scholarly journals Fatty acids induce release of Ca2+ from acidosomal stores and activate capacitative Ca2+ entry in Dictyostelium discoideum

1998 ◽  
Vol 332 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Ralph SCHALOSKE ◽  
Jürgen SONNEMANN ◽  
Dieter MALCHOW ◽  
Christina SCHLATTERER
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Dictyostelium Discoideum ◽  
Knockout Mutant ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Inhibition Studies ◽  
Higher Doses ◽  
Phospholipase A2 Activity ◽  
Entry Mechanism

cAMP-induced Ca2+ fluxes in Dictyostelium discoideum largely depend on phospholipase A2 activity generating non-esterified fatty acids [Schaloske and Malchow (1997) Biochem. J. 327, 233–238]. In the present study the effect of fatty acids on Ca2+ homoeostasis in D. discoideum was investigated. Cytosolic free Ca2+ concentration ([Ca2+]i) was analysed by digital imaging of single fura 2–dextran-loaded cells. Arachidonic acid and linoleic acid induced a transient increase in [Ca2+]i. The concentration of arachidonic acid determined the percentage of responding cells, with the mean height of the increase being dose-independent. In nominally Ca2+-free medium or in the presence of bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid (BAPTA), no [Ca2+]i transient was detectable. In spite of this, we found that (1) arachidonic acid induced Ca2+ release from permeabilized cells and from vesicular fractions at concentrations that elicited Ca2+ influx in intact cells and (2) Ca2+ entry was inhibited by inhibitors of Ca2+-transport ATPases and V-type H+-ATPase, indicating that intracellular Ca2+ release precedes Ca2+ entry. Inhibition studies and mutant analysis point to the acidosomal Ca2+ stores as a target of fatty acids. Although fatty acids can substitute fully for cAMP with respect to Ca2+ influx in wild-type cells, experiments with a mutant strain revealed that cAMP also sensitizes the Ca2+-entry mechanism: cAMP-induced Ca2+ influx was normal in a phospholipase C knockout mutant but influx was fairly insensitive to arachidonic acid in this strain. This defect could be overcome by higher doses of arachidonic acid which cause sufficient Ca2+ to be released from the stores to trigger extracellular Ca2+ entry.

Arachidonic acid-induced LH release is ATP-independent and insensitive to N-ethyl maleimide

1992 ◽  
Vol 132 (1) ◽  
pp. 77-82 ◽  
Author(s):  
P. V. Kaye ◽  
P. A. van der Merwe ◽  
R. P. Millar ◽  
J. S. Davidson
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Pituitary Cells ◽  
Cell Permeabilization ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Lh Release ◽  
Stimulation Of

ABSTRACT The mechanism of arachidonic acid (AA)-induced LH release was characterized using sheep pituitary cells in primary culture permeabilized with Staphylococcal α-toxin. In intact cells, exogenous AA evoked release of LH in a manner which was partially dependent on extracellular Ca2+. At similar concentrations, AA also caused cell permeabilization as monitored by efflux of [3H]2-deoxyglucose metabolites. In α-toxin-permeabilized cells where cytosolic Ca2+ was clamped at resting levels, AA retained its ability to cause LH release. Unlike the stimulation of exocytosis produced by Ca2+, phorbol ester or cyclic AMP, AA-evoked release was independent of ATP and was not inhibited by pretreatment with N-ethyl maleimide. These findings indicated that exogenous AA does not cause LH release by Ca2+ influx or mobilization or by activating protein kinase C. The results suggest that LH release induced by exogenous AA is probably due to its detergent-like properties, and does not represent true exocytosis. Journal of Endocrinology (1992) 132, 77–82

scholarly journals Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas

1986 ◽  
Vol 238 (3) ◽  
pp. 765-772 ◽  
Author(s):  
C W Taylor ◽  
J E Merritt ◽  
J W Putney ◽  
R P Rubin
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase C ◽  
Acinar Cells ◽  
Major Effect ◽  
Arachidonic Acid Metabolism ◽  
Pancreatic Acinar Cells ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Receptor Agonists ◽  
Independent Effects

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.

scholarly journals Inhibition of protein synthesis in intact mammalian cells by arachidonic acid

1992 ◽  
Vol 282 (2) ◽  
pp. 487-494 ◽  
Author(s):  
E I Rotman ◽  
M A Brostrom ◽  
C O Brostrom
Keyword(s):  
Fatty Acids ◽  
Protein Synthesis ◽  
Arachidonic Acid ◽  
Mammalian Cells ◽  
Unsaturated Fatty Acids ◽  
Arachidic Acid ◽  
Chain Elongation ◽  
Gh3 Cells ◽  
Intact Cells ◽  
Translational Initiation

Optimal translation initiation in intact mammalian cells requires sequestered intracellular Ca2+. Arachidonic acid, which releases sequestered Ca2+ from cells and isolated organelles, was studied to assess its potential role in the regulation of protein synthesis via Ca2+ mobilization. Unsaturated fatty acids at microM concentrations inhibited protein synthesis in intact GH2 pituitary, C6 glial tumour and HeLa cells in a manner dependent on degree of unsaturation and cell number. Arachidonate was generally the most, and the fully saturated arachidic acid the least, potent of the fatty acids tested. At 2 x 10(6) GH3 cells/ml, amino incorporation into a broad spectrum of polypeptides was inhibited by 80-90% by 10-20 microM fatty acid. Inhibition was maximal at 4-8 min and was attenuated by 1-2 h and more pronounced at lower pH. Protein synthesis was maximally inhibited when arachidonate mobilized approx. 40% of cell-associated Ca2+. At lower concentrations (10 microM) arachidonate suppressed translational initiation, with the inhibition being reversed as extracellular Ca2+ concentrations were increased to supraphysiological values. At higher concentrations (20 microM) arachidonate inhibited peptide-chain elongation in a Ca(2+)-independent manner. Arachidonate also blocked elongation in reticulocyte lysates. The effects of arachidonate in intact cells were reversible with time via its metabolism or by washes containing BSA. Sufficient arachidonate appears to be synthesized during ischaemic stress to inhibit translation by either mechanism.

scholarly journals Intracellular Regulation of the Metabolism of Arachidonic Acid in Human Platelets

1977 ◽  
Author(s):  
Thomas K. Bills ◽  
J. Bryan Smith ◽  
Melvin J. Silver
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Fatty Acid ◽  
Phospholipase A2 ◽  
Unsaturated Fatty Acids ◽  
Human Platelets ◽  
Intracellular Regulation ◽  
Rate Limiting ◽  
Phospholipase A2 Activity ◽  
Oleic And Linoleic Acids

The synthesis of prostaglandins and thromboxanes by human platelets is limited by the availability of the fatty acid precursor, arachidonic acid. Although large amounts of arachidonic acid are esterified to platelet phospholipids, only the free acid can be utilized by the oxygenation pathways of platelets. Since there are only trace amounts of free arachidonic acid in platelets, the enzymatic liberation of this fatty acid from platelet phospholipids can be considered the initial and rate limiting step of these oxygenation pathways. This process is catalyzed by a phospholipase A2 whose role as the rate limiting enzyme makes it a prime target for the intracellular regulation of prostaglandin and thromboxane synthesis.A second mechanism for the regulation of prostaglandin synthesis has been hypothesized. Several commonly occurring unsaturated fatty acids, e.g., oleic and linoleic acids, are capable of inhibiting prostaglandin cyclooxygenase. If these fatty acids are liberated from phospholipids along with arachidonic acid, they could limit the production of prostaglandins and thromboxanes. Thus, the types and amounts of fatty acids released from platelet phospholipids could regulate the amounts of prostaglandins and thromboxanes produced.We have investigated these two types of intracellular regulation and have found that 1) intracellular cyclic nucleotides and divalent cations are involved in the regulation of the platelet phospholipase A2 activity stimulated by thrombin and 2) this phospholipase A2 activity catalyzes the specific release of arachidonic acid from phospholipids, thereby obviating the regulatory role of liberated oleic and linoleic acids.

scholarly journals Measurement of β-oxidation capacity of biological samples by respirometry: a review of principles and substrates

2016 ◽  
Vol 310 (9) ◽  
pp. E715-E723 ◽  
Author(s):  
Edward Ojuka ◽  
Brittany Andrew ◽  
Nicole Bezuidenhout ◽  
Siddiqah George ◽  
Gerald Maarman ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Fatty Acids ◽  
Oxygen Consumption ◽  
Biological Samples ◽  
Short Chain ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Isolated Mitochondria ◽  
Chain Acyl ◽  
Rate Of Oxygen Consumption

Oxidation of fatty acids is a major source of energy in the heart, liver, and skeletal muscle. It can be measured accurately using respirometry in isolated mitochondria, intact cells, and permeabilized cells or tissues. This technique directly measures the rate of oxygen consumption or flux at various respiratory states when appropriate substrates, uncouplers, and inhibitors are used. Acylcarnitines such as palmitoylcarnitine or octanoylcarnitine are the commonly used substrates. The β-oxidation pathway is prone to feedforward inhibition resulting from accumulation of short-chain acyl-CoA and depletion of CoA, but inclusion of malate or carnitine prevents accumulation of these intermediaries and CoA depletion.

scholarly journals Impact of n-6 Polyunsaturated Fatty Acids on Growth of Multidrug-Resistant Pseudomonas aeruginosa: Interactions with Amikacin and Ceftazidime

2000 ◽  
Vol 44 (8) ◽  
pp. 2187-2189 ◽  
Author(s):  
E. J. Giamarellos-Bourboulis ◽  
P. Grecka ◽  
A. Dionyssiou-Asteriou ◽  
H. Giamarellou
Keyword(s):  
Fatty Acids ◽  
Pseudomonas Aeruginosa ◽  
Arachidonic Acid ◽  
Experimental Infection ◽  
Multidrug Resistant ◽  
Gamma Linolenic Acid ◽  
Infection Models ◽  
Over Time ◽  
In Vitro Interactions

ABSTRACT Twenty-six multidrug-resistant Pseudomonas aeruginosaisolates were exposed over time to 300 μg of gamma-linolenic acid or arachidonic acid per ml or to the combination of both acids at 150 μg/ml each with ceftazidime and amikacin with or without albumin to observe the in vitro interactions of the antibiotics. Antibiotics and albumin were applied at their levels found in serum. Synergy between acids and antibiotics was found against 13 isolates, and it was expressed after 5 h of growth in the presence of albumin. The results indicate that further application in experimental infection models is merited.

scholarly journals Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase

1988 ◽  
Vol 250 (2) ◽  
pp. 343-348 ◽  
Author(s):  
T Matsumoto ◽  
W Tao ◽  
R I Sha'afi
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Phospholipase A2 ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Membrane Preparation ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Intact Cells

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

Effect of insulin on free fatty acid uptake by hepatocytes in the duck

1984 ◽  
Vol 102 (3) ◽  
pp. 381-386 ◽  
Author(s):  
R. Gross ◽  
P. Mialhe
Keyword(s):  
Fatty Acids ◽  
Growth Hormone ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Glucose Metabolism ◽  
Free Fatty Acids ◽  
Fatty Acid Uptake ◽  
Acid Uptake ◽  
Low Doses ◽  
Higher Doses

ABSTRACT To elucidate the hypolipacidaemic effect of insulin in ducks, its action on the uptake of free fatty acids (FFA) by duck hepatocytes was determined. At low doses (10 mu./l) insulin stimulated FFA uptake. This effect was not observed with higher doses of insulin (20, 30 and 50 mu./l). Growth hormone at physiological concentrations and corticosterone (14·4 nmol/l) decreased basal activity, probably by reducing glucose metabolism and consequently α-glycerophosphate (α-GP) supply. Insulin was able to reverse the inhibition induced by GH and corticosterone on both FFA uptake and α-GP production. These results therefore suggest that the hypolipacidaemic effect of insulin may be partly mediated by its action on hepatic FFA uptake. J. Endocr. (1984) 102, 381–386

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