Insulin action in pancreatic acini from streptozotocin-treated rats. III. Electron microscope autoradiography of 125I-insulin

1981 ◽  
Vol 240 (1) ◽  
pp. G69-G75
Author(s):  
I. D. Goldfine ◽  
B. M. Kriz ◽  
K. Y. Wong ◽  
G. Hradek ◽  
A. L. Jones ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Electron Microscope ◽  
Relative Concentration ◽  
Pancreatic Acinar Cells ◽  
Diabetic Rat ◽  
Dependent Manner ◽  
Electron Microscope Autoradiography ◽  
Intracellular Location ◽  
Pancreatic Acini ◽  
Grain Distribution

Electron microscope autoradiographs were prepared from diabetic rat pancreatic acini that had been incubated with 125I-insulin. Distribution histograms of the distance of the 125I-insulin silver grains from the nearest plasma membrane were prepared and compared with a histogram of an 125I line source. After 3 min of incubation, insulin was located predominately on the plasma membrane, but even at this early time 15% of the grains had an intracellular location. After 30 min of incubation, there was a decrease in grains on the plasma membrane and an increase (to 45%) in grains localized in the cell. At both times of incubation, a comparison of the distribution of the insulin grains overlying subcellular organelles to a theoretical random grain distribution pattern indicated that the intracellular distribution of insulin grains was nonrandom. At 3 min, there was a relative concentration of grains over the plasma membrane and vesicles with an average diameter of 100 nm. At 30 min, there was a concentration of grains over the plasma membrane, 100-nm vesicle, and Golgi. These studies suggest that 125I-insulin is internalized into pancreatic acinar cells in a time-dependent manner and then is nonrandomly distributed inside the cell.

Intracellular bile acid transport in rat liver as visualized by electron microscope autoradiography using a bile acid analogue

1983 ◽  
Vol 245 (5) ◽  
pp. G681-G689 ◽  
Author(s):  
F. J. Suchy ◽  
W. F. Balistreri ◽  
J. Hung ◽  
P. Miller ◽  
S. A. Garfield
Keyword(s):  
Plasma Membrane ◽  
Electron Microscope ◽  
Bile Acid ◽  
Golgi Apparatus ◽  
Portal Vein ◽  
Bile Acids ◽  
Electron Microscope Autoradiography ◽  
Acid Analogue ◽  
Microscope Autoradiography ◽  
Grain Distribution

The role of hepatocyte organelles in the intracellular transport and secretion of conjugated bile acids has not been defined. Therefore we studied the transport and observed the subcellular localization of the bile acid analogue 125I-cholylglycyltyrosine by electron microscope autoradiography to further understand the possible compartmentation of bile acids within the hepatocyte. 125I-cholylglycyltyrosine, which retains a net negative charge, exhibited transport properties similar to native bile acids. After portal vein injection, the compound was recovered intact from bile, and the pattern of excretion paralleled that of [14C]cholylglycine. In addition, cholylglycyltyrosine uptake by isolated hepatocytes was sodium dependent. For autoradiography the analogue was injected into the portal vein, and the liver was perfusion fixed after 30 or 300 s. Light microscope autoradiography performed 30 s after isotope injection demonstrated a steep periportal-to-centrilobular gradient for 125I-cholylglycyltyrosine uptake. At 30 s quantitative grain analysis of electron microscope autoradiographs showed predominant labeling of the plasma membrane and the smooth endoplasmic reticulum (SER). The grain distribution over the region of the plasma membrane decreased from 15% at 30 s to 7% by 300 s and was associated with a sevenfold increase in labeling of the Golgi apparatus and a sixfold increase in labeling of the pericanalicular region. Grain distribution over the SER at 300 s was the same as that noted at 30 s. The hypothesis is presented that bile acids move from the sinusoidal plasma membrane to bile via a pathway that includes the SER and Golgi apparatus.

Incorporation of Cholesterol into Peripheral Nerve Myelin

1972 ◽  
Vol 30 ◽  
pp. 334-335
Author(s):  
Frank A. Rawlins
Keyword(s):  
Electron Microscope ◽  
Sciatic Nerve ◽  
Electron Microscope Autoradiography ◽  
Myelin Sheaths ◽  
Microscope Autoradiography ◽  
Grain Distribution ◽  
Sciatic Nerves ◽  
Autoradiographic Analysis ◽  
Old Mice ◽  
Short Times

Several speculations exist as to the site of incorporation of preformed molecules into myelin. The possibility that an autoradiographic analysis of cholesterol-1,2-H3 incorporation at very short times after injection might shed some light in the solution of that problem led to the present experiment.Cholesterol-1,2-H3 was injected intraperitoneally into 24 tenday old mice. The animals were then sacrificed at 10,20,30,40,60,90,120 and 180 min after the injection and the sciatic nerves were processed for electron microscope autoradiography. To analyze the grain distribution in the autoradiograms of cross and longitudinal sections from each sciatic nerve myelin sheaths were subdivided into three compartments named: outer 1/3, middle 1/3 and inner 1/3 compartments.It was found that twenty min. after the injection of cholesterol -1.2-H3 (Figs. 1 and 2), 55% of the total number of grains (t.n.g) found in myelin were within the outer 1/3 compartment, 9% were within the middle 1/3 and 36% within the inner 1/3 compartment

CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

2003 ◽  
Vol 285 (4) ◽  
pp. G726-G734 ◽  
Author(s):  
Claus Schäfer ◽  
Hanna Steffen ◽  
Karen J. Krzykowski ◽  
Burkhard Göke ◽  
Guy E. Groblewski
Keyword(s):  
Signaling Pathways ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Dependent Manner ◽  
Calyculin A ◽  
Concentration Dependent Manner ◽  
Pancreatic Acini ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Mrna Binding

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112–2117, 2002). Investigation of the effects of phorbol ester (12- o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 μM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.

Processing of receptor-bound somatostatin: internalization and degradation by pancreatic acini

1987 ◽  
Vol 252 (4) ◽  
pp. G535-G542 ◽  
Author(s):  
N. Viguerie ◽  
J. P. Esteve ◽  
C. Susini ◽  
N. Vaysse ◽  
A. Ribet
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Specific Radioactivity ◽  
Pancreatic Acinar Cells ◽  
Nuclear Fraction ◽  
Pancreatic Acini ◽  
Specific Binding Sites ◽  
Membrane Bound ◽  
Membrane Level

We have previously demonstrated the presence of specific binding sites for somatostatin on plasma membranes from pancreatic acinar cells. In the present study we attempted to characterize the fate of receptor-bound 125I-[Tyr11]somatostatin. Internalization of somatostatin was rapid (reaching a plateau at 20% of the cell-associated specific radioactivity) and temperature dependent. To follow the processing of bound somatostatin, acini were incubated with 125I-[Tyr11]somatostatin at 5 degrees C during 16 h then, after washing, incubated at 37 degrees C for 90 min in fresh medium. Surface-bound somatostatin decreased rapidly, whereas radioactivity increased in the cell interior and the incubation medium. Intracellular and membrane-bound radioactivity was mainly intact 125I-[Tyr11]somatostatin. Degradation occurred at the plasma membrane level and led to iodotyrosine production. After 15 min of incubation, 15% of the initially surface-bound 125I-[Tyr11]somatostatin was compartmentalized within the cell, mainly in the microsomal fraction. After 30 min, a significant increase in radioactivity appeared in the nuclear fraction. These results indicate that the major part of somatostatin cellular degradation takes place at the plasma membrane level. Within the cell, somatostatin is routed to the nucleus via particular fractions sedimenting with microsomal vesicles.

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.

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.

Manganese action on pancreatic protein synthesis in normal and diabetic rats

1983 ◽  
Vol 245 (5) ◽  
pp. G628-G634 ◽  
Author(s):  
M. Korc
Keyword(s):  
Acinar Cells ◽  
Diabetic Rats ◽  
Pancreatic Acinar Cells ◽  
Diabetic Rat ◽  
Pancreatic Acini ◽  
Significant Stimulation ◽  
Biphasic Effect ◽  
Normal Rat ◽  
Streptozotocin Induced Diabetes

The effects of manganese on [3H]phenylalanine incorporation into protein were studied in pancreatic acini prepared from normal and streptozotocin-induced diabetic rats. In the absence of added Ca2+ manganese exerted a biphasic effect on [3H]phenylalanine incorporation in both groups of acini. Significant stimulation occurred at 3 X 10(-5) M manganese. At higher concentrations manganese inhibited incorporation. The magnitude of stimulation was similar in all acini, whereas the magnitude of inhibition was greater in acini from normal rats. Addition of Ca2+ to incubation media abolished the stimulatory effect of manganese in normal rat acini and greatly enhanced it in diabetic rat acini, significant stimulation now occurring at 10(-5) M manganese. The magnitude of inhibition was again greater in acini from normal rats. Insulin in vivo partially reversed the diabetes-induced alterations in acinar cell responsiveness to manganese. The present findings suggest that streptozotocin-induced diabetes is associated with postreceptor alterations in the pancreatic acinar cells.

scholarly journals Alcohols enhance caerulein-induced zymogen activation in pancreatic acinar cells

2002 ◽  
Vol 282 (3) ◽  
pp. G501-G507 ◽  
Author(s):  
Zhao Lu ◽  
Suresh Karne ◽  
Thomas Kolodecik ◽  
Fred S. Gorelick
Keyword(s):  
Acinar Cell ◽  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Dependent Manner ◽  
Zymogen Activation ◽  
Concentration Dependent Manner ◽  
Pancreatic Acini ◽  
A Chain ◽  
Peak Value

Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause zymogen activation and pancreatitis. The effects of the CCK analog, caerulein, and alcohol on trypsin and chymotrypsin activation in isolated pancreatic acini were examined. Caerulein increased markers of zymogen activation in a time- and concentration-dependent manner. Notably, trypsin activity reached a peak value within 30 min, then diminished with time, whereas chymotrypsin activity increased with time. Ethanol (35 mM) sensitized the acinar cells to the effects of caerulein (10−10 to 10−7 M) on zymogen activation but had no effect alone. The effects of ethanol were concentration dependent. Alcohols with a chain length of ≥2 also sensitized the acinar cell to caerulein; the most potent was butanol. Branched alcohols (2-propanol and 2-butanol) were less potent than aliphatic alcohols (1-propanol and 1-butanol). The structure of an alcohol is related to its ability to sensitize acinar cells to the effects of caerulein on zymogen activation.

scholarly journals PLASMA MEMBRANE AND INTERNALIZED IMMUNOGLOBULINS OF LYMPH NODE CELLS STUDIED WITH CONJUGATES OF ANTIBODY OR ITS FAB FRAGMENTS WITH HORSERADISH PEROXIDASE

1974 ◽  
Vol 63 (1) ◽  
pp. 12-23 ◽  
Author(s):  
Jean-Claude Antoine ◽  
Stratis Avrameas ◽  
Nicholas K. Gonatas ◽  
Anna Stieber ◽  
Jacqueline O. Gonatas
Keyword(s):  
Plasma Membrane ◽  
Electron Microscope ◽  
Horseradish Peroxidase ◽  
Plasma Membranes ◽  
Plasma Cells ◽  
Lymphoid Cells ◽  
Specific Staining ◽  
Cytoplasmic Staining ◽  
Electron Microscope Autoradiography ◽  
Fab Fragments

Normal rat and mouse lymphoid cells were incubated at 0°–4°C for 1 h with purified rabbit or sheep antirat (mouse) immunoglobulin (Ig)-horseradish peroxidase (PO) conjugates or with Fab fragments of antibody coupled with peroxidase. Cells were subsequently washed and incubated in fresh medium, without labeled antibody or Fab fragments for 5–30 min at 20° or 37°C. With the use of the diaminobenzidine (DAB) method, distribution of peroxidase was studied in the light and electron microscopes. Fab fragments of antirat Ig antibody were iodinated with 125I and subsequently coupled with horseradish PO. Plasma membrane and internalized immunoglobulins were detected by electron microscope autoradiography and peroxidase cytochemistry. Single- (Fab-PO), and double- ([125I]Fab-PO) labeled lymphoid cells showed identical patterns of surface or internal distribution of immunoglobulins. In the electron microscope, Fab-PO conjugates at 0°–4°C resulted in a diffuse specific staining of the plasmalemma of lymphocytes and plasma cells. Most of the small dark lymphocytes (T cells?) did not show plasma membrane Ig. Macrophages did not show plasmalemma staining, but displayed nonspecific cytoplasmic staining after incubation at 20° or 37°C with antibody or Fab-PO conjugates. Lymphocytes and plasma cells, after incubation with antibody-PO conjugates at 0°–4°C, had patchy deposits of oxidized DAB on their plasma membranes. Macrophages, similarly treated, had no plasmalemmal staining. Patch and cap formation on the plasma membrane of lymphocytes and plasma cells was seen regularly after antibody-PO incubation at 37°C. Internalization patterns were different in lymphocytes and plasma cells. In lymphocytes, peroxidase staining was observed in small round or oval vesicles clustered at one pole of the cell (30 min at 37°C). In plasma cells, peroxidase staining was seen in clusters of tubules resembling the Golgi apparatus. Internalization of plasma membrane IgG was less pronounced after antibody-PO labeling as compared to Fab-PO labeling.

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