scholarly journals Measurement of intrasynaptosomal free calcium by using the fluorescent indicator quin-2

1984 ◽  
Vol 219 (1) ◽  
pp. 149-158 ◽  
Author(s):  
R H Ashley ◽  
M J Brammer ◽  
R Marchbanks
Keyword(s):  
Cerebral Cortex ◽  
Plasma Membrane ◽  
Guinea Pig ◽  
Calcium Ionophore ◽  
Ionized Calcium ◽  
Free Calcium ◽  
Acetoxymethyl Ester ◽  
Fluorescent Indicator ◽  
Calcium Indicator ◽  
Hydrolysis Of

The recently synthesized calcium indicator quin −2 was incorporated into synaptosomes from guinea-pig cerebral cortex following uptake and internal hydrolysis of quin −2 tetra-acetoxymethyl ester. Incubation in physiological media containing 1 mM- or 2 mM-CaCl2 led to equilibrium cytosolic ionized calcium concentrations of 85 +/- 10 nM and 205 +/- 5 nM respectively (mean +/- S.E.M. from eight and eighteen preparations respectively). Cytosolic Ca2+ was elevated following increases in external Ca2+ concentration, plasma membrane depolarization, mitochondrial inhibition, calcium ionophore addition or replacement of external sodium by lithium. Preliminary experiments were performed to assess changes in cytosolic Ca2+ accompanying the release of the neurotransmitter acetylcholine.

Cytosolic free Calcium and Intracellular Calcium Stores in Neutrophils from Hypertensive Subjects

1985 ◽  
Vol 69 (2) ◽  
pp. 227-230 ◽  
Author(s):  
P. Daniel Lew ◽  
Laurent Favre ◽  
Francis A. Waldvogel ◽  
Michel B. Vallotton
Keyword(s):  
Essential Hypertension ◽  
Intracellular Calcium ◽  
Calcium Ionophore ◽  
Calcium Handling ◽  
Free Calcium ◽  
Calcium Stores ◽  
Cytosolic Free Calcium ◽  
Intact Cells ◽  
Intracellular Calcium Stores ◽  
Calcium Indicator

1. Alterations in intracellular calcium have been implicated in the pathogenesis of essential hypertension. To see whether this is a generalized phenomenon we assessed cytosolic free calcium and intracellular calcium stores in neutrophils from normo- and hyper-tensive subjects, by trapping the fluorescent calcium indicator quin2 in intact cells. 2. Ten patients with untreated essential hypertension were compared with 10 age- and sex-matched normotensive subjects. The levels of cytosolic free calcium and intracellular calcium stores releasable by the calcium ionophore ionomycin did not differ. No significant relationship was found between blood pressure and the calcium parameters in all 20 subjects studied. 3. The results indicate that essential hypertension is not associated with a membrane defect in calcium handling of all human cell systems, leading to generalized increases in resting values of cytosolic free calcium. 4. Neutrophils do not appear to be a good model for intracellular calcium handling in vascular smooth muscle.

scholarly journals The role of calcium in lymphocyte proliferation. (An interpretive review)

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 413-422
Author(s):  
AH Lichtman ◽  
GB Segel ◽  
MA Lichtman
Keyword(s):  
Plasma Membrane ◽  
Lymphocyte Proliferation ◽  
Calcium Influx ◽  
Lectin Binding ◽  
Calcium Ionophore ◽  
Total Cell ◽  
Ionized Calcium ◽  
Ionophore A23187 ◽  
Cell Calcium

A small quantity of extracellular calcium is required for the stimulation of lymphocytes by mitogens such as plant lectins. Lectin binding to the lymphocyte surface and early postbinding events that eventually lead to DNA synthesis are calcium dependent. Mitogenic lectins such as PHA and Con-A rapidly increase the size of an exchangeable pool of cell calcium and cause a smaller rise in intracellular ionized calcium. The increase in ionized calcium is so small (100–200 nM), however, that no increase in total cell calcium is measurable. When lymphocytes are stimulated by a lectin, the rate of calcium entry into the cell increases, but the plasma membrane calcium extrusion pump can prevent the total cell calcium from increasing measurably. The calcium ionophore A23187 is a lymphocyte mitogen and causes an increase in the exchangeable, ionized, and total cell calcium. The former two effects may be causal in mitogenesis; the latter effect is cytotoxic. With A23187 treatment, the rate of calcium influx exceeds the maximum rate of the plasma membrane extrusion pump and cell calcium increases in proportion to the concentration of A23187. The mitochondria, by virtue of their high membrane potential, provide a sink for the buffering of cytoplasmic calcium after A23187 treatment. Thus, the plasma membrane or mitochondria regulate the distribution of lymphocyte calcium when the cell is stimulated by mitogenic lectins or ionophores. The evidence strongly suggests that an alteration in calcium pools or an increase in cytoplasmic ionized calcium plays a role in the initiation of the biochemical reactions that lead to mitogen-induced lymphocyte proliferation in vitro and, perhaps, to the immune response.

Serotonin Modulates Glutamate Responses in Isolated Suprachiasmatic Nucleus Neurons

1999 ◽  
Vol 82 (2) ◽  
pp. 533-539 ◽  
Author(s):  
Jorge E. Quintero ◽  
Douglas G. McMahon
Keyword(s):  
Suprachiasmatic Nucleus ◽  
Cell Communication ◽  
Suppressive Effect ◽  
Cellular Level ◽  
Free Calcium ◽  
Acetoxymethyl Ester ◽  
Calcium Indicator ◽  
Camp Analogue ◽  
Retinohypothalamic Tract ◽  
Serotonergic Modulation

Two input pathways to the suprachiasmatic nucleus (SCN) of the hypothalamus are the glutamatergic retinohypothalamic tract and the serotonergic afferent from the midbrain raphe nucleus. To determine whether these two temporal signaling pathways can converge at the cellular level, we have investigated the effects of serotonin on glutamate-induced calcium responses of individual SCN neurons isolated in cell culture. Dispersed cultures were formed from the SCN of neonatal rats. The calcium indicator Fura-2 acetoxymethyl ester was used to assess the changes in [Ca2+]i by recording the 340-nm/380-nm excitation ratio. Application of glutamate (5 μM) to the culture caused a rapid (within 10 s) increase in the fluorescence ratio of neurons indicating a marked increase in the concentration of intracellular free calcium. However, when 5-hydroxytryptamine (5-HT; 5 μM) was coapplied with glutamate, 31% of neurons showed an overall 61% reduction in the peak of the glutamate-induced calcium increase. Application of the 5-HT7/1A receptor agonist, (±)-8-hydroxy-2-(di- n-propylamino)tetralin [(±)-8-OH-DPAT] (1 μM), also reduced the calcium elevation this time by 80% in 18% of the neurons tested. When the 5-HT7/2/1C receptor antagonist, ritanserin (800 nM), was coapplied with serotonin, it blocked modulation of the glutamate responses. Further support for the involvement of the 5-HT7receptor was provided by the ability of the adenylate cyclase activator, forskolin (10 μM), and the cAMP analogue, 8-Br cAMP (0.5 mM), to mimic the suppressive effect of serotonin. Blocking spike-mediated cell communication with tetrodotoxin (1 μM) did not prevent the serotonergic suppression of glutamate-induced responses. These results support the hypothesis that the serotonergic modulation of photic entraining signals can occur in SCN neurons.

ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast

1987 ◽  
Vol 252 (1) ◽  
pp. C38-C46 ◽  
Author(s):  
G. J. Fisher ◽  
L. K. Kelley ◽  
C. H. Smith
Keyword(s):  
Plasma Membrane ◽  
Calcium Transport ◽  
Plasma Membranes ◽  
Calcium Uptake ◽  
Regulatory Protein ◽  
Calcium Ionophore ◽  
Maximum Velocity ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Basal Plasma

As a first step in understanding the cellular basis of maternal-fetal calcium transfer, we examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake [K0.5 = 119 nM, maximum velocity (Vmax) = 2 nM X min-1 X mg-1]. This uptake required magnesium, was not significantly affected by Na+ or K+ (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. We conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer.

scholarly journals Net activity of phospholipase A2 in brain and the lack of stimulation of the phospholipase A2-acylation stem

1977 ◽  
Vol 164 (1) ◽  
pp. 287-288
Author(s):  
C E Rowe
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Guinea Pig ◽  
Phospholipase A2 ◽  
Synaptic Membranes ◽  
Rapid Hydrolysis ◽  
Hydrolysis Of ◽  
Stimulation Of

Certain observations reported previously from this laboratory have not proved reproducible. These are (1) the relatively rapid hydrolysis of added phosphatidylcholine by phospholipase A2 of tissue from the cerebral cortex of the guinea pig and (2) the stimulation by 10 micron-noradrenaline and by 1.0nM-cyclic AMP of the phospholipase A2-acylation system of isolated synaptic membranes.

scholarly journals The role of calcium in lymphocyte proliferation. (An interpretive review)

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 413-422 ◽  
Author(s):  
AH Lichtman ◽  
GB Segel ◽  
MA Lichtman
Keyword(s):  
Plasma Membrane ◽  
Lymphocyte Proliferation ◽  
Calcium Influx ◽  
Lectin Binding ◽  
Calcium Ionophore ◽  
Total Cell ◽  
Ionized Calcium ◽  
Ionophore A23187 ◽  
Cell Calcium

Abstract A small quantity of extracellular calcium is required for the stimulation of lymphocytes by mitogens such as plant lectins. Lectin binding to the lymphocyte surface and early postbinding events that eventually lead to DNA synthesis are calcium dependent. Mitogenic lectins such as PHA and Con-A rapidly increase the size of an exchangeable pool of cell calcium and cause a smaller rise in intracellular ionized calcium. The increase in ionized calcium is so small (100–200 nM), however, that no increase in total cell calcium is measurable. When lymphocytes are stimulated by a lectin, the rate of calcium entry into the cell increases, but the plasma membrane calcium extrusion pump can prevent the total cell calcium from increasing measurably. The calcium ionophore A23187 is a lymphocyte mitogen and causes an increase in the exchangeable, ionized, and total cell calcium. The former two effects may be causal in mitogenesis; the latter effect is cytotoxic. With A23187 treatment, the rate of calcium influx exceeds the maximum rate of the plasma membrane extrusion pump and cell calcium increases in proportion to the concentration of A23187. The mitochondria, by virtue of their high membrane potential, provide a sink for the buffering of cytoplasmic calcium after A23187 treatment. Thus, the plasma membrane or mitochondria regulate the distribution of lymphocyte calcium when the cell is stimulated by mitogenic lectins or ionophores. The evidence strongly suggests that an alteration in calcium pools or an increase in cytoplasmic ionized calcium plays a role in the initiation of the biochemical reactions that lead to mitogen-induced lymphocyte proliferation in vitro and, perhaps, to the immune response.

scholarly journals Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator.

1982 ◽  
Vol 94 (2) ◽  
pp. 325-334 ◽  
Author(s):  
R Y Tsien ◽  
T Pozzan ◽  
T J Rink
Keyword(s):  
Cell Types ◽  
Atp Depletion ◽  
Free Calcium ◽  
Fluorescence Signal ◽  
Resting Cells ◽  
Tetracarboxylic Acid ◽  
Acetoxymethyl Ester ◽  
Intact Mouse ◽  
Fluorescent Indicator ◽  
The Face

A new, fluorescent, highly selective Ca2+ indicator , "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca2+ concentrations, [Ca2+]i. Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to CA-saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. The fluorescence signal from resting cells indicates a [Ca2+]i of near 120 nM. The millimolar loadings of quin2 needed for accurately calibrated signals do not seem to perturb steady-state [Ca2+]i, but do somewhat slow or blunt [Ca2+]i transients. Loadings of up to 2mM are without serious toxic effects, though above this level some lowering of cellular ATP is observed. [Ca2+]i was well stabilized in the face of large changes in external Ca2+. Alterations of Na+ gradients, membrane potential, or intracellular pH had little effect. Mitochondrial poisons produced a small increase in [Ca2+]i, probably due mostly to the effects of severe ATP depletion on the plasma membrane. Thus intracellulary trapped chelators like quin2 offer a method to measure or buffer [Ca2+]i in hitherto intractable cell types.

A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

1994 ◽  
Vol 52 ◽  
pp. 168-169
Author(s):  
Martin Poenie ◽  
Akwasi Minta ◽  
Charles Vorndran
Keyword(s):  
Metal Binding ◽  
Acetoxymethyl Ester ◽  
Binding Properties ◽  
Fura 2 ◽  
New Family ◽  
Anion Transporter ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
High Calcium ◽  
Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

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