QUALITATIVE AND SEMIQUANTITATIVE CALCIUM CYTOCHEMISTRY IN B CELLS OF MICE TREATED WITH CYPROHEPTADINE AND MANNOHEPTULOSE

1978 ◽  
Vol 87 (4) ◽  
pp. 786-798 ◽  
Author(s):  
Günter Klöppel ◽  
Gerhard Bommer ◽  
Elke Ruttmann ◽  
Hans-Jörg Schäfer
Keyword(s):  
B Cells ◽  
B Cell ◽  
Insulin Release ◽  
Cell Membranes ◽  
Secretory Granules ◽  
Calcium Content ◽  
Calcium Handling ◽  
Cytoplasmic Matrix ◽  
Plasma Insulin Levels ◽  
Calcium Deposits

ABSTRACT The effect of cyproheptadine (CPH) and D-mannoheptulose (MH) on the function and the histo- (GBHA-method) and ultracytochemical (pyroantimonate method) calcium handling by pancreatic B cell were studied in mice. Intraperitoneal injection of CPH (45 mg/kg) or MH (1500 mg/kg) produced a hyperglycaemic syndrome, which was accompanied by decreased plasma insulin levels. CPH and MH also abolished glucose-induced insulin release. CPH led to a decrease of about 30 to 40 % in the histochemical calcium content of islets as revealed by semi-quantitative micro-densitometry, while MH did not change the calcium content of the islets. The ultracytochemical distribution pattern of calcium-rich precipitates were identical in CPH and MH treated B cells. The subcellular calcium deposits were predominantly located in the cytoplasmic matrix, but were rarely seen in granule halos and along the cell membranes. In contrast, glucose stimulation of the B cells resulted in an accumulation of the precipitates along the cell membranes and in the secretory granules. The results suggest that CPH and MH inhibit insulin release by either directly or indirectly interfering with the normal calcium handling by the B cell.

Insulin release from a cloned precursor beta cell line

1987 ◽  
Vol 113 (1) ◽  
pp. 3-NP ◽  
Author(s):  
K. W. Ng ◽  
P. R. Gummer ◽  
B. L. Grills ◽  
V. P. Michelangeli ◽  
M. E. Dunlop
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Insulin Release ◽  
Cell Lines ◽  
Neonatal Rat ◽  
Insulin Content ◽  
Morphological Evidence ◽  
Bicarbonate Buffer ◽  
Beta Cell Line

ABSTRACT This paper describes the establishment in long-term tissue culture of a functional, clonal beta (B) cell line UMR 407/3 derived from neonatal rat pancreas. Immunofluorescence demonstrated specific and uniform staining for insulin. Transmission electron microscopy showed the presence of microvilli and cytoplasmic granules. The doubling time in culture was approximately 60 h in 2% (v/v) fetal calf serum with inhibition of growth at confluence. Biochemical studies demonstrated the incorporation of [3H]leucine into proinsulin and insulin, with insulin comprising 43·6% of the total radioactivity incorporated into immune complexes. When incubated at 37 °C for 30 min with Krebs–Ringer bicarbonate buffer (pH 7·4), the amount of insulin released on stimulation by 16·7 mmol glucose/l, 20 mmol dl-glyceraldehyde/l or 20 mmol α-ketoisocaproate/l was significantly higher compared with 5·6 mmol glucose/l. The mean insulin content was equivalent to 99±0·4 fmol (s.e.m.)/5 × 105 cells. Regulated insulin release was maintained through at least 15 passages in culture. The cells showed morphological evidence of senescence after passage 26 and this was associated with significant reduction in stimulated insulin release as well as insulin content. The ability of the cells of this clonal line to grow in soft agar suggests that it is a precursor cell line. The clonal B cell lines isolated so far may thus represent variably committed rather than fully differentiated B cells in culture. These clonal non-neoplastic cell lines will be useful models with which to study the regulation of maturation/differentiation of B cells and insulin gene expression. Finally, the method described for the isolation of clonal B cell lines can be used to establish, in culture, precursor clonal lines from other normal tissues. J. Endocr. (1987) 113, 3–10

scholarly journals LOCALIZATION OF ACID PHOSPHATASE ACTIVITY AND SECRETION MECHANISM IN RABBIT PANCREATIC B-CELLS

1966 ◽  
Vol 14 (3) ◽  
pp. 233-246 ◽  
Author(s):  
SYDNEY S. LAZARUS ◽  
BRUNO W. VOLK ◽  
HERBERT BARDEN
Keyword(s):  
Acid Phosphatase ◽  
B Cells ◽  
B Cell ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Secretory Granules ◽  
Secretory Product ◽  
Cell Cytoplasm ◽  
Single Membrane ◽  
Pancreatic B Cells

Utilizing formaldehyde- or glutaraldehyde-fixed tissue and Gomori's lead method it was found by optical microscopy that rabbit pancreatic islet cell acid phosphatase activity is present in discrete, mostly perinuclear foci and that this distribution differs from that of the aldehyde fuchsin-positive secretory granules which are densely packed at the capillary pole of the cell. Electron microscopically lead reaction product was noted in dense bodies, as well as in structures thought to be Golgi vacuoles and vesicles, it was also present in the innermost of the Golgi cisternae, and at the periphery of adjacent single membrane-limited bodies whose origin can be traced from the proximal cisternae. These latter bodies in routinely prepared, osmium-fixed material show finely granular content, which is in contrast to the electron-dense, central body seen in secretory granules that appear to originate from endoplasmic reticulum. B-cell cytoplasm contained additional numerous, single membrane-limited vacuoles with pale content. These are thought also to represent secretion vacuoles but with insulin secretory product in a different physical or chemical state. The lack of acid phosphatase activity in B-cell secretion vacuoles, the dissimilarities in fine structure between the content of secretory elements and that of the Golgi-derived granular body, together with previous evidence that alteration in B-cell functional state does not result in altered number or distribution of acid phosphatase active elements in B-cell cytoplasm, indicate a lack of relationship between acid phosphatase and secretory granule formation or release in pancreatic B-cells. It is also hypothesized that the secretory vacuole with central dense granule may be a storage form while the pale vacuole is the one which liberates its content to the intercellular space.

Relationship between calbindin-D28K levels in the A and B cells of the rat endocrine pancreas and the secretion of insulin and glucagon: influence of vitamin D3 deficiency and 1,25-dihydroxyvitamin D3

1996 ◽  
Vol 148 (2) ◽  
pp. 223-232 ◽  
Author(s):  
P-M Bourlon ◽  
A Faure-Dussert ◽  
B Billaudel ◽  
B Ch J Sutter ◽  
G Tramu ◽  
...  
Keyword(s):  
Vitamin D ◽  
B Cells ◽  
B Cell ◽  
Vitamin D Deficiency ◽  
Glucagon Secretion ◽  
Calcium Handling ◽  
A Cells ◽  
Calbindin D28k ◽  
Calbindin D ◽  
A And B Cells

Abstract The pancreatic B cell is equipped with specific receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and contains vitamin D-dependent calcium binding proteins (calbindin-D). Insulin secretion is impaired by vitamin D deficiency and is restored by 1,25-(OH)2D3 (concomitantly with an improved calcium handling within B cells) but the effect of 1,25-(OH)2D3 on the pancreatic B cell via calbindin-D is unclear. Therefore we examined the relationship between calbindin-D28K or calbindin-D9K and the activity of the endocrine pancreas in normal (N), four week vitamin D-deficient (−D) and one week 1,25-(OH)2D3-replete (+D) rats. Calbindin-D9K was not found in the pancreas, neither in the islets nor in the exocrine part, of any of the groups of rats (N, −D, or +D). Surprisingly, total islet calbindin-D28K content was increased by vitamin D deficiency and partly restored by 1,25-(OH)2D3. Calbindin-D28K immunostaining was observed only on A and B cells in the endocrine part of the pancreas, the greatest staining being found in A cells. This difference in staining density was increased by vitamin D deficiency and decreased by 1,25-(OH)2D3 treatment. In vitro, 1,25-(OH)2D3 also produced a negative influence on calbindin-D28K staining in A cells, as demonstrated using pieces of pancreas incubated with the steroid for 2 h. No significant influence on labeling intensity of B cell calbindin-D28K could be shown. Plasma insulin and islet insulin release in response to 10 mm arginine stimulation were decreased in −D rats and enhanced in +D rats towards N values. In contrast, plasma glucagon and the amount of glucagon secretion, stimulated in vitro by 10 mm arginine or by low (1·7 mm) glucose concentration, was increased in −D rats and attenuated by 1,25-(OH)2D3. Thus there appears to be no relationship between the steady state level of B cell calbindin-D28K and the regulation of insulin secretion by 1,25-(OH)2D3 in vitamin D-deficient rats. However there is a correlation between A cell calbindin-D28K and glucagon secretion, which are both negatively regulated by 1,25-(OH)2D3. The predominance of calbindin-D28K in A cells raises the question as to how A and B cells interact and the role of calbindin-D28K in calcium handling. Journal of Endocrinology (1996) 148, 223–232

scholarly journals SNAP-25 is expressed in islets of Langerhans and is involved in insulin release.

1995 ◽  
Vol 128 (6) ◽  
pp. 1019-1028 ◽  
Author(s):  
K Sadoul ◽  
J Lang ◽  
C Montecucco ◽  
U Weller ◽  
R Regazzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
B Cell ◽  
Insulin Release ◽  
Islet Cells ◽  
Secretory Granules ◽  
Cell Types ◽  
Pancreatic B Cell ◽  
Pancreatic Endocrine Cells ◽  
Presynaptic Plasma Membrane

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.

scholarly journals Synergistic factors control kinase–phosphatase organization in B-cells engaged with supported bilayers

2020 ◽  
Vol 31 (7) ◽  
pp. 667-682 ◽  
Author(s):  
Marcos Francisco Núñez ◽  
Kathleen Wisser ◽  
Sarah L. Veatch
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Membranes ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Membrane Domains ◽  
Supported Bilayers ◽  
Physical Mechanisms ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

Cell membranes can influence local concentrations of signaling regulators around receptors via multiple physical mechanisms. It is shown that ordered, phase-like membrane domains can synergize with other mechanisms to enhance B-cell receptor signaling.

scholarly journals Immunoreactive GABA transaminase within the pancreatic islet is localized in mitochondria of the B-cell.

1987 ◽  
Vol 35 (8) ◽  
pp. 831-836 ◽  
Author(s):  
D J Garry ◽  
H D Coulter ◽  
T J McIntee ◽  
J Y Wu ◽  
R L Sorenson
Keyword(s):  
B Cells ◽  
Subcellular Localization ◽  
B Cell ◽  
Secretory Granules ◽  
Cell Activity ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
High Concentration ◽  
Gold Particles ◽  
Pancreatic Islets Of Langerhans

Subcellular localization of gamma aminobutyrate-alpha-ketoglutarate transaminase (GABA-T) in the pancreatic islets of Langerhans was determined by use of an electron microscopic, immunogold post-embedding protocol. The objective of this study was to define the islet cell distribution and subcellular localization of GABA-T. Within the islet, GABA-T was found only in the B-cells and was localized in mitochondria; 78 mitochondria contained 336 gold particles, whereas 245 secretory granules contained only 18 gold particles. Although studies utilizing either the isolated perfused pancreas or cultured islets have shown that exogenous GABA modulates D-cell secretion, in this study immunoreactive GABA-T, the catabolic enzyme for GABA, was not detectable in A- and D-cells of the islet. Control studies substituting normal rabbit serum for the GABA-T antiserum resulted in absence of labeling. These results indicate that the high concentration of GABA present in islet B-cells is catabolized by GABA-T in the mitochondrial compartment, consistent with the possibility that GABA functions as a mediator of B-cell activity.

scholarly journals Isolation and characterization of membrane receptors for pokeweed mitogens from mouse lymphocytes

1977 ◽  
Vol 165 (3) ◽  
pp. 431-437 ◽  
Author(s):  
K Yokoyama ◽  
T Terao ◽  
T Osawa
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Membranes ◽  
Membrane Receptors ◽  
Isolation And Characterization ◽  
Ia Antigens ◽  
Affinity Adsorbent ◽  
Cell Mitogen

The major glycoproteins that bind pokeweek B-cell mitogen (Pa-1) and pokeweed T-cell mitogen (Pa-2) were isolated and identified from bone-marrow-derived lymphocytes (B-cells) and thymus-derived lymphocytes (T-cells) of C3H/He mice. The surfaces of the cells were 125I-labelled by using the enzyme lactoperoxidase, and the plasma membranes were isolated from the 125I-labelled cells. These membranes were solubilized with Triton X-100 and subjected to affinity chromatography on the affinity adsorbent prepared by coupling mitogen Pa-1 or Pa-2 to activated Sepharose 4B. The glycoproteins specifically eluted with di-N-acetylchitobiose from the affinity adsorbents were analysed according to their mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate. These glycoproteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes, but they were not detected in the eluate from the T-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were found to be major receptor sites for the pokeweed mitogens on both B-cells and T-cells. However, mitogen Pa-1 (B-cell) has a stronger affinity to Ia antigens than does mitogen Pa-2 (T-cell).

scholarly journals Cocapture of cognate and bystander antigens can activate autoreactive B cells

2017 ◽  
Vol 114 (4) ◽  
pp. 734-739 ◽  
Author(s):  
Nicholas S. R. Sanderson ◽  
Maria Zimmermann ◽  
Luca Eilinger ◽  
Céline Gubser ◽  
Nicole Schaeren-Wiemers ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Membranes ◽  
Cell Receptor ◽  
Acute Disseminated Encephalomyelitis ◽  
Autoimmune Response ◽  
Influenza Hemagglutinin ◽  
Mog Antibodies

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with autoimmune central nervous system diseases like acute disseminated encephalomyelitis (ADEM). For ADEM, it is speculated that a preceding infection is the trigger of the autoimmune response, but the mechanism connecting the infection to the production of MOG antibodies remains a mystery. We reasoned that the ability of B cells to capture cognate antigen from cell membranes, along with small quantities of coexpressed “bystander” antigens, might enable B-cell escape from tolerance. We tested this hypothesis using influenza hemagglutinin as a model viral antigen and transgenic, MOG-specific B cells. Using flow cytometry and live and fixed cell microscopy, we show that MOG-specific B cells take up large amounts of MOG from cell membranes. Uptake of the antigen from the membrane leads to a strong activation of the capturing B cell. When influenza hemagglutinin is also present in the membrane of the target cell, it can be cocaptured with MOG by MOG-specific B cells via the B-cell receptor. Hemagglutinin and MOG are both presented to T cells, which in turn are activated and proliferate. As a consequence, MOG-specific B cells get help from hemagglutinin-specific T cells to produce anti-MOG antibodies. In vivo, the transfer of MOG-specific B cells into recipient mice after the cocapture of MOG and hemagglutinin leads to the production of class-switched anti-MOG antibodies, dependent on the presence of hemagglutinin-specific T cells. This mechanism offers a link between infection and autoimmunity.

Plasmalemma localisation of inorganic phosphate in B cells pancreas

1978 ◽  
Vol 36 (2) ◽  
pp. 528-529
Author(s):  
F. B. P. Wooding ◽  
K. Pedley ◽  
N. Freinkel ◽  
R. M. C. Dawson
Keyword(s):  
Electron Microscope ◽  
B Cells ◽  
B Cell ◽  
Pancreatic Islets ◽  
High Glucose ◽  
Lead Acetate ◽  
Inorganic Phosphate ◽  
Slight Modification ◽  
Uranyl Acetate ◽  
Lead Phosphate

Freinkel et al (1974) demonstrated that isolated perifused rat pancreatic islets reproduceably release up to 50% of their total inorganic phosphate when the concentration of glucose in the perifusion medium is raised.Using a slight modification of the Libanati and Tandler (1969) method for localising inorganic phosphate by fixation-precipitation with glutaraldehyde-lead acetate we can demonstrate there is a significant deposition of lead phosphate (identified by energy dispersive electron microscope microanalysis) at or on the plasmalemma of the B cell of the islets (Fig 1, 3). Islets after incubation in high glucose show very little precipitate at this or any other site (Fig 2). At higher magnification the precipitate seems to be intracellular (Fig 4) but since any use of osmium or uranyl acetate to increase membrane contrast removes the precipitate of lead phosphate it has not been possible to verify this as yet.

Sign in / Sign up

Export Citation Format

Share Document