The role of arachidonic acid in rat heart cell metabolism

Although it is known that arachidonic acid accumulates in the ischemic myocardium and that cardiac prostaglandin formation from the precursor arachidonic acid is altered during disease states, the role of arachidonic acid in the myocyte itself is not yet clear. Using isolated Ca-tolerant adult rat heart muscle cells, we were able to study cardiac metabolism of arachidonic acid without the effects induced by endothelial or other non-muscle tissue. Myocytes rapidly incorporate arachidonic acid as well as other fatty acids into their lipid pools, the predominant acceptor being the triacylglycerols at an extracellular fatty acid concentration of 20 microM. As exogenous arachidonic acid is decreased, the distribution pattern shifts to favor phospholipid esterification. Cardiocyte prostaglandin production from arachidonic acid added to the incubation medium was limited (less than 1% conversion of added arachidonic acid) and lipoxygenase pathway activity was not detected. Oxidation rates of arachidonic acid were 3-fold lower than for palmitic acid, indicating that it is of secondary importance in energy-yielding reactions. Our results suggest that arachidonic acid serves primarily as a structural component of myocardial membranes and that its release during ischemia would permit its use as a substrate for prostaglandin production by coronary vascular tissue.     

Role of triglycerides in endothelial cell arachidonic acid metabolism

Arachidonic acid was incorporated into triglycerides by cultured bovine endothelial cells in a time- and concentration-dependent manner. At 75 microM or higher, more arachidonic acid was incorporated into triglycerides than into phospholipids. The triglyceride content of the cells increased as much as 5.5-fold, cytoplasmic inclusions appeared, and arachidonic acid comprised 22% of the triglyceride fatty acids. Triglyceride turnover occurred during subsequent maintenance culture; there was a 60% decrease in the radioactive arachidonic acid contained in triglycerides and a 40% decrease in triglyceride content in 6 hr. Most of the radioactivity was released into the medium as free fatty acid. The turnover of arachidonic acid, but not oleic acid in cellular triglycerides, decreased when supplemental fatty acid was added to the maintenance medium. Incorporation and turnover of radioactive arachidonic acid in triglycerides also was observed in human skin fibroblasts, 3T3-L1 cells, and MDCK cells. Other fatty acids were incorporated into triglycerides by the endothelial cells; the amounts after a 16-hr incubation with 50 microM fatty acid were 20:3 greater than 20:4 greater than 18:1 greater than 18:2 greater than 22:6 greater than 16:0 greater than 20:5. These findings indicate that triglyceride formation and turnover can play a role in the fatty acid metabolism of endothelial cells and that arachidonic acid can be stored in endothelial cell triglycerides.     

Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein

The rat contains at least three homologous cytosolic proteins that bind long chain fatty acids, termed liver (L-), intestinal (I-), and heart (H-) fatty acid binding protein (FABP). I-FABP mRNA is confined to the gastrointestinal tract while L-FABP mRNA is abundantly represented in hepatocytes as well as enterocytes. We have isolated a rat heart FABP cDNA clone and determined the pattern of H-FABP mRNA accumulation in a wide variety of tissues harvested from late fetal, suckling, weaning, and adult rats. RNA blot hybridizations and primer extension analysis disclosed that the distribution of H-FABP mRNA in adult rat tissues is different from that of I- or L-FABP mRNA. H-FABP mRNA is most abundant in adult heart. This mRNA was also present in an adult slow twitch (type I) skeletal muscle (soleus, 63% of the concentration in heart), testes (28%), a fast twitch skeletal muscle (psoas, 17%), brain (10%), kidney (5%), and adrenal gland (5%). H-FABP mRNA was not detected in adult small intestine, colon, spleen, lung, or liver RNA. Distinct patterns of developmental change in H-FABP mRNA accumulation were documented in heart, placenta, brain, kidney, and testes. Myocardial H-FABP mRNA levels rise rapidly during the 48 h prior to and after birth, reaching peak levels by the early weaning period. The postnatal increase in myocardial H-FABP mRNA concentration and its relative distribution in adult fast and slow twitch skeletal muscle are consistent with its previously proposed function in facilitating mitochondrial beta-oxidation of fatty acids. However, the presence of H-FABP mRNA in brain, a tissue which does not normally significantly oxidize fatty acids in late postnatal life, suggests that H-FABP may play a wider role in fatty acid metabolism than previously realized. Mouse-hamster somatic cell hybrids were utilized to map H-FABP. Using stringencies which did not produce cross-hybridization between L-, I-, and H-FABP DNA sequences, we found at least three loci in the mouse genome, each located on different chromosomes, which reacted with our cloned H-FABP cDNA. None of these H-FABP-related loci were linked to the gene which specifies a highly homologous adipocyte-specific protein termed aP2 or to genes encoding two other members of this protein family, cellular retinol binding protein and cellular retinol binding protein II.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipid transfer between endothelial and smooth muscle cells in coculture

A coculture system was employed to study the interactions between endothelium and vascular smooth muscle cells in arachidonic acid metabolism. Bovine aortic endothelial cells grown on micropore filters impregnated with gelatin and coated with fibronectin are mounted on polystyrene chambers and suspended over confluent smooth muscle cultures. The endothelial basal laminae are oriented toward the underlying smooth muscle, and the two layers are separated by only 1 mm. Each cell layer was assayed individually: apical and basolateral fluid also was collected separately for assay. Fatty acids, including arachidonic acid, are readily transferred between the endothelial and smooth muscle cells in this system. Distribution of the incorporated fatty acids among the lipids of each cell is the same as when the fatty acid is added directly to the culture medium. Arachidonic acid released from endothelial cells is available as a substrate for prostaglandin production by smooth muscle. In addition, fatty acids released from the smooth muscle cells can pass through the endothelium and accumulate in the fluid bathing the endothelial apical surface. These fatty acid interchanges may be involved in cell-cell signaling within the vascular wall, the clearance of lipids from the vascular wall, or the redistribution of arachidonic acid and other polyunsaturated fatty acids between adjacent cell types. Furthermore, the findings suggest that prostaglandin production by smooth muscle cells can occur in response to stimuli that cause arachidonic acid release from endothelial cells.     

Fatty acids in striated muscle of the rat treated with toxic radicals

In the striated muscle of the growing and adult rat CCl4 poisoning increases all the saturated fatty acids and decreases the mono-unsaturated and arachidonic fatty acids. The supplementation with poly-unsaturated fatty acids increases, in rat's striated muscle, mono-unsaturated acids and decreases saturated, arachidonic, docosaexahenoic acids.     

Effect of arachidonic and other fatty acids on the intracellular calcium concentration and calcium signaling in peritoneal macrophages

Effects of arachidonic and other fatty acids on the intracellular Ca2+ concentration ([Ca2+]i) in rat peritoneal macrophages was investigated. It has been shown that cis-polyunsaturated arachidonic and linoleic induce a significant and dose-dependent increase in [Ca2+]i, which is due to depletion of thapsigargin-sensitive Ca2+ store and to stimulation of Ca2+ entry from the extracellular medium. Pharmacological characteristics of Ca2+ entry induced by arachidonic acid appeared to be similar to those of store-dependent Ca2+ entry activated by thapsigargin or cyclopiazonic acid; Ca2+ entry is attenuated by the same Ca2+ channel inhibitors, by tyrosine kinase inhibitor genistein and epoxygenase inhibitor proadifen. Cis-monounsaturated oleic and saturated myristic acids appeared to be less effective and induced only a slight increase in [Ca2+]i at much higher concentrations. Arachidonic and other fatty acids can also stimulate Ca(2+)-ATPase in the macrophage plasma membrane. The data are compatible with the important role played by arachidonic and other free fatty acids in the regulation of [Ca2+]i in peritoneal macrophages.     

Essential fatty acid uptake and esterification in primary culture of rat hepatocytes

Primary cultures of adult rat hepatocytes were used to compare the uptake and esterification of essential polyunsaturated fatty acids (18:2, 20:3 and 20:4 of the n-6 series) with those of palmitic and oleic acids. The uptake of unesterified fatty acids was linearly related to the free fatty acid/albumin molar ratio for 14 h and did not depend on the unbound free fatty acid level. Whatever the initial free fatty acid/albumin molar ratio, it dropped to 0.5 +/- 0.1 mM after 14 h, thus showing that hepatocytes have a high capacity for clearing free fatty acids from the medium at high free fatty acid/albumin molar ratios. The free fatty acid uptake become saturable when the free fatty acid and albumin concentrations were raised and the free fatty acid/albumin ratio remained constant. This strongly suggests that albumin-hepatocyte interaction mediates free fatty acid uptake. This uptake was identical whatever the fatty acid tested and did not depend on the relative amounts of fatty acids when they were added simultaneously. Triacylglycerol accumulation and synthesis, monitored by labelled fatty acids, were related to the free fatty acid/albumin molar ratio and exhibited no specificity for the series of fatty acids tested. Triacylglycerols were enriched in all the fatty acids tested by up to 60%, and fatty acid incorporation into diacylglycerols and triacylglycerols reflected the free fatty acid composition of the medium. By contrast, neither the level nor the synthesis of phospholipids varied with free fatty acid/albumin, but the rate of phospholipid turnover depended on the fatty acids tested. Accumulation of these acids was smaller in phospholipids than in triacylglycerols. When linoleic and arachidonic acids were added together, phospholipids (especially phosphatidylethanolamine and phosphatidylinositol) were more enriched in arachidonic acid than triacylglycerols. This might be due to the specificity for fatty acid of the enzymes involved in phospholipid metabolism.     

Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids

Although the three distinct forms of protein kinase C isolated from rat brain soluble fraction are structurally very similar, they respond differently to free unsaturated fatty acids such as arachidonic acid to exhibit their catalytic activity. Type I enzyme encoded by gamma-sequence, as predicted by cDNA clone analysis, responds to these fatty acids only slightly, whereas Type III enzyme determined by alpha-sequence is activated by free unsaturated fatty acids in the presence of Ca2+ in a comparable manner to phosphatidylserine plus diacylglycerol. Type II, a mixture of two enzymes encoded by beta I- and beta II-sequence, resulting from alternative splicing, shows properties in between those of Type I and Type III. Some of these forms of protein kinase C may function at a relatively later phase of cellular responses when large amounts of unsaturated fatty acids and Ca2+ are mobilized.     

The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.     

Content of arachidonic and eicosapentaenoic acids in polar lipids from Gracilaria (Gracilariales,Rhodophyta)

Fatty acid composition, especially the distribution of eicosapolyenoic acids in several species of Gracilaria, was analyzed in relation to their taxonomy. The species have been grouped into two types based on distribution of these polyenoic acids: Type 1, which contains palmitic, oleic and arachidonic acids as the major components, and Type II, which contains eicosapentaenoic acid in addition to Type I fatty acids. Octadecapolyenoic acids were detected only in trace amounts in each Type. A similar remarkable difference also was observed in the fatty acid composition of lipid classes. The major component of eicosapolyenoic acids in Type I was arachidonic acid in all lipid classes. In Type II, eicosapentaenoic acid was the major component in monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. Arachidonic and eicosapentaenoic acids were contained in large amounts in Type II phosphatidylcholine. Grouping of Gracilaria species into Type I and Type II is not entirely consistent with morphological and taxonomic features, but the difference in fatty acid composition is likely due to genetic rather than to environmental factors.     

The circadian clock within the cardiomyocyte is essential for responsiveness of the heart to fatty acids

Cells/organs must respond both rapidly and appropriately to increased fatty acid availability; failure to do so is associated with the development of skeletal muscle and hepatic insulin resistance, pancreatic beta-cell dysfunction, and myocardial contractile dysfunction. Here we tested the hypothesis that the intrinsic circadian clock within the cardiomyocytes of the heart allows rapid and appropriate adaptation of this organ to fatty acids by investigating the following: 1) whether circadian rhythms in fatty acid responsiveness persist in isolated adult rat cardiomyocytes, and 2) whether manipulation of the circadian clock within the heart, either through light/dark (L/D) cycle or genetic disruptions, impairs responsiveness of the heart to fasting in vivo. We report that both the intramyocellular circadian clock and diurnal variations in fatty acid responsiveness observed in the intact rat heart in vivo persist in adult rat cardiomyocytes. Reversal of the 12-h/12-h L/D cycle was associated with a re-entrainment of the circadian clock within the rat heart, which required 5-8 days for completion. Fasting rats resulted in the induction of fatty acid-responsive genes, an effect that was dramatically attenuated 2 days after L/D cycle reversal. Similarly, a targeted disruption of the circadian clock within the heart, through overexpression of a dominant negative CLOCK mutant, severely attenuated induction of myocardial fatty acid-responsive genes during fasting. These studies expose a causal relationship between the circadian clock within the cardiomyocyte with responsiveness of the heart to fatty acids and myocardial triglyceride metabolism.     

Cholesterol esters in developing rat brain: concentration and fatty acid composition

Abstract— The contents and the fatty acid composition of cholesterol esters were analysed in developing rat brain. The total content did not exceed 20 μg/brain throughout development. Elimination of serum by adequate perfusion was essential for accurate results. Two separate events appeared to affect the levels of cholesterol esters in developing rat brain, one probably reflecting general developmental changes and the other apparently related to myelination. On either a unit weight or a whole brain basis, the curves appeared to be a superimposition of the two events. There was an underlying developmental change, which was characterized on a unit weight basis by the highest level of cholesterol esters immediately after birth and a steady decline to the adult level by 30 days of age or which on the basis of whole brain was characterized by a steady increase throughout the development. A period of transient increase was superimposed on this underlying developmental change between the ages of 7 and 27 days and corresponded to the period of active myelination. The major fatty acids of rat brain cholesterol esters were palmitic, palmitoleic, oleic and arachidonic acids. Palmitic and palmitoleic acids decreased in proportion while oleic acid increased, as the animal matured. The fatty acid composition of serum cholesterol esters was distinctly different from that of brain cholesterol esters; those from serum contained much higher proportions of linoleic and arachidonic acids and much less palmitoleic and oleic acids.     

Regulation of the proliferation of primary rat hepatocytes by eicosanoids

DNA synthesis in primary adult rat hepatocyte cultures was promoted by epidermal growth factor (EGF), arachidonic acid, and prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). Growth promotion by EGF was blocked by 0.1 mM indomethacin and 1 mM aspirin, without affecting cell viability. If verapamil was present in the medium when EGF was added, the growth response was inhibited. Hepatocytes stimulated by EGF or arachidonic acid released PGE2 and PGF2 alpha into the culture medium. This was diminished if 0.1 mM indomethacin was also in the medium. The importance of autocrine regulation of hepatocyte growth by prostaglandins is discussed.     

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k₁ = 0.003 hr^?1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k₁ = 0.145 and 0.162 hr^?1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A₂ may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. © 1995 wiley-Liss, Inc.     

The influence of insulin on glucose and fatty acid metabolism in the isolated perfused rat hind quarter.

Glucose and fatty acid metabolism of resting skeletal muscle were studied by perfusion of the isolated rat hind leg with a hemoglobin-free medium. Tissue integrity was demonstrated by normal ATP, ADP and creatine phosphate levels, by a sufficient oxygen supply, and by a normal appearance of perfused muscle specimens under the electron microscope. The rates of glucose and fatty acid uptake, and of lactate, alanine, glycerol and fatty acid release were constant over a perfusion period of 60 min. Insulin (1 unit/l) caused a more than threefold increase in glucose uptake, a stimulation of lactate production, and a 20% increase in the muscular glycogen levels. Fatty acids and alanine release were significantly diminished by insulin, but glycerol release did not change. The uptake of oleate by the rat hind leg was dependent on the medium concentration in a range of 0.7-1.9mM oleate, and was stimulated by insulin. Glucose uptake was not influenced by oleate, whether sodium was present or not. When the leg was perfused with [1-14C]oleate, 75% of the incorporated fatty acids were found in muscle lipids, 10% were oxidized to CO2, and 5% were recovered in bone lipids. The absolute amount of oleate oxidation was not altered by insulin. In all experiments with and without glucose in the medium, 70-80% of the 14C label incorporated into muscle lipids was found in the triglyceride fraction. In the presence of glucose, insulin significantly increased the incorporation of [1-14C]oleate into muscle triglycerides, whereas no insulin effect, either on fatty acid uptake or on triglyceride formation, could be observed when glucose was omitted from the perfusate. The present results indicate that a "glucose-fatty acid cycle" as found in rat heart muscle does not operate in resting peripheral skeletal muscle tissue. They also demonstrate that the stimulating effect of insulin on muscular fatty acid uptake and triglyceride synthesis is dependent on glucose supply. This finding can be intrepreted as a stimulation of fatty acid esterification by sn-glycerol 3-phosphate derived from an increased glucose turnover, which is in turn due to insulin.     

Diacylglycerols interfere in normal phase HPLC analysis of lipoxygenase products of docosahexaenoic or arachidonic acids

A methodological problem with the normal phase high performance liquid chromatography (HPLC) of hydroxylated products of docosahexaenoic and arachidonic acids is described. Diacylglycerols present in lipid extracts of rat retina co-elute with monohydroxy derivatives of docosahexaenoic or arachidonic acid, when samples are applied to uPorosil columns and eluted with hexane/isopropanol/acetic acid. Analysis of fatty acid composition of diacylglycerols which were acetone-extracted from the incubation medium showed a profile similar to diacylglycerols extracted from the tissue by hexane/isopropanol, although acetone extraction resulted in extremely variable recovery of diacylglycerols. This co-elution of diacylglycerols with monohydroxy polyunsaturated fatty acids can lead to a significant error in estimation of lipoxygenation activity by conversion of radiolabeled precursors, because the incorporation of fatty acids into diacylglycerols is very active in many tissues. An alternative extraction method and reverse phase HPLC procedures that result in the complete separation of hydroxy fatty acids and diacylglycerols are described.     

Stimulation of polyunsaturated fatty acid oxidation in myocytes by regulating its cellular uptake. On the rate limiting step of polyunsaturated fatty acid oxidation in heart.

In order to investigate the regulation of polyunsaturated fatty acid oxidation in the heart, the effect of the phosphodiesterase inhibitor enoximone on the oxidation of [1-14C] arachidonic acid, and [1-14C] arachidonyl-CoA, were studied in adult rat myocytes, and isolated rat heart mitochondria. Enoximone stimulated arachidonate oxidation by 94%, at a concentration of 0.25 mM. The apparent Vmax value of arachidonate oxidation in the presence of enoximone (6.98 nmol/mg protein/30 min), was approximately 75% higher than the value observed with the control (4.0 nmol/mg protein/30 min) in isolated myocytes. Also, enoximone stimulated arachidonate uptake by 27% at a concentration of 0.25 mM. On the other hand, enoximone had no effect on the oxidation of [1-14C] arachidonyl-CoA in isolated rat heart mitochondria. These results suggest that the oxidation of polyunsaturated fatty acids in myocytes is regulated by the rate of uptake of these acids across sarcolemmal membranes.     

Hormone selective lipase activation in the isolated rabbit heart

The synthesis and release of PGs by the isolated perfused rabbit heart upon bradykinin stimulation results from lipase stimulation which liberates arachidonic acid for PG biosynthesis. The [¹⁴C]-labelled fatty acids, arachidonate, linoleate, and oleate, when infused into the heart preparation, were efficiently incorporated into the phospholipid pool in the heart, mostly in the 2-position of phosphatidylcholine. On the other hand, [¹⁴C]-palmitate was esterified into both the 1- and the 2-position. Bradykinin released bioassayable PG when injected into the rabbit hearts regardless of which fatty acid label was incorporated into the phospholipid pool. However, only [¹⁴C]-arachidonic acid (but not [¹⁴C]-linoleate, oleate or palmitate) was liberated from the variously labelled hearts upon hormone stimulation. This selective bradykinin effect on fatty acid release suggests that hormone stimulation either activates a specific lipase that distinguishes different fatty acids in the 2-position or activates lipase which is selectively compartmented with arachidonate-containing phospholipids. Ischemia, on the other hand, appeared to non-specifically stimulate tissue lipases, resulting in a non-selective release of oleic as well as arachidonic acid. A disproportionally large release of arachidonic acid was observed accompanying a relatively small PG (10:1 arachidonate: PG ratio) production during ischemia, as compared to bradykinin (3:1 ratio), suggesting distinct mechanisms for PG biosynthesis induced by bradykinin and ischemia.This work was supported by NIH grants: SCOR-HL-17646, HE-14397, HL-20787, and Experimental Pathology training grant (WH) 5 TO1 GM00897-16. Address correspondence to Dr. Philip Needleman, Department of Pharmacology, Washington University Medical School, St. Louis, Missouri 63110.