scholarly journals 3D holotomographic monitoring of Ca++ dynamics during ionophore-induced Neospora caninum tachyzoite egress from primary bovine host endothelial cells

2021 ◽  
Author(s):  
C. Larrazabal ◽  
C. Hermosilla ◽  
A. Taubert ◽  
I. Conejeros
Keyword(s):  
Endothelial Cells ◽  
Neospora Caninum ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Live Cell ◽  
Lytic Cycle ◽  
Host Cells ◽  
Ionophore A23187 ◽  
Cell Functions

AbstractNeospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.

scholarly journals Transcellular sulfidopeptide leukotriene biosynthetic capacity of vascular cells

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 703-707 ◽  
Author(s):  
J Maclouf ◽  
RC Murphy ◽  
PM Henson
Keyword(s):  
Endothelial Cells ◽  
Cell Activation ◽  
Calcium Ionophore ◽  
Cell Types ◽  
Fixed Number ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Mixed Cell ◽  
Wide Range

Abstract Cells in the vasculature, including polymorphonuclear leukocytes, platelets, and endothelial cells, have been shown to be jointly involved in the biosynthesis of active lipid mediators derived from arachidonic acid. Stimulation of neutrophils with the calcium ionophore A23187 as a model for cell activation results in production of leukotriene (LT)A4 with subsequent intracellular conversion into LTB4. When platelets or endothelial cells were present in the incubation system, LTC4 was produced from the neutrophil-derived LTA4. Whereas production and release of LTA4 under resting conditions in vivo might be expected to be quite low, under pathologic conditions, LTA4 production could be markedly increased. Therefore, the metabolism of exogenous LTA4 by platelets and endothelial cells was studied at a wide range of LTA4 concentrations. The production of LTC4 during coincubation of neutrophils with platelets was found to be dependent on neutrophil number ranging from 2 x 10(5) to 2 x 10(7) cells/mL. When a fixed number of neutrophils were stimulated with platelets alone or with mixtures of platelets and endothelial cells, LTC4 production was observed to be dependent on both acceptor cell types. These results suggest that mixed cell populations, which are likely to occur in vivo, may be critical determinants of the profile of eicosanoids produced in pathophysiologic circumstances. We suggest that both endothelial cells and platelets, in the presence of neutrophils, contribute large quantities of sulfidopeptide leukotrienes to inflammatory and thrombotic situations. Furthermore, platelets, because of their quantity and reactivity, may play a pivotal role in transcellular biosynthesis of eicosanoids.

scholarly journals The effect of acute feeding of carnitine, acetyl carnitine and propionyl carnitine on basal and A23187-stimulated eicosanoid release from rat carrageenan-elicited peritoneal macrophages

1990 ◽  
Vol 64 (2) ◽  
pp. 497-503 ◽  
Author(s):  
G. R. Elliott ◽  
A. P. M. Lauwen ◽  
I. L. Bonta
Keyword(s):  
Calcium Ionophore ◽  
Acute Effect ◽  
Peritoneal Macrophages ◽  
Calcium Ionophore A23187 ◽  
Prostaglandin E ◽  
Ionophore A23187 ◽  
Cell Functions ◽  
Prostaglandin F ◽  
Propionyl Carnitine

Little is known about the ability of carnitine to modulate cell functions. As carnitine plays an important role in lipid metabolism we investigated the acute effect of L-carnitine, L-acetyl carnitine and L-propionyl carnitine (300 mg/kg per d; 4 d) on the basal and calcium-ionophore (A23187)-stimulated release of arachidonic acid metabolites from rat carrageenan-elicited peritoneal macrophages. A decrease in the number of peritoneal carrageenan-elicited macrophages was observed after feeding all three compounds. The basal release of prostaglandin E2, 6 keto-prostaglandin F1α and leukotriene B4 was stimulated by all treatments. In contrast, thromboxane B2 production was diminished by feeding carnitine and acetyl carnitine. A23187-stimulated synthesis of 6 keto-prostaglandin F1α and leukotriene B4 was further enhanced by all three compounds. Acetyl carnitine and propionyl carnitine also enhanced thromboxane B2 synthesis. However, no effects on prostaglandin E2 formation were detected. The 6 keto-prostaglandin F1α: thromboxane B2 ratio, calculated from the basal and A23187-stimulated values, was increased by carnitine treatment. In the presence of A23187 there was also an increase in the 6 keto-prostaglandin F1α: leukotriene B4 ratio. We conclude that carnitine, and possibly some of its derivatives, could modify the macrophage component of an inflammation in vivo.

scholarly journals Ionophore-Resistant Mutants of Toxoplasma gondii Reveal Host Cell Permeabilization as an Early Event in Egress

2000 ◽  
Vol 20 (24) ◽  
pp. 9399-9408 ◽  
Author(s):  
Michael W. Black ◽  
Gustavo Arrizabalaga ◽  
John C. Boothroyd
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Lytic Cycle ◽  
Host Cells ◽  
Infected Host ◽  
Early Event ◽  
Ionophore A23187 ◽  
Cell Permeabilization

ABSTRACT Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca2+]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly lose infectivity (termed ionophore-induced death [IID]). From among several Iie− mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The association between the Iie− and Iid− phenotypes is supported by the observation that two-thirds of mutants selected as Iid− are also Iie−. Characterization of three distinct classes of IIE and IID mutants revealed that the Iie− phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized just prior to egress. Iie−parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming that this step is deficient in the Iie− mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading to parasite egress. The fact that Iie−mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and IIE.

scholarly journals Human leukocyte cathepsin G and elastase specifically suppress thrombin- induced prostacyclin production in human endothelial cells

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1627-1634 ◽  
Author(s):  
BB Weksler ◽  
EA Jaffe ◽  
MS Brower ◽  
OF Cole
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Human Leukocyte ◽  
Calcium Ionophore A23187 ◽  
Cathepsin G ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Prostacyclin Production

Polymorphonuclear leukocytes (PMN) when activated release products that can potentially injure endothelial cells or alter endothelial function. Exposure of cultured human umbilical vein endothelial cells to cathepsin G and elastase isolated from human PMN at concentrations reached in vivo (100 ng/mL to 10 micrograms/mL) selectively inhibited thrombin-induced prostacyclin production and the thrombin-induced rise in cytosolic free calcium ([Ca++]i) concentration. These proteases also blocked thrombin-induced release of arachidonic acid from prelabeled endothelial cells (EC). In contrast, induction of prostacyclin (PGI2) production by arachidonate, histamine, or the calcium ionophore A23187 was not altered by treatment of EC with these proteases. The effects of the proteases were concentration-dependent, were blocked by serum or serum protease inhibitors, and were reversed when the endothelial cells were further cultured for 24 hours in the absence of the proteases. Elastase, but not cathepsin G, also produced detachment of endothelial cells. Thus, the major leukocyte proteases selectively suppress thrombin-induced prostacyclin production by human vascular endothelial cells and may alter the hemostatic balance at sites of PMN activation.

Transcellular sulfidopeptide leukotriene biosynthetic capacity of vascular cells

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 703-707 ◽  
Author(s):  
J Maclouf ◽  
RC Murphy ◽  
PM Henson
Keyword(s):  
Endothelial Cells ◽  
Cell Activation ◽  
Calcium Ionophore ◽  
Cell Types ◽  
Fixed Number ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Mixed Cell ◽  
Wide Range

Cells in the vasculature, including polymorphonuclear leukocytes, platelets, and endothelial cells, have been shown to be jointly involved in the biosynthesis of active lipid mediators derived from arachidonic acid. Stimulation of neutrophils with the calcium ionophore A23187 as a model for cell activation results in production of leukotriene (LT)A4 with subsequent intracellular conversion into LTB4. When platelets or endothelial cells were present in the incubation system, LTC4 was produced from the neutrophil-derived LTA4. Whereas production and release of LTA4 under resting conditions in vivo might be expected to be quite low, under pathologic conditions, LTA4 production could be markedly increased. Therefore, the metabolism of exogenous LTA4 by platelets and endothelial cells was studied at a wide range of LTA4 concentrations. The production of LTC4 during coincubation of neutrophils with platelets was found to be dependent on neutrophil number ranging from 2 x 10(5) to 2 x 10(7) cells/mL. When a fixed number of neutrophils were stimulated with platelets alone or with mixtures of platelets and endothelial cells, LTC4 production was observed to be dependent on both acceptor cell types. These results suggest that mixed cell populations, which are likely to occur in vivo, may be critical determinants of the profile of eicosanoids produced in pathophysiologic circumstances. We suggest that both endothelial cells and platelets, in the presence of neutrophils, contribute large quantities of sulfidopeptide leukotrienes to inflammatory and thrombotic situations. Furthermore, platelets, because of their quantity and reactivity, may play a pivotal role in transcellular biosynthesis of eicosanoids.

scholarly journals Human leukocyte cathepsin G and elastase specifically suppress thrombin- induced prostacyclin production in human endothelial cells

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1627-1634 ◽  
Author(s):  
BB Weksler ◽  
EA Jaffe ◽  
MS Brower ◽  
OF Cole
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Human Leukocyte ◽  
Calcium Ionophore A23187 ◽  
Cathepsin G ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Prostacyclin Production

Abstract Polymorphonuclear leukocytes (PMN) when activated release products that can potentially injure endothelial cells or alter endothelial function. Exposure of cultured human umbilical vein endothelial cells to cathepsin G and elastase isolated from human PMN at concentrations reached in vivo (100 ng/mL to 10 micrograms/mL) selectively inhibited thrombin-induced prostacyclin production and the thrombin-induced rise in cytosolic free calcium ([Ca++]i) concentration. These proteases also blocked thrombin-induced release of arachidonic acid from prelabeled endothelial cells (EC). In contrast, induction of prostacyclin (PGI2) production by arachidonate, histamine, or the calcium ionophore A23187 was not altered by treatment of EC with these proteases. The effects of the proteases were concentration-dependent, were blocked by serum or serum protease inhibitors, and were reversed when the endothelial cells were further cultured for 24 hours in the absence of the proteases. Elastase, but not cathepsin G, also produced detachment of endothelial cells. Thus, the major leukocyte proteases selectively suppress thrombin-induced prostacyclin production by human vascular endothelial cells and may alter the hemostatic balance at sites of PMN activation.

Effects of glucocorticoids on prostaglandin formation by human amnion

1990 ◽  
Vol 68 (6) ◽  
pp. 671-676 ◽  
Author(s):  
William Gibb ◽  
Jean-Claude Lavoie
Keyword(s):  
Calcium Ionophore ◽  
Inhibitory Effect ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Isolated Cells ◽  
Stimulatory Action ◽  
Human Amnion ◽  
Human Labor ◽  
Epidermal Growth

The human amnion may be an important source of prostaglandins involved in the onset of human labor and therefore it is important to define the factors that regulate their formation in this tissue. In the present study we demonstrate that glucocorticoids inhibit prostaglandin production by freshly isolated amnion cells. The inhibitory action of the glucocorticoids, however, changes to a stimulatory action when the cells are maintained in primary culture for a few days. For both inhibition and stimulation, concentrations of 10−8 M dexamethasone or greater were required to give significant effects, and estradiol and progesterone had no effect on the prostaglandin output of the cells. Epidermal growth factor (EGF), which has previously been found to stimulate prostaglandin output by confluent amnion cells, did not alter prostaglandin output of cells initially placed in culture. Furthermore, the stimulatory action of EGF and dexamethasone appeared additive. The calcium ionophore A23187 stimulated prostaglandin output in freshly isolated cells and accentuated the inhibitory effect of dexamethasone. These studies indicate that prostaglandin formation by human amnion during pregnancy could be regulated by glucocorticoids. These steroids are easily available to the amnion by way of cortisone conversion to Cortisol by the maternal decidua. The results also indicate that amnion is capable of responding to glucocorticoids in both a stimulatory and inhibitory fashion and whether one or both actions are of importance in vivo is a question that is as yet unresolved.Key words: prostaglandins, amnion, fetal membranes, glucocorticoids, labor, pregnancy.

scholarly journals Tulathromycin Exerts Proresolving Effects in Bovine Neutrophils by Inhibiting Phospholipases and Altering Leukotriene B4, Prostaglandin E2, and Lipoxin A4Production

2014 ◽  
Vol 58 (8) ◽  
pp. 4298-4307 ◽  
Author(s):  
Carrie D. Fischer ◽  
Stephanie C. Duquette ◽  
Bernard S. Renaux ◽  
Troy D. Feener ◽  
Douglas W. Morck ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Lipid Signaling ◽  
Prostaglandin E ◽  
Ionophore A23187 ◽  
Proinflammatory Mediators ◽  
Bovine Neutrophils

ABSTRACTThe accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4(LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4[LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytesin vitroand inMannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammationin vivoand characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4and prostaglandin E2(PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2(PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

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