by which three enzymes (peroxidase, acid phosphatase, and arylsulfatase) are segregated and packaged into specific granules of eosinophils was investigated by cytochemistry and electron microscopy. During the myelocyte stage, peroxidase is present within (a) all rough ER cisternae, including transitional elements and the perinuclear cisterna; (b) clusters of smooth vesicles at the periphery of the Golgi complex; (c) all Golgi cisternae; and (d) all immature and mature specific granules. At later stages, after granule formation has ceased, peroxidase is not seen in ER or Golgi elements and is demonstrable only in granules. The distribution of acid phosphatase and arylsulfatase was similar, except that the reaction was more variable and fully condensed (mature) granules were not reactive. These results are in accord with the general pathway for intracellular transport of secretory proteins demonstrated in the pancreas exocrine cell by Palade and coworkers. The findings also demonstrate (a) that in the eosinophil the stacked Golgi cisternae participate in the segregation of secretory proteins and (b) that the entire rough ER and all the Golgi cisternae are involved in the simultaneous segregation and packaging of several proteins.
Article
Since in the pancreatic exocrine cell synthesis and intracellular transport of secretory proteins can be uncoupled (1), it is possible to examine separately the metabolic requirements of the latter process. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-(3)H for 3 min and incubated post-pulse for 37 min in chase medium containing 5 x 10(-4)M cycloheximide and inhibitors of glycolysis, respiration, or oxidative phosphorylation. In each case, the effect on transport was assessed by measuring the amount of labeled secretory proteins found in zymogen granule fractions isolated from the corresponding slices. This assay is actually a measure of the efficiency of transport of secretory proteins from the cisternae of the rough endoplasmic reticulum (RER) to the condensing vacuoles of the Golgi complex which are recovered in the zymogen granule fraction (16). The results indicate that transport is insensitive to glycolytic inhibitors (fluoride, iodoacetate) but is blocked by respiratory inhibitors (N(2), cyanide, Antimycin A) and by inhibitors of oxidative phosphorylation (dinitrophenol, oligomycin). Except for Antimycin A, the effect is reversible. Parallel radioautographic studies and cell fractionation procedures applied to microsomal subfractions have indicated that the energy-dependent step is located between the transitional elements of the RER and the small, smooth-surfaced vesicles at the periphery of the Golgi complex. Radiorespirometric data indicate that the substrates oxidized to support transport are endogenous long-chain fatty acids.
Article
Experiments have been carried out to determine whether intracellular transport of pancreatic secretory proteins is obligatorily coupled to protein synthesis or whether it is a separable process which can be independently regulated. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-(3)H for 3 min and incubated post-pulse for 37 min in chase medium containing cycloheximide up to concentrations sufficient to inhibit protein synthesis by 98%. In controls, newly synthesized secretory proteins are transported over this interval to condensing vacuoles of the Golgi complex. Since the latter are recovered in the zymogen granule fraction upon cell fractionation, intracellular transport was assayed by measuring the amount of protein radioactivity found in the zymogen granule fraction after a (3 + 37) min incubation. The results indicated that at maximum inhibition of protein synthesis (5 x 10(-4)M cycloheximide), transport proceeded with an efficiency approximately 80% of control. Parallel radioautographic studies on intact slices confirmed these data and further indicated that all the steps of intracellular transport, including discharge to the acinar lumen, were independent of protein synthesis. We conclude that: (1) transport and protein synthesis are separable processes; (2) intracellular transport is not the result of a continuous delivery of secretory proteins from attached polysomes to the cisternae of the rough endoplasmic reticulum; and (3) transport is not dependent on the synthesis of "specific" nonsecretory proteins within the time limits tested.
Article
Rats were starved for 48 hrs and stimulated to secrete by means of pilocarpine. The rate of synthesis of protein was determined both biochemically and autoradiographically by the assessment of leucine-3H incorporation during 10 min after its administration at various intervals (up to 7 hrs) after the pilocarpine stimulation. Care was taken to assess the incorporation with reference to the number of cells present, not to the total dry matter content nor to the protein content, as the latter tend to vary with the physiological state of the organ. Depletion of a pancreas filled with zymogen granules by means of pilocarpine gives rise to a transitory decrease in its rate of protein synthesis. After 1 hr the rate of synthesis reverts to the value found in fasting, unstimulated rats. Studies of pancreas slices taken from the rats after killing show that the in vitro synthetic capacity (leucine-14C) of the gland is unaffected by either fasting or stimulation. Peri-insular acini do not lose their zymogen granules upon pilocarpine stimulation as do the other acini. Yet, the rate of protein synthesis in both types of acini follows the same course after fasting and subsequent stimulation. Protein synthesized during fasting is largely lost from the gland during the next 24 hrs of continued fasting, either through catabolism or secretion. The results are discussed with a view on conflicting reports in the literature on the possibility of enhancing the rate of protein synthesis in the pancreas by stimulation of the secretion. A concept is given of a secretory cycle combined with a continuous protein synthesis at a constant rate in the cells of the rat pancreas, the cycle being divided into four phases.
Article
Synchronization of the secretory cycle in vivo was obtained by injecting isoprenaline as an inducer of secretion. A quantitative correlation between enzyme release, its subsequent reaccumulation, and the sequence of ultrastructural changes was found. At the ultrastructural level secretion was paralleled by depletion of zymogen granules through fusion of the granule membrane with the lumen membrane and discharge of the content. Each zymogen granule membrane, once connected with the lumen, acted as a lumen membrane. Fusion was thus sequential and resulted in a dramatic enlargement of the lumen space. During the entire process the passage between the lumen and the intercellular space remained blocked by the tight junctions, as shown by their impenetrability to ferritin. Reduction of the lumen size following enzyme discharge seemed to be achieved by withdrawal of lumen membrane in the form of small smooth vesicles which appeared mostly in the apical part of the cell. At the same time, the cell retracted towards the lumen, the whole process being completed within 2 hr from onset of secretion. Disappearance of the smooth vesicle followed, concomitant with formation of many condensing vacuoles and appearance of mature zymogen granules. The fate of the zymogen granule membrane, including its fusion with the lumen membrane, resorption in the form of small smooth vesicles, and its eventual reutilization mediated by the Golgi system, is discussed.
Article
Electron microscopic studies were performed on exocrine pancreas biopsy specimens from eight dogs, following continuous infusion of secretin and injection of pancreozymin. The excreting process of the proteins by acinar cells is increased after the injection of pancreozymin: there is an enlargement of the acinar lumina, a multiplication of the apical vacuoles, an apical localization and usually a marked depletion of the zymogen granules within the cells. On the other hand, the dense content of the cisternae of the endoplasmic reticulum, the enlargement of the Golgi complex, the abundance of presumed immature zymogen granules are the witnesses of an excreting process which seems to be followed by a rise in the rate of the protein synthesis. The abundance of lysosomal structures chiefly after the pancreozymin injection, can be attributed to an increase of the protein synthesis or to the toxic effects of the drugs. In the washing out period, the emptiness of the acinar lumen can be explained by the diminution of the protein secretion. The main result of this experiment is to show that a presumed specific surstimulation is capable of evoking ultra-structural alterations of the acinar cells which are similar to those observed after experimental intoxication and under pathological conditions.Copyright © 1969 S. Karger AG, Basel
Article
Alterations in net uptake and exchange of free amino acids and in their subsequent incorporation into protein were produced in rat-liver slices by addition of varying amounts of one amino acid to the incubation medium. When the initial extracellular concentration of l-phenylalanine was changed from subnormal (2 μM) or normal (50 μM) levels to a supranormal level (155 μM), hepatic uptake and flux of this amino acid, as well as the flux of free valine, were increased. Concomitantly, hepatic exchange of leucine was depressed and that of lysine was unchanged. The effects of elevated intracellular levels of phenylalanine on net uptake of the other essential amino acids were masked by a general increase in their accumulation and overall utilization.Elevated levels of phenylalanine were associated with enhanced incorporation or flux of leucine, valine, and lysine into hepatic protein, even though the effect on hepatic exchange of each of these amino acids differed. Graded increases in extracellular levels of l-threonine of l-valine also enhanced incorporation of leucine into hepatic protein. The results indicate that protein synthesis and degradation were stimulated in the presence of elevated intracellular levels of one amino acid.
Article
1. Canine pancreas slices were incubated with [6-(14)C]orotic acid and the rate of its incorporation into RNA was measured. RNA was fractionated by shaking homogenates with phenol at 2 degrees , 50 degrees , 65 degrees and 80 degrees . Cytoplasmic RNA was extracted at the lowest temperature and nuclear RNA at the higher temperatures. The samples were centrifuged through sucrose gradients and the E(260) and (14)C-sedimentation patterns determined. Incorporation of orotic acid was very rapid into cytoplasmic 4s RNA. This probably represents end-group turnover. No incorporation into cytoplasmic ribosomal RNA was observed. 2. The nuclear 50 degrees -RNA exhibited two E(260) peaks, at 18s and 28s. This portion of the sample contained but moderate amounts of [(14)C]RNA. The highly labelled material had sedimentation coefficients in the range 35-50s. The nuclear 65 degrees -RNA showed an E(260) peak at 16s. The [(14)C]RNA peak occurred at 25-35s and this portion demonstrated the highest specific activity of any RNA fraction. 3. The 50 degrees -RNA, 65 degrees -RNA and 80 degrees -RNA were hydrolysed and their base compositions were determined. All three samples possess a ribosomal type of composition (G+C)/(A+U)=(1.4-1.7). For this reason they are considered to contain ribosomal precursor RNA as their major constituent. 4. Actinomycin D (0.5mug./ml.) in the incubation medium inhibited incorporation of orotic acid into both nuclear fractions but not into 4s RNA. 5. The cholinergic drug Urecholine inhibited incorporation into the heavy, high-specific-activity portions of the nuclear fractions but did not inhibit incorporation into the ribosomal precursor type of nuclear RNA. A similar result was also obtained with the hormone pancreozymin. Moderate inhibition of incorporation of orotic acid into 4s RNA likewise resulted from the presence of the drug and the hormone.
Article
In the previous paper we described an in vitro system of guinea pig pancreatic slices whose secretory proteins can be pulse-labeled with radioactive amino acids. From kinetic experiments performed on smooth and rough microsomes isolated by gradient centrifugation from such slices, we obtained direct evidence that secretory proteins are transported from the cisternae of the rough endoplasmic reticulum to condensing vacuoles of the Golgi complex via small vesicles located in the periphery of the complex. Since condensing vacuoles ultimately become zymogen granules, it was of interest to study this phase of the secretory cycle in pulse-labeled slices. To this intent, a zymogen granule fraction was isolated by differential centrifugation from slices at the end of a 3-min pulse with leucine-(14)C and after varying times of incubation in chase medium. At the end of the pulse, few radioactive proteins were found in this fraction; after +17 min in chaser, its proteins were half maximally labeled; they became maximally labeled between +37 and +57 min. Parallel electron microscopic radioautography of intact cells in slices pulse labeled with leucine-(3)H showed, however, that zymogen granules become labeled, at the earliest, +57 min post-pulse. We assumed that the discrepancy between the two sets of results was due to the presence of rapidly labeled condensing vacuoles in the zymogen granule fraction. To test this assumption, electron microscopic radioautography was performed on sections of zymogen granule pellets isolated from slices pulse labeled with leucine-(3)H and subsequently incubated in chaser. The results showed that the early labeling of the zymogen granule fractions was, indeed, due to the presence of highly labeled condensing vacuoles among the components of these fractions.
Article
It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-(14)C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.
Article
Relationship between the dose of the stimulants pancreozymin and methacholine and the secretion of amylase by the pancreas has been studied. The nembutalized dogs were given a continuous intravenous infusion of secretin during the entire course of the experiments and samples of pancreatic juice were collected every 10 minutes for amylase determination. The average minimal dose required for elevation of enzyme output was 0.025 mg and 0.008 µg/kg body weight for pancreozymin and methacholine, respectively. The response to these doses at the start and end of the experimental period were quite similar. Over a wide dosage-range of both methacholine and pancreozymin, the absolute rise in amylase output was a linear function of the logarithm of the dose. Within a small portion of the S-shaped curve obtained by correlating the absolute increase in output with the absolute value of the dose, an apparent linear proportionality existed at low dosage levels. No synergism between pancreozymin and methacholine was found; the combined effect was additive. The gland was practically inexhaustible by continuous stimulation for 20 hours and the enzyme could be made at the rate secreted within the dosage-range of the stimulants used. Practical suggestions have been made for the bioassay of stimulants of pancreatic enzyme output.
Dense, homogeneous granules of 250 to 350 mmicro in diameter have been found inside the cavities of the endoplasmic reticulum in the acinar cells of the pancreas of the guinea pig. They apparently correspond to the fine granules described in the same material in light microscopy by Mankowski and Bensley. The location of these granules indicates that the endoplasmic reticulum is a cavitary system and suggests its participation in secretory processes.
Article
Changes in the intracellular distribution of amylase and in the biochemical properties of the microsome and supernatant fluid fractions of rat pancreas have been studied during a complete secretory cycle. In general, the amylase activity reached a minimum about two hours after the injection of pilocarpine returning gradually to normal at about eight hours after the injection. The most extensive early loss of amylase activity occurred in the secretory granules; the smallest relative loss occurred in the microsome fraction. Essentially no change was observed in the protein, ribose nucleic acid or phospholipid of the microsome and supernatant fluid fractions, but extensive changes were observed in the quantity of secretory granule material. Apparently the structural components of the microsome and supernatant fluid fractions are conserved throughout the secretory cycle, while the substance of the secretory granules undergoes marked changes in quantity.
Article
Fixed tissue is dehydrated with tertiary butyl alcohol overnight. The following day it is cleared in toluene, infiltrated and embedded in Araldite resin-hardener-accelerator mixture without dibutyl phthalate, and polymerized at 60° C. More rapid than previous techniques, this method gives blocks which do not fracture unduly on trimming and provides sections of soft tissues at 1 μ for phase contrast microscopy, as well as ultrathin sections which cut as easily with glass knives as sections of methacrylate. Araldite manufactured in the U.S.A. and in England are different. Satisfactory proportions for the American are: hardener DDSA, 3.5 ml; casting resin 6005, 5.0 ml; accelerator B, 0.12 ml. For the British product, these are: hardener 964 B, 5.0 ml; casting resin M, 5.0 ml; accelerator 964 C, 0.25 ml. The use of 2% agar for orienting small specimens in Araldite is feasible. Mallory's borax-methylene blue has been applied to the staining of Araldite sections as thin as 0.5 μ mounted on glass slides.
Article
Beef pancreas slices constitute a very active amino acid incorporating system. Rate of uptake of valine-C14 and tryptophan-C14 into protein indicates that ribosomes are the site of synthesis. Valine-C14 has also been shown to be incorporated into the chromatographed proteins trypsinogen, chymotrypsinogen A, and ribonuclease during short (2 and 15 minute) incubations. The specific radioactivities of the latter two purified proteins in the microsomal solubilized portion were much greater than that of total protein in this fraction after a 2-minute incubation. This large difference in specific radioactivity was not found in the ribosomes themselves. The data suggest either that "export" proteins are synthesized many times more rapidly than total protein or that the sites of synthesis of the two types of protein are different.
Article
This paper presents a brief review of the ultrastructure of some of the structural elaborations of the plasma membrane that may be regarded as adaptations for specific cell functions. Among the relatively stable specializations of the free surface, it considers the striated border of the intestinal mucosa, the brush border in the nephron, and filamentous coatings on the membranes of certain cells of the gastric mucosa. It then turns to those transient configurations of cell surfaces involved in the dynamic processes of pinocytosis, phagocytosis, and liberation of secretory products and considers the turnover of membranes associated with these phenomena. Discussion of specializations of the contact surfaces includes the desmosomes and terminal bars and the present status of intercellular cement and interfacial canals. A section on specializations of the cell base considers the basement membrane and basal infoldings of the plasma membrane in cells engaged in active transport. And finally, there is a description of the terminal web and the marginal band of nucleated erythrocytes—specializations of the superficial cytoplasm concerned with maintenance of cell shape.
Article
Methods used in obtaining high resolution in autoradiography, with special emphasis on the technique of electron microscopic autoradiography, are described, together with control experiments designed to establish the optimum conditions or procedures. On the basis of these experiments the emulsion selected was Ilford L-4, with a crystal size slightly larger than 0.1 micron. It is applied to the specimen in the form of a gelled film consisting of a monolayer of silver halide crystals. Background, when present, can be eradicated by a simple method. The preparations can be stored, in presence of a drying agent, at room temperature or in a refrigerator. Photographic development is done in Microdol, or in a special fine grain "physical" developer. For examination in the electron microscope the sections are stained with uranyl or lead stains. These methods give a good localization of the label, at the subcellular level, and good reproducibility in relative grain counts.
Article
The dog pancreas isolated in situ was perfused with oxygenated dog blood and stimulated with pancreozymin, secretin, or both. There were no significant changes in the fine structure of the acinar, centroacinar, or duct cells attributable to the perfusion. Combined glutaraldehyde and osmium fixation gave good preservation of the secretory products of the acinar cell. Before stimulation, the lumen of the acini is filled with material similar in texture to the content of the zymogen granules, but of somewhat lower density. Release of secretion commonly takes place by coalescence of the limiting membrane of zymogen granules with the plasmalemma, but one granule opening at the surface may frequently be joined by others coalescing with its membrane and forming an interconnected series all with contents having the same texture as the released zymogen. Such a mechanism seems to permit a more rapid release of secretory product than discharge of individual granules. Pancreozymin stimulation caused marked depletion of zymogen granules, but no obvious changes in the Golgi apparatus. It is clear, therefore, that this hormone exerts its effect upon release of granules rather than upon their formation. Secretin stimulation of water and bicarbonate secretion caused a marked washing out of the luminal contents, but had little detectable effect on cellular structure.
CHYMOTRYPSINOGEN SYNTHESIS IN THE EXOCRINE CELL OF THE GUINEA PIG PANCREAS WAS STUDIED UNDER THE FOLLOWING CONDITIONS: Animals fed after a fast of approximately 48 hours received approximately 1 hour after feeding an intravenous injection of DL-leucine-1-C(14). At various time intervals (1 to 45 minutes) after the injection, the glands were removed and fractionated into a series of cell fractions of known cytological significance. Ten to twelve animals were used for each time point. From each cell fraction, the chymotrypsinogen was isolated by acid extraction and purified by (NH(4))(2)SO(4) fractionation, isoelectric precipitation, and chromatography. Because of the minuteness of the quantities involved, chymotrypsinogen amounts were calculated from enzymatic activity figures, and a carrier method was used to precipitate and count the enzyme. The chymotrypsinogen isolated from the attached ribonucleoprotein particles of the microsomal fraction had the highest specific radioactivity at the early time points (1 to 3 minutes). After long intervals (at 15 to 45 minutes), the specific radioactivity of the enzyme increased in the microsomal contents and finally in the zymogen granules. The results are compatible with the view that the chymotrypsinogen is synthesized in or on the attached RNP particles and subsequently transported to other cell compartments.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
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