Inhibition by BN 52021 (ginkgolide B) of the binding of [3H]-platelet-activating factor to human neutrophil granulocytes

The inhibitory effect of BN 52021, a specific antagonist of platelet-activating factor (PAF) on PAF-induced activation of human polymorphonuclear granulocytes (PMNL) and on the binding of [3H]-PAF to neutrophils were examined. BN 52021 over the range of 10(-9)-10(-4) M inhibited PAF-induced degranulation and superoxide production of PMNLs in a dose-dependent manner with Kd values of 0.6 +/- 0.1 x 10(-6) M and 0.4 +/- 0.1 x 10(-6) M, respectively. BN 52021 (up to 1 mM) did not show any agonistic activity and it did not affect neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine or leukotriene B4. The Ki value of BN 52021 for the specific binding of [3H]-PAF to neutrophils was 1.3 +/- 0.5 x 10(-6) M versus a Ki of 1.1 +/- 0.3 x 10(-7) M for PAF itself. BN 52021 did not affect metabolism of PAF by PMNL. These studies indicate that BN 52021 inhibits neutrophil responses to PAF by inhibiting binding of PAF to its specific PMNL receptor.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

Interference of the PAF-acether antagonist BN 52021 with endotoxin-induced hypotension in the guinea-pig

Because of the potential role of PAF-acether in the pathogenesis of endotoxin shock, we examined the preventive and curative effects of BN 52021, a new PAF-acether antagonist in guinea-pig challenged with S. Typhimurium endotoxin. A biphasic reduction of mean arterial pressure was elicited by i.v. endotoxin (300 micrograms/kg) in control animals, with a rapid drop of blood pressure (maximal decrease within 10 min), partial recovery at 20 min and a second gradual decrease after 30 min. Treatment with BN 52021 injected 15 min prior to endotoxin reduced the initial rapid drop of blood pressure from 38.5 +/- 5 mmHg in vehicle-treated controls (n = 15) to 17 +/- 3 mmHg (p less than 0.01) in animals treated with 1 mg/kg BN 52021(n = 10) and to 9.5 +/- 8 mmHg (p less than 0.01) in guinea-pigs treated with 6 mg/kg BN 52021 (n = 5). The early hypotensive phase was associated with severe thrombocytopenia-leukopenia; only the thrombocytopenia was reduced by BN 52021. The prolonged secondary phase of hypotension was reduced by BN 52021 pretreatment whereas a small increase of hematocrit persisted. The two phases of the arterial pressure profile during endotoxic shock were not observed in animals previously made thrombopenic by rabbit and anti-platelet serum and only the late hypotensive phase persisted. This late hypotension induced by endotoxin in thrombopenic animals was suppressed by BN 52021 pretreatment suggesting that BN 52021 may act via a platelet-independent mechanism. The intravenous injection of BN 52021 during the prolonged secondary phase of shock was followed by an immediate increase of the depressed blood pressure. This increase of blood pressure was dose-dependent, maximum at 6 mg/kg BN 52021, and observed in normal and thrombopenic animals. The interference of BN 52021 with endotoxin shock may be related to its PAF-acether antagonist properties and suggests that PAF-acether is an important participant in endotoxic shock.     

Interference of the PAF-acether antagonist BN 52021 with passive anaphylaxis in the guinea-pig

PAF-acether may be involved in anaphylaxis and asthma. We tested the new PAF-acether antagonist BN 52021 against the effects of antigen in passively sensitized guinea-pigs. Bronchoconstriction by ovalbumin administered i.v. (1 mg/kg) or by aerosol (1 or 10 mg/ml for a period of 1 min) was significantly reduced by BN 52021 (1–10 mg/kg), which did not inhibit drop of leukocyte counts after the i.v. challenge. In both cases, when the guinea-pigs were pretreated by propranolol, high amounts of BN 52021 became ineffective against shock. The reduction of the anaphylactic bronchoconstriction, induced by the combination of mepyramine, aspirin and FPL 55712 was not improved by BN 52021. Tested on isolated lung strips from passively sensitized guinea-pig, BN 52021, at a concentration which inhibits PAF-induced contraction (0.1 mM), did not inhibit the anaphylactic contraction triggered by the administration of ovalbumin (10 μg/ml) nor the accompanying release of histamine and thromboxane. In contrast, BN 52021 (30 μM) significantly reduced the anaphylactic release of histamine and of thromboxane from perfused lungs of passively sensitized guinea-pigs. The results with the isolated lung strips and the propranolol-treated guinea-pigs in vivo suggest a dissociation between the anti-anaphylactic and the anti-PAF-acether properties of BN 52021.     

Metabolism of platelet-activating factor in primary cultured adult rat hepatocytes by a new pathway involving phospholipase C and alkyl monooxygenase

The metabolic fate of 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether) upon interaction with primary cultured adult rat hepatocytes was investigated. [3H]PAF-acether was transformed time-dependently into [3H]lyso-PAF-acether, 1-O-[3H]alkylglycerol and finally converted to 3H-labeled fatty aldehyde. 1-O-[3H]Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) was formed after a long incubation time and with a smaller amount compared with that formed in platelets and neutrophils. When lipids from cells, cell surfaces and incubation medium were analyzed separately, most of the transformed products of [3H]PAF-acether remained in the cells. When 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine was incubated with hepatocytes, it was mainly converted into 1-O-[3H]alkylglycerol. 3H-labeled fatty aldehyde and [3H]alkylacyl-GPC were also found. Hepatocytes metabolized slowly from 1-O-[1-14C]hexadecylglycerol to 3H-labeled fatty aldehyde and 3H-labeled phospholipid. These findings suggest that cultured hepatocytes mainly catabolize exogeneous PAF-acether by removing the acetyl residue and the polar head group and, finally, by cleaving an ether bond. The deacetylation-reacylation step, which is important in platelets and neutrophils, was not shown to be a main metabolic pathway of PAF-acether in cultured hepatocytes.     

Platelet activating factor (PAF-acether) is released into rat pulmonary alveolar fluid as a consequence of hypoxia

Hypoxia provokes pulmonary constriction and because PAF-acether is a very strong pulmonary constrictor, we looked for PAF-acether in lung alveolar lavage (LAL) with a biological method based on the measurement of rabbit platelet aggregation. We first demonstrated a PAF-acether secretion during bronchoalveolar lavage with sterile isotonic NaCl (pH 7.2). PAF-acether secretion was completely suppressed with isotonic NaCl containing 5 mM EDTA but lyso-PAF-acether was still present (1.9 +/- 0.55 nmoles). Upon hypobaric hypoxia, PAF-acether was detected in LAL (1.05 +/- 0.25 10(-2)nmoles). The amount of lyso-PAF-acether increased by 6 times (12.1 +/- 4.1 nmoles). These results are given for 10(4) nmoles phospholipids of LAL. They indicate that alveolar macrophages might be activated by hypobaric hypoxia, so they produce PAF-acether in the alveole. Such a process could be involved in the well-known bronchoconstriction accompanying hypoxia.     

Possible origins of PAF-acether and lyso-PAF-acether in rat lung alveoli secondary to hypoxia

After 4 h hypoxia, platelet activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and its deacetylated derivative, lyso-PAF-acether, accumulate in rat lung surfactant, the latter in a 1000-fold excess (Prévost, M.C., Cariven, C., Simon, M.F., Chap, H. and Douste-Blazy, L. (1984) Biochem. Biophys. Res. Commun. 119, 58-63). In order to determine the origin of these two phospholipids, rat lung alveolar lavages and rat lung macrophages were examined for phospholipid composition before and after 4 h of hypoxic treatment. Our data indicate an activation of phospholipase A2 in both compartments, as detected by the accumulation of lysophosphatidylcholine. The main effect was observed in lung surfactant, where phosphatidylcholine hydrolysis attained 13%. This change was concomitant with the activation of a calcium-independent phospholipase A2 present in lung alveolar lavages, which might be responsible for the accumulation of some lyso-PAF-acether, alkylacylcholine glycerophospholipids being present in low but significant amounts in lung surfactant. However, the main source of PAF and lyso-PAF-acether appears to be alveolar macrophages, which secreted significant amounts of the two phospholipids upon in vitro hypoxic treatment, although the participation of other cells, such as type II pneumocytes, cannot be excluded. The relative amounts of the two compounds might be regulated by both an intracellular and an extracellular acetylhydrolase, the two enzymes being distinct proteins on the basis of their different isoelectric points.     

Interference of the PAF-acether antagonist BN 52021 with passive anaphylaxis in the guinea-pig

PAF-acether may be involved in anaphylaxis and asthma. We tested the new PAF-acether antagonist BN 52021 against the effects of antigen in passively sensitized guinea-pigs. Bronchoconstriction by ovalbumin administered i.v. (1 mg/kg) or by aerosol (1 or 10 mg/ml for a period of 1 min) was significantly reduced by BN 52021 (1-10 mg/kg), which did not inhibit drop of leukocyte counts after the i.v. challenge. In both cases, when the guinea-pigs were pretreated by propranolol, high amounts of BN 52021 became ineffective against shock. The reduction of the anaphylactic bronchoconstriction, induced by the combination of mepyramine, aspirin and FPL 55712 was not improved by BN 52021. Tested on isolated lung strips from passively sensitized guinea-pig, BN 52021, at a concentration which inhibits PAF-induced contraction (0.1 mM), did not inhibit the anaphylactic contraction triggered by the administration of ovalbumin (10 micrograms/ml) nor the accompanying release of histamine and thromboxane. In contrast, BN 52021 (30 microM) significantly reduced the anaphylactic release of histamine and of thromboxane from perfused lungs of passively sensitized guinea-pigs. The results with the isolated lung strips and the propranolol-treated guinea-pigs in vivo suggest a dissociation between the anti-anaphylactic and the anti-PAF-acether properties of BN 52021.     

Pharmacologic profile of BN 50739, a new PAF antagonist in vitro and in vivo

The effect of a new PAF antagonist BN 50739 was studied on PAF-induced [3H]-serotonin release from washed rabbit platelets in vitro and on PAF-induced hypotension in vivo. BN 50739 competitively inhibited PAF-induced [3H]-serotonin release from the platelets in a dose-dependent manner. In the presence of 4, 10 and 50 nM of BN 50739, the concentration of PAF inducing 50% maximal [3H]-serotonin release from the platelets (EC50) increased from 2.15 nM to 5.10, 45.10 and 900 nM, respectively. The IC50 of BN 50739 for PAF (10 nM) induced [3H]-serotonin release was 3.67 nM. Under the same experimental condition, the IC50s of BN 50726, BN 50730, BN 50741, WEB 2086, SRI 63-441 and BN 52021 were 5.40, 4.61, 6.88, 5.98, 40.90 nM and 14.90 microM, respectively. PAF-induced hypotension in conscious rats was also inhibited dose-dependently by i.p. pretreatment of BN 50739 (3 and 10 mg/kg). PAF-induced hypotension was diminished both in magnitude and duration in rats pretreated with BN 50739. These data taken together indicate that BN 50739 is a most potent PAF antagonist in vitro and in vivo.     

Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes

PAF-acether, at doses ranging from 1pM to 0.1μM did not induce a significative release and/or synthesis of IL1 from monocytes. In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed. PAF-acether at 1pM increased IL1 release by 120 ± 39% and synthesis by 87 ± 27% whereas at 0.1μM a decrease of IL1 release of 52 ± 9% and synthesis of 46 ± 6% were observed. BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase or inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1μM of the autacoid, respectively. No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted. These results indicate that PAF-acether modulates monocyte functions, possibly specific binding sites.     

1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine is the precursor of platelet-activating factor in stimulated rabbit platelets. Evidence for an alkylacetyl-glycerophosphorylcholine cycle

The metabolism of [3H]PAF-acether ([1',2'-3H]alkyl-2-acetyl-sn-glycero-3-phosphorylcholine ([3H]alkylacetyl-GPC)) by rabbit platelets was investigated using thin-layer chromatography and high-performance liquid chromatography followed by radioactivity detection. After 2 h of incubation at 37 degrees C, 90 +/- 5.3% of [3H]PAF-acether taken up by the platelets were converted into a product identified as sn-2 long-chain acyl analogue ([3H]alkylacyl-GPC) which was incorporated in the membranes. This conversion was independent from extracellular calcium and was completely inhibited by platelet pre-exposure to 2 mM phenylmethylsulfonyl fluoride, a serine hydrolase inhibitor, which failed to inhibit the uptake of [3H]PAF-acether by the cells. The 2-deacetylated derivative, lyso-[3H]PAF-acether was found to be an intermediate of the conversion of [3H]PAF-acether into [3H]alkylacyl-GPC in platelet homogenates. Platelet stimulation with 2.5 U/ml of thrombin induced a reduction (16.5 +/- 2.2%) of its content of [3H]alkylacyl-GPC, accompanied by the release of [3H]PAF-acether and lyso-[3H]PAF-acether to the medium. These effects were suppressed by the phospholipase A2 inhibitor, p-bromophenacyl bromide. Our results demonstrate that intact platelets convert exogenous PAF-acether into alkylacyl-GPC, which can serve as the precursor of PAF-acether released during stimulation. The existence of a metabolic cycle for the uptake, the release and the inactivation of PAF-acether by platelets is suggested.     

Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes

PAF-acether, at doses ranging from 1pM to 0.1 microM did not induce a significative release and/or synthesis of IL1 from monocytes. In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed. PAF-acether at 1pM increased IL1 release by 120 +/- 39% and synthesis by 87 +/- 27% whereas at 0.1 microM a decrease of IL1 release of 52 +/- 9% and synthesis of 46 +/- 6% were observed. BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase of inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1 microM of the autacoid, respectively. No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted. These results indicate that PAF-acether modulates monocytes functions, possibly via specific binding sites.     

Antigen-initiated release of platelet-activating factor (PAF-acether) from mouse bone marrow-derived mast cells sensitized with monoclonal IgE

Mouse bone marrow mast cells sensitized with monoclonal IgE and activated with specific antigen released 2.8 +/- 0.5 ng of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (PAF-acether)/ 10(6) cells. The PAF-acether was identified by its ability to aggregate fully aspirin-treated washed rabbit platelets in the presence of an adenosine diphosphate (ADP)-scavenger complex, by its co-chromatography with [3H]-labeled semi-synthetic PAF-acether and synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, and by its inactivation by phospholipases A2, C, and D and not by lipase A1. The antigen-initiated release of PAF-acether, leukotriene C4 (LTC4), and leukotriene B4 (LTB4), and the secretion of the granule marker beta-hexosaminidase were not diminished by washing the cells before challenge, indicating that they were due to the interaction of antigen with the IgE fixed on the cell membrane and not to phagocytosis of immune complexes formed in the fluid phase. The parallel antigen-induced dose-response relationship, along with the superimposable time-course of the extracellular appearance, of beta-hexosaminidase, PAF-acether, and both leukotrienes indicated that the origin of these diverse mediators was from a common cell type with IgE-Fc receptors. Ethanol extraction of antigen-stimulated bone marrow-derived mast cells revealed the early transient appearance of a cell-associated platelet-aggregating activity, the action of which on platelets, like PAF-acether, was independent of ADP and arachidonic acid metabolism. The cell-associated activity contained a novel product that eluted at 13 min during high performance liquid chromatography (HPLC) (solvent hexane:n-propanol:water, 46:46:8), permitting resolution from PAF-acether and lyso-PAF-acether (1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which eluted at 29 min and 30 min, respectively. The cell-associated material, which differs from lyso-PAF-acether, the putative precursor of PAF-acether, in being active in the bioassay on platelets may represent a newly recognized intermediate in the generation of PAF-acether. As the transiently present cell-associated intermediate has not been previously recognized, its detection may depend upon the relatively unique properties of the bone marrow-derived mast cell system in which IgE-dependent activation leading to product generation is complete within 5 min.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transient increase of cytosolic free calcium in cultured human vascular endothelial cells by platelet-activating factor

The effect of platelet-activating factor (PAF-acether) on cytosolic free calcium, [Ca2+]i, in adherent human vascular endothelial cells in culture was directly determined using a new fluorescent calcium indicator, fura-2. It was found that PAF-acether but not lyso PAF-acether induced a rapid and transient increase in [Ca2+]i in endothelial cells. Restimulation with PAF-acether after the first challenge did not cause further response, while the cells were able to respond to thrombin. In the absence of extracellular calcium, PAF-acether evoked a similar transient increase, suggesting that PAF-acether raises [Ca2+]i mainly by discharging calcium from intracellular pools. PAF-acether-induced rise in [Ca2+]i was completely blocked by a specific antagonist, BN 52021. These results suggest the receptor-mediated increase in [Ca2+]i as an early event in PAF-acether activation of human vascular endothelial cells.     

Application of quantitative deuterium NMR to the study of isotope fractionation in the conversion of saccharides to ethanols

³H-PAF-acether (Alkyl-[1′,2′-³H]-2-acetyl-sn-glyceryl-3-phosphorylcholine) was time-dependently transformed by plasma-free rabbit platelets into an unknown product, “PX”, which was distinct from lyso-PAF-acether. This effect was specific for platelets since plasma only converted ³H-PAF-acether into lyso-³H-PAF-acether and platelets were not effective in metabolizing lyso-³H-PAF-acether. Platelet aggregation did not interfere with the formation of “PX”. The latter is a novel platelet metabolite of PAF-acether with as yet unknown biological properties.     

Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets.

The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82 X 10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87 X 10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.     

Effects of platelet activating factor (PAF) and its receptor antagonist BN 52021 on isolated perfused guinea-pig heart

The cardiac effects of PAF and its antagonist BN 52021 have been investigated on the isolated perfused guinea-pig heart maintained at a constant hydrostatic perfusion pressure of 80 cm water. In this model, PAF (1 x 10(-11) to 1 x 10(-7) moles) induced a dose-dependent coronary vasoconstriction, a decrease in heart rate and a fall in contractile force. BN 52021 (1 x 10(-6) to 2 x 10(-4) M) dose-dependently inhibited the vasospasm induced by PAF (1 x 10(-10) moles). BN 52021 also antagonized the decrease in coronary flow and heart rate, but not that of contractile force induced by a high dose of PAF (1 x 10(-7) moles). This dose of PAF also significantly (p less than 0.001) provoked a marked release of TxB2 but did not alter the generation of 6 Keto PGF1 alpha, PGE2 or LTC4. The PAF-induced increase in TxB2 release was completely abolished by BN 52021.     

Failure of platelet-activating factor (PAF-acether) to induce decidualization in mice and failure of antagonists of PAF to inhibit implantation

The possible role of platelet-activating factor (PAF) in the uterine responses associated with implantation was investigated. Attempts to trigger a decidual cell response in the uteri of hormonally sensitized, ovariectomized mice by instilling PAF-acether (1-1000 ng) intraluminally were unsuccessful. The effect of PAF antagonists on implantation was investigated in females ovariectomized on Day 3 of pregnancy and treated with progesterone. Implantation was induced in these females by injection of 10 ng oestradiol-17 beta on Day 8. Hourly intraperitoneal injections of three PAF antagonists (WEB 2086, CV 3988 and BN 52021 at doses of 1.2-1.4 mg/kg) given over a 24-h period starting 1 h before the injection of oestradiol-17 beta had no significant effect on the occurrence of implantation sites. Intraluminal injection of WEB 2086 (15 micrograms) or BN 52021 (5 micrograms) either 3 h before or 6 h after the nidatory oestradiol also had no significant inhibitory effect on implantation. SRI 63-441 given once daily over the first 4 days of pregnancy at a dose of 40 micrograms/30 g body weight had no inhibitory effect on the establishment of pregnancy. These results are not consistent with a critical role for PAF in implantation in mice.     

Desensitization and antagonism of rat polymorphonuclear leukocytes stimulated with PAF acether

The activation of rat polymorphonuclear leukocytes (PMN) with PAF-acether (platelet-activating factor) was blocked by two antagonists: 48740 RP and BN 52021. The release of beta glucuronidase was usually better inhibited than that of lysozyme suggesting that the tested antagonists interfere rather with PAF-acether exocytosis of azurophil granules. Inhibition was relatively specific, even though a moderate effect (below 34%) upon PMN activation by the two unrelated agonist n-formyl-Methionyl-Leucyl-Phenylalanine (fMLP) and leukotriene B4 sometimes reached significance. The partial persistence of inhibition to stimulation with PAF-acether after elimination of both inhibitors suggests that they do not act at the PAF-acether receptor only. Furthermore, the observation of cross desensitization between PAF-acether and fMLP may be related to common pathways and/or metabolites, including PAF-acether itself.     

Sequence of prolactin effects on phospholipid synthesis in mouse mammary gland explants

In cultured mouse mammary gland explants derived from 12-14 day pregnant mice, the effect of prolactin (PRL) on the rate of incorporation of several precursors into neutral lipids and phospholipids was determined. Employing [14C]-acetate as a substrate, PRL stimulates its incorporation into a) neutral lipids by 4-6 hours, b) phosphatidyl choline (PC) and phosphatidyl inositol-phosphatidyl serine (PI-PS) by 1-2 hours, and c) phosphatidyl ethanolamine (PE) by 2-4 hours. Using [3H]-glycerol as a substrate, the temporal response to PRL for its incorporation into the neutral lipids was the same as that for [14C]-acetate, however, PRL did not enhance the rate of [3H]-glycerol incorporation into the phospholipids at any time through 16 hours. PRL similarly had no effect on the rates of [3H]-choline, [3H]-serine, [3H]-ethanolamine, or [32P]O4 incorporation into the phospholipids at hormone exposure periods of 8 hours or more. And finally, PRL had no effect on the rates of [3H]-arachidonate or [14C]-linoleate incorporation into neutral lipids or phospholipids at culture periods up to 18 hours. These data suggest that the early effect of PRL on [14C]-acetate incorporation into the phospholipids is due to either the insertion of newly synthesized fatty acids and/or the extension of fatty acids contained in the phospholipids.