Exocytosis in mammalian cells. II. Topography of ferritin-concanavalin a conjugate and ruthenium red binding sites on the luminal surface of pancreatic acinar cells

1978 ◽  
Vol 63 (2) ◽  
pp. 170-177 ◽  
Author(s):  
A.R. Beaudoin ◽  
M.R. Paquet ◽  
A. Lord ◽  
L. Dionne
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Acinar Cells ◽  
Ruthenium Red ◽  
Pancreatic Acinar Cells ◽  
Luminal Surface

scholarly journals Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

1975 ◽  
Vol 23 (8) ◽  
pp. 607-617 ◽  
Author(s):  
T Amakawa ◽  
T Barka
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Concanavalin A ◽  
Binding Sites ◽  
Lectin Binding ◽  
Single Cells ◽  
Apical Cell ◽  
Acinar Cells ◽  
Con A ◽  
Dissociated Cells

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.

scholarly journals Synthesis and application of photoaffinity analogues of inositol 1,4,5-trisphosphate selectively substituted at the 1-phosphate group

1990 ◽  
Vol 272 (3) ◽  
pp. 817-825 ◽  
Author(s):  
R Schäfer ◽  
M Nehls-Sahabandu ◽  
B Grabowsky ◽  
M Dehlinger-Kremer ◽  
I Schulz ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Inositol Phosphates ◽  
Specific Binding ◽  
Acinar Cells ◽  
Phosphate Group ◽  
Pancreatic Acinar Cells ◽  
Maximal Effect ◽  
Microsomal Preparation ◽  
Specific Binding Sites

We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P3 selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P3-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AbaIP3) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3), bind to high affinity Ins(1,4,5)P3-specific binding sites at a 9-fold lower affinity (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 45 nM]. Ca2+ release activity of the inositol derivatives was tested with AbaIP3. It induced a rapid and concentration-dependent Ca2+ release from the ER fraction [EC50 (dose producing half-maximal effect) = 3.1 microM] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 = 0.3 microM). From the two radioactive labelled analogues ([3H]AbaIP3 and 125I-AsIP3) synthesized, the radioiodinated derivative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P3 binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P3-inducible Ca2+ release (fraction P1). The other two Ins(1,4,5)P3-binding proteins, of 49 and 31 kDa, were obtained in fraction P2, in which Ins(1,4,5)P3-induced Ca2+ release was half of that obtained in fraction P1. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)P3-induced Ca2+ release from the ER of rat pancreatic acinar cells.

scholarly journals Sialoglycoconjugates of a pancreatic tumor: markers for cell polarity, membrane fluidity, and possible role in exocytosis.

1986 ◽  
Vol 34 (10) ◽  
pp. 1265-1270 ◽  
Author(s):  
Z Mureşan ◽  
V Mureşan ◽  
V Iwanij ◽  
J D Jamieson
Keyword(s):  
Tumor Cells ◽  
Binding Sites ◽  
Pancreatic Tumor ◽  
Acinar Cells ◽  
Basement Membranes ◽  
Pancreatic Acinar Cells ◽  
Asymmetric Distribution ◽  
Neoplastic Cells ◽  
Dissociated Cells

The distribution and nature of sialoglycoconjugates on the surface of cells of a pancreatic carcinoma and their behavior when interacting with the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph) were compared to those of normal pancreatic acinar cells. Fluorescence microscopy of frozen sections, using rhodaminated LPA (Rh-LPA), revealed protease-resistant binding sites evenly distributed over the cell surface of neoplastic cells, contrasting with the asymmetric distribution of sialoglycoconjugates on normal acinar cells. An asymmetric staining pattern, resembling that of normal acinar cells, was occasionally observed in tumor cells that had regained their structural polarity when in contact with the basement membranes of blood vessels. Cytochemistry, using horseradish peroxidase-conjugated LPA (HRP-LPA), showed that the binding of limulin to neoplastic cells was less intense than that to any plasmalemmal domain of normal acinar cells. In tumor cells, local intensification of LPA binding was systematically observed on plasmalemmal regions adjacent to zymogen granules. Fixed dissociated cells, both tumor and normal, treated with Rh-LPA, retained the fluorescence distribution of Rh-LPA observed in situ. Nonfixed neoplastic cells showed lectin-induced patching of limulin binding sites and were more susceptible to agglutination by LPA than normal acinar cells.

scholarly journals Identification and localization of cholecystokinin-binding sites on rat pancreatic plasma membranes and acinar cells: a biochemical and autoradiographic study.

1983 ◽  
Vol 96 (5) ◽  
pp. 1288-1297 ◽  
Author(s):  
S A Rosenzweig ◽  
L J Miller ◽  
J D Jamieson
Keyword(s):  
Acinar Cell ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Cross Linking ◽  
Dependent Manner ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Pancreatic Acini

Using the combined approaches of affinity labeling and light and electron microscopic autoradiography, we investigated the identification and localization of cholecystokinin (CCK)-binding sites on rat pancreatic acinar cells. To define the molecular properties of the CCK-binding site, we incubated rat pancreatic plasma membranes with 125-I-CCK-33 for 15 min at 23 degrees C followed by washing and cross-linking with disuccinimidyl suberate. Specific labeling of a major Mr 85,000 component was revealed as assessed by SDS PAGE under reducing conditions and autoradiography of the dried gels. Components of Mr greater than 200,000, Mr 130,000-140,000, and, Mr 55,000 were labeled under maximal cross-linking conditions. The labeling of all components was specifically inhibited by CCK-8 in a dose-dependent manner (Kd approximately 9 nM). The Mr 85,000 component had identical electrophoretic mobilities under reducing and nonreducing conditions indicating that it likely does not contain intramolecular disulfide bonds. The larger labeled species may be cross-linked oligomers of this binding protein or complexes between it and neighboring polypeptides. For studies on the distribution of CCK-binding sites, pancreatic acini were incubated with 125I-CCK-33 (0.1 nM) in the absence or presence of CCK-8 (1 microM) for 2 or 15 min at 37 degrees C, washed, and fixed in 2% glutaraldehyde. Quantitative autoradiographic analysis indicated that approximately 60% of the total grains were located within +/- 1 HD (1 HD = 100 nm) of the lateral and basal plasmalemma with little or no labeling of the apical plasmalemma. From these data, it was estimated that each acinar cell possesses at least 5,000-10,000 CCK-binding sites on its basolateral plasmalemma. The remaining grains showed no preferential concentration over the cytoplasm or nucleus. Together, these data indicate that CCK interacts with a Mr 85,000 protein located on the basolateral plasmalemma of the pancreatic acinar cell.

scholarly journals Dual sensitivity of sarcoplasmic/endoplasmic Ca2+-ATPase to cytosolic and endoplasmic reticulum Ca2+ as a mechanism of modulating cytosolic Ca2+ oscillations

2004 ◽  
Vol 383 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Kojiro YANO ◽  
Ole H. PETERSEN ◽  
Alexei V. TEPIKIN
Keyword(s):  
Mathematical Model ◽  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Acinar Cells ◽  
Spike Frequency ◽  
Pancreatic Acinar Cells ◽  
Single Type ◽  
Store Depletion ◽  
The Mathematical Model ◽  
Conformation State

The effects of ER (endoplasmic reticulum) Ca2+ on cytosolic Ca2+ oscillations in pancreatic acinar cells were investigated using mathematical models of the Ca2+ oscillations. We first examined the mathematical model of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) to reproduce the highly co-operative inhibitory effect of Ca2+ in the ER lumen on ER Ca2+ uptake in the acinar cells. The model predicts that luminal Ca2+ would most probably inhibit the conversion of the conformation state with luminal Ca2+-binding sites (E2) into the conformation state with cytoplasmic Ca2+-binding sites (E1). The SERCA model derived from this prediction showed dose–response relationships to cytosolic and luminal Ca2+ concentrations that were consistent with the experimental data from the acinar cells. According to a mathematical model of cytosolic Ca2+ oscillations based on the modified SERCA model, a small decrease in the concentration of endoplasmic reticulum Ca2+ (approx. 20% of the total) was sufficient to abolish the oscillations. When a single type of IP3R (IP3 receptor) was included in the model, store depletion decreased the spike frequency. However, the frequency became less sensitive to store depletion when we added another type of IP3R with higher sensitivity to the concentration of free Ca2+ in the cytosol. Bifurcation analysis of the mathematical model showed that the loss of Ca2+ from the ER lumen decreased the sensitivity of cytosolic Ca2+ oscillations to IP3 [Ins(1,4,5)P3]. The addition of a high-affinity IP3R did not alter this property, but significantly decreased the sensitivity of the spike frequency to IP3. Our mathematical model demonstrates how luminal Ca2+, through its effect on Ca2+ uptake, can control cytosolic Ca2+ oscillations.

Ruthenium red selectively depletes inositol 1,4,5-trisphosphate-sensitive calcium stores in permeabilized rabbit pancreatic acinar cells

1993 ◽  
Vol 135 (2) ◽  
pp. 153-163 ◽  
Author(s):  
Frans H. M. M. van de Put ◽  
Joost G. J. Hoenderop ◽  
Jan Joep H. H. M. De Pont ◽  
Peter H. G. M. Willems
Keyword(s):  
Acinar Cells ◽  
Ruthenium Red ◽  
Pancreatic Acinar Cells ◽  
Calcium Stores ◽  
Inositol 1,4,5 Trisphosphate

scholarly journals Adaptive regulation of pancreatic acinar mitochondrial thiamin pyrophosphate uptake process: possible involvement of epigenetic mechanism(s)

2017 ◽  
Vol 313 (5) ◽  
pp. G448-G455 ◽  
Author(s):  
Subrata Sabui ◽  
Veedamali S. Subramanian ◽  
Rubina Kapadia ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Mammalian Cells ◽  
Acinar Cells ◽  
Epigenetic Mechanism ◽  
Pancreatic Acinar Cells ◽  
Adaptive Regulation ◽  
Oxidative Energy Metabolism ◽  
Uptake Process ◽  
First Time ◽  
Thiamin Pyrophosphate

The essentiality of thiamin stems from its roles as a cofactor [mainly in the form of thiamin pyrophosphate (TPP)] in critical metabolic reactions including oxidative energy metabolism and reduction of cellular oxidative stress. Like other mammalian cells, pancreatic acinar cells (PAC) obtain thiamin from their surroundings and convert it to TPP; mitochondria then take up TPP by a carrier-mediated process that involves the mitochondrial TPP (MTPP) transporter (MTPPT; product of SLC25A19 gene). Previous studies have characterized different physiological/biological aspects of the MTPP uptake process, but little is known about its possible adaptive regulation. We addressed this issue using pancreatic acinar 266-6 cells (PAC 266-6) maintained under thiamin-deficient (DEF) and oversupplemented (OS) conditions, as well as thiamin-DEF and -OS transgenic mice carrying the SLC25A19 promoter. We found that maintaining PAC 266-6 under the thiamin-DEF condition leads to a significant induction in mitochondrial [3H]TPP uptake, as well as in the level of expression of the MTPPT protein and mRNA compared with thiamin-OS cells. Similar findings were observed in mitochondria from thiamin-DEF mice compared with thiamin-OS. Subsequently, we demonstrated that adaptive regulation of MTTP protein was partly mediated via transcriptional mechanism(s) via studies with PAC 266-6 transfected with the SLC25A19 promoter and transgenic mice carrying the SLC25A19 promoter. This transcriptional regulation appeared to be, at least in part, mediated via epigenetic mechanism(s) involving histone modifications. These studies report, for the first time, that the PAC mitochondrial TPP uptake process is adaptively regulated by the prevailing thiamin level and that this regulation is transcriptionally mediated and involves epigenetic mechanism(s). NEW & NOTEWORTHY Our findings show, for the first time, that the mitochondrial thiamin pyrophosphate (MTPP) uptake process is adaptively regulated by the prevailing thiamin level in pancreatic acinar cells and this regulation is mediated, at least in part, by transcriptional and epigenetic mechanism(s) affecting the SLC25A19 promoter.

scholarly journals Uptake of ascorbic acid by pancreatic acinar cells is negatively impacted by chronic alcohol exposure

2016 ◽  
Vol 311 (1) ◽  
pp. C129-C135 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Hamid M. Said
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Mammalian Cells ◽  
Marked Reduction ◽  
Acinar Cells ◽  
Alcohol Exposure ◽  
Epigenetic Mechanism ◽  
Pancreatic Acinar Cells ◽  
Chronic Alcohol ◽  
Chronic Alcohol Exposure

Vitamin C (ascorbic acid, AA) is indispensable for normal metabolism of all mammalian cells including pancreatic acinar cells (PACs). PACs obtain AA from their surroundings via transport across the cell membrane. Chronic alcohol exposure negatively affects body AA homeostasis; it also inhibits uptake of other micronutrients into PACs, but its effect on AA uptake is not clear. We examined this issue using both in vitro (266-6 cells) and in vivo (mice) models of chronic alcohol exposure. First, we determined the relative expression of the AA transporters 1 and 2 [i.e., sodium-dependent vitamin C transporter-1 (SVCT-1) and SVCT-2] in mouse and human PACs and found SVCT-2 to be the predominant transporter. Chronic exposure of 266-6 cells to alcohol significantly inhibited AA uptake and caused a marked reduction in SVCT-2 expression at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Similarly, chronic alcohol feeding of mice significantly inhibited AA uptake and caused a marked reduction in level of expression of the SVCT-2 protein, mRNA, and hnRNA. These findings suggest possible involvement of transcriptional mechanism(s) in mediating chronic alcohol effect on AA uptake by PACs. We also observed significant epigenetic changes (histone modifications) in the Slc23a2 gene (reduction in H3K4me3 level and an increase in H3K27me3 level) in the alcohol-exposed 266-6 cells. These findings show that chronic alcohol exposure inhibits PAC AA uptake and that the effect is mediated, in part, at the level of transcription of the Slc23a2 gene and may involve epigenetic mechanism(s).

Control of Ca2+ wave propagation in mouse pancreatic acinar cells

1998 ◽  
Vol 274 (3) ◽  
pp. C663-C672 ◽  
Author(s):  
Fatima Pfeiffer ◽  
Lutz Sternfeld ◽  
Andreas Schmid ◽  
Irene Schulz
Keyword(s):  
Laser Scanning ◽  
Basolateral Membrane ◽  
Acinar Cells ◽  
Ruthenium Red ◽  
Luminal Cell ◽  
Pancreatic Acinar Cells ◽  
Confocal Laser ◽  
Control Mechanisms ◽  
Cell Pole ◽  
Pkc Activation

We have investigated control mechanisms involved in the propagation of agonist-induced Ca2+ waves in isolated mouse pancreatic acinar cells. Using a confocal laser-scanning microscope, we were able to show that maximal stimulation of cells with acetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initial Ca2+ release of comparable amounts with both agonists at the luminal cell pole. Subsequent Ca2+ spreading to the basolateral membrane was faster with ACh (17.3 ± 5.4 μm/s) than with bombesin (8.0 ± 2.2 μm/s). The speed of bombesin-induced Ca2+ waves could be increased up to the speed of ACh-induced Ca2+waves by inhibition of protein kinase C (PKC). Activation of PKC significantly decreased the speed of ACh-induced Ca2+ waves but had only little effect on bombesin-evoked Ca2+waves. Within 3 s after stimulation, production of inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3] was higher in the presence of ACh compared with bombesin, whereas bombesin induced higher levels of diacylglycerol (DAG) than ACh. These data suggest that the slower propagation speed of bombesin-induced Ca2+ waves is due to higher activation of PKC in the presence of bombesin compared with ACh. The higher increase in bombesin- compared with ACh-induced DAG production is probably due to activation of phospholipase D (PLD). Inhibition of the PLD-dependent DAG production by preincubation with 0.3% butanol led to an acceleration of the bombesin-induced Ca2+ wave. In further experiments, we could show that ruthenium red (100 μM), an inhibitor of Ca2+-induced Ca2+ release in skeletal muscle, also decreased the speed of ACh-induced Ca2+ waves. The effect of ruthenium red was not additive to the effect of PKC activation. From the data, we conclude that, following Ins(1,4,5) P 3-induced Ca2+ release in the luminal cell pole, secondary Ca2+ release from stores, which are located in series between the luminal and the basal plasma membrane, modifies Ca2+spreading toward the basolateral cell side by Ca2+-induced Ca2+ release. Activation of PKC leads to a reduction in Ca2+release from these stores and therefore could explain the slower propagation of Ca2+ waves in the presence of bombesin compared with ACh.

Sign in / Sign up

Export Citation Format

Share Document