thapsigargin and 5-6-epoxy-8-11-14-eicosatrienoic-acid

A major pathway for Ca(2+) entry in non-excitable cells is activated following depletion of intracellular Ca(2+) stores. A de novo conformational coupling between elements in the plasma membrane (PM) and Ca(2+) stores has been proposed as the most likely mechanism to activate this capacitative Ca(2+) entry (CCE) in several cell types, including platelets. Here we report that a cytochrome P450 metabolite, 5,6-EET, might be a component of the de novo conformational coupling in human platelets. In these cells, 5,6-EET induces divalent cation entry without having any detectable effect on Ca(2+) store depletion. 5,6-EET-induced Ca(2+) entry was sensitive to the CCE blockers 2-APB, lanthanum, SKF-96365 and nickel and impaired by incubation with anti-hTRPC1 antibody. Ca(2+) entry stimulated by low concentrations of thapsigargin, which selectively depletes the dense tubular system and induces EET production, was impaired by the cytochrome P450 inhibitor 17-ODYA, which has no effect on CCE mediated by depletion of the acidic stores using 2,5-di-(tert-butyl)-1,4-hydroquinone. We have found that 5,6-EET-induced Ca(2+) entry requires basal levels of H(2)O(2), which might maintain a redox state favourable for this event. Finally, our results indicate that 5,6-EET induces the activation of tyrosine kinase proteins and the reorganization of the actin cytoskeleton, which might provide a support for the transport of portions of the Ca(2+) store towards the PM to facilitate de novo coupling between IP(3)R type II and hTRPC1 detected by coimmunoprecipitation. We propose that the involvement of 5,6-EET in TG-induced coupling between IP(3)R type II and hTRPC1 and subsequently CCE is compatible with the de novo conformational coupling in human platelets.

Topics: 8,11,14-Eicosatrienoic Acid; Actins; Antibodies; Blood Platelets; Boron Compounds; Calcium; Calcium Channels; Cell Membrane; Cell Membrane Permeability; Cytochalasin D; Cytochrome P-450 Enzyme System; Cytoskeleton; Enzyme Activation; Humans; Hydrogen Peroxide; Imidazoles; Inositol 1,4,5-Trisphosphate Receptors; Lanthanum; Manganese; Nickel; Protein-Tyrosine Kinases; Receptors, Cytoplasmic and Nuclear; Thapsigargin; TRPC Cation Channels

We present evidence in astrocytes that 5,6-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid, may be a component of calcium influx factor, the elusive link between release of Ca2+ from intracellular stores and capacitative Ca2+ influx. Capacitative influx of extracellular Ca2+ was inhibited by blockade of the two critical steps in epoxyeicosatrienoic acid synthesis: release of arachidonic acid from phospholipid stores by cytosolic phospholipase A2 and cytochrome P450 metabolism of arachidonic acid. AAOCF3, which inhibits cytosolic phospholipase A2, blocked thapsigargin-stimulated release of arachidonic acid as well as thapsigargin-stimulated elevation of intracellular free calcium. Inhibition of P450 arachidonic acid metabolism with SKF525A, econazole, or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide, a substrate inhibitor of P450 arachidonic acid metabolism, also blocked thapsigargin-stimulated Ca2+ influx. Nano- to picomolar 5, 6-epoxyeicosatrienoic acid induced [Ca2+]i elevation consistent with capacitative Ca2+ influx. We have previously shown that 5, 6-epoxyeicosatrienoic acid is synthesized and released by astrocytes. When 5,6-epoxyeicosatrienoic acid was applied to the rat brain surface, it induced vasodilation, suggesting that calcium influx factor may also serve a paracrine function. In summary, our results suggest that 5,6-epoxyeicosatrienoic acid may be a component of calcium influx factor and may participate in regulation of cerebral vascular tone.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Astrocytes; Calcium; Cytochrome P-450 Enzyme Inhibitors; Enzyme Inhibitors; Ion Transport; Phospholipases A; Phospholipases A2; Rats; Signal Transduction; Thapsigargin

Products of cytochrome P-450 enzymes may play a role in capacitative Ca2+ entry in endothelial cells, which can promote a rise in vascular permeability. Thapsigargin (150 nM) stimulated capacitative Ca2+ entry and increased the capillary filtration coefficient (Kf,c) in isolated normal canine lung lobes. Pretreatment of the lobes with cytochrome P-450 inhibitors clotrimazole (10 microM) or 17-octadecynoic acid (5 microM) abolished the thapsigargin-induced increases in Kf,c. Because clotrimazole also blocks Ca2+-activated K+ channels, the K+-channel blocker tetraethylammonium (10 mM) was used to ensure that permeability was not influenced by this mechanism. Tetraethylammonium did not affect thapsigargin-induced permeability. The effects of the cytochrome P-450 arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (EET) were also investigated in lobes taken from control dogs and dogs with pacing-induced heart failure (paced at 245 beats/min for 4 wk). 5,6-EET (10 microM) significantly increased Kf,c in lobes from the control but not from the paced animals. We conclude that cytochrome P-450 metabolites are involved in mediating microvascular permeability in normal canine lungs, but an absence of 5,6-EET after heart failure does not explain the resistance of lungs from these animals to permeability changes.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Capillary Permeability; Clotrimazole; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dogs; Endothelium, Vascular; Fatty Acids, Unsaturated; In Vitro Techniques; Lung; Microcirculation; Potassium Channels; Pulmonary Circulation; Tetraethylammonium; Thapsigargin