Intestinal absorption of ester and ether glycerophospholipids in guinea pig. Role of a phospholipase A2 from brush border membrane

In vivo intestinal perfusion was used to follow the absorption of three different choline glycerophospholipids (CGP) in guinea pig. These included 1-[³H]palmitoyl-2-acyl-sn-glycero-3-phosphocholine (diacyl-GPC), 1-[³H]-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) and 1,2-di-O-hexadecyl-sn-glycero-3-phospho-[³H]-choline (dialkyl-GPC). About 80% of diacyl-GPC was absorbed within 4 hr, compared to 60% of alkylacyl-GPC and 30% of dialkyl-GPC. The radioactivity disappearing from the perfusion fluid was recovered in intestinal lipids, mostly triacylglycerol, free fatty acid and CGP from diacyl-GPC, CGP from alkylacyl-GPC and dialkyl-GPC. These results indicated that the nonhydrolyzable substrate dialkyl-GPC was much less absorbed, whereas diacyl-GPC, which released over 80% of [³H]palmitic acid in the perfusion fluid, displayed the highest absorption rate. The intermediate picture observed for alkylacyl-GPC suggested the possible involvement of a phospholipase A₂, which was detected in the entire intestinal tract. This enzyme was further found to concentrate in villus cells, where it is localized in the brush border membrane, as shown using two different subcellular fractionation procedures. These data suggest a possible role of this new enzyme in the digestion of alimentary phospholipids.     

Regulation of the metabolism of linoleic acid to arachidonic acid in rat hepatocytes

When 5×10⁶ hepatocytes were incubated for 40 min with from 0.15 to 0.60 mM [1-¹⁴C]linoleic acid, [1-¹⁴C]6,9,12-octadecatrienoic acid, or [1-¹⁴C]8,11,14-eicosatrienoic acid, there was a concentration-dependent acylation of radioactive metabolites into both triglycerides and phospholipids. When the concentration of either [1-¹⁴C]linoleic acid or [1-¹⁴C]8,11,14-eicosatrienoic acid exceeded 0.3 mM, there was no further increase in the metabolism of either fatty acid to other (n−6) metabolites. When the concentration of [1-¹⁴C]6,9,12-octadecatrienoic acid exceeded 0.15 mM, there was an apparent substrate-induced inhibition in its metabolism to 8,11,14-eicosatrienoic acid. With all three substrates (0.3 mM), there was time-dependent metabolism to other (n−6) acids. Cells then were incubated simultaneously with 0.3 mM [1-¹⁴C]linoleic acid along with 0.15 to 0.45 mM 6,9,12-octadecatrienoic acid or 8,11,14-eicosatrienoic acid. These exogenous nonradioactive (n−6) acids suppressed but did not abolish the conversion of [1-¹⁴C]linoleate to radioactive arachidonate. These findings suggest that some linoleate is converted to arachidonate without intracellular mixing of 6,8,12-octadecatrienoic or 8,11,14-eicosatrienoic acids. This hypothesis is supported by the finding that exogenous linoleate did not markedly affect the metabolism of [1-¹⁴C]6,9,12-octadecatrienoic or [1-¹⁴C]8,11,14-eicosatrienoic acid by microsomal chain elongating or desaturating enzymes.     

Effect of phagocytosis and lonophores on release and metabolism of arachidonic acid from human neutrophils

Challenge of human neutrophils prelabeled with [³H]arachidonate and [¹⁴C] palmitate or [¹⁴C]-stearate with opsonized zymosan or the Ca²⁺ ionophores A23187 or Ionomycin caused the release of [³H], but not [¹⁴C], fatty acid. With the ionophores, but not zymosan, considerable conversion of the [³H] arachidonate to hydroxyeicosatetraenoates occurred. Although various isomers were recovered, the 5-hydroxyeicosatetraenoate appeared to be the major product. In these experiments, no [¹⁴C] products were detected such as lysophospholipid, diglyceride or monoglyceride. Although no definitive statement can be made about the mechanism of release of arachidonate, our data are most easily interpreted as the result of the action of a phospholipase A₂.     

The questionable role of a microsomal °8 acyl-CoA-dependent desaturase in the biosynthesis of polyunsaturated fatty acids

Several experimental approaches were used to determine whether rat liver and testes express an acyl-CoA-dependent δ8 desaturase. When [1-¹⁴C]5, 11, 14-eicosatrienoic acid was injected via the tail vein, or directly into testes, it was incorporated into liver and testes phospholipids, but it was not metabolized to other labeled fatty acids. When [1-¹⁴C]11, 14-eicosadienoic acid was injected, via the tail vein or directly into testes, or incubated with microsomes from both tissues, it was only metabolized to 5,11, 14-eicosatrienoic acid. When ethyl 5,5,11,11,14,14-d₆-5,11,14-eicosatrienoate was fed to rats maintained on a diet devoid of fat, it primarily replaced esteri-fied 5,8,11-eicosatrienoic acid, but not arachidonic acid. No labeled linoleate or arachidonate were detected. Dietary ethyl linoleate and ethyl 19,19,20,20-d₄-1,2-¹³C-11,14-eicosadienoate were about equally effective as precursors of esterified arachidonate. The doubly labeled 11,14-eicosadienoate was metabolized primarily by conversion to 17,17,18,18-d₄-9,12-ocatdeca-dienoic acid, followed by its conversion to yield esterified arachidonate, with a mass four units greater than endogenous arachidonate. In addition, the doubly labeled substrate gave rise to a small amount of arachidonate, six mass units greater than endogenous arachidonate. No evidence was obtained, with the radiolabeled substrates, for the presence of a δ8 desaturase. However, the presence of an ion, six mass units greater than endogenous arachidonate when doubly labeled 11, 14-eicosa-dienoate was fed, suggests that a small amount of the substrate may have been metabolized by the sequential use of δ8 and δ5 desaturases.     

The effects oftrans fatty acids on fatty acyl Δ5 desaturation by human skin fibroblasts

The effectiveness of different fatty acids as inhibitors of fatty acyl Δ5 desaturation activity in human skin fibroblasts has been investigated. When incubated with 2.25 μM [¹⁴C] eicosatrienoate (20∶3ω6) in otherwise lipid-free medium, these cells rapidly incorporate the radiolabeled fatty acid into cellular glycerolipids and desaturate it to produce both [¹⁴C] arachidonate and [¹⁴C] docosatetraenoate. The Δ5 desaturation activity can be enhanced by prior growth of the cells without serum lipids. Elaidate (9t–18∶1) is a potent inhibitor of Δ5 desaturation whiletrans-vaccenate (11t–18∶1) is virtually without effect. Oleate and linoleate are only mildly inhibitory. Linoelaidate (9t, 12t–18∶2) is more inhibitory than linoleate but significantly less effective than elaidate. The effects of elaidate can be readily overcome by increasing the concentration of exogenous eicosatrienoate. Studies with a variety oftrans monounsaturates of differing chain lengths indicate that the ω9trans fatty acids are potent inhibitors of Δ5 desaturation, while ω7trans fatty acids are relatively ineffective. Intact human fibroblasts could thus be important in characterizing novel fatty acids as selective inhibitors of arachidonate synthesis in vivo.     

Cardiac and renal lipases and prostaglandin biosynthesis

The tissue phospholipids of isolated Krebs perfused rabbit hearts and kidneys can be efficiently labeled with [¹⁴C]arachidonic acid. Subsequent stimulation of the prelabeled organ with hormones or ischemia results in release of [¹⁴C] prostaglandins (PG). There is a highly efficient acylation mechanism existing in these perfused organs. Thus, tissue lipase activity can only be quantitiatively assessed by measuring the [¹⁴C]arachidonic acid in the venous effluent in the presence of albumin infusion which “traps” the released fatty acid. Two types of tissue lipases appear to exist. One is a highly specific phospholipase which is hormone sensitive, thus selectively liberates [¹⁴C]arachidonate (but not [¹⁴C]oleate, linoleate, and palmitate) from tissue lipids. The other nonspecifically responds to noxious stimuli such as ischemia and releases all fatty acids (e.g., including arachidonic and oleic acid) from tissue lipids. The hormone sensitive lipase appears to be predominantly localized in the vascular tissue (in hearts and kidney cortex), while nonspecific lipase activity is widely distributed in myocardial cells and renal tubules. Therefore, hormone stimulation elicits tightly coupled PG synthesis in the vasculature which possess both phospholipase and the cyclooxygenase while ischemia induced deacylation in the entire, but the majority of the arachidonic acid released is not converted to PG due to a relative lack of cyclooxygenase activity in myocytes and renal tubules.     

Transfer of arachidonate from phosphatidylcholine to phosphatidylethanolamine and triacylglycerol in guinea pig alveolar macrophages

Guinea pig alveolar macrophages were labeled by incubation with either arachidonate or linoleate. Arachidonate labeled phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG) equally well, with each lipid containing about 30% of total cellular radioactivity. In comparison to arachidonate, linoleate was recovered significantly less in PE (7%) and more in TG (47%). To investigate whether redistributions of acyl chains among lipid classes took place, the macrophages were incubated with 1-acyl-2-[1-¹⁴C]arachidonoyl PC or 1-acyl-2-[1-¹⁴C]linoleoyl PC. After harvesting, the cells incubated with 1-acyl-2-[1-¹⁴C]linoleoyl PC contained 86% of the recovered cellular radioactivity in PC, with only small amounts of label being transferred to PE and TG (3 and 6%, respectively). More extensive redistributions were observed with arachidonate-labeled PC. In this case, only 60% of cellular radioactivity was still associated with PC, while 22 and 12%, respectively, had been transferred to PE and TG. Arachidonate transfer from PC to PE was unaffected by an excess of free arachidonate which inhibited this transfer to TG for over 90%, indicating that different mechanisms or arachidonoyl CoA pools were involved in the transfer of arachidonate from PC to PE and TG. Cells prelabeled with 1-acyl-2-[1-¹⁴C]arachidonoyl PC released¹⁴C-label into the medium upon further incubation. This release was slightly stimulated by zymosan and threefold higher in the presence of the Ca²⁺-ionophore A₂₃₁₈₇. Labeling of macrophages with intact phospholipid molecules appears to be a suitable method for studying acyl chain redistribution and release reactions.     

The effects of hypophysectomy and testosterone treatment on the composition and metabolism of testicular lipids

The effects of hypophysectomy and of testosterone administration on lipid composition and metabolism of rat testicular tissue have been investigated. Increased concentrations of triacylglycerols and cholesterol were observed in testes of hypophysectomized compared to control (non-hypophysectomized) rats on the eighth day posthypophysectomy. Administration of testosterone maintained the concentrations of these lipids at about normal levels. The concentration of phospholipids was not affected by the hypophysectomy. Incorporation of¹⁴C from 1-[¹⁴C] linoleate into testicular lipids was determined 24 hours after intratesticular injection. In hypophysectomized compared to control rats there was more¹⁴C in C 16∶0, C 20∶2 and C 20∶3 and less¹⁴C in C 20∶4 and C 22∶4 of both phospholipids and triacylglycerols. After intratesticular injection of 1-[¹⁴C] eicosatrienoate there was more¹⁴C in C 16∶0 and C 20∶3 and less¹⁴C in C 20∶4 and C 22∶4 of both phospholipids and triacylglycerols of hypophysectomized compared to control rats. Intratesticular injection of 1-[¹⁴C]-arachidonate resulted in less¹⁴C incorporation in C 22∶4 in testes of hypophysectomized than in those of control rats. Treatment with testosterone did not affect the metabolism of any of the¹⁴C-substrates. These results indicate that the testicular desaturation of C 20∶3 to arachidonate, requiring a Δ5 desaturase, is inhibited by hypophysectomy and that testosterone by itself may control the concentrations of some testicular lipid classes but not the metabolism of the polyenoic acids.     

Metabolism of linoleic acid in the cat

Cats fed a diet containing linoleate as the only polyunsaturated fatty acid showed extremely low levels of arachidonate in the plasma lipids, as well as an increase in linoleate, eicosadienoate and an unknown fatty acid. Administration of [1-¹⁴C] linoleic acid and [2-¹⁴C] eicosa-8,11,14-trienoic acid to cats showed that in the liver there was no conversion of the [1-¹⁴C] 18∶2 to arachidonate, whereas there was significant metabolism of [2-¹⁴C] 20∶3 to arachidonate. It was found when methyl-γ-linolenate was fed to cats that the level of 20∶3ω6 and 20∶4ω6 in the erythrocytes increased significantly. These results show that there is no significant Δ6 desaturase activity in the cat, whereas chain elongation and Δ5 desaturase enzymes are operative. The unknown fatty acid was isolated from the liver lipids and shown to be a 20-carbon fatty acid with 3 double bonds and which by gas liquid chromatography could be separated from 20∶3ω9 and 20∶3ω6. The presence of the Δ5-desaturase activity and the results of the ozonolysis studies indicated that this unknown fatty acid was eicosa-5,11,14-trienoic acid.     

Enzymic regulation of arachidonate metabolism in brain membrane phosphoglycerides

The metabolism of arachidonate in brain membrane phosphoglycerides was investigated in vivo by intracerebral injection of labeled arachidonate and by in vitro assay of enzymic systems associated with the metabolism. After intracerebral injection, labeled arachidonate was incorporated rapidly into brain phosphoglycerides with radioactivity distributed mainly in diacyl-sn-glycero-3-phosphoinositols (GPI) and diacyl-sn-glycero-3-phosphocholines (GPC). Some evidence of a metabolic relationship between diacyl-sn-glycerophosphoinositols (diacyl-GPI) and diacylglycerols was observed. Among the phosphoglycerides labeled with [¹⁴C] arachidonoyl groups, diacyl-GPI were most rapidly metabolized in brain microsomal and synaptosomal fractions. The decay of diacyl-GPI in brain synaptosomes may be represented by two pools with half-lives of 5 hr and 5 days. Three types of enzymic systems related to metabolism of the polyunsaturated fatty acids in brain were investigated. The first system involves the cyclic events relating the ATP-dependent activation of polyunsaturated fatty acids (PUFA) to their acylCoA by the acylCoA ligase and subsequent hydrolysis of acylCoA to free fatty acids by the acylCoA hydrolase. It is apparent that fatty acid activation and hydrolysis is under strigent control in order to maitain suitable levels of free fatty acids and acylCoA in the brain tissue for various metabolic use. Factors involved in the regulation may include the level of ATP, divalent cations and the nature of substrates. The second enzymic system pertains to deacylation via phospholipase A₂ and reacylation via the acyltransferase of membrane phosphoglycerides. In brain tissue, activity of the acyl transferase is generally higher than that of the phospholipase A₂. Factors known to affect specificity of the acyltransferase include substrate concentration and the nature of the acyl groups and lysophosphoglycerides. The acyltranferase(s) in brain preferentially transfers arachidonate to 1-acyl-GPI. Activity of the acyltransferase can be inhited by a number of lypophilic compounds including local anesthetics and cell surface agents. Activity of the phospholipase A₂ in brain may depend on the physical form of the substrates, i.e., whether the substrates are in monomeric or micellar form. The third process is associated with the degradation of diacyl-GPI by enzymes present in brain subcellular membranes. Incubation of brain subcellular membranes with 1-acyl-2-[¹⁴C] arachidonoyl-GPI yielded labeled diacylglycerols and arachidonate. The phospholipase C action is specific for hydrolysis of diacyl-GPI. The arachidonate released from incubation of labeled diacyl-GPI may be the result of phospholipase A₂ action which is not specific for diacyl-GPI or the hydrolysis by lipase acting on the diacylglycerols formed from the phospholipase C activity. Enzymic hydrolysis of diacyl-GPI is most active in the microsomal fraction, but uoon disruption of synaptosomes, enzyme in synaptic plasma membranes is also active in degradating this glycerophospholipid. In general, the results of in vitro studies are in good agreement with those observed in vivo and the information yielded has contributed towards understanding the metabolism of polyunsaturated fatty acids in brain subcellular membranes.     

Metabolism of arachidonate and stearate injected simultaneously into the mouse brain

The metabolism of a polyunsaturated and a saturated fatty acid in brain membrane phosphoglycerides was examined by injecting simultaneously a mixture of¹⁴C-arachidonate and³H-stearate into the mouse brain and isolating the microsomal and synaptosomal fractions at 1–40 min after injections. Both types of labeled fatty acids were utilized more readily in the microsomal than the synaptosomal fractions in brain. However, labeled arachidonate was incorporated more rapidly into membrane phosphoglycerides than was stearate. In both subcellular fractions, the relative specific radioactivity (³H and¹⁴C) of diacyl-glycerophosphorylinositol (diacyl-GPI) was higher than other types of phosphoglycerides such as diacyl-glycerophosphorylcholine (diacyl-GPC) and diacyl-glycerophosphorylethanolamine (diacyl-GPE). Furthermore, the apparent rates of incorporation of radioactivity into diacyl-GPI was more rapid for the¹⁴C-arachidonate than for the³H-stearate. Results of the experiment have demonstrated obvious differences in metabolism between stearate and arachidonate in brain. The more rapid transfer of arachidonate to diacyl-GPI is probably due to the presence of an acyl transferase system specially active for the transfer of arachidonyl groups to diacyl-GPI.     

Reciprocal interactions in the desaturation of linoleic acid into γ-linolenic and eicosa-8,11,14-trienoic into arachidonic

Variable concentrations of [I¹⁴C] linoleic acid and [I¹⁴C] eicosa-8,11,14-trienoic acid were incubated with liver microsomes in a medium containing the necessary cofactors for fatty acid desaturation. The conversion of linoleic into γ-linolenic acid and eicosatrienoic into arachidonic acid were mutually inhibited and the inhibition depended on the concentration of the fatty acids incubated.     

Biosynthesis of [14C]arachidonic acid from [14C]linoleate in primary cultures of rat Sertoli cells

The conversion of [¹⁴C]linoleate to [¹⁴C]arachidonate by rat Sertoli cells was established by use of primary cultures. Most of the¹⁴C from [1-¹⁴C]linoleate was located in C-3 of the synthesized arachidonate, indicating that the labeled tetraene had originated largely by elongation and desaturation of the intact labeled substrate rather than by mere addition of¹⁴C-acetate generated by bio-oxidation of the radioactive substrate to an already existing 18-carbon precursor. Although a relatively small amount of¹⁴C was present in 18∶3ω6 and a relatively large amount of¹⁴C was present in 20∶2, it was not possible from these data to establish the relative importance of 20∶2 in the biosynthesis of arachidonic acid in rat Sertoli cells.     

Determination of phospholipase D-mediated hydrolysis of phosphatidylethanolamine

While phospholipase D-mediated hydrolysis of phosphatidylcholine is well documented, we have recently shown that phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) [Kiss, Z., and Anderson, W.B.,J. Biol. Chem. 264, 1483–1487 (1989);J. Biol. Chem. 265, 7345–7350 (1990)] is equally prominent. This made it necessary to define in detail the conditions required for the detection of agonist-stimulated PtdEtn hydrolysis. Using the [¹⁴C]ethanolamine-prelabeled rat-1 fibroblast model and 12-O-tetradecanoylphorbor 13-acetate (TPA) as a model compound with the known ability to stimulate phospholipase D, we demonstrated that optimal detection of TPA-induced ethanolamine release requires i) fractionation of water-soluble ethanolamine products; ii) addition of unlabeled ethanolamine to quench the phosphorylation of newly formed [¹⁴C]ethanolamine; and/or iii) prolonged preincubation of prelabeled cells in an isotope-free medium before the addition of TPA. This preincubation step reduces the cellular content of unincorporated¹⁴C-labeled ethanolamine metabolites and improves the signal-to-noise ratio.     

Biosynthesis of prostaglandin E1 by human seminal vesicles

[1-¹⁴C] 8,11,14-Eicosatrienoic acid was converted into prostaglandin E₁ when incubated with a homogenate of human seminal vesicles. No conversion could be detected with homogenates of human prostate or testis.     

Trans fatty acids: Positional specificity in brain lecithin

Fifteen-day-old rats were divided into three groups: one group received an intracerebral injection of 5 μ Ci of 9-trans, 12-trans [1-¹⁴C] octadecadienoic acid; the second group was given 5 μCi of the same compound plus an equal wt of nonradioactive allcis arachidonic acid; the third group was given 5 μCi of 9-trans [1-¹⁴C] octadecenoic acid. All animals were sacrificed 8 hr after injection. Glycerophosphocholine (GPC) was isolated and partically deacylated with phospholipase A₂ fromCrotalus Adamanteus venom. The results of this study were as follows: 1) aftert [1-¹⁴C] 18∶1 injection, there was twice as much radioactivity in the 1-position as in the 2-position; 2) whentt [1-¹⁴C] 18∶2 was injected, more than 90% of the total radioactivity was found in the 2-position; 3) followingtt[1-¹⁴C]-18∶2 +nonradioactive arachidonate injection, ca. 75% of the total radioactivity still remained in the 2-position; and 4) all of the injected [1-¹⁴C]-tracers showed evidence of undergoing β-oxidation to form acetyl-CoA, which was converted to radioactive palmitate. The possibility is discussed that the observed distribution pattern of the injected radioactive tracers may be attributed to tissue metabolic specificity. Ramifications of the deposition of dietarytrans fatty acids in the brain during the developmental stage of the central nervous system are also discussed.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat adrenal gland

Delta-6 and Δ5 desaturation activity of rat adrenal gland microsomes was studied to determine the effect of microsomal protein and the substrate saturation curves. This tissue has a very active Δ6 desaturase for linoleic and α-linoleic acids and a Δ5 desaturase for eicosa-8,11,14-trienoic acid. The administration of epinephrine (1 mg/kg body weight) 12 hr before killing, produced approximately a 50% decrease in desaturation of [1-¹⁴C]linoleic acid to γ-linolenic acid, [1-¹⁴C]α-linolenic acid to octadeca-6,9,12,15-tetraenoic acid and [1-¹⁴C]eicosa-8,11,14-trienoic acid to arachidonic acid. A 30% decrease in Δ5 desaturation activity was also shown after 7 hr of epinephrine treatment. The changes on the oxidative desaturation of the same fatty acids in liver microsomes were similar. No changes were observed in the total fatty acid composition of adrenal microsomes 12 hr after epinephrine treatment. Mechanisms of action of the hormone on the biosynthesis of polyunsaturated fatty acids in the adrenal gland are discussed.     

Different pools of esterified arachidonic acid in rabbit kidney medulla: Relationship to Ca2+-stimulated prostaglandin biosynthesis

We investigated the effect of Ca²⁺ ions on renal medulla metabolism of endogenous esterified arachidonic acid in contrast to that of radioactive arachidonate incorporated into medullary lipids. Some striking differences between the release of unlabeled prostaglandin E₂ and of¹⁴C-labeled prostaglandin E₂ and arachidonic acid were seen in incubations in absence or presence of Ca²⁺ ions. These differences indicated that exogenous [¹⁴C] arachidonate incubated with medulla slices is incorporated into both Ca²⁺-sensitive and Ca²⁺-insensitive lipid pools of esterified arachidonate and furthermore, the Ca²⁺-sensitive pool is itself heterogeneous and consists of at least 2 functionally different lipid pools of esterified arachidonate. The first Ca²⁺-sensitive pool is characterized by a higher arachidonate turnover rate and incorporates more rapidly added radioactive arachidonate. The acylhydrolase activity which releases arachidonate from this pool is not efficiently coupled to prostaglandin endoperoxide synthase. In contrast, the second Ca²⁺-sensitive lipid pool has a slower arachidonate turnover rate and, consequently, a slower incorporation of added¹⁴C-acid. The acylhydrolase activity associated with this pool is more tightly coupled to prostaglandin endoperoxide synthase, so that a higher portion of released arachidonate is converted to prostaglandin E₂. Studies on arachidonic acid metabolic transformations using exogenously radioactive free acid added to tissues should therefore be inerpreted with caution because the results obtained may not reflect accurately the metabolic fate of endogenous, lipid-esterified arachidonate which is released and metabolized under physiological conditions in vivo.     

Metabolism of phosphatidylinositol in plasma membranes and synaptosomes of rat cerebral cortex: A comparison between endogenous Vs exogenous substrate pools

The metabolism of phosphatidylinositols (PI) labeled with [¹⁴C]arachidonic acid within plasma membranes or synaptosomes was compared to the metabolism of PI prelabeled with [¹⁴C]arachidonic acid and added exogenously to the same membranes. Incubation of membranes containing the endogenously-labeled PI pool in the presence of Ca²⁺ resulted in the release of labeled arachidonic acid, as well as a small amount of labeled diacylglycerol. Labeled arachidonic acid was effectively reutilized and returned to the membrane phospholipids in the presence of adenosine triphosphate (ATP), CoA, and lysoPI. Although Ca²⁺ promoted the release of labeled diacylglycerol from prelabeled plasma membranes, this amount was only 17% of the maximal release, i.e., release in the presence of deoxycholate and Ca²⁺. This latter condition is known to fully activate the PI-phospholipase C, and incubation of prelabeled plasma membranes resulted in a six-fold increase in labeled diacylglycerols. On the other hand, when exogenously labeled PI were incubated with plasma membranes in the presence of Ca²⁺, the labeled diacylglycerols released were 59% of that compared to the fully activated condition. The phospholipase C action was calcium-dependent, regardless of whether exogenous or endogenous substrates were used in the incubation. In contrast to plasma membranes, intact synaptosomes had limited ability to metabolize exogenous PI even in the presence of Ca²⁺, although the activity of phospholipase C was similar to that in the plasma membranes when assayed in the presence of deoxycholate and Ca²⁺. These results suggest that discrete pools of PI are present in plasma membranes, and that the pool associated with the acyltransferase is apparently not readily accessible to hydrolysis by phospholipase C.     

Molecular species of PC and PE formed during castor oil biosynthesis

As part of a program to elucidate castor oil biosynthesis, we have identified 36 molecular species of PC and 35 molecular species of PE isolated from castor microsomes after incubations with [¹⁴C]-labeled FA. The six [¹⁴C]FA studied were ricinoleate, stearate, oleate, linoleate, linolenate, and palmitate, which were the only FA identified in castor microsomal incubations. The incorporation of each of the six FA into PC was better than that into PE. The [¹⁴C]FA were incorporated almost exclusively into the sn-2 position of both PC and PE. The incorporation of [¹⁴C]stearate and [¹⁴C]palmitate into 2-acyl-PC was slower compared to the other four [¹⁴C]FA. The incorporation does not show any selectivity for the various lysoPC molecular species. The level of incorporation of [¹⁴C]FA in PC was in the order of: oleate>linolenate>palmitate>linoleate >stearate>ricinoleate, and in PE: linoleate>linolenate> oleate>palmitate>stearate>ricinoleate. In general, at the sn-1 position of both PC and PE, linoleate was the most abundant FA, palmitate was the next, and oleate, linolenate, stearate, and ricinoleate were minor FA. The activities of oleoyl-12-hydroxylase, oleoyl-12-desaturase seem unaffected by the FA at the sn-1 position of 2-oleoyl-PC. The FA in the sn-1 position of PC does not significantly affect the activity of phospholipase A₂, whereas ricinoleate is preferentially removed from the sn-2 position of PC. The results show that (i) [¹⁴C]oleate is most actively incorporated to form 2-oleoyl-PC, the immediate substrate of oleoyl-12-hydroxylase; (ii) 2-ricinoleoyl-PC is formed mostly by the hydroxylation of 2-oleoyl-PC, not from the incorporation of ricinoleate into 2-ricinoleoyl-PC; and (iii) 2-oleoyl-PF is less actively formed than 2-oleoyl-PC.