Effects of dietary omega 3 fatty acids on vascular contractility in preanoxic and postanoxic aortic rings.

BACKGROUND: Vasomotor reactivity may contribute to the pathophysiology of ischemic injury. The atherosclerotic vessel may be particularly susceptible to vasoconstriction because of the damaged endothelial layer with resultant loss of vasodilatory factors. While dietary omega 3 fatty acids have been proposed to protect against vascular occlusion, it is not clear to what extent this results from alterations in the function of platelets or from changes intrinsic to the blood vessel itself. METHODS AND RESULTS: The effects of dietary supplementation with fish oils on vascular contractility were examined in endothelialized and de-endothelialized aortic rings under pre- and postanoxic conditions. De-endothelialization was defined functionally by the loss of acetylcholine-induced vasodilation in norepinephrine-preconstricted aortic rings from rats fed normal rat chow. Three groups of rats were fed diets containing either 20% menhaden oil or 20% beef tallow, both supplemented with 3% corn oil or 23% corn oil for longer than 4 weeks. All animals received vitamin E. Under well-oxygenated conditions, de-endothelialized aortic rings from rats fed fish oil and corn oil contracted to similar extents with norepinephrine and vasopressin and less than rings from rats fed beef tallow. Endothelialized (intact) and de-endothelialized rings from rats fed fish oil relaxed more in response to acetylcholine than rings from rats fed beef tallow and corn oil. After anoxic exposure and reoxygenation, KCl-induced contraction of intact rings from rats fed fish oil and corn oil was similar and less than rings from rats fed beef tallow. Intact and de-endothelialized rings from rats fed fish oil relaxed more to acetylcholine than did rings from rats fed beef tallow and corn oil. CONCLUSIONS: Under preanoxic or postanoxic conditions, rings from rats fed fish oil and corn oil contracted less than rings from rats fed beef tallow. The relaxation response to acetylcholine, however, was greater in rings from rats fed fish oil than from rats fed either corn oil or beef tallow. These vascular effects of fish oil feeding may result in increased blood flow to ischemic and reperfused tissues in vivo.

Incorporation of marine lipids into mitochondrial membranes increases susceptibility to damage by calcium and reactive oxygen species: evidence for enhanced activation of phospholipase A2 in mitochondria enriched with n-3 fatty acids.

Experiments were designed to evaluate the susceptibility of mitochondrial membranes enriched with n-3 fatty acids to damage by Ca2+ and reactive oxygen species. Fatty acid content and respiratory function were assessed in renal cortical mitochondria isolated from fish-oil- and beef-tallow-fed rats. Dietary fish oils were readily incorporated into mitochondrial membranes. After exposure to Ca2+ and reactive oxygen species, mitochondria enriched in n-3 fatty acids, and using pyruvate and malate as substrates, had significantly greater changes in state 3 and uncoupled respirations, when compared with mitochondria from rats fed beef tallow. Mitochondrial site 1 (NADH coenzyme Q reductase) activity was reduced to 45 and 85% of control values in fish-oil- and beef-tallow-fed groups, respectively. Exposure to Ca2+ and reactive oxygen species enhance the release of polyunsaturated fatty acids enriched at the sn-2 position of phospholipids from mitochondria of fish-oil-fed rats when compared with similarly treated mitochondria of beef-tallow-fed rats. This release of fatty acids was partially inhibited by dibucaine, the phospholipase A2 inhibitor, which we have previously shown to protect mitochondria against damage associated with Ca2+ and reactive oxygen species. The results indicate that phospholipase A2 is activated in mitochondria exposed to Ca2+ and reactive oxygen species and is responsible, at least in part, for the impairment of respiratory function. Phospholipase A2 activity and mitochondrial damage are enhanced when mitochondrial membranes are enriched with n-3 fatty acids.

n-3 fatty acids increase postischemic blood flow but do not reduce myocardial necrosis.

The effects of a fish oil-supplemented diet on infarct size and regional myocardial blood flow were examined in a rat model of acute ischemia followed by reperfusion. Thirty-five rats were fed a diet containing 20% by weight: fish oil (FO), rich in n-3 polyunsaturated fatty acids; corn oil (CO), with predominantly n-6 polyunsaturated fatty acids; or beef tallow (BT), containing large amounts of saturated fatty acids. After 6-12 wk on the diet, animals underwent 40 min of left coronary artery occlusion followed by 2 h of reperfusion. Regional transmural myocardial blood flow was determined with radioactive microspheres at 30 min of occlusion and again 30 min after reperfusion. Infarct size was determined with triphenyltetrazolium chloride. Blood flow was virtually undetectable within the ischemic zone in all groups during occlusion. With reperfusion, however, ischemic zone absolute blood flow and relative flow (normalized to nonischemic zone flow) were significantly greater in the fish oil group [2.4 +/- 0.25 ml.min-1.g-1, 44 +/- 4% vs. 1.7 +/- 0.3, 29 +/- 5% for CO (P less than 0.05 vs. FO), and 1.4 +/- 0.3, 29 +/- 5% for BT (P less than 0.05 vs. FO)]. Despite differences in reperfusion blood flow, average percent transmural extent of infarction was nearly identical (68 +/- 4, 68 +/- 5, and 64 +/- 3%) and overall infarct size was similar (38 +/- 3, 36 +/- 4, and 29 +/- 3%) for FO, CO, and BT groups, respectively. In conclusion, dietary supplementation with fish oils increases postischemic blood flow but has no effect on extent of myocardial infarction in this ischemia-reperfusion model in rats.

Susceptibility of mitochondrial membranes to calcium and reactive oxygen species: implications for ischemic and toxic tissue damage.

In summary, with post ischemic and toxic injury, reactive oxygen species together with the Ca2+ activation of phospholipase A2, can produce injury to mitochondria. Reactive oxygen species damage mitochondria by enhancing membrane permeability and decreasing F1F0ATPase activity. With exposure of mitochondria to Ca2+ and reactive oxygen species, there is a synergistic injurious effect manifested by a marked increase in membrane permeability, a profound reduction in the electron transport chain respiratory function at site I, and a pronounced reduction in F1F0ATPase and adenine nucleotide translocase activities. Dibucaine, a PLA2 inhibitor, protected mitochondria exposed to Ca2+ and reactive oxygen species by preventing the electron transport defect, partially preserving F1F0ATPase activity, and restoring adenine nucleotide translocase activity to control levels. Mitochondrial function is important in generating ATP necessary for energy-dependent transport and restorative synthetic processes during the recovery state subsequent to ischemic or toxic injury. Understanding the cellular pathophysiology of ischemic and toxic mitochondrial damage will likely lead to the development of pharmacological approaches aimed at the enhancement of mitochondrial function and hence tissue survival and function after ischemic or toxic exposure.

Mechanisms of mitochondrial photosensitization by the cationic dye, N,N-bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC): preferential inactivation of complex I in the electron transport chain.

We investigated mechanisms of mitochondrial phototoxicity caused by the cationic cyanine dye N,N'-bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC), examining the role of the mitochondrial membrane potential on the dye uptake by carcinoma cells in vitro, and both the dark and photosensitizing effects of the dye on the function of isolated mouse liver mitochondria. When human bladder carcinoma cells (EJ) were pretreated with 2,4-dinitrophenol or nigericin, cellular uptake of EDKC decreased or increased, respectively, consistent with dye uptake that is dependent on membrane potentials. In isolated liver mitochondria, during NADH linked substrate oxidation (using glutamate plus malate or beta-hydroxybutyrate as substrates), low concentrations of the dye (0.25-0.5 microM) sensitized mitochondria to illumination with long wavelength light and inhibited both basal and ADP-stimulated respiration. Similar effects were observed during succinate oxidation, but only at higher concentrations of EDKC (greater than 5 microM) and at 10-fold greater light doses. NADH coenzyme Q reductase (Complex I) activity was inhibited by dye with or without light to an extent comparable to the inhibition of glutamate plus malate oxidation. Activity of cytochrome c oxidase, the terminal enzyme in the electron transport chain, was photosensitized with high dye doses (greater than 5 microM) and light, but the extent of inhibition was much less than the inhibition of respiration with succinate as substrate. ATP synthetase (F0F1 ATPase) activity was minimally affected by 4.0 microM EDKC with or without 24 J/cm2 light. We conclude that at low concentrations of dye, respiratory Complex I is a primary target for EDKC dark and light-induced toxicities. If Complex I is bypassed by using succinate as a respiratory substrate, the mitochondria can tolerate much higher dye concentrations and light doses.

Mechanism of calcium potentiation of oxygen free radical injury to renal mitochondria. A model for post-ischemic and toxic mitochondrial damage.

With a variety of forms of ischemic and toxic tissue injury, cellular accumulation of Ca2+ and generation of oxygen free radicals may have adverse effects upon cellular and, in particular, mitochondrial membranes. Damage to mitochondria, resulting in impaired ATP synthesis and diminished activity of cellular energy-dependent processes, could contribute to cell death. In order to model, in vitro, conditions present post-ischemia or during toxin exposure, the interactions between Ca2+ and oxygen free radicals on isolated renal mitochondria were characterized. The oxygen free radicals were generated by hypoxanthine and xanthine oxidase to simulate in vitro one of the sources of oxygen free radicals in the early post-ischemic period in vivo. With site I substrates, pyruvate and malate, Ca2+ pretreatment, followed by exposure to oxygen free radicals, resulted in an inhibition of electron transport chain function and complete uncoupling of oxidative phosphorylation. These effects were partially mitigated by dibucaine, a phospholipase A2 inhibitor. With the site II substrate, succinate, the electron transport chain defect was not manifest and respiration remained partially coupled. The electron transport chain defect produced by Ca2+ and oxygen free radicals was localized to NADH CoQ reductase. Calcium and oxygen free radicals reduced mitochondrial ATPase activity by 55% and adenine nucleotide translocase activity by 65%. By contrast oxygen free radicals alone reduced ATPase activity by 32% and had no deleterious effects on translocase activity. Dibucaine partially prevented the Ca2+-dependent reduction in ATPase activity and totally prevented the Ca2+-dependent translocase damage observed in the presence of oxygen free radicals. These findings indicate that calcium potentiates oxygen free radical injury to mitochondria. The Ca2+-induced potentiation of oxygen free radical injury likely is due in part to activation of phospholipase A2. This detrimental interaction associated with Ca2+ uptake by mitochondria and exposure of the mitochondria to oxygen free radicals may explain the enhanced cellular injury observed during post-ischemic reperfusion.

Nephrotoxicity of lysine and of a single dose of aminoglycoside in rats given lysine.

Hyperalimentation solutions have been shown to increase aminoglycoside nephrotoxicity in rats and rabbits. Lysine is a major constituent of hyperalimentation solutions and is known to inhibit tubular reabsorption of protein. To test the effects of lysine on renal function and structure and on aminoglycoside nephrotoxicity, three groups of rats were prepared. Groups 1 and 2 were infused with lysine (55 mumol/kg/min, 1.9 gm/kg total) for 4 hours. In group 2, gentamicin (60 mg/kg) was also infused during the third hour. In group 3, dextrose was given instead of lysine, and gentamicin was given as in group 2. In group 1 (lysine-saline solution), there was a decrease in glomerular filtration rate (GFR) and an increase in 125I-albumin clearance factored by GFR. In group 2 (lysine-gentamicin), the same effects were seen, but the reduction in GFR was significantly greater. Group 3 (dextrose-gentamicin) showed no change in GFR over the 4-hour period, but did show an increase in 125I-albumin clearance factored by GFR. Fractional excretion of sodium rose in group 2 but not in groups 1 and 3. A gradual mild (20%) and nonsignificant fall in renal blood flow followed the combined administration of lysine and gentamicin. In separate 20-hour studies, lysine (1.9 gm/kg intraperitoneally) or gentamicin or tobramycin (60 mg/kg subcutaneously) produced mild renal failure, but the combination of lysine and an aminoglycoside produced substantially greater renal failure. Serum creatinine in experimental groups was significantly correlated with medullary cast formation and tubular necrosis (p less than 0.001). Giant lysosomes with crystalloid inclusions in proximal tubular cells, individual cell necrosis in the pars recta, and casts in the thin limb of the loop of Henle were seen in rats given lysine. We conclude that lysine alone and single large doses of aminoglycosides alone are nephrotoxic, and when the two are combined, toxicity is additive. The nephrotoxicity of lysine may be related to direct tubular toxicity and to tubular obstruction.

Effects of verapamil in models of ischemic acute renal failure in the rat.

This study was designed to determine whether verapamil protects renal function in experimental ischemia in the rat and, if so, whether the protection is mediated by verapamil's vasodilatory action or by an effect on renal cells independent of vascular perfusion. Inulin clearance (CIn) was examined for 3 h subsequent to 40 min of unilateral intrarenal infusion of norepinephrine (0.75 microgram X kg-1 X min-1) and 3 and 48 h subsequent to 40 min of unilateral renal pedicle clamp. In norepinephrine-induced ischemia CIn fell to 0.8 +/- 0.4% of preischemic values in saline-treated kidneys and 0.5 +/- 0.3% in verapamil post-treated kidneys. By contrast, CIn fell only to 52.3 +/- 6.5% of preischemic values in verapamil-pretreated kidneys. Verapamil pretreatment significantly counteracted the intrarenal vasoconstriction produced by norepinephrine, sustaining renal blood flow during the norepinephrine infusion. In pedicle clamp-induced ischemia verapamil pre- and posttreatment had no beneficial effect on preservation of glomerular filtration rate, whereas mannitol pretreatment was beneficial. Parallel studies in the isolated perfused rat kidney confirmed the in vivo observations. In conclusion, verapamil exerts no protective effect on renal function at 3 or 48 h when ischemia is induced by renal pedicle clamp. Likewise, verapamil administration subsequent to norepinephrine-induced ischemia is ineffective in preserving renal function. Verapamil pretreatment in norepinephrine-induced ischemia preserves renal function probably by attenuating the vasoconstrictive ischemic insult due to norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphocitrate inhibits mitochondrial and cytosolic accumulation of calcium in kidney cells in vivo.

Synthetic 3-phosphocitrate, an extremely potent inhibitor of calcium phosphate crystallization as determined in a nonbiological physical-chemical assay, has many similarities to a mitochondrial factor that inhibits crystallization of nondiffracting amorphous calcium phosphate. In order to determine whether phosphocitrate can prevent uptake and crystallization of calcium phosphate in mitochondria in vivo, it was administered intraperitoneally to animals given large daily doses of calcium gluconate or parathyroid hormone, a regimen that causes massive accumulation and crystallization of calcium phosphate in the mitochondria and cytosol of renal tubule cells in vivo. Administration of phosphocitrate greatly reduced the net uptake of Ca2+ by the kidneys and prevented the appearance of apatite-like crystalline structures within the mitochondrial matrix and cytosol of renal tubule cells. Phosphocitrate, which is a poor chelator of Ca2+, did not reduce the hypercalcemia induced by either agent. These in vivo observations therefore indicate that phosphocitrate acts primarily at the cellular level to prevent the extensive accumulation of calcium phosphate in kidney cells by inhibiting the mitochondrial accumulation or crystallization of calcium phosphate.