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.

scholarly journals Identification of cell surface receptors for the Act-2 cytokine.

1990 ◽  
Vol 172 (1) ◽  
pp. 285-289 ◽  
Author(s):  
M Napolitano ◽  
K B Seamon ◽  
W J Leonard
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
K562 Cells ◽  
Peripheral Blood Lymphocytes ◽  
Polyclonal Antiserum ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Rabbit Polyclonal Antiserum ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd

We have identified cell surface receptors for Act-2, a secreted protein expressed upon activation of T cells, B cells, and monocytes. Although 125I-Act-2 showed little, if any, specific binding to resting peripheral blood lymphocytes (PBL) receptors were readily detected on PHA/PMA-activated PBL and a variety of cell lines including MT-2, HL60, DMSO differentiated HL60, HeLa, and K562 cells. The equilibrium dissociation constant (Kd) is 3-12 nM for MT-2, K562, and PBL activated with PHA/PMA for 40-80 h. We have also identified a rabbit polyclonal antiserum that can block Act-2 binding to its receptors. The ability to detect specific Act-2 receptors and the development of a blocking antiserum should prove valuable in efforts to molecularly clone the Act-2 receptor and to dissect the biological actions of Act-2.

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 Cholecystokinin octapeptide inhibits Ca2+-dependent amylase secretion from permeabilized pancreatic acini by blocking the MgATP-dependent priming of exocytosis

1998 ◽  
Vol 330 (1) ◽  
pp. 329-334 ◽  
Author(s):  
J. Philip PADFIELD ◽  
Ninder PANESAR
Keyword(s):  
Acinar Cells ◽  
Secretory Response ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
Rapid Phase ◽  
Pancreatic Acini ◽  
Cholecystokinin Octapeptide ◽  
Cholecystokinin Receptor ◽  
Specific Antagonist

At present little is known about how the low-affinity cholecystokinin receptor inhibits secretagogue-stimulated amylase secretion from pancreatic acinar cells. To examine this question we have determined how cholecystokinin octapeptide (CCK8) influences Ca2+-dependent amylase secretion from α-toxin-permeabilized pancreatic acini. CCK8 significantly inhibited Ca2+-stimulated amylase secretion. The inhibitory actions of CCK8 were completely blocked by the addition of JMV-180, a specific antagonist for the low-affinity CCK8 receptor. Previous studies have shown that Ca2+-dependent amylase secretion from α-toxin-permeabilized acini has two distinct phases [Padfield and Panesar (1997) Am. J. Physiol. 36, G655-660]. There is an initial rapid phase of secretion which represents release from exocytotic sites primed by MgATP prior to permeabilization. This is followed by a slower sustained phase of secretion which, in part, reflects the MgATP-dependent repriming of the exocytotic machinery. CCK8 did not influence the initial rapid phase of the Ca2+-dependent secretory response, but inhibited the second slower sustained phase. Moreover, CCK8 was shown to inhibit the MgATP-dependent priming of exocytosis in the acini. These results indicate that the low-affinity CCK receptor blocks stimulated amylase secretion by inhibiting the MgATP-dependent repriming of exocytosis.

scholarly journals HSV-1 Virions Engineered for Specific Binding to Cell Surface Receptors

2004 ◽  
Vol 9 (3) ◽  
pp. 419-427 ◽  
Author(s):  
Paola Grandi ◽  
Samuel Wang ◽  
Deborah Schuback ◽  
Victor Krasnykh ◽  
Matthew Spear ◽  
...  
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Hsv 1

scholarly journals Distribution of cell surface saccharides on pancreatic cells

1979 ◽  
Vol 80 (1) ◽  
pp. 69-76 ◽  
Author(s):  
M Maylie-Pfenninger ◽  
JD Jamieson
Keyword(s):  
Cell Surface ◽  
General Procedure ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Affinity Constant ◽  
Affinity Column ◽  
Gel Chromatography ◽  
Binding Studies ◽  
Binding Properties ◽  
Pancreatic Cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a "universal" affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.

Quantitative electron microscope autoradiographs of 125I-cholecystokinin in pancreatic acini

1982 ◽  
Vol 243 (4) ◽  
pp. G291-G296 ◽  
Author(s):  
J. A. Williams ◽  
H. Sankaran ◽  
E. Roach ◽  
I. D. Goldfine
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Specific Binding ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Pancreatic Acini ◽  
Time Points ◽  
Basolateral Plasma Membrane ◽  
Specific Receptors ◽  
Silver Grains

To morphologically evaluate the interaction of cholecystokinin (CCK) with its receptors on pancreatic acinar cells, we incubated isolated mouse acini at 37 degrees C with radioiodinated CCK and then prepared quantitative electron microscope autoradiographs. Specific binding of CCK to acini was one-half maximal at 2 min of incubation and maximal after 10 min. The cell-associated radioactivity was extracted and analyzed on Sephadex G-50. After 2 min, 90% of the total cellular radioactivity remained as intact CCK; after 30 min, the intact radioactivity decreased to 65% of total. At 2 min, the fraction of bound hormone that fixed to acini was 84% of total; this amount decreased to 78% after 30 min. Thus, the majority of radioactivity in the autoradiographs at both time points was intact CCK; however, at 30 min, a small amount was also degraded hormone. After both 2 and 30 min of incubation, silver grains were highly concentrated over the basolateral plasma membrane. A significant number of grains were in the cell interior at both time points, increasing from 13% of total grains at 2 min to 42% at 30 min. At both times, the largest fraction of internalized grains was localized over the endoplasmic reticulum. At 30 min, a significant concentration of CCK grains was observed over multivesicular bodies. The present study demonstrates, therefore, that CCK binds to specific receptors on the basolateral surface of pancreatic acinar cells. After binding, the hormone is internalized, locates predominantly on the endoplasmic reticulum, and is then degraded.

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.

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