Patterns of calcium localization in pancreatic endocrine cells

1976 ◽  
Vol 21 (1) ◽  
pp. 107-117
Author(s):  
M. Ravazzola ◽  
F. Malaisse-Lagae ◽  
M. Amherdt ◽  
A. Perrelet ◽  
W.J. Malaisse ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Endocrine Cells ◽  
Secretory Granules ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Calcium Localization ◽  
A Cells ◽  
D Cells

Subcellular calcium localization in the dndocrine cells of rat pancreas was studied by the pyroantimonate precipitation technique. Calcium-containing electron-dense deposits in the endocrine cells were mostly found within secretory granules and along the plasma membrane, but their pattern of distribution in A-, B- and D-cells displayed qualitative and quantitative differences. In B-cells, numerous secretory granules contained deposits located in the halo surrounding the granule core. In A-cells, only few granules contained precipitates in their halo, whereas in D-cells, deposits were situated in the dense core of the secretory granules. Deposits along the plasma membrane occurred generally on the outer leaflet of the plasma membrane of B- and D-cells and on the inner leaflet of that of A-cells. In islets incubated at a high glucose concentration or in the presence of the calcium ionophore A23187, the number of beta granules containing precipitates was significantly increased. By contrast, only few deposits were observed in B-cells incubated in calcium-deprived medium enriched with EGTA. These findings indicate that: the pattern of calcium localization varies in different islet cell types; in B-cells the secretory granules represent one of the major stores of intracellular calcium; and that this store undergoes changes in conditions which alter insulin release.

Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa

2005 ◽  
Vol 17 (4) ◽  
pp. 467 ◽  
Author(s):  
H. D. Guthrie ◽  
G. R. Welch
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Calcium Ionophore ◽  
Fluorescein Isothiocyanate ◽  
Calcium Ionophore A23187 ◽  
Membrane Phospholipid ◽  
Ionophore A23187 ◽  
Liquid Storage ◽  
Boar Spermatozoa ◽  
Plasma Membrane Phospholipid

Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin–fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3–8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction.

scholarly journals Early plasma-membrane-potential changes during stimulation of lymphocytes by concanavalin A.

1983 ◽  
Vol 210 (3) ◽  
pp. 885-891 ◽  
Author(s):  
S M Felber ◽  
M D Brand
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Concanavalin A ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
K Channel ◽  
Ionophore A23187 ◽  
Resting Cells ◽  
Plasma Membrane Potential ◽  
Stimulation Of

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.

Apoptosis Might Contribute to the Thrombocytopenia in Heparin-Induced Thrombocytopenia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2545-2545
Author(s):  
Tatiana A. Nevzorova ◽  
Elmira R. Mordakhanova ◽  
Anastasia A. Ponomareva ◽  
Izabella A. Andrianova ◽  
Rustem I. Litvinov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Immune Complexes ◽  
Secretory Granules ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Time Dependent ◽  
Ionophore A23187 ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is a prothrombotic autoimmune complication of heparin therapy. Thrombocytopenia and thrombosis in HIT patients are caused by immune complexes containing pathogenic antibodies against platelet factor 4 (PF4)/glycosaminoglycan complexes. Mechanisms of platelet activation and/or destruction in HIT are not fully understood. Phosphatidylserine expression is a marker of platelet activation that contributes to the procoagulant function. On the other hand, phosphatidylserine expression is generally an early marker of cell apoptosis, which, similarly to other cells, controls platelet life span. The aim of this study was to investigate if apoptosis might play a role in HIT. Gel-filtered normal platelets were incubated for 15 or 60 min with recombinant PF4 (10 µg/ml) and KKO antibodies (50 µg/ml) and studied by electron microscopy and flow cytometry using fluorescently labeled markers of cell activation and apoptosis, such as annexin V, antibodies to CD62P (P-selectin) and MitoTracker DeepRed FM. Platelets and platelet-derived microparticles were identified by flow cytometry using labeled antibodies to CD41 and electron microscopy. Calcium ionophore A23187 (10 µM) was used as a positive control. Incubation of platelets with PF4+KKO caused fast expression of P-selectin on platelets comparable with calcium ionophore A23187 stimulation, suggesting that platelets were fully activated by PF4+KKO within 15 min, when they also started to produce CD41 and annexin-positive microparticles. Activation of platelets with PF4+KKO for 60 minutes led to a further increase in phosphatidylserine expression on their surface, with a time-dependent reduction of mitochondrial membrane potential, which reflects a disturbance of energy metabolism and is characteristic of cell apoptosis. Scanning electron microscopy showed that platelets treated with PF4+KKO or A23187, unlike untreated cells, displayed dramatic morphological changes with a loss of discoid shape, formation of filopodia, and microvesiculation. By transmission electron microscopy, the PF4+KKO-treated platelets had an irregular shape due to formation of plasma membrane invaginations and pseudopodia. Formation of an increasing number of intracellular vacuoles and enlargement of the lumen of the open canalicular system were observed. Some vacuoles contained various inclusions, such as secretory granules, membrane components, and grainy particles. The number of secretory granules in the PF4+KKO-treated cells was dramatically reduced. In all cases, formation of microparticles of various shapes and sizes was observed. These results indicate that the PF4-containing pathogenic immune complexes induce strong and time-dependent platelet activation leading to procoagulant microparticle formation that may contribute to thrombosis. At the same time, the results strongly suggest that the HIT-like immune complexes likely induce platelet apoptosis that can be an important mechanism of thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Microfilaments and microtubules in calcium ionophore-induced secretion of lysosomal enzymes from human polymorphonuclear leukocytes.

1978 ◽  
Vol 78 (3) ◽  
pp. 769-781 ◽  
Author(s):  
S Hoffstein ◽  
G Weissmann
Keyword(s):  
Plasma Membrane ◽  
Lysosomal Enzymes ◽  
Cytochalasin B ◽  
Human Peripheral Blood ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Microtubule Assembly ◽  
Ionophore A23187 ◽  
Enzyme Release ◽  
Experimental Conditions

Human peripheral blood leukocytes (PMN) are induced to release lysosomal enzymes by the calcium ionophore A23187 in the presence but not the absence of extracellular Ca++. Whereas secretion induced by particulate or immune stimuli is accompanied by an increase in visible microtubules and is inhibitable by colchicine, secretion induced by A23187 and Ca++ was not accompanied by an increase in microtubule numbers and was not inhibited by colchicine. Ca++ did not appear to regulate microtubule assembly in these cells since resting PMN had a mean of 22.3 +/- 2.0 microtubules in the centriolar region as compared to 22.3 +/- 1.1 in ionophore-treated cells and 24.9 +/- 1.5 in cells exposed to ionophore and 1 mM Ca++. Bipolar filaments, 10 nm thick and 300--400 nm long, were numerous in the pericortical cytoplasm of cells exposed to both reagents. Microtubules in these cells were decorated with an electron-opaque fibrillar material. PMN exposed to A23187 and Ca++ were contracted in two directions at right angles to each other: (a) Contractions parallel to the plasma membrane resulted in extensive plication of the cell membrane. The cytoplasm subjacent to the plicae contained dense filamentous webs. Plication was prevented by cytochalasin B or reversed by subsequent exposure to an endocytic stimulus such as zymosan. (b) Contractions perpendicular to the plasma membrane, toward the cytocenter, resulted in the formation of vacuoles in normal PMN and of membrane invaginations in cytochalasin B-treated PMN. Whereas contractions parallel to the plasma membrane could occur in the absence of enzyme release (ionophore alone) and enzyme release could occur in the absence of such contractions (ionophore plus calcium plus cytochalasin B), contraction toward the cytocenter occurred in all experimental conditions in which significant enzyme release was obtained. Thus, lysosomal enzyme secretion in PMN involves contractile movements in the plasma membrane toward the lysosomes rather than the reverse. These calcium-mediated contractile events are mediated by cytochalasin B-insensitive microfilaments but not by microtubule assembly.

scholarly journals Cdc42 and Rac Stimulate Exocytosis of Secretory Granules by Activating the Ip3/Calcium Pathway in Rbl-2h3 Mast Cells

2000 ◽  
Vol 148 (3) ◽  
pp. 481-494 ◽  
Author(s):  
Elizabeth Hong-Geller ◽  
Richard A. Cerione
Keyword(s):  
Calcium Release ◽  
Calcium Influx ◽  
Secretory Granules ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Dominant Negative ◽  
Ionophore A23187 ◽  
Ip3 Receptor ◽  
Extracellular Medium

We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP3 production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP3 receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCγ1, the enzyme immediately upstream of IP3 formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP3 formation and calcium mobilization upon antigen stimulation.

Growth factors and the primary cilium in BALB/c 3T3 cells

1987 ◽  
Vol 45 ◽  
pp. 830-831
Author(s):  
R. W. Tucker ◽  
N. S. More ◽  
S. Jayaraman
Keyword(s):  
Growth Factors ◽  
Primary Cilium ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Calcium Ionophore ◽  
Growth Stimulation ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
3T3 Cells ◽  
Microtubule Depolymerization

The mechanisms by which polypeptide growth factors Induce DNA synthesis in cultured cells is not understood, but morphological changes Induced by growth factors have been used as clues to Intracellular messengers responsible for growth stimulation. One such morphological change has been the transient disappearance of the primary cilium, a “9 + 0” cilium formed by the perinuclear centriole in interphase cells. Since calcium ionophore A23187 also produced both mitogenesis and ciliary changes, microtubule depolymerization might explain ciliary disappearance monitored by indirect immunofluorescence with anti-tubulin antibody. However, complete resorption and subsequent reformation of the primary cilium occurs at mitosis, and might also account for ciliary disappearance induced by growth factors. To settle this issue, we investigated the ultrastructure of the primary cilium using serial thin-section electron microscopy of quiescent BALB/c 3T3 cells before and after stimulation with serum.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

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