scholarly journals GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability

1987 ◽  
Vol 248 (3) ◽  
pp. 741-747 ◽  
Author(s):  
C V Nicchitta ◽  
S K Joseph ◽  
J R Williamson
Keyword(s):  
Membrane Permeability ◽  
Time Course ◽  
Liver Microsomes ◽  
Subsequent Treatment ◽  
Complete Inhibition ◽  
Hill Coefficient ◽  
Release Process ◽  
Thiol Reagent ◽  
Mannose 6 Phosphate ◽  
Poly Ethylene

Guanine nucleotides have been reported to stimulate reticular Ca2+ release. By using the structure-linked latency of microsomal mannose-6-phosphate phosphatase as an index of microsomal permeability [Arion, Ballas, Lange & Wallin (1976) J. Biol. Chem. 251, 4901-4907], the effects of GTP on Ca2+ release and membrane permeability were compared in liver microsomes. In a stripped rough-microsome preparation, GTP caused a dose-dependent increase in mannose 6-phosphate permeability. Half-maximal and maximal effects were observed at 3 microM- and 10 microM-GTP respectively. The time course of the change in membrane permeability coincided with the time course of GTP-dependent Ca2+ release. This increase in microsomal permeability displayed positive to-operativity with respect to GTP (Hill coefficient = 1.8). By analogy to the GTP-dependent Ca2+ release process, guanosine 5′-[gamma-thio]triphosphate and guanosine 5′-[beta gamma-imido]-triphosphate inhibited the ability of GTP to alter microsomal permeability, but were without effect when added alone. In the presence of 50 microM-GTP, complete inhibition of the GTP-dependent increase in microsomal permeability was achieved with 10 microM-guanosine 5′-[gamma-thio]triphosphate, whereas a 25% inhibition was observed with 10 microM-guanosine 5′-[beta gamma-imido]triphosphate. In contrast with previous observations in crude microsomal preparations, GTP-dependent Ca2+ release in the stripped rough-microsome preparation did not require the addition of poly(ethylene glycol), although the latter did stimulate the rate of Ca2+ release. The ability of GTP to alter microsomal permeability was blocked by prior treatment with the thiol reagent p-hydroxymercuribenzoate; complete inhibition was observed after a 10 min exposure to 50 microM. Inhibition was reversed by subsequent treatment with dithiothreitol. The marked similarities between the two GTP-sensitive processes indicate that they may function via the same mechanism.

scholarly journals Histone II-A stimulates glucose-6-phosphatase and reveals mannose-6-phosphatase activities without permeabilization of liver microsomes

1995 ◽  
Vol 310 (1) ◽  
pp. 221-224 ◽  
Author(s):  
J F St-Denis ◽  
B Annabi ◽  
H Khoury ◽  
G van de Werve
Keyword(s):  
Substrate Specificity ◽  
Membrane Permeability ◽  
Phosphatase Activity ◽  
Liver Microsomes ◽  
Microsomal Membrane ◽  
Phosphatase Activities ◽  
Phosphate Hydrolysis ◽  
Mannose 6 Phosphate ◽  
Stimulation Of

The effect of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase activities was investigated in relation to microsomal membrane permeability. It was found that glucose-6-phosphatase activity in histone II-A-pretreated liver microsomes was stimulated to the same extent as in detergent-permeabilized microsomes, and that the substrate specificity of the enzyme for glucose 6-phosphate was lost in histone II-A-pretreated microsomes, as [U-14C]glucose-6-phosphate hydrolysis was inhibited by mannose 6-phosphate and [U-14C]mannose 6-phosphate hydrolysis was increased. The accumulation of [U-14C]glucose from [U-14C]glucose 6-phosphate into untreated microsomes was completely abolished in detergent-treated vesicles, but was increased in histone II-A-treated microsomes, accounting for the increased glucose-6-phosphatase activity, and demonstrating that the microsomal membrane was still intact. The stimulation of glucose-6-phosphatase and mannose-6-phosphatase activities by histone II-A was found to be reversed by EGTA. It is concluded that the effects of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase are not caused by the permeabilization of the microsomal membrane. The measurement of mannose-6-phosphatase latency to evaluate the intactness of the vesicles is therefore inappropriate.

Stimulus-Dependent Alterations in Quantal Neurotransmitter Release

2006 ◽  
Vol 96 (6) ◽  
pp. 3082-3087 ◽  
Author(s):  
Chad P. Grabner ◽  
Aaron P. Fox
Keyword(s):  
Neurotransmitter Release ◽  
Chromaffin Cells ◽  
Time Course ◽  
Ion Concentration ◽  
Fusion Pore ◽  
Release Process ◽  
Large Dense Core Vesicles ◽  
Intracellular Calcium Ion ◽  
Individual Vesicle ◽  
Digitonin Permeabilization

Neurotransmitter release is a steep function of the intracellular calcium ion concentration ([Ca2+]i) at the release sites. Both the Ca2+ amplitude and the time course appear to be important for specifying neurotransmitter release. Ca2+ influx regulates the number of vesicles exocytosed as well as the amount of neurotransmitter each individual vesicle releases. In our study we stimulated mouse chromaffin cells in two different ways to alter Ca2+ presentation at the release sites. One method, digitonin permeabilization followed by exposure to Ca2+, allows for a large uniform global elevation of [Ca2+]i, whereas the second method, application of nicotine, depolarizes chromaffin cells and activates voltage-dependent Ca2+ channels, thereby producing more phasic and localized changes in [Ca2+]i. Using amperometry to monitor catecholamine release, we show that both kinds of stimuli elicit the exocytosis of similar quantities of neurotransmitter per large dense core vesicles (LDCVs) released. Even so, the release process was quite different for each stimulus; nicotine-elicited events were small and slow, whereas digitonin events were, in comparison, large and fast. In addition, the transient opening of the fusion pore, called the “foot,” was essentially absent in digitonin-stimulated cells, but was quite common in nicotine-stimulated cells. Thus even though both strong stimuli used in this study elicited the release of many vesicles it appears that the differences in the Ca2+ levels at the release sites were key determinants for the fusion and release of individual vesicles.

scholarly journals Effect of dual agonists on phosphoinositide pools in WRK-1 cells

1990 ◽  
Vol 269 (3) ◽  
pp. 633-637 ◽  
Author(s):  
M E Monaco ◽  
M Attinasi ◽  
K Koréh
Keyword(s):  
Time Course ◽  
Inositol Phosphates ◽  
Subsequent Treatment ◽  
Tumour Cells ◽  
The Other ◽  
Mammary Tumour ◽  
Lipid Pool ◽  
Phosphoinositide Cycle ◽  
Heterologous Desensitization ◽  
Hormone Sensitive

Both vasopressin and bradykinin activate the phosphoinositide cycle in WRK-1 rat mammary tumour cells. When the two agonists are added simultaneously, partial additivity is observed with respect to disappearance of prelabelled phosphoinositides and accumulation of inositol phosphates; no additivity is observed with respect to resynthesis of phosphatidylinositol as assessed by monitoring [32P]Pi incorporation. Lack of complete additivity can be explained, at least in part, by heterologous desensitization. In order to determine whether the two agonists were accessing a common or individual hormone-sensitive phosphoinositide pools, cells were incubated with [32P]Pi in the presence of either vasopressin or bradykinin and subsequently restimulated with the alternative agonist. The lipid pool labelled in the presence of either agonist was sensitive to subsequent treatment by the other ligand, suggesting a common phosphoinositide pool. However, when cells were incubated with [32P]Pi in the absence of agonists, the time course of labelling of the hormone-sensitive pool was different for bradykinin and vasopressin, with that for bradykinin becoming labelled within a much shorter time. Thus although there is a significant overlap between the phosphoinositide pools responding to vasopressin and bradykinin, there is a small fraction of the hormone-sensitive lipid which responds only to bradykinin.

The ovotestis of Aplysia californica: anatomy and egg release

1980 ◽  
Vol 58 (12) ◽  
pp. 2220-2229 ◽  
Author(s):  
F. Edward Dudek ◽  
Hampik S. Injeyan ◽  
Bonnie Soutar ◽  
Greg Weir ◽  
Stephen S. Tobe
Keyword(s):  
Time Course ◽  
Aplysia Californica ◽  
Cell Extract ◽  
Continuous Exposure ◽  
Gradual Reduction ◽  
Release Process ◽  
Cell Extracts ◽  
High K ◽  
Scanning Electron

Egg release from the ovotestis of Aplysia californica has been studied using ovotestis fragments and bag cell extracts. Light and scanning electron microscopy showed clusters of follicles surrounded by muscle cells. Mature oocytes observed in egg masses and those released from ovotestis fragments were 90 μm in diameter. The number of mature releasable oocytes was relatively constant throughout the ovotestis, although a gradual reduction occurred with increasing distance from the small hermaphroditic duct.Bag cell induced egg release was detectable in vitro within 30 min and was complete by 180 min. The time course of egg release was similar under conditions of either continuous exposure or a 30-min pulse of bag cell extract. Artificial seawater (ASW) solutions with high K+ (110 mM) did not stimulate egg release unless bag cell extract was present. ASW with no Ca2+ and 3 mM EGTA or ASW containing Co2+ (10 mM) inhibited both bag cell induced and spontaneous (ASW alone) egg release.Therefore, brief exposure to bag cell peptide can trigger the egg release process, which is long lasting (~ 3 h) and Ca2+ dependent. The observation that high K+ did not stimulate egg release challenges the muscle contraction hypothesis of egg release.

scholarly journals Studies on cell adhesion and recognition. III. The occurrence of α-mannosidase at the fibroblast cell surface, and its possible role in cell recognition

1981 ◽  
Vol 88 (1) ◽  
pp. 149-159 ◽  
Author(s):  
H Rauvala ◽  
S Hakomori
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Inhibitory Activity ◽  
Time Course ◽  
Gas Chromatography Mass Spectrometry ◽  
Hydrolytic Cleavage ◽  
Intact Cells ◽  
Cell Monolayers ◽  
Mannose 6 Phosphate ◽  
Hydrolysis Of

The occurrence of α-mannosidase activity at the surface of hamster embryo (NIL) fibroblasts is indicated by the following findings: (a) When NIL cells were incubated on the glass surfaces on which ovalbumin glycopeptides were covalently linked, a rapid release of free mannose from ovalbumin glycopeptides was observed as evidenced by analysis on gas chromatography/mass spectrometry. (b) Cell suspensions as well as intact cell monolayers hydrolyzed rapidly p-nitrophenyl-α-D-mannoside, and the time-course of the hydrolytic cleavage was linear from the moment of mixing of the substrate with the cells. The hydrolysis of the nitrophenyl glycosides of β-D-mannose, α-D-galactose, β-D-galactose, α-L-fucose, β-D-glucose, β-D-N-acetylgalactosamine and β-D-N-acetylglucosamine was negligible or more than ten times lower as compared with the hydolysis of α-D-mannoside. (c) No released or secreted activity of mannosidase could be detected under the conditions used. (d) Studies using known proportions of broken cells in the incubation mixture indicated that more than 90 percent of the mannosidase activity measured was attributable to intact cells and not to broken cells or cell fragments. (e) Hydrolysis of p-nitrophenyl-α-D-mannoside by cell monolayers was inhibited, in the order of decreasing inhibitory activity, by yeast mannan, ovalbumin, α-1,4-L-mannonolactone, α-methylmannoside, and mannose-6-phosphate. High inhibitory activity of the mannan polysaccharide and of ovalbumin favored the presence of the mannosidase activity at the cell surface, as these substrates may not penetrate rapidly into the cells. The following findings indicated that the cell surface mannosidase is mediating the cell adhesion based on the recognition of high-mannose-type glycopeptide: (a) Ovalbumin- coated plastic surfaces strongly promoted attachment and spreading of NIL fibroblasts, whereas the same ovalbumin coat did not promote attachment and spreading of some other cell types (BALB/c 3T3 fibroblasts and freshly prepared rat liver cells). (b) Digestion of ovalbumin with α-mannosidase greatly reduced the adhesion-mediating activity. (c) Cell adhesion to ovalbumin-coated surfaces was strongly inhibited by mannose tetrasaccharides, moderately by α-1,4-L-mannonolactone, and weakly by α- methylmannoside and mannose-6-phosphate. This order of the inhibitory activity for cell attachment is the same as that for the inhibition of mannosidic hydrolysis. The interpretation that the cell surface mannosidase is able to mediate cell adhesion is in agreement with previous studies suggesting that polyvalent glycosidase surfaces can promote cell adhesion to a degree similar to that caused by fibronectin and several lectins by interacting with their cell surface substrate site (the accompanying papers of this series).

Time course of oxidative benz[a]anthracene metabolism by liver microsomes of normal and PCB-treated rats

1981 ◽  
Vol 2 (5) ◽  
pp. 395-401 ◽  
Author(s):  
Jürgen Jacob ◽  
Achim Schmoldt ◽  
Gernot Grimmer
Keyword(s):  
Time Course ◽  
Liver Microsomes

scholarly journals Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder).

1992 ◽  
Vol 99 (6) ◽  
pp. 841-862 ◽  
Author(s):  
F Markwardt ◽  
G Isenberg
Keyword(s):  
Steady State ◽  
Time Course ◽  
Boltzmann Distribution ◽  
Hill Coefficient ◽  
K Channel ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
K Channels ◽  
The Mean ◽  
Inside Out

Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.

Voltage-clamp analysis of currents produced by glutamate and some glutamate analogues on horizontal cells isolated from the catfish retina

1986 ◽  
Vol 56 (1) ◽  
pp. 19-31 ◽  
Author(s):  
G. Hals ◽  
B. N. Christensen ◽  
T. O'Dell ◽  
M. Christensen ◽  
R. Shingai
Keyword(s):  
Membrane Potential ◽  
Amino Acid ◽  
Membrane Permeability ◽  
Time Course ◽  
Reversal Potential ◽  
Horizontal Cells ◽  
Resting Potential ◽  
Bathing Solution ◽  
Patch Type ◽  
Highly Nonlinear

Horizontal cells isolated from the catfish retina were exposed to radiolabeled glutamate, glycine, gamma-aminobutyric acid (GABA), and sucrose to determine if the enzymatic dissociation procedure altered the high-affinity uptake mechanism for GABA and generally reduced membrane selectivity. As in the intact retina, isolated cells could transport GABA but not the other substances. The horizontal cells were voltage clamped using a single low-resistance patch-type electrode. The acidic amino acid L-glutamate, and its analogues kainate and quisqualate, were applied to the cell by pressure ejection from a nearby pipette. All three agonists produced inward currents that reversed near O mV. Quisqualate produced a current with a similar time course as glutamate, but the time course of the response to kainate was faster. The agonists N-methyl-D-aspartate and L-aspartate had little effect on the membrane conductance. The current-to-voltage (I-V) relationship for all three agonists was nonlinear when the membrane potential was hyperpolarized. The nonlinearity was, at least in part, a result of the decreased response to the three agonists. Removal of Mg did not alter this nonlinear relationship. When the inward potassium rectifier was blocked with 100 microM Ba, the response to glutamate was increased compared with the control experiment before block by barium; however, the I-V relationship was still highly nonlinear. Thus glutamate block of the inward potassium current cannot account entirely for the nonlinear I-V. The increase in membrane permeability to specific ions in the presence of an agonist was determined by ion substitution experiments and measuring the shift in the reversal potential. The three agonists appear to increase the membrane permeability to cations but not to anions. The amino acid antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and D-glutamyl glycine (DGG) were bath applied to test their ability to block the depolarizing effects of glutamate. DGG had no measureable effect at 100 microM concentration, whereas PDA reversibly reduced the glutamate response at 1 mM concentration although block was incomplete. Isolated horizontal cells responded to bath-applied glutamate in concentrations of 10-500 microM. In concentrations of glutamate greater than 50 microM, when the membrane potential was held at the resting potential, the inward current reached a maximum followed by a decrease to a steady-state level. This apparent time-dependent desensitization at high agonist concentrations was at least partially removed when Mg was removed from the bathing solution.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Control of oxygen affinity of hemoglobin in K562 cells induced by hemin

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1447-1452 ◽  
Author(s):  
Y Wu ◽  
A Dean ◽  
W Egan ◽  
AN Schechter
Keyword(s):  
Time Course ◽  
31P Nmr ◽  
Oxygen Affinity ◽  
K562 Cells ◽  
Hill Coefficient ◽  
Oxygen Dissociation ◽  
Oxygen Affinity Of Hemoglobin ◽  
Dissociation Curves ◽  
The Relationship ◽  
2,3 Dpg

Abstract The oxygen affinity of hemoglobin in K562 cells induced by hemin and the relationship between levels of 2,3-diphosphoglycerate (2,3-DPG) and hemoglobin have been investigated. Absorption spectra of induced cells indicate that the hemoglobin is oxygenated; oxygen dissociation curves are symmetric, with a P50 of 20 +/- 0.9 mm Hg, Hill coefficient of 2.5, and a normal temperature dependence. The intracellular pH measured by phosphorus 31 nuclear magnetic resonance (NMR) was 7.3. The amount of 2,3-DPG was determined by an enzymatic method and by 31P NMR. The level of 2,3-DPG in uninduced K562 cells, containing 0.5 pg of hemoglobin per cell, was low (5 +/- 0.5 mumole/10(8) cells), but increased to 64 +/- 5 mumole/10(8) cells upon induction of hemoglobin accumulation (to a final level of 20 pg hemoglobin/cell). For several experiments, there was a closely coordinated relationship between 2,3-DPG and hemoglobin levels, at about 1:1 stoichiometry of the two molecules. The time course of induction of hemoglobin, and of 2,3-DPG levels, are very similar; both processes are reversible. These data suggest that induction of hemoglobin synthesis in K562 cells by hemin results in hemoglobin-containing cells with normal oxygenation properties and that 2,3-DPG and hemoglobin levels are coordinately controlled in these cells. Elucidation of the mechanism of this effect should be of importance in understanding the erythroid-like differentiation of these cells.

Membrane permeability to the molecular and ionic forms of DMO in barnacle muscle

1981 ◽  
Vol 240 (1) ◽  
pp. C73-C79 ◽  
Author(s):  
D. W. Keifer ◽  
A. Roos
Keyword(s):  
Membrane Permeability ◽  
Time Course ◽  
Experimental Approach ◽  
Weak Acid ◽  
Mathematical Treatment ◽  
Permeability Ratio ◽  
Barnacle Muscle ◽  
Membrane Permeabilities ◽  
Barnacle Muscle Fibers

The time course of the intracellular pH (pHi) of barnacle muscle fibers was followed using microelectrodes while the fibers were exposed to 15 mM of the weak acid 5,5-dimethyloxazolidine-2,4-dione (DMO) at pH 6.5. The rapid initial fall in pHi was used to determine the membrane permeability to the DMO molecule, while the much slower fall during the subsequent plateau phase yielded the permeability to the DMO ion. This experimental approach and the mathematical treatment of the data can be used to obtain the membrane permeabilities to other weak acids or bases. We found values of 1.9 X 10(-4) cm/s for the permeability to the DMO molecule and 1.5 X 10(-7) cm/s for the permeability to the ion, assuming the fiber to be a cylinder. Thus the permeability of the neutral form is about 10(3) times that of the ionized form. At commonly encountered conditions of pHi = 7.3, outside pH = 7.5, and membrane potential = -52 mV, this permeability ratio introduces an error of only -0.01 to -0.02 into the determination of pHi based on the distribution of DMO.

Sign in / Sign up

Export Citation Format

Share Document