scholarly journals Influence of the calcium-sensitive fluorophore, Quin 2, on platelet function

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 354-361 ◽  
Author(s):  
GH Rao ◽  
JD Peller ◽  
CP Semba ◽  
JG White
Keyword(s):  
Platelet Function ◽  
Light Emission ◽  
Cytosolic Calcium ◽  
Functional Restoration ◽  
Calcium Flux ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Experimental Conditions ◽  
Calcium Dependent ◽  
Dose Dependent Increase

Abstract Recent investigations using Quin 2, a fluorophore used to monitor cytosolic free calcium shifts, have shown that strong agonists cause a dramatic dose-dependent increase in platelet fluorescence. However, weak agonists stimulated little or no increase in light emission of Quin 2-loaded platelets, suggesting that calcium flux is not involved in activation by these agents. The present study has sought an alternative explanation for the failure of weak stimuli to cause a rise in cytosolic free calcium in platelets containing Quin 2. Conditions used to prepare, wash, load, gel-filter, and evaluate the fluorophore- filled cells were examined for their compatibility with retention of sensitivity to activation by weak agonists. The technique used to measure shifts in cytosolic calcium with Quin 2 requires multiply washed, unstirred platelets. Under these conditions, platelets do not aggregate or secrete in response to weak agonists. Quin 2, at concentrations greater than 40 mumol/L, inhibits the response of platelets to strong agonists, and completely blocks their reaction to weak agonists. Quin 2 inhibition of platelet function appears related to high buffering capacity for free calcium, although other mechanisms cannot be ruled out. This suggestion is supported by the observation that Quin 2-induced blockade can be overcome by membrane modulation, which is a calcium-dependent process. However, since both agonists are weak, significant elevation in cytosolic calcium concurrent with functional restoration could not be demonstrated under the experimental conditions used for monitoring calcium. Thus, the conditions used to prepare platelets for Quin 2 evaluation and Quin 2 itself appear to be responsible for the failure of weak agonists to cause evidence of a calcium shift in fluorophore-loaded cells.

scholarly journals Influence of the calcium-sensitive fluorophore, Quin 2, on platelet function

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 354-361
Author(s):  
GH Rao ◽  
JD Peller ◽  
CP Semba ◽  
JG White
Keyword(s):  
Platelet Function ◽  
Light Emission ◽  
Cytosolic Calcium ◽  
Functional Restoration ◽  
Calcium Flux ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Experimental Conditions ◽  
Calcium Dependent ◽  
Dose Dependent Increase

Recent investigations using Quin 2, a fluorophore used to monitor cytosolic free calcium shifts, have shown that strong agonists cause a dramatic dose-dependent increase in platelet fluorescence. However, weak agonists stimulated little or no increase in light emission of Quin 2-loaded platelets, suggesting that calcium flux is not involved in activation by these agents. The present study has sought an alternative explanation for the failure of weak stimuli to cause a rise in cytosolic free calcium in platelets containing Quin 2. Conditions used to prepare, wash, load, gel-filter, and evaluate the fluorophore- filled cells were examined for their compatibility with retention of sensitivity to activation by weak agonists. The technique used to measure shifts in cytosolic calcium with Quin 2 requires multiply washed, unstirred platelets. Under these conditions, platelets do not aggregate or secrete in response to weak agonists. Quin 2, at concentrations greater than 40 mumol/L, inhibits the response of platelets to strong agonists, and completely blocks their reaction to weak agonists. Quin 2 inhibition of platelet function appears related to high buffering capacity for free calcium, although other mechanisms cannot be ruled out. This suggestion is supported by the observation that Quin 2-induced blockade can be overcome by membrane modulation, which is a calcium-dependent process. However, since both agonists are weak, significant elevation in cytosolic calcium concurrent with functional restoration could not be demonstrated under the experimental conditions used for monitoring calcium. Thus, the conditions used to prepare platelets for Quin 2 evaluation and Quin 2 itself appear to be responsible for the failure of weak agonists to cause evidence of a calcium shift in fluorophore-loaded cells.

Transepithelial calcium transport in the chick chorioallantoic membrane. II. Compartmentalization of calcium during uptake

1993 ◽  
Vol 105 (2) ◽  
pp. 381-388 ◽  
Author(s):  
R.E. Akins ◽  
R.S. Tuan
Keyword(s):  
Calcium Transport ◽  
Chorioallantoic Membrane ◽  
Cytosolic Calcium ◽  
Calcium Flux ◽  
Free Calcium ◽  
Extracellular Medium ◽  
Calcium Accumulation ◽  
Chick Chorioallantoic Membrane ◽  
Cellular Compartments ◽  
Endosomal Vesicles

Calcium transport from the eggshell to the developing chick embryo is carried out by the ectoderm cells of the chick chorioallantoic membrane. Primary cells isolated from chick chorioallantoic membrane ectoderm were used to analyze the subcellular distribution of 45Ca2+ accumulated from the extracellular medium. We present evidence suggesting that calcium may be sequestered into endosome-like vesicles during the initial phase of uptake. A combination of techniques were utilized to monitor calcium fluxes and calcium compartmentalization in the cultured chorioallantoic membrane cells: (1) fura-2 fluorescence was used to indicate cytosolic free calcium concentrations, (2) 45Ca2+ tracer was used to follow calcium accumulation in all cellular compartments, and (3) digitonin was used to differentially permeabilize subcellular membranes in order to localize 45Ca2+ by following tracer release profiles. Differences between cytosolic calcium flux and whole cell calcium accumulation suggested that the pathway of calcium uptake from the medium involves sequestration into an internal compartment separate from the cytosol. Kinetic analysis of the digitonin-mediated release of specific subcellular markers (lactate dehydrogenase, NAD-dependent isocitrate dehydrogenase, [3H]inulin, and [3H]-2-deoxyglucose) and preloaded 45Ca2+ indicated that calcium was localized in a compartment similar to endosomal vesicles. Our results are consistent with a transcytotic mechanism for chorioallantoic membrane calcium transport.

Angiotensin II causes sustained elevations in cytosolic calcium in glomerulosa cells

1988 ◽  
Vol 255 (3) ◽  
pp. E338-E346 ◽  
Author(s):  
R. E. Kramer
Keyword(s):  
Angiotensin Ii ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Calcium Signal ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Cytosolic Calcium Concentration ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Glomerulosa Cells

Studies were conducted to examine the effects of angiotensin II on cytosolic free calcium concentration in bovine adrenal glomerulosa cells maintained in primary culture. The calcium indicator, fura-2, and discontinuous dual-wavelength fluorescence spectroscopy were used to measure cytosolic free calcium in superfused adherent cell monolayers. Basal cytosolic free calcium concentration was 63.7 +/- 3.3 nM. The threshold concentration for angiotensin II-stimulated increases in cytosolic calcium was 10(-14)-10(-13) M, and maximal elevation of cytosolic calcium was produced by 10(-9) M angiotensin II. Angiotensin II (10(-13) M) produced a gradual increase in cytosolic calcium concentration that plateaued after 3-5 min of superfusion at a level approximately 1.2 times that of control cells. The calcium signal invoked by a maximal concentration (10(-9) M) of angiotensin II, in contrast, was characterized by an immediate, intense (approximately 8-fold) increase in cytosolic calcium concentration that decayed within 5 min to a lower, but sustained, level 2.5-3 times that of control cells. The calcium signals invoked by intermediate concentrations (10(-12)-10(-10) M) of angiotensin II exhibited dose-dependent increases in magnitude and a gradual transition in nature between those invoked by threshold and maximal concentrations of the peptide. The effect of angiotensin II to increase cytosolic calcium concentration was accompanied by an increase in aldosterone output. The increase in steroidogenesis was most closely correlated with the magnitude of the initial calcium signal. At high concentrations (10(-10) and 10(-9) M) of angiotensin II, there was a clear dissociation between aldosterone output and the magnitude of the sustained calcium signal.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Hypertension Secondary to PHPT: Cause or Coincidence?

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Helmut Schiffl ◽  
Susanne M. Lang
Keyword(s):  
Cardiovascular Reactivity ◽  
Vascular Reactivity ◽  
Cytosolic Calcium ◽  
Free Calcium ◽  
Normal Blood Pressure ◽  
Cytosolic Free Calcium ◽  
Hypertensive Patients ◽  
Before And After ◽  
Underlying Mechanisms ◽  
Normal Controls

Primary hyperparathyroidism (PHPT) may be associated with arterial hypertension. The underlying mechanisms are not fully understood and reversibility by parathyroid surgery is controversial. This study aimed to characterize pressor hormones, vascular reactivity to norepinephrine, and cytosolic-free calcium in platelets in 15 hypertensive patients with hypercalcaemic PHPT before and after successful parathyroidectomy and to compare them with 5 pre-hypertensive patients with normocalcaemic PHPT, 8 normotensive patients with hypercalcaemic PHPT and 15 normal controls. Hypertensive patients with hypercalcaemic PHPT had slightly higher levels of pressor hormones (), enhanced cardiovascular reactivity to norepinephrine () and increased cytosolic calcium in platelets () than controls. Pre-hypertensive patients with normocalcaemic PHPT had intermediate values of increased cardiovascular reactivity and cytosolic calcium. Normotensive patients with hypercalcaemic PHPT and normotensive controls had comparable pressor hormone concentrations and intracellular calcium levels. Successful parathyroidectomy was associated with normal blood pressure values and normalisation of pressor hormone concentrations, cardiovascular pressor reactivity and cytosolic free calcium. Our results suggest that parathyroid hypertension is mediated/maintained, at least in part, by functional alterations of vascular smooth muscle cells and can be cured by parathyroidectomy in those patients who do not have primary hypertension.

scholarly journals Calcium Regulates the Association between Mitochondria and a Smooth Subdomain of the Endoplasmic Reticulum

2000 ◽  
Vol 150 (6) ◽  
pp. 1489-1498 ◽  
Author(s):  
Hui-Jun Wang ◽  
Ginette Guay ◽  
Liviu Pogan ◽  
Remy Sauvé ◽  
Ivan R. Nabi
Keyword(s):  
Confocal Microscopy ◽  
Mdck Cells ◽  
Close Association ◽  
Cytosolic Calcium ◽  
Free Calcium ◽  
Autocrine Motility Factor ◽  
Cytosolic Free Calcium ◽  
Motility Factor ◽  
Rat Liver Cytosol ◽  
Close Contacts

Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca2+ concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 μM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.

Control of cytosolic free calcium in cultured human pancreatic beta-cells occurs by external calcium-dependent and independent mechanisms.

Endocrinology ◽  
1994 ◽  
Vol 134 (4) ◽  
pp. 1771-1781 ◽  
Author(s):  
E Rojas ◽  
P B Carroll ◽  
C Ricordi ◽  
A C Boschero ◽  
S S Stojilkovic ◽  
...  
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Calcium Dependent ◽  
External Calcium

Influence of Quin 2, Fura 2 and Indo 1 on Platelet Function and on the Measurement of Cytosolic Free Calcium Ions

Platelets ◽  
1991 ◽  
Vol 2 (1) ◽  
pp. 19-24 ◽  
Author(s):  
O.K. Bellinger ◽  
C.M. Kirchmaier ◽  
A. Schirmer ◽  
H.K. Breddin
Keyword(s):  
Platelet Function ◽  
Calcium Ions ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Fura 2

Intracellular calcium and phospholipid turnover are not involved in the inhibition of iodothyronine 5'-deiodinase type II activity by T4

1991 ◽  
Vol 124 (1) ◽  
pp. 67-75
Author(s):  
Cristiana Juge-Aubry ◽  
Pierre Dôme ◽  
Catherine A. Siegrist-Kaiser ◽  
Alessandro M. Capponi ◽  
Albert G. Burger
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Enzyme Inactivation ◽  
Free Calcium ◽  
Type Ii ◽  
Cytosolic Free Calcium ◽  
Experimental Conditions ◽  
Phospholipid Turnover ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration

Abstract. In glial cell cultures, iodothyronine 5'-deiodinase type II is stimulated by dibutyryl cAMP. Serum-free medium increases enzyme activity and prolongs the half-life of the enzyme. T4 and rT3 specifically inhibit this activity. We tested whether enzyme inactivation by T4 was mediated by changes in cytosolic free calcium concentration and/or phospholipid turnover. Intracellular calcium concentration was decreased either by chelation of extracellular calcium or by chelation of extracellular and intracellular calcium. Neither basal hypothyroid 5'-deiodinase activity nor its inactivation by T4 were modified in such experimental conditions, compared with control cells incubated in normal calcium-containing medium. T4 by itself had no effect on the cytosolic free calcium concentration for up to 20 min. Studies on phospholipid turnover included norepinephrine in parallel to T4 as positive stimulation control. While norepinephrine clearly accelerated phosphoinositide turnover, there was no effect of T4 on any phospholipid turnover. These results suggest that neither cytosolic free calcium nor phospholipid turnover is involved in T4-dependent modulation of 5'-deiodinase type II activity in astrocytes in culture.

Patterns of free calcium in multicellular stages of Dictyostelium expressing jellyfish apoaequorin

Development ◽  
1995 ◽  
Vol 121 (8) ◽  
pp. 2291-2301 ◽  
Author(s):  
A.B. Cubitt ◽  
R.A. Firtel ◽  
G. Fischer ◽  
L.F. Jaffe ◽  
A.L. Miller
Keyword(s):  
Doubling Time ◽  
Cytosolic Calcium ◽  
Free Calcium ◽  
Calcium Waves ◽  
Calcium Spikes ◽  
Multicellular Development ◽  
Previous Evidence ◽  
Calcium Dependent ◽  
High Calcium

To examine the patterns of high free cytosolic calcium or [Ca2+]i during Dictyostelium's development, we expressed apoaequorin in D. discoideum, reconstituted aequorin and observed the resultant patterns of calcium-dependent luminescence. Specific, high calcium zones are seen throughout normal multicellular development and are roughly coincident with those regions that later differentiate into stalk or stalk-like cells. A slug, for example, shows a primary high calcium zone within its front quarter and a secondary one around its tail; while a mound shows such a zone around the periphery of its base. Combined with previous evidence, our findings support the hypothesis that high [Ca2+]i feeds back to favor the stalk pathway. We also discovered several high calcium zones within the mound's base that do not coincide with any known prepatterns in D. discoideum. These include two, relatively persistent, antipodal strips along the mound's periphery. These various persistent zones of high calcium are largely made up of frequent, 10 to 30 second long, semiperiodic calcium spikes. Each of these spikes generates a correspondingly short-lived, 200 to 500 microns long, high calcium band which extends along the nearby surface. Similar, but relatively large and infrequent, spikes generate cross bands which extend across migrating slugs and just behind their advancing tips as well as across the peripheries of rotating mounds and midway between their antipodal strips. Moreover, calcium has a doubling time of about a second as various spikes rise. This last observation suggests that the calcium bands seen in Dictyostelium may be generated by so-called fast calcium waves.

Effect of 17 β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

1990 ◽  
Vol 10 (1) ◽  
pp. 73-78 ◽  
Author(s):  
Tiziana Bellini ◽  
Diana Degani ◽  
Maurizio Matteuzzi ◽  
Franco Dallocchio
Keyword(s):  
Cell Surface ◽  
Steroid Hormones ◽  
Human Lymphocytes ◽  
Cytosolic Calcium ◽  
Free Calcium ◽  
Cytosolic Free Calcium ◽  
Calcium Response ◽  
Pre Treatment ◽  
Preincubation Time ◽  
Estradiol Concentration

Pre-treatment of human lymphocytes with 17 β-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17 β-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17 β-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17 β-estradiol, since the α isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5α-androstan), have no effect. Since the effect of the 17 β-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.

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