Radioactive Phospholipids Research Articles

Nuclear inositol lipids. Relationship between growth factor induced metabolic changes and protein kinase C activity

We have sought to establish the effect of mitogen treatment on nuclear inositol lipids and the relationship between inositol cycle products and hyperphosphorylation of nuclear proteins via PKC during the lag phase leading to the onset of DNA synthesis. Swiss 3T3 cells were labelled for 36 hr with high levels of [ 3H]-myo-inositol and the radioactivity in nuclear inositol phospholipids was measured. Treatment of cells for 2 min, but not for 4 hr, with mitogenic concentrations of insulin-like growth factor I and bombesin caused a marked decrease in PtdInsP and PtdInsP 2. Moreover, in vivo phosphorylation of some nuclear proteins occurs later on. Among these proteins, histone H1 and 0.75 m PCA soluble polypeptide, with an apparent M r of 21,000 as revealed by electrophoretic analysis, are phosphorylated in vitro by protein kinase C in isolated nuclei purified from 3T3 cells treated for 90 min with IGF-I and bombesin. Since these phosphorylative events follow the earlier changes in nuclear polyphoinositide metabolism induced by the same mitogen combination, it seems possible that these two phenomena are related to each other and trigger the synthetic machinery responsible for replicating DNA.

Accumulation of inositol phosphates induced by chlorpromazine in C6 glioma cells.

Chlorpromazine, a cationic amphiphilic drug known to affect phospholipid metabolism, greatly increases the generation of inositol phosphates in C6 glioma cells. When a pulse-chase protocol with myo-[2-3H]inositol as the radioactive precursor was used, the peak increase in radioactivity of inositol phosphates was observed at 20 min. The drug decreased inositol tetrakisphosphate labeling as a percentage of inositol trisphosphate in a dose-dependent manner. It also increased the labeling of the inositol-containing phospholipids, the precursors of the inositol phosphates. The increase in radioactivity of both phospholipids and inositol phosphates was dose-dependent, but appeared also to be a function of the time of exposure of the cultures to the drug, suggesting that the concentration of chlorpromazine in the cell, and not that in the medium, is the critical factor. The optimum concentration for maximum phospholipid labeling was lower than that eliciting maximum generation of inositol phosphates. The data suggest that the mechanism probably does not involve cell-surface receptors, but rather may consist of a direct effect of chlorpromazine on phosphoinositidase C and possibly other enzymatic reactions concerned with the metabolism of inositol phosphates.

Selective hepatic enrichment of polyunsaturated phosphatidylcholines after intravenous administration of dimethylethanolamine in the rat

The content of polyunsaturated phosphatidylcholines (PCs) is one of the parameters which regulate membrane functions. Polyunsaturated PCs are preferentially synthesized in the liver by the microsomal enzyme phosphatidylethanolamine N-methyltransferase. The activity of this enzyme may be stimulated in vitro in isolated rat hepatocytes by supplementation with dimethylethanolamine (DME), the polar head group of the precursor of PC along this pathway. The aim of this study was to evaluate in vivo the effect of an intravenous infusion of DME in the rat on the hepatic phospholipid composition. Bile fistula rats were intravenously infused for 15 h with sodium taurocholate (1 μmol/kg per min), with or without the addition of 0.3 mg/kg per min of [ 14C]DME. The concentration per gram of wet liver of individual phospholipid classes, PC molecular species and of total triacylglycerols, as well as the distribution of radioactivity in liver phospholipids, in rat tissues and body fluids were analyzed. A significant ( P < 0.01) enrichment in PC was found in the liver of DME-infused rats with respect to controls. No differences in the other phospholipid classes were found. DME-infused rats showed a significant ( P < 0.01) decrease in the hepatic concentration of triacylglycerols. At HPLC analysis, the enrichment in PC in DME-infused rats was found to be selectively due to three molecular species (i.e., sn-1stearoyl/sn-2arachidonoyl, sn-1stearoyl/sn-2linoleoyl, sn-1stearoyl/sn-2docosahexanoyl molecular species). In agreement with quantitative data, more than 70% of hepatic radioactivity was recovered in polyunsaturated PC species, with the highest specific activity in the sn-1 stearoyl PCs. The specific activity of hepatic PC approximates that of phosphatidyldimethylethanolamine. This finding together with the effective incorporation of DME in PC suggests that this amino base is methylated after its incorporation into phosphatidyldimethylethanolamine, throughout the stimulation of hepatic N-methyltransferase activity. The selective hepatic enrichment with polyunsaturated PC species after DME infusion may offer a new experimental tool for studying hepatic membrane metabolism.

Intravenous injection of [1‐14C]arachidonate to examine regional brain lipid metabolism in unanesthetized rats

We examined the metabolic disposition and brain distribution of an unsaturated fatty acid, [1-14C]arachidonate, between 5 and 240 min following its intravenous bolus injection in unanesthetized adult rats. Injected [1-14C]arachidonate was cleared rapidly from plasma, with less than 10% remaining by 2 min. Total brain radioactivity, 0.2% of the injected dose, was near maximal by 5 min, reached a peak by 15 min, then slowly declined. Radioactivity in brain lipids constituted greater than 82% of the total brain radioactivity at all times. Radioactivity in aqueous-soluble metabolites was greatest at 5 min (13% of total) and declined to 5% by 240 min. Protein pellet-associated radioactivity gradually rose to a peak of 7% by 120 min. Within the lipid fraction, more than 92% of radioactivity was in glycerolipids, with greater than 81% in phospholipids. Radioactivity in inositol phosphoglyceride was maximal at 5 min (47% of phospholipid radioactivity); and declined to 34% by 20 min, whereas radioactivity in choline phosphoglyceride peaked at 15 min (41% of phospholipid radioactivity) and was constant thereafter. In contrast, radioactivity in ethanolamine phosphoglycerides increased from 7 to 17% during the course of the experiment. Quantitative autoradiography of brain sections indicated incorporation of [1-14C]arachidonate into gray-matter regions was 1.5- to threefold that into white-matter regions. The data were analyzed in terms of a model for brain fatty acid uptake from plasma. Estimates of unidirectional transfer constants, k, for [1-14]arachidonate from plasma to brain regions with an intact blood-brain barrier ranged from 0.0005 to 0.0015 ml.sec-1.g-1 and were correlated with those for [9,10-3H]palmitate. The results indicate that brain phospholipid metabolism in awake animals can be examined regionally and quantitatively using intravenous injection of [1-14C]-arachidonate combined with quantitative autoradiography and biochemical analysis.

Studies on the incorporation and transacylation of various fatty acids in choline and ethanolamine-containing phosphoacyglycerol subclasses in human neutrophils

The incorporation of eight 14C-labeled fatty acids into human neutrophil phospholipids was investigated and the results were expressed as percent of the total phospholipid associated 14C-labeled substrate incorporated after an initial 15 min labeling and a subsequent 2 h reincubation in fatty acid-free buffer. In all cases, the PC fraction accounted for more than 40% of the total phospholipid radioactivity. The inositol-containing phosphoacylglycerols were also labeled well by all the fatty acids except 22:6(n - 3) and 16:0; however, a greater percentage of [14C]22:6(n - 3) was found in PE than that of any other labeled fatty acid substrate. In all cases, most of the radioactivity in PC after 15 min was in the diacyl subclass. After 2 h, there was a shift of [14C]20:4(n - 6), [14C]20:5(n - 3), [14C]22:6(n - 3) and [14C]18:4(n - 4) into the ether-linked subclass. No such shift was observed for [14C]16:0 or [14C]18:2(n - 6) and, although there was an increase in the percent radioactive 20:3(n - 6) and 20:3(n - 9) in ether-linked PC after 2 h, the total radioactivity in this fraction remained low by comparison. A similar shift in label occurred in the plasmalogenic-linked PE subspecies in cells labeled with [14C]20:4(n - 6), [14C]20:5(n - 3) and [14C]22:6(n - 3).

Phosphatidylcholine storage in mucolipidosis IV

The accumulation of phosphatidylcholine (PC) in cultured fibroblasts of mucolipidosis IV (MLIV) patients was studied by subcellular fractionation on percoll gradients. Labelled PC accumulated in secondary lysosomes of the MLIV cells in significantly higher rates when compared to normal controls incubated with the precursors [ 32P]phosphate or [ 14C]choline. This accumulation was noted after 4 days of pulse and became more profound after 7 days of chase resulting in a 30-fold increase of this substance in the lysosomal fraction of MLIV compared to normal controls. On the other hand, no significant increase of radioactive PC was demonstrated in the buoyant fraction of the affected cells, and similarly the rate of disappearance of labeled PC from this fraction was identical in MLIV and controls. The retention of PC in lysosomes of MLIV could also be demonstrated following incubation of cultured fibroblasts with the radioactive phospholipid itself. In these experiments increased labelled PC in MLIV was also noted in endosomes, which are involved in the uptake process of PC enroute to the lysosomes. The metabolic defect leading to this storage in MLIV has not yet been identified, but these data indicate impairment in the lysosomal catabolism of phospholipids in MLIV.

Changes in nuclear inositol phospholipids induced in intact cells by insulin-like growth factor I

Swiss 3T3 cells were labelled for 36 hours with high levels of 3H-myo-inositol and the radioactivity in nuclear inositol phospholipids was measured. Treatment of cells for 2 minutes, but not for 4 hours, with mitogenic concentrations of insulin-like growth factor I and bombesin caused a slight decrease in PtdIns and more marked decreases in PtdIns P and PtdIns P 2. These effects were not seen if isolated nuclei were incubated with IGF-I and bombesin. We interpret these results to mean that rapid mass changes occur in nuclear inositol phospholipids in the early stages of the mitotic response.

Inositol trisphosphate, inositol phospholipid metabolism, and germinal vesicle breakdown in surf clam oocytes

Others have reported that microinjection of inositol 1,4,5-trisphosphate (InsP 3) releases stored intracellular Ca 2+ and causes fertilization envelope elevation, part of the activation process normally initiated by fertilization in deuterostome eggs. In the protostome, Spisula solidissima, germinal vesicle breakdown (GVBD) is the first visible response of the egg to fertilization. To test the effects of InsP 3 on egg activation in this organism, we microinjected the compound into oocytes. Microinjection of 0.4−7.0 × 10 −21 moles of InsP 3 (equivalent to 5–80 p M if distributed throughout the cell) elicited GVBD in a dose-dependent manner, demonstrating that increased oocyte InsP 3 can mimic part of the activation process in this protostome. Synthesis of InsP 3 occurs in vivo when phosphatidylinositol 4,5-bisphosphate (PtdInsP 2) is hydrolyzed by phospholipase C. To determine whether stimulus-induced synthesis of InsP 3 occurs after fertilization of Spisula oocytes, we labeled oocyte lipids with [ 32P]orthophosphate and measured the radioactivity in phospholipids after insemination. Fertilization resulted in a rapid, transient loss of radioactivity from PtdInsP 2. Because the radioactivity in phosphatidylinositol 4-phosphate and other phospholipids did not change, the loss of radioactivity from PtdInsP 2 is most likely due to its hydrolysis, yielding InsP 3 and diacylglycerol. The latter compound activates protein kinase C which has also been shown to be involved in regulating Spisula oocyte GVBD. Since both of these compounds appear to be early products of fertilization, they could coordinately activate Ca 2+- and protein kinase C-dependent processes involved in Spisula oocyte GVBD. These data indicate that egg activation in this protostome includes pathways similar to those found in deuterostome eggs and in other eukaryotic cells.

Inhibition by colchicine of phospholipid secretion induced by lung distension.

The effect of colchicine, a microtubule disruptor, on phospholipid secretion stimulated by distension of fetal rabbit lungs was investigated. After colchicine injection and breathing for 45 min, pups were killed and their lungs were lavaged with colchicine. Controls were injected and lavaged with saline. All lungs were given static air inflation and a final lavage, and the returns were analyzed for phospholipid DNA, and lactate dehydrogenase. The first lavage after breathing yielded 33% less phospholipid with colchicine, 3.83 compared with 5.72 mg/g dry lung wt (P less than 0.05). The postinflation phospholipid yield was also significantly reduced with colchicine from 1.04 to 0.70 mg/g dry lung wt (P less than 0.05). The postinflation DNA was significantly reduced with colchicine, from 1.26 to 0.44 micrograms (P less than 0.01), suggesting reduced alveolar macrophages. Colchicine did not change the recovery by lavage of exogenous radioactive phospholipid. As reflected by ATP and lactate levels, tissue metabolism was well maintained. The results are interpreted to mean that colchicine reduced simultaneously lavage-associated phospholipid secretion, inflation-produced phospholipid secretion, and macrophage migration.

Effects of phospholipid composition on the metabolism of triacylglycerol, cholesteryl ester and phosphatidylcholine from lipid emulsions injected intravenously in rats

Lipid emulsions were prepared with a similar size and lipid composition to natural lymph chylomicrons, but in which the surface phospholipid was either egg phosphatidylcholine, dioleoyl-, dimyristoyl-, dipalmitoyl- or 1-palmitoyl-2-oleoylphosphatidylcholine (EYPC, DOPC, DMPC, DPPC or POPC). When injected into the bloodstream of conscious rats, the emulsions containing EYPC or POPC were metabolized similarly to natural chylomicrons, consistent with rapid lipoprotein lipase-mediated hydrolysis of triacylglycerols, followed by hepatic uptake of the remnants derived from the emulsions. Phospholipids from the injected emulsions were removed more slowly and became associated with the high-density lipoprotein fractions of the plasma. Emulsions containing DPPC were metabolized differently. Triacylglycerols disappeared very slowly from plasma, indicating lack of hydrolysis by lipoprotein lipase, and phospholipid radioactivity did not transfer to high-density lipoprotein. With emulsions containing DMPC, the plasma removal rates for emulsion triacylglycerols and cholesteryl esters were fast, but phospholipid radioactivity failed to transfer to the high-density lipoprotein fractions of plasma. With DOPC emulsions, clearances were slower than EYPC or POPC emulsions, but transfer to high-density lipoproteins was efficient. Therefore, an unsaturated chain at the glycerol 2-position was necessary for rapid hydrolysis by lipoprotein lipase and for efficient transfer of phospholipids to high-density lipoproteins. With an unsaturated chain at the glycerol 2-position, a saturated chain at the glycerol 1-position optimized the rate of remnant removal from the plasma.

Rates of production and consumption of phosphatidic acid upon thrombin stimulation of human platelets.

Human platelets were labelled with [32P]Pi and [3H]glycerol before gel filtration. In unstimulated cells, the specific 32P radioactivity in phosphatidic acid (PtdOH) was similar to that of phosphatidylinositol (PtdIns) but only 4% of that of the gamma-phosphate of ATP. Upon 3 min of stimulation with 0.5 U/ml of thrombin, there was a 20-fold increase in specific 32P radioactivity of PtdOH which approached that of the ATP gamma-phosphate. Based on constant rates of synthesis and removal, this thrombin-induced increase in specific 32P radioactivity in PtdOH allowed us to calculate the flux of phosphate through PtdOH upon stimulation. Synthesis and removal occurred at rates of 107 and 52 nmol min-1/10(11) cells, respectively. The specific [3H]glycerol radioactivity was similar in PtdIns, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in unstimulated platelets. In PtdOH, it was 50% of that of the inositol phospholipids. Thrombin stimulation induced no changes in the specific 3H radioactivity of the inositol phospholipids whereas specific [3H]PtdOH increased to the level of these lipids. It is concluded that PtdIns, PtdInsP and PtdInsP2 exist in a metabolic homogenous pool in human platelets.

Age-related changes of RNA and lipid synthesis in vitro by retina and optic nerve of the rat

We examined the effects of age on RNA and lipid formation by whole retina and optic nerve in vitro. Male Wistar rats, aged 4, 12, and 24 mo, were used. From the results obtained the following conclusions may be drawn: 1. In assaying the lipid biosynthesis during aging, a striking difference between the retina and optic nerve clearly emerged; 2. In isolated retina, [3H]uridine incorporation into RNA was relatively constant at the three ages, whereas both [14C]palmitate and [3H]choline incorporation into lipids showed a substantial increase in rats at 24 mo of age compared with those at 4 mo; 3. In contrast, in the optic nerve of the oldest rats, compared with the youngest, a significant decrease of [14C]acetate and [14C]palmitate incorporation into acylglycerols, cerebrosides, and phospholipids was found. Each fatty acid precursor label was incorporated to a proportion that reflected the typical acyl group composition of individual lipids; 4. Following labeling of the optic nerve with [3H]choline, the specific radioactivity of choline-containing phospholipids was drastically decreased with increasing rat age; and 5. The incorporation of [2-3H]glycerol into optic nerve diacylglycerols, PtdEtn, and PtdIns declined with age, whereas no significant change took place in the incorporation into PtdCho. The results strongly support the concept that RNA metabolism of rat retina (most likely photoreceptor cell layer) is not altered during aging; on the contrary, phospholipid synthesis is stimulated in comparison with that of the optic nerve, for which a serious impairment was concomitantly observed. The physiological significance of these responses, and the mechanism by which retinal tissue is spared from the general age derangement of the nervous system, remain to be defined.

A significant increase of lysophosphatidylinositol 4-phosphate with insulin in isolated rat fat cells

We studied the effects of insulin on the incorporation of 32P i into phospholipids in rat fat cells. When the cells were treated with insulin, a new radioactive phospholipid was detected on thin layer chromatography. The substance migrated slower than phosphatidylinositol 4,5-bisphosphate and was hardly detectable in the absence of insulin. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 120 μU/ml. Pretreatment of insulin with anti-insulin antibody or the cells with anti-insulin receptor anti-body inhibited the effect of insulin. The product of phosphatidylinositol 4-phosphate hydrolysed by phospholipase A 2 was coincided with the substance on thin layer chromatography. Quinacrine inhibited the formation of the substance in a dose-dependent manner. These results suggested that insulin stimulates the generation of lysophosphatidylinositol 4-phosphate through the insulin-receptor interaction.

Selective Uptake of [3H]Arachidonic Acid Into the Dense Tubular System of Human Platelets

We have used quantitative electron microscopic autoradiography to characterize the subcellular distribution of arachidonoyl phospholipids following brief (5 minutes) exposure of unstimulated human platelets to [3H]arachidonic acid. Labeled arachidonate was taken up rapidly and incorporated into phospholipids. Phospholipid radioactivity was preserved and spatially fixed during tissue processing for electron microscopy. Analysis of autoradiographs showed that following a brief exposure to 750 nmol/L [3H]arachidonate, there is selective labeling of an internal membrane compartment composed of the dense tubular system and the open canalicular system. The plasma membrane, platelet granules, and nonmembranous cytoplasm were not labeled. Since the open canalicular system is continuous with the plasma membrane and since phospholipids in continuous membranes are freely diffusible, our observations indicate that [3H]arachidonate was incorporated into phospholipids within the dense tubular system and not the open canalicular system. Thus, the dense tubular system, known to contain cyclooxygenase activity, incorporates arachidonate selectively following brief exposure to this fatty acid, presumably to concentrate it in proximity to enzymes for icosanoid synthesis.

Selective uptake of [3H]arachidonic acid into the dense tubular system of human platelets

We have used quantitative electron microscopic autoradiography to characterize the subcellular distribution of arachidonoyl phospholipids following brief (5 minutes) exposure of unstimulated human platelets to [3H]arachidonic acid. Labeled arachidonate was taken up rapidly and incorporated into phospholipids. Phospholipid radioactivity was preserved and spatially fixed during tissue processing for electron microscopy. Analysis of autoradiographs showed that following a brief exposure to 750 nmol/L [3H]arachidonate, there is selective labeling of an internal membrane compartment composed of the dense tubular system and the open canalicular system. The plasma membrane, platelet granules, and nonmembranous cytoplasm were not labeled. Since the open canalicular system is continuous with the plasma membrane and since phospholipids in continuous membranes are freely diffusible, our observations indicate that [3H]arachidonate was incorporated into phospholipids within the dense tubular system and not the open canalicular system. Thus, the dense tubular system, known to contain cyclooxygenase activity, incorporates arachidonate selectively following brief exposure to this fatty acid, presumably to concentrate it in proximity to enzymes for icosanoid synthesis.

Effects of norepinephrine and 5-hydroxytryptamine on phosphoinositide-PO 4 turnover in rabbit cornea

We have investigated: (a) Phospholipid composition and phosphoinositide-PO4 turnover in rabbit cornea tissues; and (b) the effects of adrenergic and serotonergic agonists on breakdown of phosphoinositides in the rabbit cornea. The data obtained from these studies can be summarized as follows: (1) in the cornea phosphatidylcholine and phosphatidylethanolamine constitute about 55%, phosphatidylinositol (PI) 10%, and phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) comprise about 1% each of the total phospholipids; (2) incubation of cornea in 32Pi-containing medium resulted in incorporation of radioactivity in tissue phospholipids. The radioactivity was highest in PIP2 (39%), followed by PI (19%), PIP (16%) and PA (5% of the total radioactivity). When compared with stroma and endothelium, the cornea epithelium was most active in phosphoinositide metabolism; (3) addition of norepinephrine (NE) or 5-hydroxytryptamine (5-HT), 200 microM each, to 32P-labeled cornea resulted in a loss of radioactivity in PIP and PIP2 by about 12- and 20%, respectively. Concomitantly, the radioactivity in PA and PI was increased by 44- and 66%, respectively. The effects of the neurotransmitters were time- and concentration-dependent. When added to the cornea labeled with myo [3H] inositol, NE and 5-HT increased the production of labeled myo-inositol phosphates; (4) prazosin (20 microM), but not yohimbine or propranolol, blocked the effects of NE. Similarly, the effects of 5-HT were antagonized by methysergide (20 microM) and ketanserin (10 microM) but not by prazosin. These data demonstrate that NE and 5-HT stimulate phospholipase C-mediated hydrolysis of PIP2 into diacylglycerol (DG) and myo-inositol trisphosphate (IP3). Furthermore, the effects of NE and 5-HT are mediated by alpha 1-adrenergic and 5-HT2 receptors, respectively. It is suggested that IP3, by releasing Ca2+ from ER, and DG, by activating protein kinase C, may function as second-messenger molecules which may participate in agonist-induced functional responses, including chloride transport, in the cornea epithelium.

Histamine acting on H2 receptors stimulates phospholipid methylation in synaptic membranes of rat brain.

Histamine stimulated [3H]methyl group incorporation into phospholipids in crude synaptic membranes of rat whole brain (without cerebellum) in modified Krebs-Ringer solution containing the methyl donor S-adenosyl-[methyl-3H]methionine. The transient increase of [3H]methyl incorporation into lipids peaked within 45 s after addition of histamine (5 or 10 microM) and decreased the basal level in 60 s. Histamine-stimulated [3H]methyl incorporation was increased linearly in a protein concentration-dependent manner. The stimulation was temperature and histamine concentration dependent. TLC analysis of a chloroform/methanol extract indicated that radioactive phospholipids (phosphatidylcholine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidyl-N-monomethylethanolamine) accounted for 60-65% of the total radioactivity recovered. The synaptosomal fraction had the highest specific activity of all the subfractions of crude synaptic membranes (P2). Histamine-induced [3H]methyl incorporation was inhibited by addition of cimetidine (0.01-10 microM) or famotidine (0.01-1.0 microM) in a concentration-dependent manner but not by mepyramine (0.1-10 microM) or diphenhydramine (0.1-10 microM). The stimulation of [3H]methyl incorporation was also observed by addition of impromidine (0.01-10 microM) or dimaprit (1.0 microM-1.0 mM) in a concentration-dependent manner but not by 2-pyridylethylamine (1.0 microM-1.0 mM). These results indicate that phospholipid methylation is induced by histamine acting on H2 receptors in rat brain synaptosomes.

Regulation of arachidonic acid metabolism in resident and BCG-activated alveolar macrophages: role of lyso(bis)phosphatidic acid.

To dissect mechanisms of arachidonic acid (20:4) metabolism in pulmonary alveolar macrophages (PAM), two distinct cell populations were investigated, resident and BCG-activated rabbit alveolar macrophages. After purified resident PAM were labeled overnight with [3H]20:4, radioactivity was localized primarily within lyso(bis)phosphatidic acid (L(bis)PA) (13.1% +/- 1.7), phosphatidylethanolamine (PE) (22.8% +/- 0.8), and phosphatidylcholine (PC) (26.7% +/- 1.7), with lesser amounts recovered in phosphatidylserine plus phosphatidylinositol (PS/PI) (9.2 +/- 0.8%). By contrast, analysis of the phospholipid classes from prelabeled BCG-activated PAM revealed that the amount of [3H]20:4 contained in L(bis)PA was profoundly decreased (4.7% +/- 0.4), p less than 0.003), whereas [3H]20:4 contained within other BCG phospholipids remained unchanged. Moreover, L(bis)PA, which composed 18.6% +/- 1.2 of the total phospholipid phosphorus of resident PAM, was reduced to 4.1% +/- 0.1 in BCG-activated macrophages (p less than 0.01). Phospholipase A2 from snake venom or from pancreas failed to release 20:4 from L(bis)PA, and lipase (phospholipase A1) from Rhizopus delmar liberated no more than one-third of this arachidonate. These results suggest that much of the arachidonate is not mobilized by classical phospholipases A1 and A2. When [3H]20:4-labeled PAM were stimulated with 1 microM 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a loss of [3H]20:4 was observed from L(bis)PA, PE, PC, and PS/PI, with a concomitant increase in the synthesis of Hete and leukotriene C4. BCG-activated PAM exposed to either TPA or 3.8 microM calcium ionophore A23187 liberated [3H]20:4 solely from PE and PC, with diminished 20:4 oxidative metabolism. Analysis of the specific radioactivities of phospholipids obtained from resident PAM prelabeled with [3H]20:4 or [32P]i demonstrated that the specific activity of [32P]L(bis)PA was negligible, whereas that of [3H]20:4 was quite high. In addition, L(bis)PA deacylation induced by TPA in resident PAM was always accompanied by a corresponding loss of [3H]20:4 from phosphatidylinositol (PI), suggesting that metabolism of this novel phospholipid proceeded by a deacylation-reacylation reaction rather than by de novo synthesis. BCG-activated PAM, which exhibited depressed eicosanoid formation, consistently failed to deacylate [3H]20:4 from L(bis)PA or PI. These studies demonstrate that, unlike 20:4 derived from PE and PC by BCG-activated PAM, L(bis)PA may indeed provide a novel source of 20:4 that is tightly coupled to the lipoxygenase pathway.

Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of liposomes by type II cells.

The alveolar type II cell which synthesizes and secretes surfactant also plays a major role in the reuptake of surfactant lipids. In a recent in vivo study we found that the subfractions of natural surfactant that contained the surfactant protein with molecular weights of 26,000-36,000 (SP-26-36) were preferentially taken up into lamellar bodies of type II cells to a greater extent than were fractions that did not contain SP-26-36. Because the subfractions of natural surfactant in that study differed in other properties than the presence or absence of SP-26-36, the current study was undertaken to determine whether purified SP-26-36 enhanced the uptake of surfactant-like lipids by freshly isolated type II cells. SP-26-36 increased the uptake of label in radioactive surfactant-like lipids by up to 10-fold, and the effect of SP-26-36 was dependent on time, protein concentration, and temperature. The enhancement was inhibited by heat-treating the protein, by a polyclonal antibody against SP-26-36, and by metabolic inhibitors. The distribution of radioactivity in cell-associated phospholipids differed if cells were incubated with or without SP-26-36. If SP-26-36 was present during the incubation, greater than 96% of the radioactivity remained associated with phosphatidylcholine. In the absence of SP-26-36, only 85% of the radioactivity remained associated with phosphatidylcholine and 7% of the label appeared in phosphatidylglycerol. We hypothesize that SP-26-36 may act as a ligand to direct surfactant lipids to type II cells, perhaps to different metabolic pathways, and to regulate recycling and surfactant homeostasis.

The roles of haemolymphatic lipoproteins in the oogenesis of Rhodnius prolixus.

The fates of purified 32P-vitellin and 32P-lipophorin were followed in vitellogenic females of Rhodnius prolixus. While the radioactivity from 32P-vitellin 6 hours after injection was found almost exclusively in the ovary, the radioactivity from injected 32P-lipophorin was found distributed among several organs. In the ovary, the radioactivity from 32P-vitellin was associated with the contents of the yolk granules. 32P-lipophorin delivered a great amount of radioactive phospholipids to the ovary with no accumulation of its protein moiety, as observed after its iodination with 131I. The delivery of phospholipids was inhibited at 0 degrees C and by the metabolic inhibitors, sodium azide and sodium fluoride. Comparison of the radioactivity incorporation from 32P-lipophorin with that of 14C-inulin suggests that the 32P-phospholipids from lipophorin are not taken up by fluid phase endocytosis. The data presented here are compatible with the concept of lipophorin as a carrier of lipids in insects and provide evidence that lipophorin transports phospholipids as shown previously for other classes of lipids. The utilization by the oocytes of the phospholipids transported by lipophorin is discussed.