We have earlier shown that enterocyte mitochondria contain a phospholipase D (PLD) activity which can be stimulated by oxygen free radicals, divalent cations and polyamines. The functional ...significance of this enzyme in mitochondria is not known but it can be investigated using selective inhibitors. In the present study, mitochondrial PLD was activated by exposure to oxidants (X + XO or menadione), calcium or polyamines and the effect of antimalarial drugs, chloroquine, amodiaquin and primaquine on PLD activity was studied. Chloroquine and amodiaquine inhibited Ca
2+ stimulated PLD activity in dose dependent manner whereas these drugs had no significant effect on PLD activated by oxidants or polyamines. Increasing the calcium concentration relieved the PLD inhibition by these drugs. Primaquine did not have any effect on calcium stimulated PLD activity whereas it slightly activated the enzyme. These results indicate that chloroquine and amodiaquine may bind with calcium making it unavailable for PLD activation.
Inositol 1,4,5-trisphosphate receptors (IP
3
Rs) mediate cytosolic free calcium concentration (Ca
2+
c
) signals in response to a variety of agonists that stimulate mesangial cell contraction and ...proliferation. In the present study, we demonstrate that mesangial cells express both type I and III IP
3
Rs and that these receptors occupy different cellular locations. Chronic treatment with transforming growth factor-β1 (TGF-β1; 10 ng/ml, 24 h) leads to downregulation of both type I and III IP
3
Rs as measured by immunoblot and confocal analysis. TGF-β1 treatment does not affect IP
3
levels, and downregulation of type I IP
3
R is not due to enhanced degradation of the protein, as the half-life of type I IP
3
R is unchanged in the presence or absence of TGF-β1. Functional effects of TGF-β1-induced downregulation of the IP
3
Rs were evaluated by measuring Ca
2+
c
changes in response to epidermal growth factor (EGF) in intact cells and sensitivity of Ca
2+
c
release to IP
3
in permeabilized cells. TGF-β1 pretreatment led to a significant decrease of Ca
2+
c
release induced by EGF in intact cells and by submaximal IP
3
(400 nm) in permeabilized cells. Total IP
3
-sensitive Ca
2+
c
stores were not changed, as assessed by stimulation with maximal doses of IP
3
(10.5 μm) and thapsigargin-mediated calcium release in permeabilized cells. We conclude that prolonged exposure to TGF-β1 leads to downregulation of both type I and III IP
3
Rs in mesangial cells and this is associated with impaired sensitivity to IP
3
.
Inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate cytosolic free calcium concentration (Ca2+c) signals in response to a variety of agonists that stimulate mesangial cell contraction and ...proliferation. In the present study, we demonstrate that mesangial cells express both type I and III IP3Rs and that these receptors occupy different cellular locations.
Inflammation is associated with oxidative stress and altered cellular calcium homeostasis. Our earlier studies have shown that, increased phosphatidic acid (PA) formation occurred in enterocyte ...mitochondria when exposed to superoxide, divalent metal ions or polyamines resulting in altered lipid composition. Since aminosalicylates are the drug of choice for gut inflammation, we have tested the effect of aminosalicylates on PA formation by enterocyte mitochondria. When stimulated by superoxide, Ca
2+ or spermine, phosphatidylethanolamine (PE) degradation and PA formation occurred in enterocyte mitochondria which can be inhibited by aminosalicylates. The inhibition was 50–60% at 0.5-mM concentration and at 1- or 2-mM final concentration, complete inhibition was observed. Both 5-aminosalicylate (5-ASA) and 4-aminosalicylate (4-ASA) showed similar effects. The stimulation of PA formation by calcium or spermine was not due to increased generation of superoxide by mitochondria which was confirmed by measurement of superoxide production by the mitochondria. These studies suggest that in addition to other cellular effects, aminosalicylates may prevent the enterocyte mitochondrial damage by inhibition of PA formation and PE degradation and alteration of mitochondrial lipid composition.
Mitochondrial swelling and calcium cycling occurs during oxidative stress and can be prevented by cyclosporin A (CysA). Our earlier work has shown that enterocyte mitochondria contains a ...phospholipase D (PLD) which can be activated by superoxide or calcium. In this study, we have shown that enterocyte mitochondrial PLD activated by these agents can be inhibited by cyclosporin A. This was clearly shown by the absence of phosphatidic acid (PA) formation and phosphatidylethanolamine (PE) degradation. Since this PLD specifically utilizes PE as substrate, PLD activity was also assessed by ethanolamine formation which was inhibited by CysA. CysA also inhibited the cabbage PLD activity as judged by phosphatidylethanol formation. These results suggest that cyclosporin A is an inhibitor of PLD and this may be one of the mechanism by which CysA protects enterocyte mitochondria from oxidative stress.
Intestinal mitochondria have a phospholipase D (PLD) activity which was stimulated by polyamines and monoamines resulting in the formation of phosphatidic acid (PA) from endogenous phospholipids. ...When stimulated by polyamines, mitochondrial PLD utilized endogenous phosphatidylethanolamine (PE) as substrate whereas stimulated by monoamines, both PE and phosphatidylcholine (PC) were hydrolysed. Stimulation of PA formation by spermine was enhanced by the presence of calcium. Since polyamines are known to alter the calcium transport by mitochondria and PA is known to possess an ionophore effect, stimulation of PA formation in mitochondria by polyamines suggests that polyamine-induced alteration in calcium homeostasis might involve a PA related mechanism.
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