Production and physiological actions of anandamide in the vasculature of the rat kidney.

The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects.

Acylation of dog heart lysophosphatidylserine by transacylase activity.

Dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of phosphatidylcholine (PC) to lysophosphatidylserine (lysoPS) in the presence of coenzyme A (CoA) at pH optima of 4.5-5.0 and 7.5. Acyl transfer activity at acidic pH is about three times higher than at neutral pH. Transacylation of lysoPS by acyl transfer from PC with dog heart microsomes at neutral pH favors arachidonate over linoleate by a factor of 2.1, whereas free linoleic acid is favored by a factor of 3.7 over arachidonic acid for lysoPS acylation in the presence of acyl-CoA-generating cofactors. Considering the location and acyl composition of myocardial PS, it appears that both acyl transfer from PC and utilization of unesterified fatty acids may be involved in the acylation of lysoPS at its sn-2 position.

N-Acylation of ethanolamine phospholipids by acyl transfer does not involve hydrolysis.

N-Acylethanolamine phospholipids occur in infarcted but not in normal canine myocardium. Their synthesis is catalyzed by a membrane-bound, Ca2+-requiring N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of various phospholipids including phosphatidylethanolamine to the amino group of ethanolamine phospholipids. When dog heart mitochondria are incubated in media containing Ca2+ and H2(18)O, the resulting N-acylethanolamine phospholipids do not accumulate 18O in either the amide or 1-O-acyl groups. The results indicate that acyl transfer occurs without hydrolysis, most likely through an acyl-enzyme complex which may be covalently linked.

Coenzyme A-dependent and -independent acyl transfer between dog heart microsomal phospholipids.

We have recently shown that dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of exogenous phosphatidylcholine to lysophosphatidylethanolamine with strong preference for arachidonate over linoleate (Biochem. Biophys. Res. Commun. 129, 381-388 (1985)). We now report that the addition of 0.5 mM CoA enhances the acyl transfer activity 3-4-fold but reduces the selectivity for arachidonate. Acyl transfer in the absence of CoA exhibits a pH optimum of 7.5-8.5, whereas two pH optima (7.5 and 4.5) are observed in the presence of CoA with transfer activity at pH 4.5 exceeding that of pH 7.5 by 4-5-fold. The plasmalogen (alkenyl) analog of lysophosphatidylethanolamine is an equally effective acyl acceptor in the absence of CoA but less effective in its presence. The microsomal acyl-CoA/lysophosphatidylethanolamine acyltransferase does not favor arachidonate over linoleate. Therefore, transacylation from phosphatidylcholine may account for the high arachidonate content of dog heart microsomal phosphatidylethanolamine and its plasmalogen analog. In fact, acyl transfer from endogenous lipids to 1-[1'-14C]palmitoyl-2-lyso-sn-glycerophosphoethanolamine results in the generation of mostly (over 80%) tetraunsaturated phosphatidylethanolamine. This proportion is reduced by the addition of CoA and, even more, by CoA plus acyl-CoA-generating cofactors. We conclude that in dog heart microsomes, lysophosphatidylethanolamine can be acylated by different mechanisms, of which the CoA-independent transacylase exhibits the greatest acyl selectivity.

Metabolism of long-chain isoprenoid alcohols. Incorporation of phytol and dihydrophytol into the lipids of rat brain.

[U-14-C]Phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol) and [U-14-C]dihydrophytol (3,7,11,15-tetramethylhexadecanol) were administered intracerebrally to 18-day-old rats and incorporation of radioactivity into brain lipids was determined after 6 and 24 h. Radioactivity from [U-14-C]phytol was found in free phytenic (3,7,11,15-tetramethylhexadec-2-enoic), phytanic (3,7,11,15-tetramethylhexadecanoic) and pristanic (2,6,10,14-tetramethylpentadecanoic) acids, in phytanic and pristanic acid moieties of neutral and polar lipids, and in esters of phytol. In addition, evidence is presented for the utilization of phytol to form 1-O-phytenyl-2-acyl glycerophosphatides. Radioactivity from [U-14-C]dihydrophytol was found in free phytanic and pristanic acids, the corresponding acyl groups of neutral and polar lipids, esters of dihydrophytol and 1-O-phytanyl-2-acyl glycerophosphatides. Incorporation of either substrate into O-alkylglycerols was very low, and labeled branched-chain alk-1-enylglycerols could not be detected.

N-acylethanolamine phospholipid metabolism in normal and ischemic rat brain.

N-Acylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per mumol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca2+ for maximal (0.2 mM Ca2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1-3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.

Alterations of fatty acyl turnover in macrophage glycerolipids induced by stimulation. Evidence for enhanced recycling of arachidonic acid.

Glycerophospholipid biosynthesis by the de novo pathway was assessed in mouse peritoneal macrophages by pulse-labeling with [U-14C]glycerol. Phosphatidylcholine (PC), which amounts to about 35% of total cellular phospholipids, exhibited the highest rate of glycerol uptake, followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Remodeling of PC molecular species by deacylation/reacylation was established by determining the redistribution of glycerol label over 2 h after a 1 h pulse of [U-14C]glycerol and by determining incorporation of 18O from H2(18)O-containing media. These data suggest that stearic and arachidonic acid enter PC primarily by the remodeling pathway but that small amounts of highly unsaturated molecular species, including 1,2-diarachidonoyl PC, are rapidly synthesized de novo, and subsequently remodeled or degraded. Treatment of the cells with the ionophore A23187 resulted in the selective enhancement of arachidonate turnover in PC, PI and neutral lipid, as well as enhanced de novo PI synthesis. [U-14C]Glycerol labeling experiments suggest that arachidonic acid liberated by Ca(2+)-dependent phospholipase A2 activity is also reacylated in part through de novo glycerolipid biosynthesis, leading to the formation and remodeling of 1,2-diarachidonoyl PC and other highly polyunsaturated molecular species.

Diabetic heart and kidney exhibit increased resistance to lipid peroxidation.

Alloxan-diabetic rats and age-matched controls were killed after 6 weeks of diabetes; heart and kidneys were removed and assayed for thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides, lipid phosphorus, total fatty acid composition and glutathione. Tissue homogenates from a second group of diabetic and control rats were incubated in oxygen-saturated buffer with and without the free radical generating system Fe2+/ascorbate (0.1/1.0 mM) and were assayed for lipid peroxidation. Diabetic hearts contained markedly lower levels of TBARS and lipid hydroperoxides (40% and 18%, respectively) than control hearts, whereas differences in TBARS were less pronounced in kidneys (9%). Incubation of homogenates of both organs in the presence or absence of Fe2+/ascorbate for up to 2 h yielded significantly lower levels of TBARS and lipid hydroperoxides with diabetic tissue. Diabetic hearts and kidneys contained higher levels of glutathione (28% and 13% over controls) and both diabetic tissues showed much higher linoleate/arachidonate ratios than did the controls (9.86 vs. 2.56 for heart, 2.01 vs. 0.86 for kidney). We conclude that diabetic tissues develop enhanced defense systems against oxidative stress and we assume tha the lower levels of arachidonate contribute to their resistance to lipid peroxidation as well.

Agonist-stimulated glycerophospholipid acyl turnover in alveolar macrophages.

The inflammatory compounds beta-glucan, a particulate agonist, and tannin, a soluble agonist, are present in cotton dust and, when inhaled, cause massive arachidonic acid release from alveolar macrophages. Earlier work had shown that these agonists exhibit different effects on arachidonate liberation and release, and that only tannin inhibits the uptake and incorporation of exogenous arachidonic acid, suggesting inhibition of reacylation. Here we have used the time-dependent incorporation of 18O from H218O-containing media into glycerophospholipid acyl groups as an indicator of acyl turnover in resting and agonist-treated rabbit alveolar macrophages. Highest turnover rates were seen in phosphatidylinositol ( approximately 30% per hour) and in choline phospholipids (10-20% per hour). Both beta-glucan and tannin stimulated acyl turnover, especially arachidonic acid turnover, in these and other lipid classes by a factor of 2 or more. We conclude that neither agonist promotes arachidonic acid accumulation in and release from alveolar macrophages by inhibiting reacylation.

Substrate specificity in plasmalogen biosynthesis. Desaturation of 1-O-hexadecyloxyethyl-2-acylglycero-3-phosphoethanolamine in developing rat brain.

1-O-[1'-14C]Hexadecyloxyethyl rac-glycerol was administered to 18-day-old rats by intracerebral injection, and incorporation of radioactivity into the brain lipids was determined after 6, 24 and 48 h. Some of the substrate was catabolized by oxidative cleavage of either of the two ether bonds. Cleavage in the hexadecyloxyethyl moiety yielded labeled palmitic acid, whereas oxidative cleaveage of the glycol glycerol ether bond produced O-hexadecyl glycolic acid. The substrate was also incorporated as such into both ethanolamine and choline phospholipids. Evidence is presented for the desaturation by rat brain of 1-O-hexadecyloxyethyl-2-acyl-sn-glycero-3-phosphoethanolamine to the plasmalogen analogue, while the corresponding choline phospholipid was not desaturated.

The role of cardiolipin as an acyl donor in dog heart N-acylethanolamine phospholipid biosynthesis.

N-Acylethanolamine phospholipids were produced from endogenous substrates with dog heart mitochondrial and microsomal preparations. With mitochondria the N-acyl group contained 13.8% linoleate, with microsomes only 3.6%. Cardiolipin comprised 18.5% of mitochondrial and 3.3% of microsomal lipid P and contained 93.7 and 72.4% linoleic acid, respectively. Incubation of dog heart subcellular fractions with [1-14C]linoleoyl cardiolipin in the presence of Ca2+ resulted in the formation of N-acylethanolamine phospholipids labeled primarily in the N-acyl and 1-O-acyl moieties. The data indicate that cardiolipin is the major source of linoleic acid used in the N-acylation of ethanolamine phospholipids by transacylase activity.

N-Acylation of dog heart ethanolamine phospholipids by transacylase activity.

Radioactive N-acylethanolamine phospholipids were produced in dog heart homogenates incubated with acyl-labeled phosphatidylcholine in the presence of 5 mM Ca2+ and Triton X-100. 70-80% of the label in the N-acylethanolamine phospholipids was recovered in the N-acyl groups and most of the remainder was in the 1-O-acyl groups. Incubations with 1,2-dipalmitoylPC and 1-palmitoyl-2-linoleoylPC labeled in either the 1-O-acyl or 2-O-acyl moiety showed the predominant utilization of the acyl groups at the sn-1 position, indicating transacylation by phospholipase A1 (or lysophospholipase) activity. It is suggested that intramolecular transacylation from 1-O-acyl to N-acyl groups of phosphatidylethanolamine also occurred to some extent, thus providing a free primary hydroxy group as an additional acyl acceptor for the transacylation reaction.

N-Acylation of ethanolamine phospholipids in canine myocardium.

N-Acylethanolamine phospholipids, which are found in infarcted but not in normal canine myocardium, were produced by preparations of normal myocardial tissue incubated in the presence of millimolar concentrations of Ca2+. Biosynthetic activity from endogenous substrates was associated with both microsomal and mitochondrial fractions and exhibited an alkaline pH optimum. The time course of N-acylethanolamine phospholipid synthesis and degradation was followed after pulse labeling of myocardial ethanolamine phospholipids with [1,2-14C]ethanolamine. Enzymic N-acylation of both phosphatidylethanolamine and lysophosphatidylethanolamine was demonstrated by incubating these substrates with homogenates of myocardial tissue. Neither free fatty acids nor acyl-CoA derivatives served as acyl donor but some of the constituent fatty acids of exogenous phosphatidylethanolamine were recovered among the amide-linked fatty acids of the N-acylethanolamine phospholipids. N-Acylation may thus occur by the transfer of O-acyl groups catalyzed by a Ca2+ -dependent transacylase. Since N-acylethanolamine phospholipids are precursors of the biologically active N-acylethanolamines, their biosynthesis may constitute an important injury-induced metabolic event aimed at the protection of ischemic myocardial tissue.

Structure and metabolism of phospholipids in bovine epididymal spermatozoa.

Lipid analysis of bovine epididymal spermatozoa showed relatively large amounts of alkylacyl- and alk-1-enylacylglycerols in their choline and ethanol-amine phospholipids and alkylacylglycerol as the major constituent of a glycolipid tentatively identified as a monogalactosyl sulfate. The ether lipids exhibited remarkably simple molecular structures, i.e., the phospholipids had only 16:0 as alkyl and alk-1-enyl groups and their constituent fatty acids were almost exclusively 22:5 (n-6) and 22:6 (n-3). The glycolipid had mainly 16:0 as both alkyl and acyl moieties. In contrast, the diacyl choline and ethanolamine phosphoglycerides exhibited a much more complex fatty acid composition. 1,2-Diacylglycerols were the major nonpolar glycerolipid class and their acyl groups consisted almost exclusively of 14:0, 16:0 and 18:0. Labeled glycerol and dihydroxyacetone added to the incubation medium were readily incorporated into sperm lipids under both aerobic and anaerobic conditions. In each case, diacylcholine phosphoglycerides, diacylglycerols and phosphatidic acid were the major labeled lipids. Distribution of label among the molecular species of diacylglycerols and choline phosphoglycerides resembled somewhat their natural abundance. No radioactivity was found in alkylacyl or alk-1-enylacyl glycerolipids. The ether lipids may provide stable structural components of sperm membrane while the diacyl analogs undergo degradation and resynthesis.

On the biosynthesis and metabolism of N-acylethanolamine phospholipids in infarcted dog heart.

Minced tissues from both infarcted and apparently normal areas of canine myocardium 6 h after ligation of the left descending branch of the coronary artery were incubated with [1,2-(14)C]ethanolamine. In each case substantial amounts of radioactivity were incorporated into both phosphatidylethanolamine and N-acylphosphatidylethanolamine. Incubation of homogenates from the same tissues with N-[1-(14)C]palmitoyl phosphatidylethanolamine yielded labeled N-palmitoyl ethanolamine. The data support the concept that N-acylethanolamines, pharmacologically active compounds which accumulate in infarcted myocardium, are produced by N-acylation of ethanolamine phospholipids followed by phosphodiesterase action.

Occurrence of N-acylethanolamine phospholipids in fish brain and spinal cord.

N-Acylethanolamine phospholipids were identified in the central nervous system of the fresh water fish, pike (Esox lucius) and carp (Cyprinus carpio), at levels ranging from 0.1 to 0.9% of total phospholipid. The N-acylethanolamine phospholipids of carp brain were isolated and characterized by chemical, biochemical and spectroscopic methods. Two major species, 1,2-diacyl-sn-glycero-3-phospho(N-acyl)ethanolamines (approx. 30%) and 1-O-(1'-alkenyl)-2-acyl-sn-glycero-3-phospho(N-acyl)ethanolamines (approx. 70%) were identified. The N-acyl groups of each species consisted primarily of 16:0 (approx. 60%) but also contained 16:1, 18:0 and 18:1 (approx. 10% each) and a number of trace constituents. The N-acylethanolamine phospholipids had O-acyl and O-alkenyl group compositions similar but not identical to those of the ethanolamine phospholipids of the same tissue. N-Acylethanolamine phospholipids were present in all subcellular fractions of carp brain, except mitochondria.

Ether lipid metabolism. Incorporation of O-hexadecyl ethanediol into rat brain lipids.

1-O-[1'-14C]Hexadecyl ethanediol was administered intracerebrally to myelinating rat brain, and incorporation of radioactivity into brain lipids was followed over a 48-h period: (1) O-Hexadecyl ethanediol was metabolized primarily through oxidative ether bond cleavage, and much of the label was recovered in phospholipid acyl groups. (2) Substantial amounts of radioactivity were also found in choline and ethanolamine phospholipids having an O-hexadecyloxyethyl glycerol backbone. This means that alkyl ethanediol was used in glycerol ether biosynthesis as are long-chain primary alcohols. (3) Acidic hydrolysis of the ethanolamine glycerophosphatide fraction yielded also labeled hexadecanol which may indicate desaturation of 1-O-hexadecyloxyethyl 2-acyl glycerophosphoryl ethanolamine to the plasmalogen analogue. (4) Small amounts of the substrate were oxidized to O-hexadecyl glycolic acid and incorporated into the phospholipids. The substrate did not serve as precursor of O-hexadecyl ethanediol phosphorylcholine or phosphorylethanolamine in the brain.

Properties of canine myocardial phosphatidylethanolamine N-acyltransferase.

Dog heart contains a membrane bound N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of membrane phospholipids to the amino group of ethanolamine phospholipids in the presence of millimolar Ca2+ concentrations. Using crude membrane preparations, we found this N-acyltransferase activity to be heat sensitive and inhibited by sulfhydryl reagents. Pretreatment of a membrane fraction with trypsin reduced N-acyltransferase activity to 60% while pretreatment with trypsin and Triton X-100 together reduced it to 30% of the control value. At pH 8.0 both Sr2+ and Mn2+ could fully substitute for Ca2+ with respect to optimum ion concentration and molecular species of the product formed in dog heart membranes from endogenous substrates. Ba2+ was equally effective in achieving N-acylation of ethanolamine phospholipids while other divalent cations were less effective or ineffective. The reaction exhibited a pH optimum of 8.5 to 9.0 with both Ca2+ and Sr2+ while Mn2+ precipitated above pH 8.0 resulting in decreased N-acylation activity. Both phosphatidylcholine and 1-acyl lysophosphatidylcholine could serve as acyl donors. Triton X-100 at a concentration of 0.1% stimulated acyl transfer from exogenous phosphatidylcholine but inhibited acyl transfer from lysophosphatidylcholine.

Activation of PAF receptors results in enhanced synthesis of 2-arachidonoylglycerol (2-AG) in immune cells.

The endocannabinoid signaling system is believed to play a down-regulatory role in the control of cell functions. However, little is known about the factors activating endocannabinoid synthesis and which of two known endocannabinoids, 2-arachidonoylglycerol (2-AG) or N-arachidonoylethanolamine (20:4n-6 NAE, anandamide), is of physiological importance. We approached these questions by studying a possible link between cell activation with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF) and the generation of 2-AG and anandamide in human platelets and mouse P388D1 macrophages. Human platelets responded to stimulation with the production of various 1- and 2-monoacylglycerols, including 2-AG, whereas stimulation of P388D1 macrophages induced the rapid and selective generation of 2-AG, which was immediately released into the medium. The effect of PAF was receptor mediated, as PAF receptor antagonist BN52021 blocked the effect. The treatment did not change the content of anandamide in either macrophages or platelet-rich plasma. The inhibitors of PI- and PC-specific phospholipases C (U73122 and D609) as well as PI3-kinase inhibitor (wortmannin) attenuated PAF-induced 2-AG production in macrophages. These data suggest a direct role for the endocannabinoid system in controlling immune cell activation status and indicate that 2-AG rather than anandamide is the endocannabinoid rapidly produced in response to proinflammatory stimulation of immune cells.

Interstitial hyperosmolarity may cause axis cylinder shrinkage in streptozotocin diabetic nerve.

Maximal conduction velocity values of nerves of diabetic rats 20 weeks after streptozotocin intoxication were found to be intermediate between those of onset-control and those of end-control groups. The abnormality of conduction velocity of the streptozotocin group might therefore be attributed to a failure of maturation. Detailed electron microscopic morphometry of myelinated fibers (MFs) indicates that more than lack of maturation is involved. Whereas the number of lamellae and the perimeter of axis cylinders of myelinated fibers of the three study groups suggested that growth continues, cross-sectional area of the axis cylinders of the streptozotocin group was smaller than those of either control group. Scored evaluation of fiber shape and the measured index of circularity, which related perimeter and transverse axis cylinder area, also indicated that a selective shrinkage of axis cylinders had occurred. This selective alteration in size and shape of axis cylinders is identical to that described after hyperosmolar fixation. Compared with that of controls, the serum of streptozotocin rats is hyperosmolar. It would seem reasonable to attribute the axis cylinder changes to shrinkage. Whether an additional maturational effect is operative as well cannot be resolved from our data.