Synthesis of platelet activating factor and metabolism of related lipids in embryonic cells.

Primary cultures of mouse embryo palate mesenchyme (MEPM) cells incubated with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H])lyso-PAF) incorporated radiolabel into 1-radyl-2-acyl-sn-glycero-3-phosphocholine (PC) and -phosphoethanolamine (PE). The radiolabeled PC was insensitive to hydrolysis with HCl fumes, whereas at least 82% of the 3H found in the PE was hydrolyzed to 3H-aldehydes by such treatment. Treatment of the PC with Vitride produced [3H]alkylglycerol; similar treatment of the PE produced [3H]alk-1-enylglycerol. None of the radiolabeled products yielded fatty alcohol upon reduction with Vitride. These findings indicate the radiolabeled PC was 1-O-alkyl-linked whereas the PE contained predominantly 1-O-alk-1'-enyl species with smaller amounts of 1-O-alkyl species. Homogenates of MEPM cells which had been prelabeled with [3H]lyso-PAF and [14C]arachidonic acid produced 14C-fatty acid, [3H]lyso-PC, and [3H]alkylglycerol when incubated at selected values of pH and concentrations of calcium. There was no accumulation of [3H]lyso-PE in the various incubation mixtures. Stimulation of MEPM cells with the ionophore A23187 in the presence of calcium and [3H]acetate resulted in the production of 3H-platelet-activating factor (PAF), identified by its migration with authentic PAF and its conversion to 1-O-[3H]alkyl-2,3-diacetylglycerol upon treatment with phospholipase C and acetic anhydride. These studies demonstrate that: (i) MEPM cells are able to incorporate [3H]lyso-PAF into 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, the storage form of PAF, and into 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (PE plasmalogen); (ii) endogenous 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine can serve as a substrate for phospholipase A2 in homogenates; and (iii) MEPM cells have the ability to synthesize PAF, thus raising the possibility that this compound may play a role in modulating the physiology of these embryonic cells.

Phospholipase A activities in embryonic palate mesenchyme cells in vitro.

Incubation of palate mesenchyme cells in vitro in the presence of [3H]arachidonate and [14C]palmitate resulted in incorporation of radiolabel into all major families of phospholipids. Almost all (more than 90%) of the 3H and about one-half of the 14C in the phospholipids were in the sn-2 position. The [14C] fatty acid in the sn-2 position was saturate. When [14C]stearate was added to the culture medium with [3H]arachidonate, 30-40% of the total 14C in phospholipids was in the sn-2 position. 80% of the 14C in the sn-2 position was found in unsaturated fatty acids. Hydrolysis of phospholipids could be demonstrated at acid, neutral and alkaline pH. Calcium stimulated phospholipase activity at neutral and alkaline pH, but inhibited hydrolytic activity at acid pH. Radiolabeled lysophospholipid indicative of phospholipase A2 activities accumulated at acid pH, whereas little, if any, radiolabeled lysophospholipids accumulated at neutral and alkaline pH. Quantitative analysis revealed the production of some lysophosphatidylethanolamine at alkaline pH.

Radioimmunologic identification of prostaglandins produced by serum-stimulated mouse embryo palate mesenchyme cells.

High-performance liquid chromatography and radioimmunoassay were used to identify the prostaglandins synthesized by mouse embryo palate mesenchyme cells. Serum stimulated the release of several different metabolites of arachidonic acid including 6-ketoprostaglandin F1 alpha (the stable product of prostacyclin, prostaglandin I2), prostaglandin E2 and prostaglandin F2 alpha. Compared to control cells, the serum-stimulated cells produce elevated levels of prostaglandin E2 (36-fold), 6-ketoprostaglandin F1 alpha (15-fold) and prostaglandin F2 alpha (7-fold). The acetylenic analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid prevented this accelerated synthesis.

Oxidant-sensitive protein phosphorylation in endothelial cells.

Reactive oxygen is an important regulator of vascular cell biology; however, the mechanisms involved in transducing signals from oxidants in endothelial cells are poorly defined. Because protein phosphorylation is a major mechanism for signal transduction, cultured aortic endothelial cells were exposed to nonlethal concentrations of H2O2 to examine oxidant-sensitive changes in phosphorylation state. Addition of H2O2 increases the phosphorylation of the heat shock protein 27 (HSP27) within 2 min. This response is maximal by 20 min and remains constant for more than 45 min. Levels of intracellular free Ca2+ in endothelial cells did not change following addition of 100 microM H2O2, nor did the ability of the cells to respond to bradykinin. H2O2-induced phosphorylations were either not affected or were slightly increased in cells pretreated with PKC inhibitors (H-8, staurosporin, or calphostin c). Two-dimensional analysis of phosphoproteins from homogenates of 32P-labeled cells revealed that phorbol myristate acetate (PMA) did not cause the same degree of HSP27 phosphorylation as H2O2. Simultaneous addition of 10 eta M PMA and 50 microM H2O2 decreased the oxidant-stimulated phosphorylation of the most acidic HSP27 isoform. These data suggest that signal transduction for H2O2-sensitive endothelial cell responses are not only independent of PKC, but may also be suppressed by the action of the kinase.

The annexins: specific markers of midline structures and sensory neurons in the developing murine central nervous system.

The annexins are a family of cytoplasmic proteins that have been shown to have numerous actions within a cell. Recent evidence suggests that at least one of these proteins plays a role in the development of the central nervous system (CNS). The present study examines the temporal expression and spatial distribution of annexins I, II, IV, V, and VI during development and at maturity in the murine CNS by immunocytochemical analysis. The results demonstrate that annexins I, II and IV exhibit clear immunolabeling in the murine CNS with distinct patterns of temporal and spatial expression. Annexin IV is the first annexin to be expressed on embryonic day (E) 9.5 while annexin I is the last to be expressed (E11.5). Annexins I, II and IV are found in the floor plate region, but to differing rostrocaudal extents. Annexin I has a very restricted distribution, only present in the midline raphe of the brainstem. Annexin II is present in the spinal cord, brainstem and mesencephalon. Annexin IV has the widest midline distribution, being observed in the floor and roof plates of the developing CNS. Additionally, antibodies against annexin II and IV immunolabel most dorsal root and sensory ganglion cells and their axons. During early postnatal development, immunolabeling with each antibody gradually disappears in many structures, and only first order sensory neurons and their fibers are immunopositive for annexins II and IV at weaning. Three functions of the annexins are suggested by the present findings: (1) to help establish the midline structures of the floor and roof plates, (2) to help direct the decussation of sensory fibers, and (3) to regulate some aspect of sensory neuron processing, such as signal transduction.

Effects of retinoic acid treatment on release of arachidonic acid by chondrogenic cells in response to ionophore A23187.

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid in a dose-dependent fashion. Cells prelabeled with (3H) arachidonic acid were treated with 0.3 microgram/ml retinoic acid. Treatment with retinoic acid increased the (3H) fatty acid in the triglyceride fraction. Furthermore, treatment with retinoic acid enhanced the release of (3H) fatty acid upon stimulation of these cells with the divalent ionophore A23187. These data permit the suggestion that there may be a correlation between altered lipid metabolism and retinoic acid's ability to disrupt chondrogenic differentiation.

Epidermal growth factor modulates release of arachidonic acid from embryonic cells.

The calcium ionophore A23187 stimulates release of free [3H]arachidonic acids from radiolabeled cultures of MEPM cells which are growing, but not from those which are confluent. However, when confluent MEPM cells are pretreated with EGF or PMA, release of [3H]arachidonic acids does occur in response to A23187. Since neither EGF nor PMA themselves stimulate release of [3H]arachidonic acids from these cells, but do activate protein kinase C, these data support the hypothesis that protein kinase C modulates the activities of phospholipid hydrolases in MEPM cells.

Cyclic adenosine-3',5'-monophosphate alters hydrolysis of phospholipids by mouse embryo palate mesenchyme cells.

The purpose of this study was to determine whether inhibition of release of arachidonic acid from mouse embryo palate mesenchyme (MEPM) cells in response to cAMP is due to a selected or generalized inhibition of hydrolysis of esterified pools of arachidonic acid. The calcium ionophore A23187 proved to be a useful probe of phospholipid hydrolases in MEPM cells, since it stimulated release of radiolabeled fatty acids from phospholipids of prelabeled MEPM cells as a function of the length of exposure, concentration, and concentration of Ca2+ in the medium. Elevation of intracellular levels of cAMP by treatment with (-) isoproterenol resulted in the inhibition of release of radiolabeled arachidonic acid in response to A23187. Analysis by quantitative gas-liquid chromatography revealed the source of the arachidonic acid released in response to the ionophore to be 1,2-diradyl-sn-glycero-3-phosphoethanolamine; elevation of intracellular levels of cAMP inhibited hydrolysis of this substrate, but may have stimulated hydrolysis of 1,2-diradyl-sn-glycero-3-phosphocholine. These findings permit the conclusions that 1) the ionophore stimulates activities of selected phospholipases A in MEPM cells and 2) cAMP modulates certain phospholipases A in MEPM cells in a specific manner.

Effects of epidermal growth factor and phorbol 12-myristate 13-acetate on protein phosphorylation in mouse embryo palate mesenchyme cells in vitro.

Epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulated mouse embryo palate mesenchyme (MEPM) cells to incorporate [32P]O(3-)4 into a protein with an apparent molecular weight of 80 kDa, in vitro. Agents known to elevate intracellular levels of cyclic AMP did not stimulate phosphorylation of this phosphoprotein. Since there is a significant amount of evidence obtained with other cells indicating that phosphorylation of such an 80-kDa phosphoprotein reflects specifically the activation of protein kinase C in response to PMA and other agents, including mitogens, these findings raise the possibility that EGF may activate protein kinase C in MEPM cells.

Epidermal growth factor alters metabolism of inositol lipids and activity of protein kinase C in mouse embryo palate mesenchyme cells.

Epidermal growth factor (EGF) stimulated mouse embryo palate mesenchyme (MEPM) cells (1) to incorporate [32P]O4(3-) into phosphatidylinositol (PI), phosphatidylcholine, and phosphatidic acid over a period of 60 min; 2) to incorporate [32P]O4(3-) into polyphosphoinositides as a function of time; and 3) to incorporate [32P]O4(-3) into PI, only, as a function of concentration when the period of stimulation was kept short. EGF stimulated the release of radiolabeled inositol phosphates from MEPM cells that had been radiolabeled with [3H]myoinositol. The release of inositol 1-phosphate was sustained over a period of at least 60 min, whereas the release of inositol 1,4-bisphosphate and inositol trisphosphate peaked during the first 10 min of stimulation. EGF also stimulated phosphorylation of an Mr 80,000 protein whose pI, phosphopeptide map, and phosphoamino acid pattern were identical to those of an Mr 80,000 protein phosphorylated in response to phorbol 12-myristate 13-acetate. Mobilization or metabolism of arachidonic acid was not stimulated under the same conditions that permitted EGF to alter inositol lipid metabolism. We interpret these data to mean that 1) in contrast to the findings with some cell lines, alterations in inositol lipid metabolism may be part of the signalling mechanism for EGF in embryonic cells; 2) EGF is capable of activating inositol-dependent signalling pathways leading to activation of protein kinase C in MEPM cells; and 3) mobilization and metabolism of arachidonic acid are not an inherent part of this signalling mechanism.

Metabolism of prostaglandin E2 by mouse embryo palate mesenchyme cells in vitro.

Mouse embryo palate mesenchyme cells synthesize a number of prostaglandins, particularly prostaglandin E2 (PGE2). However, the ability of such cells to metabolize prostaglandins was unknown. By use of radiolabeled PGE2 we determined that palate mesenchyme cells have little ability to degrade that prostaglandin in vitro but are able to metabolize products formed from its spontaneous degradation.

Cyclic AMP inhibits the release of prostaglandins and arachidonic acid from cultures of mouse embryo palate mesenchyme cells.

Mouse embryo palate mesenchyme (MEPM) cells are able to synthesize and respond to prostaglandins. However, mechanisms that regulate their synthesis in these cells are not known. Cyclic adenosine 3',5' monophosphate (cAMP) has been implicated as being involved in differentiation of the palate, accumulates in MEPM cells in response to stimulation with selected prostaglandins, and has been found to modulate synthesis of prostaglandins by other cells and tissues. Therefore, we have investigated whether cAMP modulates synthesis of prostaglandins by MEPM mesenchyme cells and partially characterized the metabolic site at which such modulation occurs. We found that treatment of MEPM cells with various agents to stimulate a seven- to 100-fold increase in intracellular levels of cAMP inhibited release of various prostaglandins by at least 50%. Similarly, elevation of intracellular levels of cAMP inhibited release of radiolabeled arachidonic acid from membrane phospholipids by as much as 27%. The inhibitory effects of cAMP on release of prostaglandins from MEPM cells could be almost completely overcome by the addition of arachidonic acid to the culture medium. We interpret these data to mean that there is a regulatory cycle in MEPM cells in which intracellular levels of cAMP regulates synthesis of prostaglandins and prostaglandins regulate accumulation of cAMP and regulation of synthesis of prostaglandins by cAMP is predominantly through inhibition of a phospholipase.

Characterization and comparison of two plasma membrane reference enzymes with those of other organelles during mammalian embryogenesis.

1. Optimal assay conditions for two plasma membrane reference enzymes, alkaline phosphodiesterase 1 and 5'-nucleotidase, were determined in homogenates of rat embryos obtained on the 14th day of gestation and were found to be different than those reported for adult tissues. 2. Measurements of various organelle reference enzyme activities on the 14th and 15th days of gestation revealed a pattern of selective rates of subcellular organelle biogenesis during this period: lysosomes greater than mitochondria greater than endoplasmic reticulum = plasma membranes.

Phospholipase C activity in palate mesenchyme cells: calcium and pH requirements, substrate specificity, and subcellular localization.

Primary cultures of mouse embryo palate mesenchyme cells were incubated with [3H]arachidonic acid and [14C]stearic acid in order to radiolabel their lipids. The cells were then washed, collected by centrifugation, and homogenized. Incubation of the homogenates under various conditions revealed that deoxycholate inhibited phospholipase A activity and stimulated a phospholipase C activity in these cells which preferentially degraded phosphatidylinositol (PI) compared to phosphatidylcholine (PC), -ethanolamine (PE), and -serine (PS). Expression of this phospholipase C (E.C. 3.1.4.10) activity was dependent on Ca2+ and had a pH optimum of no more than 7.0-7.5. Centrifugation of the homogenates at 105,000g for 30 min produced a membranous fraction that contained phospholipase C activity with characteristics similar to those of the enzyme found in the supernatant. Such a dual distribution of this enzyme may reflect that mouse embryo palate mesenchyme cells are neural crest in origin.

Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ cultures.

Because products of arachidonic acid metabolism, particularly the PG, have been implicated as modulators of growth and differentiation of adult thymocytes, we investigated relations between metabolism of arachidonic acid and growth, as well as differentiation, of thymocytes during fetal thymic organ culture. Fetal thymic cells synthesized immunoreactive PGE2 during organ culture and were found to be capable of metabolizing exogenous arachidonic acid to products that cochromatographed with authentic 6-keto-PGF1 alpha, PGE2, PGF2 alpha. Synthesis of these products and growth and expression of Thy-1 and Lyt-1 Ag were inhibited by culture of fetal thymic lobes with indomethacin, a cyclooxygenase inhibitor, as well as meclofenamate and eicosatetraynoic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Only indomethacin inhibited expression of Lyt-2. Culture with eicosatetraynoic acid also inhibited the capacity of thymic lobes to synthesize 15-hydroxyeicosatetraenoic acid-like products. The inhibitory effects of indomethacin on growth and expression of Thy-1 were partially reversed by simultaneous addition of arachidonic acid. Thus, fetal thymic cells appear to require an intact cyclooxygenase, and possibly lipoxygenase, pathway of arachidonic acid metabolism for growth and differentiation. These data also provide evidence that Lyt-1 and Lyt-2 may be regulated by different requirements with respect to arachidonic acid metabolism.

Dexamethasone does not interfere with hormone-sensitive PI hydrolysis.

Serum, but not epidermal growth factor (EGF), stimulated the release of radiolabeled inositol phosphates from human embryo palate mesenchyme (HEPM) cells prelabeled with [3H]-myoinositol. Pretreatment of cells with 10(-6) M dexamethasone (DEX) for 48 h had no effect on the release of inositol phosphates in response to serum. Furthermore, although treatment of the glucocorticoid-sensitive A/J strain of mouse embryo palate mesenchyme (MEPM) cells with 10(-6) M DEX inhibited their proliferation by 40%, it had no effect on the activity of phospholipase(s) C. However, DEX did enhance the incorporation of [3H]-myoinositol into membrane lipids. We interpret these data to mean that 1) serum factors enhance metabolism of inositol lipids in HEPM cells, 2) DEX does not interfere with the primary events by which agonists utilize metabolism of inositol lipids as a mechanism for transmembrane signaling, and 3) DEX may affect synthesis of phosphoinositides, as reported by Grove et al. (Biochem. Biophys. Res. Commun. 110:200-207, 1983; J. Craniofac. Genet. Dev. Biol. Suppl. 2:285-292, 1986).

Acyl composition and phospholipase activities in plasma membranes isolated from rat embryos.

A subcellular fraction enriched in plasma membranes and relatively poor in other subcellular membranes was isolated from homogenates of rat embryos obtained on the 15th day of gestation. Characterization of this fraction revealed a paucity of stearate, arachidonate and long chain polyunsaturated fatty acids relative to palmitic, oleic, linoleic and linolenic acids, in total phospholipids. Estimation of phospholipase A activity revealed that phospholipase A1 and A2 were present in plasma membranes from rat embryos. A relatively high lysophospholipase activity was also found in the PM-rf, and may be the metabolic basis for the paucity of long chain polyunsaturated fatty acids in the embryo-derived plasma membranes.

Proliferation of cells undergoing chondrogenesis in vitro.

Continuous exposure of chicken embryo limb bud mesenchyme cells undergoing chondrogenesis in vitro to [3H] thymidine thymidine [(3H]TdR) revealed that more than 90% of the cells synthesized DNA at least once during 120 h of culture. When cells were exposed to [3H]TdR for 24 h beginning at various times throughout the culture period, the percentage of cells which incorporated [3H]TdR during each period was approximately 92%. However, when the period for incorporation of radioisotope was limited to two hours, the number of cells which incorporated [3H]TdR was found to decline during chondrogenesis in vitro. This decline was coincident with the appearance of extracellular matrix material and occurred in those cells which had, and had not, expressed the cartilage phenotype. We conclude from these studies that (1) practically all of the cells continue to proliferate while chondrogenesis is occurring in vitro, (2) there is an increase in the length of the cell cycle during chondrogenesis in vitro, and (3) withdrawal from the cell cycle is not required for differentiation of mesenchyme into cartilage.

Effects of dexamethasone on phospholipase activities in palate mesenchyme cells in vitro.

Treatment of primary cultures of palate mesenchyme cells from AJAX strain embryos with dexamethasone inhibited only phospholipase activity expressed at pH 7.5. A similar treatment did not have such an effect on palate mesenchyme cells from C57BL/6J strain embryos. Since the AJAX strain embryo is sensitive to the induction of cleft palate by exogenous glucocorticoids and the C57BL/6J strain is less so, these data allow consideration of phospholipase activity as a site of regulation for development of the palate.