scholarly journals Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide.

1985 ◽  
Vol 101 (4) ◽  
pp. 1191-1197 ◽  
Author(s):  
J L Slonczewski ◽  
M W Wilde ◽  
S H Zigmond
Keyword(s):  
Glycogen Phosphorylase ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Dose Dependence ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cytoplasmic Calcium ◽  
Chemotactic Peptide ◽  
Transient Activation ◽  
Fold Activation

The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels.

Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes

1985 ◽  
Vol 5 (6) ◽  
pp. 1212-1219
Author(s):  
E Resendez ◽  
J W Attenello ◽  
A Grafsky ◽  
C S Chang ◽  
A S Lee
Keyword(s):  
Gene Transfection ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Induced Response ◽  
Specific Gene ◽  
Regulatory Sequence ◽  
Cdna Clones ◽  
Ionophore A23187 ◽  
Flanking Sequence

Using two cDNA clones which encode hamster genes specifically induced by glucose starvation, we demonstrated that an 8- and 30-fold increase, respectively, in the transcription rates of these genes was coordinately effected by calcium ionophore A23187 treatment, resulting in a similar increase in the steady-state levels of their mRNAs. This response was observed within several hours of ionophore treatment in several mammalian cell types and appeared to be specifically mediated by A23187 but not by other ionophores in general. To define the regulatory sequence which mediates this Ca2+-induced response, we showed by gene transfection techniques that the 5' flanking sequence of a rat glucose-regulated gene contained the region for induction by A23187. The system reported here offers attractive features for the study of specific gene regulation by Ca2+.

Amplification of LTB4 generation in AM-PMN cocultures: transcellular 5-lipoxygenase metabolism

1991 ◽  
Vol 261 (2) ◽  
pp. L195-L203 ◽  
Author(s):  
F. Grimminger ◽  
U. Sibelius ◽  
W. Seeger
Keyword(s):  
Hydrolysis Product ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Metabolite Profile ◽  
Product Formation ◽  
Calcium Ionophore A23187 ◽  
Oxidation Products ◽  
Ionophore A23187 ◽  
Lipoxygenase Product ◽  
Isolated Cell

The generation of arachidonic acid (AA) metabolites by human polymorphonuclear leukocytes (PMN) and by rabbit alveolar macrophages (AM) was investigated and compared with that produced under conditions of coculture. Incubation of PMN with the calcium ionophore A23187 resulted in rapid generation of leukotriene (LT) B4 and its omega-oxidation products, paralleled by substantial secretion of 5-hydroxyeicosatetraenoic acid (HETE) and intact LTA4. Rapid LTA4 decay to nonenzymatic hydrolysis products in the extracellular space ensued. Exogenous AA, offered simultaneously with the ionophore, markedly increased 5-lipoxygenase product formation. Incubation of AM with A23187 evoked protracted generation of LTB4 in the absence of omega-oxidation, with concomitant liberation of 5-HETE, 15-HETE, free AA, and minor amounts of AA cyclooxygenase products. Exogenously offered LTA4 was avidly taken up and converted into LTB4 by these cells. Costimulation of AM and PMN with the ionophore resulted in an approximately 2.5-fold increase in the generation of LTB4 and its metabolites (compared with the summed amounts of the isolated cell experiments), whereas 5-HETE and nonenzymatic LTA4, hydrolysis product formation were markedly reduced. This change in metabolite profile was dependent on the AM-to-PMN ratio. Acetylsalicylic acid increased 5-lipoxygenase product formation in the coculture studies but not in the isolated cell experiments. AA prelabeling of either PMN or AM resulted in radioactivity detection in all AA lipoxygenase products except for 15-HETE.(ABSTRACT TRUNCATED AT 250 WORDS)

INFLUENCE OF CALCIUM FLUX ON STABILITY OF PLATELET MICROTUBULE COILS

1987 ◽  
Author(s):  
Gundu H R Rao ◽  
James G White
Keyword(s):  
Shape Change ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Human Platelets ◽  
Calcium Flux ◽  
Osmic Acid ◽  
Ionophore A23187 ◽  
Cytoplasmic Calcium ◽  
Fura 2 ◽  
Morphological Studies

Resting human platelets have a characteristic discoid form supported by a circumferential microtubule (MT). Ultrastructural, immunocytochemical and immunofluorescence studies have shown that the circumferential MT is a stable structure which undergoes constriction following exposure of platelets to aggregating agents. However, some biochemical and morphological studies suggested that MT coils dissolved almost completely within seconds after exposure to aggregating agents, then reassembled 1-4 minutes later. One investigation suggested that disappearance of the MT coils was associated with calcium flux caused by agonist stimulation or exposure to the ionophore, A23187. The present study has examined the latter hypothesis directly. Fura 2, a calcium sensitive fluorophore, was loaded into washed platelets at a concentration of 1 μM, the cells washed once and resuspended in HEPES buffer. Fluorescence changes were monitored in a Fluorolog spectrofluorometer.Thrombin stimulation of Fura 2 loaded platelets resulted in an immediate eight fold increase in the level of cytoplasmic calcium. The calcium ionophore, ionomycin, stimulated a 15 fold rise in the cytoplasmic calcium of Fura 2 loaded cells.Samples of thrombin and ionomycin stimulated platelets were fixed in glutaraldehyde and osmic acid at the peak of calcium flux indicated by the rise in fluorescence. Examination of activated platelets in the electron microscope revealed shape change and internal transformation. MT coils were constricted, but did not disappear from platelets fixed during the maximum elevation of cytoplasmic calcium. The findings do not support the concept that the calcium rise produced in platelets following exposure to potent agonists causes disappearance of the circumferential MT.

scholarly journals Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes.

1985 ◽  
Vol 5 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
E Resendez ◽  
J W Attenello ◽  
A Grafsky ◽  
C S Chang ◽  
A S Lee
Keyword(s):  
Gene Transfection ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Induced Response ◽  
Specific Gene ◽  
Regulatory Sequence ◽  
Cdna Clones ◽  
Ionophore A23187 ◽  
Flanking Sequence

Using two cDNA clones which encode hamster genes specifically induced by glucose starvation, we demonstrated that an 8- and 30-fold increase, respectively, in the transcription rates of these genes was coordinately effected by calcium ionophore A23187 treatment, resulting in a similar increase in the steady-state levels of their mRNAs. This response was observed within several hours of ionophore treatment in several mammalian cell types and appeared to be specifically mediated by A23187 but not by other ionophores in general. To define the regulatory sequence which mediates this Ca2+-induced response, we showed by gene transfection techniques that the 5' flanking sequence of a rat glucose-regulated gene contained the region for induction by A23187. The system reported here offers attractive features for the study of specific gene regulation by Ca2+.

cAMP-independent stimulation of glycogen phosphorylase in newborn rat hepatocytes

1985 ◽  
Vol 248 (5) ◽  
pp. E560-E566
Author(s):  
A. Noguchi ◽  
P. A. Jett ◽  
A. H. Gold
Keyword(s):  
Glycogen Phosphorylase ◽  
Calcium Ionophore ◽  
Rat Hepatocytes ◽  
Calcium Ionophore A23187 ◽  
Membrane Receptors ◽  
Ionophore A23187 ◽  
Adrenergic Stimulation ◽  
Adult Rats ◽  
Isolated Rat Hepatocytes ◽  
Beta Adrenergic

Postnatal development of glycogen phosphorylase activation by the cAMP-independent pathway was examined in isolated rat hepatocytes from control and propylthiouracil-treated congenital hypothyroid rat pups. At 5 days postnatum there was complete phosphorylase activation by beta-adrenergic stimulation, glucagon, and the calcium ionophore A23187, but no activation by alpha-adrenergic stimulation. Activation of phosphorylase by angiotensin or vasopressin was less than in hepatocytes from adult rats (P less than 0.01). At 28 days postnatum activation by all of these hormones was complete. In the propylthiouracil-treated group hormone responsiveness was similar to the control at 5 days postnatum. However, alpha-adrenergic (P less than 0.025), angiotensin, and vasopressin (P less than 0.05) activation was decreased at 28 days postnatum, and beta-adrenergic, glucagon, and A23187 activation was complete. The attenuated responses were restored by thyroxine replacement from 15 days postnatum. [32P]Pi incorporation into phosphatidylinositol by epinephrine and vasopressin in 28-day propylthiouracil-treated rats was lower than the control (P less than 0.01). We speculate that the diminished phosphorylase response of hepatocytes to alpha-adrenergic, vasopressin, or angiotensin stimuli in the early neonatal period could be related to low receptor numbers and the weaker phosphoinositide response during this period. Also, the depressed phosphorylase response to alpha-adrenergic, vasopressin, and angiotensin stimulation in congenital hypothyroidism at 28 days postnatum could be related to a decrease in number of plasma membrane receptors for these agonists.

scholarly journals The activation of protein degradation in muscle by Ca2+ or muscle injury does not involve a lysosomal mechanism

1986 ◽  
Vol 237 (3) ◽  
pp. 859-864 ◽  
Author(s):  
K Furuno ◽  
A L Goldberg
Keyword(s):  
Muscle Injury ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Muscle Fibres ◽  
Complete Medium ◽  
Ionophore A23187 ◽  
Protein Breakdown ◽  
Thiol Proteinases ◽  
Thiol Proteinase

By use of different inhibitors, we distinguished three proteolytic processes in rat skeletal muscle. When soleus muscles maintained under tension were exposed to the calcium ionophore A23187 or were incubated under no tension in the presence of Ca2+, net protein breakdown increased by 50-80%. Although leupeptin and E-64 inhibit this acceleration of protein breakdown almost completely, other agents that prevent lysosomal function, such as methylamine or leucine methyl ester, did not inhibit this effect. A similar increase in net proteolysis occurred in muscle fibres injured by cutting, and this response was also inhibited by leupeptin, but not by methylamine. In contrast, all these inhibitors markedly decreased the 2-fold increase in protein breakdown induced by incubating muscles without insulin and leucine, isoleucine and valine. In addition, the low rate of proteolysis seen in muscles under passive tension in complete medium was not affected by any of these inhibitors. Thus the basal degradative process in muscle does not involve lysosomes or thiol proteinases, and muscle can enhance protein breakdown by two mechanisms: lack of insulin and nutrients enhances a lysosomal process in muscle, as in other cells, whereas Ca2+ and muscle injury activate a distinct pathway involving cytosolic thiol proteinase(s).

scholarly journals Changes in cytoplasmic calcium determine the secretory response to extracellular cations in human parathyroid cells: a confocal microscopy study using FM1-43 dye

2000 ◽  
Vol 352 (2) ◽  
pp. 353-361 ◽  
Author(s):  
Radu MIHAI ◽  
Teresa LAI ◽  
George J. SCHOFIELD ◽  
John R. FARNDON
Keyword(s):  
Confocal Microscopy ◽  
Plasma Membranes ◽  
Calcium Concentration ◽  
Membrane Surface ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cytoplasmic Calcium ◽  
Acetoxymethyl Ester ◽  
Prolonged Incubation

Whether activation of the calcium receptor (CaR) modulates secretory events was investigated by real-time fluorescence and confocal microscopy using fura 2 and FM1-43 fluorescent dye. Two paradigms were used: human parathyroid cells, which are stimulated by a step from a high to a low extracellular calcium concentration ([Ca2+]ext), and rMTC6-23 cells, a rat medullary thyroid carcinoma cell line whose secretion is stimulated by an increase in [Ca2+]ext. Parathyroid cells were dispersed from parathyroid adenomas removed from 18 patients with primary hyperparathyroidism. In both cell types, incubation with FM1-43 (2µM) resulted in staining of the plasma membranes, which was rapidly increased following changes in [Ca2+]ext known to stimulate secretion. A high [Ca2+]ext and lanthanum (La3+) decreased the membrane-associated FM1-43 fluorescence. Prolonged incubation (5–30min) in the presence of FM1-43 resulted in accumulation of the dye in the cytoplasm, its granular distribution suggesting targeting of the secretory compartment. These data suggest that FM1-43 fluorescence is determined by: (i) changes in cell membrane surface area associated with secretion-associated events, (ii) displacement/quenching by extracellular cations and (iii) endocytosis of the dye. In parathyroid cells, a rise in FM1-43 fluorescence occurred during incubation in a high (inhibitory) [Ca2+]ext if the cytoplasmic calcium concentration ([Ca2+]i) was decreased by the calcium chelator BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,Nƀ,Nƀ-tetra-acetic acid tetrakis(acetoxymethyl ester)] (10–50µM). Alternatively, the expected rise in FM1-43 fluorescence did not occur during incubation in a low (stimulatory) [Ca2+]ext if [Ca2+]i was increased by addition of the calcium ionophore A23187 (10–25µM). These data suggest that [Ca2+]i, rather than the absolute value of [Ca2+]ext, is the main modulator of secretion from parathyroid cells.

scholarly journals Adenosine signaling in outer medullary descending vasa recta

2001 ◽  
Vol 280 (3) ◽  
pp. R854-R861 ◽  
Author(s):  
Erik P. Silldorff ◽  
Thomas L. Pallone
Keyword(s):  
Calcium Ionophore ◽  
Receptor Activation ◽  
Calcium Ionophore A23187 ◽  
No Synthase ◽  
Ionophore A23187 ◽  
Ang Ii ◽  
Cytoplasmic Calcium ◽  
Camp Production ◽  
Camp Analog ◽  
Vasa Recta

We tested whether dilation of outer medullary descending vasa recta (OMDVR) is mediated by cAMP, nitric oxide (NO), and cyclooxygenase (COX). Adenosine (A; 10−6 M)-induced vasodilation of ANG II (10−9 M)-preconstricted OMDVR was mimicked by the cAMP analog 8-bromoadenosine 3′,5′-cyclic monophosphate (10−10to 10−4 M) and reversed by the adenylate cyclase inhibitor SQ-22536. Adenosine (10−4 M) stimulated OMDVR cAMP production greater than threefold. NO synthase blockade with N G-nitro-l-arginine methyl ester and N G-monomethyl-l-arginine (10−4 M) did not affect adenosine vasodilation. Adenosine induced endothelial cytoplasmic calcium transients that were small. Indomethacin (10−6 M) reversed adenonsine-induced dilation of OMDVR preconstricted with ANG II, endothelin, 4-bromo-calcium ionophore A23187, or carbocyclic thromboxane A2. In contrast, selective A2-receptor activation dilated endothelin-preconstricted OMDVR even in the presence of indomethacin. We conclude that OMDVR vasodilation by adenosine involves cAMP and COX but not NO. COX blockade does not fully inhibit selective A2 receptor-mediated OMDVR dilation.

Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 187-195 ◽  
Author(s):  
B. Ciapa ◽  
B. Borg ◽  
M. Whitaker
Keyword(s):  
Sea Urchin ◽  
Calcium Release ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Calcium Signal ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Isotopic Equilibrium ◽  
Sea Urchin Eggs

A transient increase in intracellular free calcium is believed to be the signal responsible for the stimulation of the egg metabolism at fertilization and the resumption of the cell cycle. We have studied how the polyphosphoinositides (PPI) turn over at fertilization in sea urchin eggs, in order to determine the relationship between the metabolism of these lipids and the calcium signal. We compare the patterns of PPI turnover that occur during the first minute following fertilization in eggs in which PPI are labelled to steady state with [3H]inositol or [3H]arachidonate with that in which PPI are labelled for a shorter period with [3H]inositol. When eggs are labelled to apparent isotopic equilibrium with either [3H]inositol or [3H]arachidonate, no early increase in [3H]PtdInsP2 occurs while PtdIns decreases slightly. On the contrary, when not labelled to isotopic equilibrium, all [3H]PPI increase during the first 15 seconds following fertilization. We find that, within seconds, fertilization triggers a 600-fold increase in the turnover of PPI, producing an amount of InsP3 apparently sufficient to trigger calcium release. We suggest that phosphoinositidase C and PtdInsP kinase, responsible respectively for the hydrolysis and synthesis of PtdInsP2, are both stimulated to a comparable degree in the first 30 seconds following fertilization and that net changes in the amount of PtdInsP2 at fertilization are very sensitive to the relative levels of activation of the two enzymes. Activating the eggs with the calcium ionophore A23187 showed that both these enzymes are sensitive to calcium, suggesting that calcium-dependent InsP3 production might play a role in the initiation and/or the propagation of the fertilization calcium wave.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Oxygen-dependent 1,25-dihydroxycholecalciferol-induced calcium ion transport in rat intestine

1981 ◽  
Vol 194 (1) ◽  
pp. 178-186 ◽  
Author(s):  
N C Kendrick ◽  
B Kabakoff ◽  
H F DeLuca
Keyword(s):  
Vitamin D ◽  
Calcium Ionophore ◽  
Maximum Velocity ◽  
Fold Increase ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Mucosal Surfaces ◽  
Calcium Ion Transport ◽  
Disc Assay

O2-dependent CA2+ uptake by rat duodenal discs has been characterized and used in a revised assay for 1,25-dihydroxycholecalciferol-induced intestinal Ca2+ transport. Although both muscle and mucosal surfaces are exposed in this free-floating-disc assay, the Ca2+ influx across the muscle surface is small, not O2- or vitamin D-dependent, and can be subtracted out. Depriving the animals of food for 9-14 h before assay increases the O2-dependent uptake by about 75%. Half-saturation values for O2-dependent Ca2+ uptake as determined with this assay are: 0.8mM-Ca2+ (fed) and 0.5mM-Ca2+ (food-deprived) for vitamin D-deficient rats, and 0.9mM-Ca2+ (fed) and 1.5mM-Ca2+ (food-deprived) for rats dosed with 1,25-dihydroxycholecalciferol. The maximum velocity of uptake varies from 6.7nmol of Ca2+ per cm2/min (fed) to 7.0nmol of Ca2+ per cm2/min (food-deprived) for vitamin D-deficient rats and 16.7nmol of Ca2+ per cm2/min (fed) to 29 nmol of Ca2+ per cm2/min (food-deprived) for 1,25-dihydroxycholecalciferol-treated rats. By using a 5 min preincubation and 15 min incubation with 1.0mM-Ca2+, duodenal tissue taken from vitamin D-treated rats shows about a 3-fold increase in O2-dependent Ca2+ uptake when compared with tissue taken from vitamin D-deficient animals. The calcium ionophore A23187, depending on concentration, either has no significant effect on or inhibits the O2-dependent uptake, rather than increasing it. Actinomycin D, at a dose of 2 micrograms/g, inhibits the O2-dependent uptake in intestinal discs from both vitamin D-deficient and vitamin D-treated rats by 58 and 80% respectively, when administered in vivo 3 1/2 h before assay.

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