维生素C衍生物对癌细胞浸润转移能力的抑制作用机理研究

前期研究表明Asc2P6Plm能够有效地抑制癌细胞的浸润转移，本文试图以Asc2P6Plm对人成纤维瘤细胞浸润转移作用探讨维生素C衍生物对癌细胞转移能力抑制的机理，对HT-1080细胞分别以50-300μmol/LAsc2P6Plm处理1h，随着Asc2P6Plm浓度的增大，细胞移动的数目明显减少，Asc2P6Plm对HT-1080细胞移动的抑制作用呈现出量效关系，Asc2P6Plm对ROS的清除作用，通过自旋捕集剂DMPO以电子自旋共振方法进行研究，HT-1080细胞经Asc2P6Plm处理后，细胞内的自由基水平与对照组相比有显著的降低，用F-actin的分子探针NBD研究表明，随处理时间延长，细胞内荧光强度与对照组相比显著降低，Western blots研究表明，细胞核内的RhoA蛋白量随Asc2P6Plm处理时间延长而逐渐增加，研究提示，Asc2P6Plm对癌细胞浸润转移能力的抑制作用是与抑制癌细胞内的ROS，提高细胞核内RhoA水平，降低细胞质内F-actin相关。     

Release of eicosanoids from cultured rat aortic endothelial cells; studies with arachidonic acid and calcium ionophore A23187

The release of prostanoids from the three different vascular cell types derived from rat aortic explants has been studied in vitro. Under resting conditions and when incubated with exogenous arachidonic acid (AA, 10 microM), the endothelial cells (EC) produced the highest concentration of prostacyclin (PGI2 PGE2 PGF2 alpha TxA2). In contrast, PGE2 was the major prostanoid produced by the smooth muscle cells and fibroblasts. Pretreatment of EC with aspirin (10 microM) or indomethacin (10 microM) effectively inhibited the production of prostanoids by these cells. Incubation with the calcium ionophore A23187 (10 microM) did not stimulate production of PGI2 or leukotriene B4 (LTB4) by EC. However, treatment of EC with a combination of A23187 and AA led to production of amounts of both PGI2 and LTB4 which were greater than the summed values for the different drug treatments. These findings indicate that the concentration of substrate, AA, is a limiting factor in prostanoid formation by these cultured vascular cells but that rat EC are relatively poor in the enzymes required for leukotriene formation.     

Enhanced prostaglandin synthesis as a mechanism for inhibition of melanoma cell growth by ascorbic acid

Both ascorbic acid and the 1-series prostaglandins have been reported to be important regulators of cell growth and since ascorbic acid also increases the synthesis of the 1-series prostaglandins, it is possible that the effects of ascorbic acid on cell growth might be mediated by changes in 1-series prostaglandin synthesis induced by ascorbic acid. This study attempted to examine this possible relationship. The effects of ascorbic acid, prostaglandin E1 and the essential fatty acid precursors of the prostaglandins, linoleic acid and gamma-linolenic acid on the in vitro growth of transformed BL6 murine melanoma cells and untransformed monkey kidney (LLCMK) cells was determined. The effects of ascorbic acid addition on the growth inhibitory effect of the essential fatty acids and on the activity of delta-6-desaturase, a key enzyme in 1-series prostaglandin synthesis were also examined. Addition of ascorbic acid, prostaglandin E1 and both essential fatty acids was found to reduce BL6 growth while PGE1 and to a lesser extent the essential fatty acids reduced LLCMK cell growth. The growth inhibitory effect of the essential fatty acids was enhanced by ascorbic acid which was also found to stimulate delta-6-desaturase activity in BL6 cells. The growth inhibitory effect of ascorbic acid on BL6 cells may thus be mediated by changes in prostaglandin synthesis through an association with the metabolism of the essential fatty acid precursors of the prostaglandins.     

Reduced levels of PGE2 uptake by intact reno-medullary collecting tubule cells isolated from the spontaneously hypertensive rat and the identification of an intracellular PGE2 receptor/binding protein

Prostaglandin E2 (PGE2) is thought to be involved in the control of NaCl loading in the kidney. Since the ability to balance salt concentrations across the nephron appears to be impaired in the Spontaneously Hypertensive Rat (SHR), the uptakes of PGE by isolated medullary collecting tubule cells (MCT) from both SH and normotensive (NT) rats were compared. A rabbit antiserum directed against PGE2 revealed by flow cytometry the active internalisation of exogenous ligand by a high density fraction of intact MCT cells from NT tissue. Conversely, PGE2 uptake by the same fraction was markedly reduced. The monoclonal antibodies (MoAbs) W2.44 and SH6.6 raised against a 45,000 X g fraction of medullary tissue and which blocked binding of the anti-PGE2 serum, identified by Western blotting, an intracellular PGE2 receptor or binding protein (PGER) of 16 k daltons. No alteration in the structure or level of expression of this antigen could be detected in the MCT fractions isolated from the SHR. It is suggested that an impairment of PGE2 membrane transport in the renal medulla may be a contributory factor to the SH condition.     

Enhanced Protection Against Peroxidation-Induced Mortality of Aortic Endothelial Cells by Ascorbic Acid-2-O-Phosphate Abundantly Accumulated in the Cell as the Dephosphorylated Form

Bovine aortic endothelial BAE-2 cells exposed to the peroxidizing agent, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mitochondrial dehydrogenase activity as assessed by fluorometry using ethidium homodimer and photometry using WST-1, respectively. The cells were protected from t-BuOOH-induced injury more markedly by L-ascorbic acid-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, which is responsible for the antioxidant ability of L-ascorbic acid (Asc), than by Asc itself. In contrast, NDE-induced membrane disruption but not mitochondrial dysfunction was prevented by Asc2P, whereas Asc exhibited no prevention against both types of injury. The amount of intracellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 cells, where the intact form Asc2P was not detected, than in Asc-administered cells as assessed by HPLC of cell extract with detection by coulometric ECD and W. During transmembrane influx into the cell, Asc2P was concentrated as highly as 70- to 90-fold relative to the extracellular Asc2P concentration, whereas Asc was 8-to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell determined by [¹⁴C]PEG/gas chromatography. Thus, Asc2P but not Asc is highly concentrated in the aqueous phase of the cell after prompt dephosphorylation, and may thereby render the cell more resistant to t-BuOOH-peroxidation assumedly via scavenging of intracellular reactive oxygen species than to peroxidation with the less hydroplulic agent NDE.     

1,25-(OH)2D3 modulates growth plate chondrocytes via membrane receptor-mediated protein kinase C by a mechanism that involves changes in phospholipid metabolism and the action of arachidonic acid and PGE2.

1,25-(OH)2D3 (1,25) exerts its effects on growth plate chondrocytes through classical vitamin D (VDR) receptor-dependent mechanisms, resulting in mineralization of the extracellular matrix. Recent studies have shown that membrane-mediated mechanisms are involved as well. 1,25 targets cells in the prehypertrophic and upper hypertrophic zones of the costochondral cartilage growth plate (GC cells), resulting in increased specific activity of alkaline phosphatase (ALP), phospholipase A2 (PLA2), and matrix metalloproteinases (MMPs). At the cellular level, 1,25 action results in rapid changes in arachidonic acid (AA) release and re-incorporation, alterations in membrane fluidity and Ca ion flux, and increased prostaglandin E1 and E2 (PGE2) production. Protein kinase C (PKC) is activated in a phospholipase C (PLC) dependent-mechanism, due in part to the increased production of diacylglycerol (DAG). In addition, AA acts directly on the cell to increase PKC specific activity. AA also provides a substrate for cyclooxygenase (COX), resulting in PGE2 production. 1,25 mediates its effects through COX-1, the constitutive enzyme, but not COX-2, the inducible enzyme. Time course studies using specific inhibitors of COX-1 show that AA stimulates PKC activity and PKC then stimulates PGE2 production. PGE2 acts as a mediator of 1,25 action on the cells, also stimulating PKC activity. The rapid effects of 1,25 on PKC are nongenomic, occurring within 3 min and reaching maximal activation by 9 min. It promotes translocation of PKC to the plasma membrane. When 1,25 is incubated directly with isolated plasma membranes, PKCalpha is stimulated although PKCzeta is also present. In contrast, when isolated matrix vesicles (MVs) are incubated with 1,25, PKCzeta is inhibited and PKCalpha is unaffected. These membrane-mediated effects are due to the presence of a specific membrane vitamin D receptor (mVDR) that is distinct from the classical cytosolic VDR. Studies using 1,25 analogs with reduced binding affinity for the classical VDR, confirm that rapid activation of PKC by 1,25 is not VDR dependent. The membrane-mediated effects of 1,25 are critical to the regulation of events in the extracellular matrix produced by the chondrocytes. MVs are extracellular organelles associated with maturation of the matrix, preparing it for mineralization. MV composition is under genomic control, involving VDR-mechanisms. In the matrix, no new gene expression or protein synthesis can occur, however. Differential distribution of PKC isoforms and their nongenomic regulation by 1,25 is one way for the chondrocyte to control events at sites distant from the cell. GC cells contain 1a-hydroxylase and produce 1,25; this production is regulated by 1,25, 24,25, and dexamethasone. 1,25 stimulates MMPs in the MVs, resulting in increased proteoglycan degradation in mineralization gels, and increased activation of latent transforming growth factor-beta 1 (TGF-beta1).     

Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells

In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis, indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.     

Trans fatty acids in partially hydrogenated soybean oil inhibit prostacyclin release by endothelial cells in presence of high level of linoleic acid

Partially hydrogenated soybean oil (PHSBO) and natural soybean oil (SBO) were obtained from a commercial source and their fatty acids were fractionated into saturates, monoenes and diene fractions. The present study compared the effect of the total, monoene and diene fatty acid fractions of PHSBO with those of the SBO on the fatty acid composition of the cell phospholipids (PL) and the prostacyclin (PGI(2)) release by endothelial cells (EC) in culture. Results showed that arachidonic acid (AA) level decreased significantly and linoleic acid (LA) significantly increased in the cells incubated with the diene fraction or the monoene fraction of PHSBO plus 18:2 at 3:1 ratio compared to the cell incubated with those fractions of SBO. These changes were attributed to the inhibition of LA conversion to AA by trans 18:1 and 18:2 isomers present in the monoene or diene fractions of PHSBO leading to a significant decrease of PGI(2) released by the cells incubated with monoene or diene fractions of PHSBO. The cells incubated with the monoene of PHSBO or SBO plus 18:2 at a 1:1 ratio showed no inhibition of LA conversion to AA and the level of AA was almost equal in their PL, but the PGI(2) released by the cells incubated with the monoene of PHSBO was significantly less than the cells incubated with the monoene of SBO. This decrease was not related to the inhibition of PGI(2) synthesizing enzymes or phospholipase (PLA(2)) activities. Our data show that trans acids in PHSBO inhibited the PGI(2) release by the cells through controlling the level of AA as substrate, either by (a) inhibiting the conversion of LA to AA or (b) by shunting the free AA released by the PLA(2) action to metabolism by another pathway leaving less AA available for PGI(2) synthesis.     

Fumonisin B1 influenced the effects of arachidonic acid, prostaglandins E2 and A2 on cell cycle progression, apoptosis induction, tyrosine- and CDC2-kinase activity in oesophageal cancer cells

In a previous study, we showed that, of a group of lipids including arachidonic acid (AA), prostaglandins E2 (PGE2) and A2 (PGA2), PGA2 had the most marked effect on the inhibition of cell growth, activation of tyrosine kinase activity, lowering of the number of G1-phase cells, and induction of p53 levels in oesophageal carcinoma (WHCO3) cells. No significant effects by the three lipids were seen in normal monkey kidney cells. In the present study, the effects of the inhibitor of ceramide synthesis, fumonisin B1 (FB1), a metabolite of Fusarium verticillioides (= F. moniliforme) which is implicated in the high incidence of oesophageal cancer, were determined on AA, PGE2 and PGA2 WHCO3 treated cells. In the presence of FB1, the lipid-enhanced tyrosine kinase activity was lowered. Flow cytometric and morphological studies showed that FB1 lowered the marked apoptosis induced by especially PGA2. FB1, however, in combination with AA, PGE2 or PGA2 increased the number of G2/M cells. AA>PGE2>PGA2 alone decreased CDC2-kinase activity, but, in the presence of FB1, CDC2-kinase activity was significantly increased. The PGA2- and AA-induced p53 levels were lowered in the presence of FB1. We concluded that FB1 diminished the cytotoxic effects of the lipids on oesophageal tumour cells.     

Incorporation and effect of arachidonic acid on the growth of human myeloma cell lines

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.     

Colony-forming capacity of porcine liver epithelial cells in culture

Previously, we reported the presence of certain nonparenchymal epithelial cells (NPECs) in adult porcine livers that demonstrate differentiation patterns including an emergence of duct-like structures (DLSs) in the colonies. In the present study, we examined the effect of supplements to the NAIR-1 medium (Dulbecco modified Eagle medium [DMEM]-F12 containing 5% fetal bovine serum [FBS] and 11 supplements) used in these cultures on formation of DLSs-emerged colonies (type I colonies). No type I colonies were observed in the cultures of the nonparenchymal cell fraction when Roswell Park Memorial Institute-1640 medium or DMEM-F12 (1:1) supplemented with 5% FBS was used as the culture medium. NAIR-1 medium without each component did not produce any significant results. No type I colonies were formed when epidermal growth factor, and hydrocortisone and insulin mixture (A) or nicotinamide and l-ascorbic acid phosphate magnesium salt (Asc2P) mixture (B) was added to the DMEM-F12 medium supplemented with 5% FBS. However, when a combination of A and B was added, colonies were formed at a significant level. Together, the number of type I colonies was increased in the combination of A and B containing a higher concentration of Asc2P. We conclude that NPECs need a mixture of Asc2P and other components as supplements for type 1 colony formation.     

Comparative study of the effects of polyunsaturated fatty acids and their metabolites on cell growth and tyrosine kinase activity in oesophageal carcinoma cells

The effects of exogenous gamma-linolenic acid (GLA), arachidonic acid (AA), prostaglandin E2 (PGE2) and prostaglandin A2 (PGA2) were evaluated on cell growth in two squamous oesophageal carcinoma cell lines, WHCO1 and WHCO3 and normal monkey kidney (NMK) cells. In both cancer cell lines all four compounds inhibited cell growth significantly. Indomethacin (I) alone, or in combination with either GLA or AA, caused marked inhibition of cell growth in WHCO3. Total tyrosine kinase (TK) activity was determined after exposure of all three cell types to the lipid compounds. Negligible differences were observed in TK activity between treated and untreated NMK cells. Small increases were noticed in WHCO1. Marked TK stimulation was observed in WHCO3. Addition of indomethacin to WHCO3 also increased TK activity above control value. Tyrosine phosphorylation status of exposed cells indicated that a band of approximately 55 kDa (approximately 55 kDa) was primarily influenced in both WHCO3 and WHCO1. PGA2 caused a decrease in tyrosine phosphorylation of the approximately 55 kDa protein in all three cell types. Negligible differences were observed in the tyrosine phosphorylation status of the approximately 55 kDa in NMK cells exposed to GLA, AA and PGE2 respectively. However, tyrosine phosphorylation of a number of other proteins (21.5-97.4 kDa) was observed in NMK cells. Flow cytometry studies showed an increase in S phase and decrease in G1 phase in WHCO3 exposed to PGE2 and PGA2. Indomethacin alone, or in combination with GLA and AA, respectively, lead to an increase in G1 and a decrease in S phase. Induction of p53 levels was observed in WHCO3 cells exposed to GLA, AA, PGA2, indomethacin and the combination of indomethacin and GLA or AA.     

Anti-apoptotic effects of arachidonic acid and prostaglandin E2 in pancreatic beta-cells.

BACKGROUND/AIMS: The polyunsaturated fatty acid arachidonic acid (AA) has been implicated in beta-cell defence mechanisms and prostaglandin (PG) products of cyclooxygenase (COX) 2 action confer resistance to alloxan-induced apoptosis in insulin-secreting RIN cells. We have now investigated the anti-apoptotic effects of AA and its metabolite, PGE(2), in the MIN6 mouse insulin-secreting beta-cell line and mouse islets. METHODS: Apoptosis was determined in MIN6 beta-cell and mouse islet extracts by measurement of capase-3 activity, and COX2 mRNA levels were quantified by real-time RT-PCR. RESULTS: Exposure of MIN6 cells to AA (3.1-12.5 microM) caused concentration-dependent reductions in apoptosis, and similar results were obtained when endogenous AA levels were elevated in cytosolic phospholipase A(2)-overexpressing MIN6 cells. 25mM glucose caused both a significant up-regulation of MIN6 cell COX2 mRNA levels and a decrease in apoptosis. Inhibition of MIN6 cell COX2 activity with a selective inhibitor, NS-398 (10-100 microM), increased apoptosis and exogenous PGE(2) (0.2-5 microM) reduced NS-398-induced apoptosis in a concentration-dependent manner. The protective effects of AA and PGE(2) were also observed in primary mouse islets. CONCLUSION: These data show that AA and its COX2-generated metabolite, PGE(2), can protect beta-cells from apoptosis.     

The protective effect of ascorbic acid derivative on PC12 cells: involvement of its ROS scavenging ability

L-ascorbic acid 2-phosphate-6-palmitate (Asc2P6P) was synthesized and its effect on the damage of PC12 cells induced by H2O2 was investigated. 200 microM H2O2 in a treatment period of 4 hours in our experiment resulted in substantial cell loss. With the increasing concentration of antioxidants, such H2O2-induced cytotoxicity was significantly prevented and the corresponding intracellular and extracellular ROS levels decreased concurrently by pre-treatment with Asc2P6P and Asc. It was found that Asc2P6P was superior to L-ascorbic acid in its protective role and showed a dose-dependent manner during a 24-hour treatment. The higher potency of Asc2P6P's protective role on PC12 cells was correlated with its more effective ROS scavenging ability. HPLC assay demonstrated that Asc2P6P could easily enter the cells and be converted into Asc persistently, which contributed to its distinguished role in protecting PC12 cells against H2O2-induced cytotoxicity.     

Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells

Sodium-dependent vitamin C transporter (SVCT) 2-mediated L-ascorbic acid (AA) uptake is required in osteoblast-like differentiation of MC3T3-E1 cells, and prostaglandin E2 (PGE2) is among the most important local factors in bone formation, but the detailed mechanism by which PGE2 induces osteoblast differentiation remains obscure. We revealed that PGE2 induced AA uptake and osteoblast-like differential markers including alkaline phosphatase, collagen, osteocalcin expression, and mineralization in MC3T3-E1 cells. Inhibition of AA uptake by SVCT2 short isoform functioning as a dominant-negative mutant not only robustly attenuated PGE2-induced markers expression and mineralization, but also decreased their basal levels. However, upregulation of AA uptake resulted from PGE2-induced plasma membrane translocation of cytoplasm SVCT2, and this effect was abolished by pretreatment with EP4 receptor antagonist, AH-23848B or cAMP-dependent protein kinase A (PKA) inhibitor, H-89. Moreover, we showed SVCT2 physically interacted with PKA in immunoprecipitates, and PKA phosphorylated SVCT2 in vitro and in intact cells at Ser402 and Ser639 sites; however, mutation of Ser402 or/and Ser639 in SVCT2 severely diminished SVCT2 translocation in response to PGE2. Together, these results suggest that PGE2-induced SVCT2 plasma membrane translocation through EP4 receptor and subsequent phosphorylation of SVCT2 at Ser402 and Ser639 sites by PKA results in an increase of AA uptake and consequent promotion of osteoblast-like differentiation in MC3T3-E1 cells.     

Phytosterols decrease prostaglandin release in cultured P388D1/MAB macrophages

Cardiovascular disease (CVD) remains the leading cause of death in Western societies. Atherosclerosis is a major cardiovascular related disorder that is responsible for 50% of all mortality in the United States. Several epidemiological studies suggest that consumption of a plant-based diet is associated with a decreased incidence of cardiovascular abnormalities. Phytosterols, especially beta-sitosterol, are plant sterols that have been shown to exert protective effects against cardiovascular diseases as well as many types of cancer. Monocyte/macrophage cells are involved with the inflammatory process. Accumulation of these cells in arteries is one of the initial events leading to atherosclerosis. Macrophages are capable of supplying the atherosclerotic vessel with substantial amounts of prostaglandins. Prostaglandins have been shown by numerous studies to play a key role in the atherosclerosis process. They can affect platelet aggregation, vasodilation or constriction of blood vessels, and the adherence of monocytes to the vessel walls. The purpose of this study was to examine the effect of phytosterols on the release of PGE(2) and PGI(2) from lipopolysaccharide (LPS)-stimulated P388D(1)/MAB macrophage cells. P388D(1)/MAB cells were supplemented with 16 microM cholesterol, beta-sitosterol or campesterol using cyclodextrin as a vehicle. Phytosterol supplementation led to a significant decrease in cellular growth at various time points throughout a 7-day treatment period, especially after 3 days of treatment. Macrophages incorporated the supplemented phytosterols into their membranes which accounted for 26% of total membrane sterols. Cholesterol supplementation at 16 microM however, had no effect on membrane sterols. Supplementation with 16 microM concentration of beta-sitosterol or campesterol resulted in a significant inhibition of PGE(2) and PGI(2) release from macrophage cells as compared to the vehicle control. Of the two phytosterols, beta-sitosterol supplementation exhibited a greater inhibitory effect. PGE(2) release was decreased 68% by beta-sitosterol and 55% by campesterol, while cholesterol supplementation was not as effective, as it led to a 37% decrease. Similarly, release of PGI(2) from macrophages was inhibited 67% by beta-sitosterol and 52% by campesterol treatment, while enrichment of the cells with cholesterol, led to a 35% decrease in PGI(2) release. The decrease in prostaglandin release was not due to alteration in the expression of cPLA(2) and COX-2 enzymes which suggests that alterations in the activities of these enzymes may be responsible for the observed changes in prostaglandin release. It was concluded that phytosterol incorporation into macrophages may offer protection from atherosclerosis by reducing their prostaglandin release and thus slowing down the atheroma development.     

Arachidonic acid regulation of prostanoid synthesis in macrophages

The dynamics of prostaglandin (PG) E2 synthesis by mouse peritoneal macrophages during the delivery of the basic substrate, arachidonic acid (AA), from different sources to the enzyme system of the cells was investigated. The dynamics of PGE2 synthesis in these cells was studied both after addition of exogenous AA and after stimulating the liberation of AA from intracellular pools with the calcium ionophore A23187. The kinetics of PGE2 synthesis when AA was supplied from intracellular and extracellular sources were absolutely different. PGE2 metabolism and the inactivation of the key enzyme of PG synthesis (PGH-synthase) during the reaction may be the regulating factors in the kinetics of PGE2 synthesis in the cells. For the different sources of AA in the cells, the rate constants of PGE2 consumption (k2) and PGH-synthase inactivation in the course of the reaction (kin) were calculated. The experimentally determined value of the apparent rate constant kin was identical to the theoretically calculated kin value for the case when AA was provided from an intracellular source. An observed deceleration in the PGE2 synthesis kinetics from exogenous AA is characterized by a 10-fold drop in the apparent kin and k2 values. The possibility of prostanoid synthesis regulation at the level of the traditional, constitutive isoenzyme PGH-synthase-1 is discussed.     

Properties of prostaglandin-sensitive adenylate cyclase system of a murine macrophage-like cell line (P388D1)

The properties of basal and prostaglandin (PG)-stimulated adenylate cyclase of membrane preparations of P388D1 cells were investigated. Three partially purified membrane fractions were obtained by sucrose density gradient centrifugation at the final step of purification from crude homogenate. About 96% of the basal and 89% of PGE2-stimulated adenylate cyclase activity in the homogenate were recovered in three membrane fractions. Two lighter membrane fractions (I and II), which were enriched 11-fold and 8.4-fold in adenylate cyclase activity over crude homogenate, were pooled and subjected to various studies. Results suggested that the basal activity of the membrane preparations has, as in many other cell types, a relatively broad pH optimum (pH 7.5 to 8.5), requires Mg2+, which must be present in excess ATP, and is inhibited by Ca2+. Highly reactive sulfhydryl group(s), which may be present in the lipid bilayer, is required for the adenylate cyclase activity. Because both fluoride ions and GTP augment the enzymatic activity, P388D1 cell membrane adenylate cyclase must possess stimulatory guanine nucleotide-binding protein. The membrane preparations respond to exogeneously added PG by 1.5-fold to 3-fold increase in adenosine 3'-5' cyclic monophosphate (cAMP) production. The magnitude of PG-responsiveness was dependent on the types of PG and the order of potency in stimulation was PGE1 greater than PGE2 greater than PGI2. PGA1, B1, B2, F1 alpha, and F2 alpha stimulated adenylate cyclase only at the highest concentration tested.     

Effects of arachidonic acid, docosahexaenoic acid and prostaglandin E(2) on cell proliferation and morphology of MG-63 and MC3T3-E1 osteoblast-like cells

During bone remodelling bone is resorbed by osteoclasts and replaced again by osteoblasts through the process of bone formation. Clinical trials and in vivo animal studies suggest that specific polyunsaturated fatty acids (PUFAs) might benefit bone health. As the number of functional osteoblasts is important for bone formation the effects of specific PUFAs on in vitro osteoblastic cell proliferation were investigated. Morphological studies were conducted to determine whether exposure of the cells to these agents caused structural damage to the cells thereby yielding invalid results. Results from this study showed that arachidonic acid (AA) and docosahexaenoic acid (DHA) both inhibit cell growth significantly at high concentrations. The anti-mitotic effect of AA is possibly independent of PGE(2) production, as PGE(2) per se had little effect on proliferation. Further study is required to determine whether reduced proliferation due to fatty acids could be due to increased differentiation of osteoblasts to the mature mineralising osteoblastic phenotype.