Regulatory roles of G-protein coupled receptors in adipose tissue metabolism and their therapeutic potential.

The high incidence of obesity has increased the need to discover new therapeutic targets to combat obesity and obesity-related metabolic diseases. Obesity is defined as an abnormal accumulation of adipose tissue, which is one of the major metabolic organs that regulate energy homeostasis. However, there are currently no approved anti-obesity therapeutics that directly target adipose tissue metabolism. With recent advances in the understanding of adipose tissue biology, molecular mechanisms involved in brown adipose tissue expansion and metabolic activation have been investigated as potential therapeutic targets to increase energy expenditure. This review focuses on G-protein coupled receptors (GPCRs) as they are the most successful class of druggable targets in human diseases and have an important role in regulating adipose tissue metabolism. We summarize recent findings on the major GPCR classes that regulate thermogenesis and mitochondrial metabolism in adipose tissue. Improved understanding of GPCR signaling pathways that regulate these processes could facilitate the development of novel pharmacological approaches to treat obesity and related metabolic disorders.

Role of S1P/S1PR3 axis in release of CCL20 from human bronchial epithelial cells.

BACKGROUND: Sphingosine kinase phosphorylates sphingosine to generate sphingosine 1 phosphate (S1P) following stimulation of the five plasma membrane G-protein-coupled receptors. The objective of this study is to clarify the role of S1P and its receptors (S1PRs), especially S1PR3 in airway epithelial cells. METHODS: The effects of S1P on asthma-related genes expression were examined with the human bronchial epithelial cells BEAS-2B and Calu-3 using a transcriptome analysis and siRNA of S1PRs. To clarify the role of CCL20 in the airway inflammation, BALB/c mice were immunized with ovalbumin (OVA) and subsequently challenged with an OVA-containing aerosol to induce asthma with or without intraperitoneal administration of anti-CCL20. Finally, the anti-inflammatory effect of VPC 23019, S1PR1/3 antagonist, in the OVA-induced asthma was examined. RESULTS: S1P induced the expression of some asthma-related genes, such as ADRB2, PTGER4, and CCL20, in the bronchial epithelial cells. The knock-down of SIPR3 suppressed the expression of S1P-inducing CCL20. Anti-CCL20 antibody significantly attenuated the eosinophil numbers in the bronchoalveolar lavage fluid (P<0.01). Upon OVA challenge, VPC23019 exhibited substantially attenuated eosinophilic inflammation. CONCLUSIONS: S1P/S1PR3 pathways have a role in release of proinflammatory cytokines from bronchial epithelial cells. Our results suggest that S1P/S1PR3 may be a possible candidate for the treatment of bronchial asthma.

Fingolimod confers neuroprotection through activation of Rac1 after experimental germinal matrix hemorrhage in rat pups.

Fingolimod, a sphingosine-1-phosphate receptor (S1PR) agonist, is clinically available to treat multiple sclerosis and is showing promise in treating stroke. We investigated if fingolimod provides long-term protection from experimental neonatal germinal matrix hemorrhage (GMH), aiming to support a potential mechanism of acute fingolimod-induced protection. GMH was induced in P7 rats by infusion of collagenase (0.3 U) into the right ganglionic eminence. Animals killed at 4 weeks post-GMH received low- or high-dose fingolimod (0.25 or 1.0 mg/kg) or vehicle, and underwent neurocognitive testing before histopathological evaluation. Subsequently, a cohort of animals killed at 72 h post-GMH received 1.0 mg/kg fingolimod; the specific S1PR1 agonist, SEW2871; or fingolimod co-administered with the S1PR1/3/4 inhibitor, VPC23019, or the Rac1 inhibitor, EHT1864. All drugs were injected intraperitoneally 1, 24, and 48 h post-surgery. At 72 h post-GMH, brain water content, extravasated Evans blue dye, and hemoglobin were measured as well as the expression levels of phospho-Akt, Akt, GTP-Rac1, Total-Rac1, ZO1, occludin, and claudin-3 determined. Fingolimod significantly improved long-term neurocognitive performance and ameliorated brain tissue loss. At 72 h post-GMH, fingolimod reduced brain water content and Evans blue dye extravasation as well as reversed GMH-induced loss of tight junctional proteins. S1PR1 agonism showed similar protection, whereas S1PR or Rac1 inhibition abolished the protective effect of fingolimod. Fingolimod treatment improved functional and morphological outcomes after GMH, in part, by tempering acute post-hemorrhagic blood-brain barrier disruption via the activation of the S1PR1/Akt/Rac1 pathway.

Regulation of endothelial nitric oxide synthase activation in endothelial cells by S1P1 and S1P3.

Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis. Lysophospholipid interaction with sphingosine 1-phosphat (S1P) receptors results in eNOS activation in different cells. In endothelial cells, eNOS activation via S1P1 or S1P3 was shown controversially. The aim of this study is to investigate the meaning of both S1P receptors for eNOS activation in human endothelial cells. Therefore, several S1P1 and S1P3 agonists in combination with antagonists and specific RNAi approach were used. eNOS activation was measured in human umbilical vein endothelial cells (HUVEC) via DAF2-DA-based fluorescence microscopy. For investigation of the signaling pathway, agonists/antagonist studies, RNAi approach, Luminex™ multiplex, and Western Blot were used. In HUVEC, both the S1P1 agonist AUY954 as well as the S1P1,3 agonist FTY720P induced eNOS activation in a time- and dose-dependent manner. Other S1P1 agonists activated eNOS to a lesser extent. The AUY954-induced eNOS activation was blocked by the S1P1 antagonist W146, the combination of W146 and the S1P3 antagonist CAY10444 and the S1P1,3 antagonist VPC23019, but not by CAY10444 indicating the meaning of S1P1 for the AUY954-induced eNOS activation. The FTY720P-induced eNOS activation was blocked only by the combination of W146 and CAY10444 and the combined S1P1,3 antagonist VPC23019, but not by W146 or CAY10444 indicating the importance of both S1P1 and S1P3 for FTY720-induced eNOS activation. These results were confirmed using specific siRNA against S1P1 and S1P3. The S1P1,3 activation results in Akt phosphorylation and subsequent activation of eNOS via phosphorylation at serine(1177) and dephosphorylation at threonine(495). Beside former investigations with rather unspecific S1P receptor activation these data show potent selective S1P1 activation by using AUY954 and with selective S1P receptor inhibition evidence was provided that both S1P1 and S1P3 lead to downstream activation of eNOS in HUVEC in the same experimental setting. Inhibition or knockdown of one of these receptor subtypes did not abolish the eNOS activation and subsequent NO production.

Regulation of Bax/mitochondria interaction by AKT.

Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated.

FcepsilonRI and Thy-1 domains have unique protein and lipid compositions.

Receptor activation leads to the dynamic remodeling of the plasma membrane. Previous work using immunoelectron microscopy showed that aggregated high-affinity receptor for immunoglobulin E (FcRI) and aggregated Thy-1, a glycerophosphoinositol (GPI)-anchored protein, have distinct membrane distributions. We now report lipidomics analysis of FcRI- and Thy-1-enriched vesicles obtained by magnetic bead isolation in the absence of detergent. Protein analyses show that FcRI domains are enriched in receptors and associated signaling molecules, whereas Thy-1 domains are devoid of FcRI subunits. Positive and negative ion electrospray mass spectrometry demonstrated that both domains retained a complex mixture of phospholipid classes and molecular species, predominantly glycerophosphocholine, glycerophosphoethanolamine (GPE), and sphingomyelin as well as glycerophosphoserine and GPI lipids. Analysis of total acyl groups showed that < 50% of fatty acids in these domains are fully saturated, inconsistent with the recruitment of aggregated receptors or GPI-anchored proteins to liquid ordered domains. However, further analysis showed that FcRI domains contain two times more sphingomyelin and a high ratio of cholesterol to total fatty acid content compared with Thy 1-enriched domains. Remarkably, plasmenyl glycerophosphoethanolamine phospholipids (plasmalogen GPE) were also 2.5-3 times more abundant in FcRI domains than in the Thy-1 microdomains, whereas most diacyl GPE molecular species were equally abundant in the two domains.

Conformationally locked isostere of phosphoSer-cis-Pro inhibits Pin1 23-fold better than phosphoSer-trans-Pro isostere.

Stereoisomeric cis and trans substrate analogues for Pin1 were designed and synthesized. The central phosphoSer-Pro core of the Pin1 substrate was replaced by cis and trans amide isosteres in Ac-Phe-Phe-pSer-Psi[(Z and E)CH=C]-Pro-Arg-NH(2), 1 and 2, peptidomimetics. They were synthesized on solid phase in 17% yield for the cis analogue 1, and 16% yield for the trans analogue 2. A second trans amide isostere with a C-terminal N-methylamide 3 was synthesized in 7% yield. The protease-coupled Pin1 assay showed that all three compounds inhibited the Pin1 peptidyl-prolyl isomerase (PPIase) enzymatic activity. The cis isostere 1 was 23 times more potent (K(i) = 1.74 +/- 0.08 muM) than its trans counterpart 2 (K(i) = 40 +/- 2 muM) in competitive inhibition of Pin1. These results suggest that the catalytic site of Pin1 binds cis substrates more tightly in aqueous solution. Antiproliferative activity toward the A2780 human ovarian cancer cell line by the cis and trans analogues correlates with Pin1 inhibition results.

Novel effect of N-palmitoyl-L-serine phosphoric acid on cytosolic Ca2+ levels in human osteoblasts.

The effect of N-palmitoyl-L-serine phosphoric acid (L-NASPA), which has been used as an inhibitor of lysophosphatidic acid receptors, on intracellular Ca2+ concentration ([Ca2+]i) in human osteosarcoma MG63 cells was measured by using fura-2. L-NASPA (0.1-10 microM) caused a rapid and transient plateau [Ca2+]i rise in a concentration-dependent manner (EC50=0.5 microM). The L-NASPA-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+ but was not altered by L-type voltage-gated Ca2+ channel blockers. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, induced a [Ca2+]i rise, after which the increasing effect of L-NASPA on [Ca2+]i was completely inhibited; also, pretreatment with L-NASPA partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phospholipase C, abolished histamine (but not L-NASPA)-induced [Ca2+]i rise. Overnight incubation with 1 microM L-NASPA did not affect cell proliferation, but 10-20 microM L-NASPA exerted 4% and 15% inhibition, respectively. Collectively, L-NASPA rapidly increased [Ca2+]i in MG63 cells by evoking both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at higher concentrations.

Amphetamine increases phosphorylation of extracellular signal-regulated kinase and transcription factors in the rat striatum via group I metabotropic glutamate receptors.

Amphetamine is an indirect dopamine receptor agonist and increases glutamate release in the striatum. Activation of group I metabotropic glutamate receptors (mGluRs) upregulates cAMP response element-binding protein (CREB) and Elk-1 phosphorylation via extracellular signal-regulated kinase 1 and 2 (ERK1/2) in the striatum in vivo. In the present study the role of mGluRs in the regulation of ERK1/2 pathways leading to CREB and Elk-1 phosphorylation by amphetamine was investigated using immunohistochemistry and Western blot in the rat dorsal striatum. Acute administration of amphetamine (5 mg/kg, i.p.) caused increases in phosphorylated (p)CREB, pElk-1, and pERK1/2 immunoreactivity. Intrastriatal blockade of group I mGluRs with N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 25 nmol) significantly attenuated amphetamine-induced pCREB, pElk-1, pERK1/2, and Fos immunoreactivity in both medial and lateral areas of the striatum. Systemic injection of an mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 10 mg/kg, i.p.), also blocked the amphetamine induction of these phosphoproteins. In contrast, intrastriatal blockade of group II/III mGluRs with (RS)-alpha-methylserine-o-phosphate monophenyl ester (MSOPPE; 25 nmol) did not affect amphetamine-induced increases in all the four markers. Similarly, intrastriatal dantrolene (2 or 20 nmol) that blocks intracellular Ca(2+) release from ryanodine-sensitive stores did not affect amphetamine effects. Injection of PHCCC, MPEP, MSOPPE, or dantrolene alone did not alter basal levels of the three phosphoproteins and Fos. These data suggest that acute amphetamine is able to facilitate the phosphorylation of CREB, Elk-1, and ERK1/2 signaling proteins and Fos gene expression via a group I mGluR-dependent mechanism in the dorsal striatum.

Selective effect of cholesterylphosphoserine on intracellular cholesterol transport.

Cholesteryl-3beta-phosphoserine (CPHS) is a synthetic steroid affecting intracellular cholesterol transport. To compare CPHS with the well-known inhibitors progesterone and U18666A, we examined cholesterol transport in three human cell lines: the monocytic U-937, the endothelial ECV-304, and the lymphoid Jurkat. Under low density lipoprotein (LDL) loading, CPHS inhibited cholesterol esterification in U-937 and ECV-304 cells but not in Jurkat cells. In contrast, CPHS inhibited the mobilization of plasma membrane cholesterol induced by 25-hydroxycholesterol, brefeldin A, or sphingomyelinase in all cell lines. In cells pulse-labeled with [3 H]cholesterol, CPHS decreased incorporation of cholesterol and inhibited its esterification. In prelabeled cells, CPHS promoted cholesterol efflux and enhanced the cyclodextrin-mediated removal of plasma membrane cholesterol. CPHS did not affect endogenous cholesterol synthesis nor acylcoenzyme A:cholesterol acyltransferase activity. These data suggest that, unlike progesterone and U18666A, CPHS inhibits intracellular cholesterol transport by specifically affecting the movements of cholesterol in the plasma membrane. Owing to this restricted site of action, CPHS may help to clarify the role of the plasma membrane in cholesterol trafficking. For example, the lack of an effect of CPHS on the esterification of LDL-derived cholesterol in Jurkat cells suggests that most of the LDL-derived cholesterol in these cells is directly delivered to the endoplasmic reticulum without cycling through the plasma membrane.

Group I metabotropic glutamate receptor activation increases phosphorylation of cAMP response element-binding protein, Elk-1, and extracellular signal-regulated kinases in rat dorsal striatum.

Cyclic AMP response element-binding protein (CREB) is a major transcriptional activator at the calcium and cAMP response-element (CaCRE). Phosphorylated (p)CREB facilitates gene expression in striatal neurons. Elk-1 is another transcriptional regulator at the serum response element in the upstream promoter region of the CaCRE. Elk-1 is phosphorylated by extracellular signal-regulated kinases (ERK) and may also contribute to the regulation of gene expression. To evaluate putative roles of group I metabotropic glutamate receptors (mGluRs) in CREB, Elk-1, and ERK phosphorylation, the group I selective agonist, 3,5-dihydroxyphenylglycine (DHPG), was infused into the dorsal striatum at doses of 125, 250, or 500 nmol in freely moving rats. Semi-quantitative immunohistochemistry demonstrated that DHPG significantly increased levels of pCREB, pElk-1, and pERK immunoreactivity of ipsilateral dorsal striatum in a dose dependent manner. The increased immunoreactivity by 500 nmol DHPG was significantly blocked by intrastriatal infusion of the group I selective antagonist, n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC, 25 nmol), but not by the group II/III antagonist, (RS)-alpha-methylserine-o-phosphate monophenyl ester (MSOPPE, 25 nmol). These data suggest that group I mGluR activation is positively linked to signaling cascades resulting in CREB, Elk-1, and ERK phosphorylation in the striatum in vivo.

Lysophosphatidic acid-independent platelet activation by low-density lipoprotein.

Mildly oxidized low-density lipoprotein activates platelets through lysophosphatidic acid (LPA). Hence, the platelet-activating properties attributed to native low-density lipoprotein (nLDL) might be caused by LPA contamination. We show that nLDL enhances thrombin receptor-activating peptide (TRAP)-induced fibrinogen binding to alpha(IIb)beta(3). The LPA receptor blocker N-palmitoyl-L-serine-phosphoric acid did not affect nLDL-enhanced fibrinogen binding induced by TRAP, but reduced TRAP-induced binding. cAMP and inhibitors of protein kinase C and Ca(2+) rises completely blocked ligand binding by TRAP and nLDL/TRAP. Inhibitors of p38(MAPK) and ADP secretion interfered only partially. Blockade of Rho-kinase increased ligand binding 2-3-fold. We conclude that nLDL enhances TRAP-induced fibrinogen binding independent of LPA.

Leaving group and gas phase neighboring group effects in the side chain losses from protonated serine and its derivatives.

The gas phase fragmentation reactions of protonated serine and its YNHCH(CH2X)CO2H derivatives, beta-chloroalanine, S-methyl cysteine, O-methyl serine, and O-phosphoserine, as well as the corresponding N-acetyl model peptides have been examined via electrospray ionization tandem mass spectrometry (MS/MS). In particular, the competition between losses from the side chain and the combined loss of H2O and CO from the C-terminal carboxyl group of the amino acids or H2O or CH2CO from the N-acetyl model peptides are compared. In this manner the effect of the leaving group (Y = H or CH3CO, vary X) or of the neighboring group can be examined. It was found that the amount of HX lost from the side chain increases with the proton affinity of X [OP(O)(OH)2 > OCH3 approximately equals OH > Cl]. The ion due to the side chain loss of H2O from the model peptide N-acetyl serine is more abundant than that from protonated serine, suggesting that the N-acetyl group is a better neighboring group than the amino group. Ab initio calculations at the MP2(FC)/6-31G*//HF/6-31G* level of theory suggest that this effect is due to the transition state barrier for water loss from protonated N-acetyl serine being lower than that for protonated serine. The mechanism for side chain loss has been examined using MS3 tandem mass spectrometry, independent synthesis of proposed product ion structures combined with MS/MS, and hydrogen/deuterium exchange. Neighboring group rather than cis 1,2 elimination processes dominate in all cases. In particular, the loss of H3PO4 from O-phosphoserine and N-acetyl O-phosphoserine is shown to yield a 3-membered aziridine ring and 5-membered oxazoline ring, respectively, and not the dehydroalanine moiety. This is in contrast to results presented by DeGnore and Qin (J. Am. Soc. Mass Spectrom. 1998, 9, 1175-1188) for the loss of H3PO4 from larger peptides, where dehydroalanine was observed. Alternate mechanisms to cis 1,2 elimination, for the formation of dehydroalanine in larger phosphoserine or phosphothreonine containing peptides, are proposed.

The immunosuppressant steroid cholesterylphosphoserine inhibits tumour necrosis factor-alpha secretion in vitro and in vivo.

The synthetic steroid cholesterylphosphoserine (CPHS) inhibited the secretion of TNF-alpha in lipopolysaccharide-challenged human monocytes. CPHS (5-20 microM) was effective when added together with the endotoxin, or after an interval (1-2 h) sufficient to have allowed for initiation of TNF-alpha synthesis. Consistently, CPHS did not alter TNF-alpha gene transcription. In contrast to its action on TNF-alpha, CPHS showed only marginal effects on interleukin 1beta secretion. Given intraperitoneally to mice 2 h before lipopolysaccharide CPHS prevented the rise in plasma TNF-alpha (IC(50): 5 mg/kg). The inhibition of TNF-alpha secretion by CPHS may contribute to the immunosuppressive activity of this steroid.

Opposite effects on cytosolic Ca2+ of antitumor phospholipids by induction of calcium influx and activation of endoplasmic reticulum Ca(2+)-ATPase.

The ability of four different antitumor phospholipids, 1-O-hexadecyl-2-chloro-2-deoxyglycero-3-phosphocholine (ET16CIPC), hexadecylphosphocholine (C16OPC), hexadecylphospho-L-serine analogs (C16OPS, C16OPS-N-Ac) and cytidine-5'-hexadecylphosphonophosphate (C16PCMP) to modulate the cytosolic Ca2+ concentration [Ca2+]i was studied in an immortalized human mammary epithelial cell line H184 A1N4. The compounds induced different modes of activity depending on their structure and concentration. ET16CIPC induced between 0.31 and 5 microM a concentration dependent transient increase which was followed by a sustained increase at 10 microM. Studies using LaCl3 and Mn2+ quench of the Fura-2 fluorescence indicated that both effects are the result of an extracellular Ca2+ influx. Low concentrations of C16OPC, C16OPS and C16OPS-N-Ac induced no, or only a small, transient increase, whereas C16PCMP caused a decrease in [Ca2+]i. Thapsigargin and cyclopiazonic acid, specific inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, prolonged the transient [Ca2+]i increase following ET16CIPC concentration dependently, increased markedly the small transient increase following C16OPC and the C16-phosphoserine analogs and converted the decrease in the basal [Ca2+]i level induced by C16PCMP to an increase. The identical effects with thapsigargin and cyclopiazonic acid provide evidence that the [Ca2+]i response observed is an expression of the balance between the ability of an analog to raise [Ca2+]i and to remove Ca2+ by activation of the endoplasmic reticulum Ca(2+)-ATPase. This behaviour might contribute to the antiproliferative effectiveness of antitumor phospholipids.

Characterization of a receptor subtype-selective lysophosphatidic acid mimetic.

Despite an intriguing cell biology and the suggestion of a role in pathophysiological responses, the mechanism of action of such lipid phosphoric acid mediators as lysophosphatidic acid (LPA) remains obscure, in part because of an underdeveloped medicinal chemistry. We report now the agonist activity of a synthetic phospholipid in which the glycerol backbone of LPA is replaced by L-serine. Like LPA, the L-serine-based lipid mobilizes calcium and inhibits activation of adenylyl cyclase in the human breast cancer cell line MDA MB231. Treatment with LPA desensitizes MDA MB231 cells to subsequent application of the L-serine compound; when the order of application is reversed, however, the L-serine compound does not prevent calcium mobilization by LPA, which might indicate the existence of two LPA receptors in these cells. The analogous D-serine-based phospholipid was distinctly less potent than the L-isomer in those assays; this finding demonstrates stereoselectivity by an LPA receptor. Unlike LPA, the L-serine-based lipid does not evoke a chloride conductance in Xenopus laevis oocytes, but injection of poly(A)+ RNA from HEK 293 cells confers this phenotype on the oocyte. The latter result has practical importance in that it allows use of the frog oocyte for expression cloning of an LPA receptor DNA, an assay system made problematic by the oocyte's strong endogenous response to LPA.

Cellular signalling as a target in cancer chemotherapy. Phospholipid analogues as inhibitors of mitogenic signal transduction.

Mitogenic signalling mechanisms emerged as novel targets for tumor chemotherapy. Current strategies for pharmacological interventions are briefly discussed. Phospholipid analogues are treated in greater detail. It is shown here that this new class of antitumor agents acts as inhibitors of mitogenic signal transduction. The common target of all phospholipid analogues studied so far is the phosphatidylinositol (PI)-specific phospholipase C (PLC). This results in an attenuated formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The reduction in IP3-levels leads to a depressed release of Ca2+ from internal stores, and the reduced formation of DAG interferes with the growth factor-induced activation of protein-kinase C (PKC). In addition to the effect on PI-specific PLC, most phospholipid analogues inhibit PKC directly by interacting with the regulatory domain of the enzyme. This effect, however, is not observed with all phospholipid analogues. Some potent growth inhibitory representatives from this group like hexadecylphosphoserine or hexadecylphosphonoserine do not affect PKC in cell-free extracts. It is concluded, therefore, that the direct inhibition of PKC is not required for the growth-inhibitory activity of these agents. The ability of phospholipid analogues to interact with PKC was also not found to be correlated the occurrence of unwanted side effects. Phospholipid analogues have also been found to act as inhibitors of phospholipase D (PLD). However, in this case the correlation to the growth inhibitory potency of various phospholipid analogues was less clear, so that the contribution of the PLD inhibition to the growth inhibitory effect of these agents still remains to be established. The inhibition of the thrombin-induced rise in cytosolic free Ca2+ by phospholipid analogues is reversible by washing the cells in phospholipid-free medium. These findings suggest that phospholipid analogues do not cause persistent membrane damage and may act as cytostatic rather than cytotoxic agents.

Analysis of phosphorylhydroxyamino acids present in hydrolyzed cell extracts using dabsyl derivatization.

We have developed a new method for the quantification of phosphoserine, phosphothreonine, and phosphotyrosine as dabsyl derivatives in acid-hydrolyzed extracts of 32P-labeled A431 cells. In the first step the phosphamino acids are concentrated using a disposable anion-exchange column. Subsequently, the phosphoamino acid fraction is treated with dabsyl reagent (28.8 mM) for 10 min at 70 degrees C. After cleanup with a second anion-exchange column followed by separation on a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC sheets using a one-dimensional solvent system. The major advantages of this method are the complete separation of dabsylated P-Ser, P-Thr, and P-Tyr on silica aluminum sheets in a very reproducible way without the interference of 32P contaminants originating from hydrolyzed cell extracts. Very clean chromatograms are obtained, enabling the fast and unambiguous quantification of the phosphoamino acids by simply cutting out the relevant spots from the aluminum sheets. A high sensitivity is achieved by the removal of the amino acids before derivatization of the sample. This allows the use of relatively low amounts of [32P]orthophosphate to load up the cells. Most important, the method allows the simultaneous analysis of dozens of samples within 1 day, making it a very convenient technique for routine analysis of the phosphorylation state of cultured cells. Consequently the method is well suited to implementation in large screenings for inhibitors of protein kinases, e.g., PTK inhibitors, in whole-cell studies.

The effect of prolonged anoxia at 3 degrees C on tissue high energy phosphates and phosphodiesters in turtles: a 31P-NMR study.

Selected tissues (skeletal muscle, heart ventrical, and liver), sampled from turtles (Chrysemys picta bellii) at 3 degrees C either under normoxic conditions or after 12 weeks of anoxic submergence were quantitatively analysed for intracellular pH and phosphorus metabolites using 31P-NMR. Plasma was tested for osmolality and for the concentrations of lactate, calcium, and magnesium to confirm anoxic stress. We hypothesized that, in the anoxic animals, tissue ATP levels would be maintained and that the increased osmolality of the body fluids of anoxic turtles would be accounted for by a corresponding increase in the concentrations of phosphodiesters. The responses observed differed among the three tissues. In muscle, ATP was unchanged by anoxia but phosphocreatine was reduced by 80%; in heart, both ATP and phosphocreatine fell by 35-40%. The reduction in phosphocreatine in heart tissue at 3 degrees C was similar to that observed in isolated, perfused working hearts from turtles maintained at 20 degrees C but no decrease in ATP occurred in the latter tissues. In liver, although analyses of several specimens were confounded by line-broadening, neither ATP nor phosphocreatine was detectable in anoxic samples. Phosphosdiesters were detected in amounts sufficient to account for 30% of normoxic cell osmotic concentration in heart and 11% and 12% in liver and muscle, respectively. The phosphodiester levels did not change in anoxia. Heart ventricular phosphodiester levels in turtles at 3 degrees C were significantly higher than those determined for whole hearts from turtles at 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple effects of antitumor alkyl-lysophospholipid analogs on the cytosolic free Ca2+ concentration in a normal and a breast cancer cell line.

Synthetic alkyl-lysophospholipids (ALP) are a new class of antitumor agents which interact with the cell membrane and the intracellular signal transduction at several sites. We studied the modulation of the intracellular calcium concentration ([Ca++]i) induced by two alkylglycerophosphocholines as well as hexadecylphosphocholine and hexadecylphosphoserine in a nontumorigenic and in a tumorigenic breast cell line. We found three distinct [Ca++]i-modulating effects: a transient increase, a decrease and a sustained increase. Their relative contribution to the observed response varies with different cell types, with the proliferation state, with the structure and with the concentration of the ALP analogs. The transient as well as the sustained increase in [Ca++]i depend mainly on extracellular Ca++; however, the Ca++ influx-inducing pathways might be different. The multiple [Ca++]i-increasing and decreasing effects induced by ALP analogs are discussed in relation to their influence on numerous Ca(++)-dependent effects, e.g. proliferation, differentiation, apoptosis and cytotoxicity.