scholarly journals Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin

1996 ◽  
Vol 314 (1) ◽  
pp. 199-203 ◽  
Author(s):  
Catherine E. KIRALY-BORRI ◽  
Alan MORGAN ◽  
Robert D. BURGOYNE ◽  
Ulrich WELLER ◽  
Claes B. WOLLHEIM ◽  
...  
Keyword(s):  
Rat Brain ◽  
Dependent Manner ◽  
Attachment Protein ◽  
Concentration Dependent Manner ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Sensitive Factor ◽  
Insulin Secreting Cells ◽  
Brain Cytosol ◽  
Soluble Components

Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5´-[γ-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide (NEM)-sensitive fraction. The NEM-sensitive factor (NSF) and the soluble NSF attachment protein (α-SNAP) were found to be expressed in HIT-T15 cells and largely lost (about 30% remaining) from porated cells. Recombinant α-SNAP partially reconstituted the Ca2+ response when added to the permeabilized cells. Moreover, α-SNAP restored the effect of NEM-treated cytosol to the level observed for untreated cytosol. In contrast, NSF was ineffective when preincubated alone or with NEM-treated cytosol. Our results indicate that both α-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.

scholarly journals An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore.

1990 ◽  
Vol 110 (3) ◽  
pp. 547-557 ◽  
Author(s):  
D D Newmeyer ◽  
D J Forbes
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Signal Sequence ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Sensitive Factor ◽  
Nuclear Protein Import ◽  
Cytosolic Factor

We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease-sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP-independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.

Ca(2+)-dependent amylase secretion from SLO-permeabilized rat pancreatic acini requires diffusible cytosolic proteins

1995 ◽  
Vol 269 (5) ◽  
pp. G647-G652 ◽  
Author(s):  
P. J. Padfield ◽  
N. Panesar
Keyword(s):  
Maximal Response ◽  
Rapid Decline ◽  
Amylase Secretion ◽  
Alpha Toxin ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Membrane Pores ◽  
Streptolysin O ◽  
The Brain ◽  
Brain Cytosol

Streptolysin O (SLO)-permeabilized pancreatic acini are now frequently used to study regulated exocytosis in the exocrine pancreas. In this paper we introduce alpha-toxin as a possible alternative permeabilization agent to SLO. Both alpha-toxin and SLO are bacterial cytolysins, but the membrane pores generated by SLO are approximately 5-10 times larger than those formed by alpha-toxin. The Ca2+ requirements for amylase secretion from both types of permeabilized acini were identical, maximal amylase secretion being obtained at 30 microM Ca2+ with an effective concentration of approximately 3-4 microM Ca2+ producing 50% of the maximal response. However, Ca(2+)-stimulated amylase secretion from the SLO-permeabilized acini stopped after 10-15 min, unlike secretion from the alpha-toxin-permeabilized cells, which continued for at least 50 min. The rapid cessation of secretion from the SLO-treated acini reflects the rapid decline in the responsiveness of the cells observed after permeabilization. This decline in Ca(2+)-dependent secretion appears to be due to the loss of cytosol, since addition of purified rat brain cytosol to nonresponsive SLO-permeabilized acini reconstituted regulated secretion. Because alpha-toxin-permeabilized acini maintained their responsiveness, the cytosolic factors lost from the SLO-permeabilized cells must be retained within the toxin-treated cells. The reconstitutive activity of the brain cytosol was nondialyzable but heat and trypsin sensitive, suggesting that the factors responsible are proteins. Of the cytosols screened (brain, liver, spleen, muscle, and lacrimal) only those prepared from brain or lacrimal gland reconstituted Ca(2+)-dependent amylase secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells

1988 ◽  
Vol 256 (2) ◽  
pp. 343-350 ◽  
Author(s):  
S Cockcroft ◽  
J Stutchfield
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dimethyl Sulphoxide ◽  
Hl60 Cell ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Hl60 Cells ◽  
Permeabilized Cells ◽  
Streptolysin O

The promyelocytic HL60 cell can be differentiated with dimethyl sulphoxide or dibutyryl cyclic AMP leading to the appearance of fMetLeuPhe receptors on the cell surface. G-protein-stimulated polyphosphoinositide phosphodiesterase (PPI-pde) activity was assessed in membranes prepared from both differentiated and non-differentiated HL60 cells. Both the extent of the response and the rank order of potency of the GTP analogues to stimulate PPI-pde activation (guanosine 5′-[gamma-thio]triphosphate (GTP[S]) greater than guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG) remains unchanged after differentiation with dimethyl sulphoxide. In comparison, differentiation by dibutyryl cyclic AMP leads to diminution of PPI-pde activity when stimulated by GTP[S] or fluoride, but not by millimolar concentrations of Ca2+. GTP[S]-stimulated PPI-pde in membranes is sensitive to the presence of Ca2+ (pCa 8-5). Pertussis-toxin pretreatment of intact HL60 cells leads to inhibition of both the secretory response and the formation of inositol phosphates when stimulated by fMetLeuPhe. In contrast, pertussis-toxin pretreatment has no effect on either GTP[S]- or fluoride-stimulated PPI-pde. Neomycin in a concentration-dependent manner inhibits both GTP[S] plus Ca2+ (pCa 5)-stimulated secretion and PPI-pde activation in streptolysin-O-permeabilized cells. The extent of PPI-pde activation in membranes compared with streptolysin-O-permeabilized cells reveals that the membrane preparation does not possess all the components that make up the inositide signalling system.

scholarly journals Inhibition of neural phospholipase D activity by aminoglycoside antibiotics

1991 ◽  
Vol 279 (1) ◽  
pp. 319-321 ◽  
Author(s):  
M Liscovitch ◽  
V Chalifa ◽  
M Danin ◽  
Y Eli
Keyword(s):  
Side Effects ◽  
Rat Brain ◽  
Phospholipase D ◽  
Aminoglycoside Antibiotics ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Brain Membranes ◽  
Rat Brain Membranes ◽  
Isolated Rat

The effects of aminoglycoside antibiotics on phospholipase D (PLD) activity were investigated in permeabilized NG108-15 cells and in isolated rat brain membranes. Neomycin inhibited guanosine 5′-[gamma-thio]triphosphate-stimulated PLD activity in digitonin-permeabilized NG108-15 cells in a concentration-dependent manner (50% inhibition at 100 microM). Neomycin similarly inhibited PLD activity present in rat brain membranes and assayed in vitro with [3H]phosphatidylcholine as substrate (50% inhibition at 65 microM). Other aminoglycosides tested (kanamycin, geneticin and streptomycin) were nearly equipotent inhibitors of rat brain PLD. These results indicate that aminoglycoside antibiotics inhibit phosphatidylcholine-PLD activity with comparable and sometimes greater potency than their well known inhibition of phosphoinositide-phospholipase C. The possibility that PLD inhibition could mediate some of the toxic side effects of aminoglycosides is suggested.

α1-Adrenergic receptor involvement in norepinephrine–ethanol inhibition of rat brain Na+-K+ ATPase and in ethanol tolerance

1985 ◽  
Vol 63 (9) ◽  
pp. 1075-1079 ◽  
Author(s):  
N. Rangaraj ◽  
H. Kalant ◽  
F. Beaugé
Keyword(s):  
Rat Brain ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Receptor Antagonists ◽  
Concentration Dependent Manner ◽  
Brain Membrane ◽  
Hypothermic Effect ◽  
Wb 4101 ◽  
Number Of Binding Sites

Norepinephrine (NE) sensitization of rat brain Na+–K+ ATPase to ethanol (EtOH) inhibition appears to be mediated by α1-adrenoreceptors, since it was reversed by prazosin and WB-4101 (α1-receptor antagonists) in a concentration-dependent manner, but not by yohimbine and piperoxan (α2-receptor antagonists). In addition, clonidine (α2-agonist) and methoxamine (central receptor type uncertain) produced very little sensitization. Chronic EtOH administration to rats for 3 weeks produced tolerance to the hypothermic effect of test doses of EtOH (3 g/kg, i.p.) and a decreased inhibitory effect of NE + EtOH on the enzyme in vitro. This inhibition was still prevented by prazosin and WB-4101. However, the binding of tritiated WB-4101 and prazosin to brain membrane preparations from control and EtOH-tolerant rats revealed that the maximum number of binding sites (Bmax) and the dissociation constant (KD) of α1-adrenoreceptors were decreased after tolerance development. These changes in numbers and binding properties of α1-adrenoreceptors probably account for the decreased NE sensitization of the ATPase to EtOH inhibition in preparations from EtOH-tolerant rats.

scholarly journals Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.

1996 ◽  
Vol 7 (3) ◽  
pp. 397-408 ◽  
Author(s):  
A J O'Sullivan ◽  
A M Brown ◽  
H N Freeman ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Peptide Sequencing ◽  
Bovine Brain ◽  
Nucleotide Exchange ◽  
Permeabilized Cells ◽  
Guanine Nucleotide Exchange ◽  
Exchange Inhibitor ◽  
Streptolysin O ◽  
Gamma S ◽  
Brain Cytosol

Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown.

scholarly journals Reconstitution of Calcium-Regulated Parathyroid Hormone Secretion from Streptolysin-O-Permeabilized Parathyroid Cells by Guanosine 5′-O-(Thio)Triphosphate*

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1170-1179 ◽  
Author(s):  
Lisa M. Matovcik ◽  
Steven S. Rhee ◽  
Jean F. Schaefer ◽  
Barbara K. Kinder
Keyword(s):  
Hormone Secretion ◽  
Dependent Manner ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Calcium Calmodulin Dependent ◽  
Streptolysin O ◽  
Dependent Protein ◽  
Vitro Model

Abstract Intracellular Ca2+ levels determine the amount of PTH secretion from parathyroid cells. Dissociated calf parathyroid cells were permeabilized with streptolysin-O (SLO) to provide an in vitro model system to examine Ca2+-dependent regulation of hormone secretion. PTH release from these cells was energy dependent and increased by cytosolic cofactors. Guanosine 5′-O-(thio)triphosphate (GTPγS) increased PTH secretion from SLO-permeabilized cells in a dose-dependent manner from 0.1–100 μm. In the absence of GTPγS there was no relationship between the ambient Ca2+ concentration and the rate of PTH secretion. However, in the presence of GTPγS, intracellular Ca2+ inhibited PTH secretion with an EC50 of approximately 0.1 μm, corresponding to physiological intracellular Ca2+ levels. Thus, the addition of GTPγS to SLO-permeabilized parathyroid cells reconstituted the inverse relationship between extracellular Ca2+ and PTH secretion that is observed in vivo and in intact cells. The data indicate that this effect is mediated at least in part by heterotrimeric guanosine triphosphatases. In addition, calcium/calmodulin-dependent protein kinase II appears to mediate low Ca2+-dependent PTH secretion from these cells.

scholarly journals Homotypic Lysosome Fusion in Macrophages: Analysis Using an In Vitro Assay

1997 ◽  
Vol 139 (3) ◽  
pp. 665-673 ◽  
Author(s):  
Diane M. Ward ◽  
Jonathan D. Leslie ◽  
Jerry Kaplan
Keyword(s):  
Fusion Reaction ◽  
In Vitro Assay ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Toxin A ◽  
Concentration Dependent Manner ◽  
Vitro Assay ◽  
Sensitive Factor

Lysosomes are dynamic structures capable of fusing with endosomes as well as other lysosomes. We examined the biochemical requirements for homotypic lysosome fusion in vitro using lysosomes obtained from rabbit alveolar macrophages or the cultured macrophage-like cell line, J774E. The in vitro assay measures the formation of a biotinylated HRP–avidin conjugate, in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis. We determined that lysosome fusion in vitro was time- and temperature-dependent and required ATP and an N-ethylmaleimide (NEM)-sensitive factor from cytosol. The NEM-sensitive factor was NSF as purified recombinant NSF could completely replace cytosol in the fusion assay whereas a dominant-negative mutant NSF inhibited fusion. Fusion in vitro was extensive; up to 30% of purified macrophage lysosomes were capable of self-fusion. Addition of GTPγs to the in vitro assay inhibited fusion in a concentration-dependent manner. Purified GDP-dissociation inhibitor inhibited homotypic lysosome fusion suggesting the involvement of rabs. Fusion was also inhibited by the heterotrimeric G protein activator mastoparan, but not by its inactive analogue Mas-17. Pertussis toxin, a Gαi activator, inhibited in vitro lysosome fusion whereas cholera toxin, a Gαs activator did not inhibit the fusion reaction. Addition of agents that either promoted or disrupted microtubule function had little effect on either the extent or rate of lysosome fusion. The high value of homotypic fusion was supported by in vivo experiments examining lysosome fusion in heterokaryons formed between cells containing fluorescently labeled lysosomes. In both macrophages and J774E cells, almost complete mixing of the lysosome labels was observed within 1–3 h of UV sendai-mediated cell fusion. These studies provide a model system for identifying the components required for lysosome fusion.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

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