scholarly journals Cell type-dependent variations in the subcellular distribution of alpha-mannosidase I and II

1993 ◽  
Vol 122 (1) ◽  
pp. 39-51 ◽  
Author(s):  
A Velasco ◽  
L Hendricks ◽  
KW Moremen ◽  
DR Tulsiani ◽  
O Touster ◽  
...  
Keyword(s):  
Cell Surface ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Cell Types ◽  
Goblet Cells ◽  
Cross Reactivity ◽  
Pancreatic Acinar Cells ◽  
Cell Type ◽  
Golgi Stack ◽  
Cell Surface Staining

alpha-mannosidases I and II (Man I and II) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis- and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man II was most commonly found in medial- and/or trans- cisternae but showed cell type-dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO cells), both medial and trans (pancreatic acinar cells, enterocytes), or trans- (goblet cells) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medial- and trans-cisternae. It also showed cell type dependent variations in its intra-Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross-reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed.

scholarly journals RNA-seq library preparation from single pancreatic acinar cells

2016 ◽  
Author(s):  
Damian Wollny ◽  
Sheng Zhao ◽  
Ana Martin-Villalba
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Acinar Cells ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Pancreatic Acinar Cells ◽  
Library Preparation ◽  
Cell Type ◽  
Promising Tool ◽  
Single Cell Rna Sequencing

Single cell RNA sequencing technology has emerged as a promising tool to uncover previously neglected cellular heterogeneity. Multiple methods and protocols have been developed to apply single cell sequencing to different cell types from various organs. However, library preparation for RNA sequencing remains challenging for cell types with high RNAse content due to rapid degradation of endogenous RNA molecules upon cell lysis. To this end, we developed a protocol based on the SMART-seq2 technology for single cell RNA sequencing of pancreatic acinar cells, the cell type with one of the highest ribonuclease concentration measured to date. This protocol reliably produces high quality libraries from single acinar cells reaching a total of 5x106 reads / cell and ∼ 80% transcript mapping rate with no detectable 3´end bias. Thus, our protocol makes single cell transcriptomics accessible to cell type with very high RNAse content.

scholarly journals Internalization of horseradish peroxidase isozymes by pancreatic acinar cells in vitro.

1989 ◽  
Vol 37 (1) ◽  
pp. 49-56 ◽  
Author(s):  
C Oliver ◽  
C L Tolbert ◽  
J F Waters
Keyword(s):  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Secretory Granules ◽  
Apical Cell ◽  
Early Time ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Peroxidase Isozymes ◽  
Apical Vesicles

We examined the uptake and fate of four horseradish peroxidase (HRP) isozymes (Type VI, VII, VIII, and IX) in isolated pancreatic acinar cells. The pattern of uptake was similar for all the isozymes examined, with the exception of Type IX. Very little Type IX HRP was internalized by the cells, and what endocytosis did occur was primarily from the apical cell surface in coated vesicles. In contrast, HRP Type VI, VII, and VIII appeared to be endocytosed largely at the basolateral cell surface. Initially, the tracer was found in smooth vesicles and tubules near the plasma membrane. The tubules resembled the basal lysosomes known to be present in these cells. At the early time points, HRP reaction product was also present in multivesicular bodies (MVBs). By 60 min, the HRP was localized in MVBs, vesicles, and tubules adjacent to the Golgi apparatus. By 12 hr after exposure to the isozymes, the tracer was present in small apical vesicles. At no time could reaction product be localized in the rough endoplasmic reticulum, Golgi saccules, or secretory granules. The results of this study suggest that the charge of a soluble-phase marker has little effect on its uptake or intracellular distribution.

scholarly journals Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

1994 ◽  
Vol 124 (1) ◽  
pp. 43-53 ◽  
Author(s):  
BP Jena ◽  
FD Gumkowski ◽  
EM Konieczko ◽  
GF von Mollard ◽  
R Jahn ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Binding Protein ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Apical Plasma Membrane ◽  
Gtp Binding Protein ◽  
Regulated Exocytosis ◽  
Gtp Binding

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

scholarly journals Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites

2002 ◽  
Vol 158 (2) ◽  
pp. 283-292 ◽  
Author(s):  
Michael C. Ashby ◽  
Madeleine Craske ◽  
Myoung Kyu Park ◽  
Oleg V. Gerasimenko ◽  
Robert D. Burgoyne ◽  
...  
Keyword(s):  
Ryanodine Receptors ◽  
Acinar Cells ◽  
Cell Types ◽  
Pancreatic Acinar Cells ◽  
Apical Region ◽  
Basal Region ◽  
Activation Pattern ◽  
Inositol Trisphosphate Receptors ◽  
Agonist Stimulation ◽  
Major Process

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.

scholarly journals Mucus formation in goblet cells

1933 ◽  
Vol 113 (784) ◽  
pp. 459-463 ◽  
Keyword(s):  
Golgi Apparatus ◽  
Salivary Glands ◽  
Secretory Granules ◽  
Goblet Cells ◽  
Neutral Red ◽  
Basal Region ◽  
Mucous Cells ◽  
Cell Type ◽  
Golgi Network ◽  
Golgi Zone

The formation of mucus in goblet cells and its relation to the Golgi apparatus has been studied by various workers. Nassanow (1923) showed clearly that the mucin granules in the goblet cells of Triton originated in the Golgi apparatus, and so brought secretion in these cells into line with his theory of the bound secretion. More recently Clara (1926) has shown in the goblet cells of birds that the mucin first appears in the region next to the nucleus, between it and the gland lumen. Florey (1932, a, b ) has considered this more extensively in two recent papers, and for a number of mammals has shown that the mucin granules of goblet cells first form in the meshes of the Golgi network. In epithelial cells of the mouse vagina, undergoing conversion into mucous cells, he has found that the same process occurs. In a recent investigation of secretory formation in the salivary glands and pancreas it was found by the present author that in every cell type examined the young secretory granules first appeared in the basal region of the cell in relation to the mitochondria. Subsequent emigration occurred into the Golgi zone, where they underwent conversion into mature secretory granules. In the mucous cells of the salivary glands it was shown that these newly formed granules might be stained intravitam by Janus green or neutral red, and that in fixed preparations they stained selectively with acid fuchsin as described by Noll (1902), In the light of this work it appeared probable that while mucin formation might occur in the Golgi zone of the goblet cells as described by these authors, the origin of the granules might lie in the basal region of the cell.

Calcium signaling in rat pancreatic acinar cells: a role for Gαq, Gα11, and Gα14

1999 ◽  
Vol 276 (1) ◽  
pp. G271-G279 ◽  
Author(s):  
David I. Yule ◽  
Christopher W. Baker ◽  
John A. Williams
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Cho Cells ◽  
Acinar Cells ◽  
Cell Types ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Pcr Products ◽  
Α Subunits ◽  
G Protein Α Subunits

Stimulus-secretion coupling in the pancreatic acinar cell is initiated by the secretagogues CCK and ACh and results in the secretion by exocytosis of the contents of zymogen granules. A key event in this pathway is the G protein-activated production of second messengers and the subsequent elevation of cytosolic-free Ca2+. The aim of this study was therefore to define the heterotrimeric G protein α-subunits present and participating in this pathway in rat pancreatic acinar cells. RT-PCR products were amplified from pancreatic acinar cell mRNA with primers specific for Gαq, Gα11, and Gα14 but were not amplified with primers specific for Gα15. The sequences of these PCR products confirmed them to be portions of the rat homologues of Gαq, Gα11, and Gα14. The pancreatic-derived cell line AR42J similarly expressed Gαq, Gα11, and Gα14; however, the Chinese hamster ovary (CHO) cell line only expressed Gα11 and Gαq. These data indicate that caution should be exercised when comparing signal transduction pathways between different cell types. The expression of these proteins in acinar cells was confirmed by immunoblotting samples of acinar membrane protein using specific antisera to the individual G protein α-subunits. The role of these proteins in Ca2+ signaling events was investigated by microinjecting a neutralizing antibody directed against a homologous sequence in Gαq, Gα11, and Gα14 into acinar cells and CHO cells. Ca2+ signaling was inhibited in acinar cells and receptor-bearing CHO cells in response to both physiological and supermaximal concentrations of agonists. The inhibition was >75% in both cell types. These data indicate a role for Gαq and/or Gα11 in intracellular Ca2+ concentration signaling in CHO cells, and in addition to Gαq and Gα11, Gα14 may also fulfill this role in rat pancreatic acinar cells.

Internalization and cellular processing of cholecystokinin in rat pancreatic acinar cells

1988 ◽  
Vol 255 (6) ◽  
pp. G738-G744
Author(s):  
R. S. Izzo ◽  
C. Pellecchia ◽  
M. Praissman
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Cholecystokinin Octapeptide ◽  
Surface Receptors ◽  
Intracellular Degradation ◽  
Lysosomotropic Agent ◽  
Cellular Processing ◽  
Intracellular Proteolysis

To evaluate the internalization of cholecystokinin, monoiodinated imidoester of cholecystokinin octapeptide [125I-(IE)-CCK-8] was bound to dispersed pancreatic acinar cells, and surface-bound and internalized radioligand were differentiated by treating with an acidified glycine buffer. The amount of internalized radioligand was four- and sevenfold greater at 24 and 37 degrees C than at 4 degrees C between 5 and 60 min of association. Specific binding of radioligand to cell surface receptors was not significantly different at these temperatures. Chloroquine, a lysosomotropic agent that blocks intracellular proteolysis, significantly increased the amount of CCK-8 internalized by 18 and 16% at 30 and 60 min of binding, respectively, compared with control. Dithiothreitol (DTT), a sulfhydryl reducing agent, also augmented the amount of CCK-8 radioligand internalized by 25 and 29% at 30 and 60 min, respectively. The effect of chloroquine and DTT on the processing of internalized radioligand was also considered after an initial 60 min of binding of radioligand to acinar cells. After 180 min of processing, the amount of radioligand internalized was significantly greater in the presence of chloroquine compared with controls, whereas the amount of radioligand declined in acinar cells treated with DTT. Internalized and released radioactivity from acinar cells was rebound to pancreatic membrane homogenates to determine the amount of intact radioligand during intracellular processing. Chloroquine significantly increased the amount of intact 125I-(IE)-CCK-8 radioligand in released and internalized radioactivity while DTT increased the amount of intact radioligand only in internalized samples. This study shows that pancreatic acinar cells rapidly internalize large amounts of CCK-8 and that chloroquine and DTT inhibit intracellular degradation.

Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles

2007 ◽  
Vol 293 (6) ◽  
pp. G1333-G1338 ◽  
Author(s):  
Svetlana G. Voronina ◽  
Mark W. Sherwood ◽  
Oleg V. Gerasimenko ◽  
Ole H. Petersen ◽  
Alexei V. Tepikin
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Acinar Cells ◽  
Cell Types ◽  
Living Cells ◽  
Pancreatic Acinar Cells ◽  
Extracellular Solution ◽  
Whole Cell Patch Clamp ◽  
Patch Pipette ◽  
Cellular Organelles

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.

Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+entry in mouse pancreatic acinar cells

2005 ◽  
Vol 288 (1) ◽  
pp. C214-C221 ◽  
Author(s):  
Juan A. Rosado ◽  
Pedro C. Redondo ◽  
Ginés M. Salido ◽  
Stewart O. Sage ◽  
Jose A. Pariente
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Cell ◽  
Botulinum Toxin A ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Amylase Secretion ◽  
Cell Type ◽  
Store Depletion

We recently reported that store-operated Ca2+entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as “secretion-like coupling.” As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

Novel localization of Rab3D in rat intestinal goblet cells and Brunner's gland acinar cells suggests a role in early Golgi trafficking

2007 ◽  
Vol 293 (1) ◽  
pp. G165-G177 ◽  
Author(s):  
Jack A. Valentijn ◽  
Laura van Weeren ◽  
Anton Ultee ◽  
Abraham J. Koster
Keyword(s):  
Laser Scanning ◽  
Secretory Granules ◽  
Physiological Role ◽  
Acinar Cells ◽  
Goblet Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Unexpected Finding ◽  
Brunner’S Gland ◽  
Brunner's Gland

Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. Here, we report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in the intestine was demonstrated by SDS-PAGE and Western blot analysis of homogenates prepared from the rat duodenum and colon. Confocal laser scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of the duodenum and colon and in Brunner's gland acinar cells. There was no colocalization between Rab3D and a trans-Golgi network marker, TGN-38. In contrast, Rab3D colocalized partially with a cis-Golgi marker, GM-130, and with a marker of cis-Golgi and coat protein complex I vesicles, β-COP. Strong colocalization was observed between Rab3D and the lectins Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of the medial and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.

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