Protection of crystal-induced polymorphonuclear leukocyte membranolysis by phosphocitrate

The protection afforded by phosphocitrate, a phosphorylated polycarboxylic acid, against crystal-induced membrane damage to polymorphonuclear leukocytes was studied in vitro. Membranolysis was assessed by nitro blue tetrazolium salt reduction, lactate dehydrogenase release, and scanning electron microscopy. Phosphocitrate protected strongly against hydroxyapatite crystal-induced damage, an action attributable to crystal surface binding of phosphocitrate rather than to the membrane. The ability of phosphocitrate to prevent hydroxyapatite crystallization, together with its membrane protective effect against preformed crystals, would suggest that the compound might have a useful future role against crystal-induced arthropathies.     

Molecular dynamics simulation studies of the effect of phosphocitrate on crystal-induced membranolysis

In this study, following our earlier work on calcium pyrophosphate dihydrate (CPPD) crystal-induced membranolysis, we demonstrate, using the CHARMM method of molecular dynamics simulation, the protective role of phosphocitrate (PC) against solvated dimyristoyl phosphatidylcholine phospholipid bilayer disintegration on contact with the CPPD crystal. Our molecular dynamics simulations studies show that coverage of the CPPD crystal with a layer of phosphocitrate molecules results in the conservation of phospholipid bilayer integrity. We show that the rupture of the lipid bilayer in presence of CPPD and the protective effect of PC are primarily due to electrostatic interactions. The protective role of PC, which may also play an important and potentially therapeutic function against crystal-induced membranolysis is also discussed.     

Inhibition of postoxidative calcium release in corn mitochondria by inorganic phosphate

Respiration drives the accumulation of a small amount of calcium in corn (Zea mays L.) mitochondria, and this calcium is released when respiration ceases. A postenergized addition of phosphate leads to phosphate uptake and enhaced calcium retention. Oligomycin, KCN, 2,4-dinitrophenol, or mersalyl are without effect on the phosphate-induced calcium retention. Addition of phosphate also inhibits the release of endogenous phosphate which normally accompanies the calcium. It is suggested that passive phosphate uptake retards the release of endogenous phosphate which is complexed with the calcium.     

Inhibition of mitogenesis in bovine lymphocytes by juvenile hormones

Phytohemagglutinin and the co-carcinogenic phorbol ester, 12-0- tetradecanoyl-phorbol-13-acetate, act synergistically in cultures of bovine lymphocytes to induce a mitogenic response. Insect juvinile hormones appear to act selectively to block or counteract an early hormones appear to act selectively to block or counteract an early event in the induction of DNA and nuclear replication while allowing other blastogenic events such as increases in RNA and protein synthesis and cell size to occur.     

Inhibition of lymphocyte mitogenesis by an arachidonic acid hydroperoxide

Incubation of murine spleen cells with the oxidation product of soybean lipoxidase-treated arachidonic acid results in profound inhibition of induction of proliferation and maturation of these cells. The active entity was shown to be the 15-hydroperoxide of arachidonic acid (15-HPAA). Inhibition of the enzymes of the cyclo-oxygenase pathway fails to disturb this effect, indicating that 15-HPAA is not a substrate for this series of enzymes. 15-HPAA produced in this manner interfered with RNA synthesis, DNA synthesis, and blastogenesis, while failing to exert cytotoxic effects on the cells themselves. A variety of lymphocyte subpopulations, distinguished by their responsiveness to a diverse group of mitogens, were all equally inhibited by the addition of 15-HPAA to culture. Addition of this agent even as late as 24 h after initiation of culture resulted in profound inhibition of the proliferative and differentiative responses of splenic B cells to bacterial lipopolysaccharide (LPS). Exposure of cells to 15-HPAA for 10–30 min was adequate to initiate inhibition, an event that exhibited marked temperature dependence. The effects of pre-incubation with 15-HPAA could not be reversed in its absence in recovery periods of up to 6 h prior to addition of LPS. The implications of these data with reference to cellular activation mechanisms are discussed.     

Precipitation of nucleotides by calcium phosphate

Earlier it has been shown that nucleic acids of high molecular weight can be introduced into cells by coprecipitation with calcium phosphate. We have studied the requirements for calcium phosphate coprecipitation of shorter nucleotides. The degree of coprecipitation of dodecanucleotides lacking terminal phosphate varied between 25 and 72%. Tetramers with a 5′-monophosphate were coprecipitated to 29–87% by calcium phosphate. A high content of guanosine residues and an increased number of terminal phosphate groups increased the degree of coprecipitation of nucleotides. The trinucleotide pppA2′p5′A2′p5′A was effectively precipitated by calcium phosphate but the monophosphate and the core structure were not.     

Free calcium rise and mitogenesis in glial cells caused by endothelin

The peptide endothelin causes a biphasic rise in intracellular free calcium levels in cultured Type-1 astrocytes and C6 glioma cells, suggesting that glial cells may be the physiological target of endothelin in the brain. Endothelin also causes a calcium-dependent increase [3H]thymidine incorporation in primary cultures of rat cerebellum, indicating that, among other possible roles, this peptide may mediate mitogenesis in brain.     

Inhibition of thymidylate synthase by pyridoxal phosphate

1. Pyridoxal phosphate (PLP) reversibly inhibited thymidylate synthase from Lactobacillus casei with a KI of 0.6-0.9 microM. 2. The inhibition was competitive with dUMP and noncompetitive with 5,10-methylenetetrahydrofolate which is consistent with an ordered addition of substrates. 3. The spectrum of PLP was altered by the addition of thymidylate synthase. The spectral changes suggest formation of a thiohemiacetal with an enzyme sulfhydryl group rather than Schiff base formation with a lysine side chain.     

Inhibition of microbial arsenate reduction by phosphate

The ratio of arsenite (As(III)) to arsenate (As(V)) in soils and natural waters is often controlled by the activity of As-transforming microorganisms. Phosphate is a chemical analog to As(V) and, consequently, may competitively inhibit microbial uptake and enzymatic binding of As(V), thus preventing its reduction to the more toxic, mobile, and bioavailable form - As(III). Five As-transforming bacteria isolated either from As-treated soil columns or from As-impacted soils were used to evaluate the effects of phosphate on As(V) reduction and As(III) oxidation. Cultures were initially spiked with various P:As ratios, incubated for approximately 48 h, and analyzed periodically for As(V) and As(III) concentration. Arsenate reduction was inhibited at high P:As ratios and completely suppressed at elevated levels of phosphate (500 and 1,000 μM; P inhibition constant (K(i))～20-100 μM). While high P:As ratios effectively shut down microbial As(V) reduction, the expression of the arsenate reductase gene (arsC) was not inhibited under these conditions in the As(V)-reducing isolate, Agrobacterium tumefaciens str. 5B. Further, high phosphate ameliorated As(V)-induced cell growth inhibition caused by high (1mM) As pressure. These results indicate that phosphate may inhibit As(V) reduction by impeding As(V) uptake by the cell via phosphate transport systems or by competitively binding to the active site of ArsC.     

Lysophosphatidic acid-induced mitogenesis is regulated by lipid phosphate phosphatases and is Edg-receptor independent

Lysophosphatidic acid (LPA) is an extracellular signaling mediator with a broad range of cellular responses. Three G-protein-coupled receptors (Edg-2, -4, and -7) have been identified as receptors for LPA. In this study, the ectophosphatase lipid phosphate phosphatase 1 (LPP1) has been shown to down-regulate LPA-mediated mitogenesis. Furthermore, using degradation-resistant phosphonate analogs of LPA and stereoselective agonists of the Edg receptors we have demonstrated that the mitogenic and platelet aggregation responses to LPA are independent of Edg-2, -4, and -7. Specifically, we found that LPA degradation is insufficient to account for the decrease in LPA potency in mitogenic assays, and the stereoselectivity observed at the Edg receptors is not reflected in mitogenesis. Additionally, RH7777 cells, which are devoid of Edg-2, -4, and -7 receptor mRNA, have a mitogenic response to LPA and LPA analogs. Finally, we have determined that the ligand selectivity of the platelet aggregation response is consistent with that of mitogenesis, but not with Edg-2, -4, and -7.     

Inhibition of bombesin-induced mitogenesis by pertussis toxin: dissociation from phospholipase C pathway

Prior incubation of quiescent cultures of Swiss 3T3 cells with pertussis toxin selectively inhibited the stimulation of DNA synthesis induced by peptides of the bombesin family. While pertussis toxin blocked mitogenesis at an early stage in the action of the peptide, the toxin did not impair the rapid stimulation of polyphosphoinositide breakdown, Ca2+ mobilization or activation of protein kinase C promoted by bombesin. Thus, inhibition of bombesin-induced mitogenesis by pertussis toxin can be dissociated from inactivation of the phospholipase C signalling pathway.     

Inhibition of acetyl-CoA carboxylase isoforms by pyridoxal phosphate

Mammalian isoforms of acetyl-CoA carboxylase (ACC-1 and ACC-2) play important roles in synthesis, elongation, and oxidation of long-chain fatty acids, and the possible significance of ACC in the development of obesity has led to interest in the development of inhibitors. Here, we demonstrate that pyridoxal phosphate (PLP) is a linear and reversible inhibitor of ACC-1 and ACC-2. ACC from rat liver and white adipose tissue (largely ACC-1) exhibited an IC50 of approximately 200 microm, whereas ACC-2 from heart or skeletal muscle exhibited an IC50 exceeding 500 microm. ACC from rat liver was equally sensitive to PLP following extensive purification by avidin affinity chromatography. When added before citrate, PLP inhibited ACC with a Ki of approximately 100 microm, reducing maximal activity >90% and increasing the Ka for citrate approximately 5-fold but having little effect on substrate Km values. Pre-treatment with citrate increased the apparent Ki for ACC inhibition by PLP by approximately 4-fold. Inhibition of ACC was reversed by removal of PLP, either by washing or by reaction with hydroxylamine or amino-oxyacetate. ACC was irreversibly inhibited and radiolabeled, to a stoichiometry of approximately 0.4 mol[H]/mol subunit, in the presence of PLP plus [3H]borohydride. Studies with structurally related compounds demonstrated that the reactive aldehyde and negatively charged substituents of PLP contribute importantly to ACC inhibition. The studies reported here suggest a rationale to develop ACC inhibitors that are not structurally related to the substrates or products of the reaction and an approach to probe the citrate-binding site of the enzyme.     

Inhibition by calcium of mammalian adenylyl cyclases

Ca(2+) regulates mammalian adenylyl cyclases in a type-specific manner. Stimulatory regulation is moderately well understood. By contrast, even the concentration range over which Ca(2+) inhibits adenylyl cyclases AC5 and AC6 is not unambiguously defined; even less so is the mechanism of inhibition. In the present study, we compared the regulation of Ca(2+)-stimulable and Ca(2+)-inhibitable adenylyl cyclases expressed in Sf9 cells with tissues that predominantly express these activities in the mouse brain. Soluble forms of AC5 containing either intact or truncated major cytosolic domains were also examined. All adenylyl cyclases, except AC2 and the soluble forms of AC5, displayed biphasic Ca(2+) responses, suggesting the presence of two Ca(2+) sites of high ( approximately 0.2 microM) and low affinity ( approximately 0.1 mM). With a high affinity, Ca(2+) (i) stimulated AC1 and cerebellar adenylyl cyclases, (ii) inhibited AC6 and striatal adenylyl cyclase, and (iii) was without effect on AC2. With a low affinity, Ca(2+) inhibited all adenylyl cyclases, including AC1, AC2, AC6, and both soluble forms of AC5. The mechanism of both high and low affinity inhibition was revealed to be competition for a stimulatory Mg(2+) site(s). A remarkable selectivity for Ca(2+) was displayed by the high affinity site, with a K(i) value of approximately 0.2 microM, in the face of a 5000-fold excess of Mg(2+). The present results show that high and low affinity inhibition by Ca(2+) can be clearly distinguished and that the inhibition occurs type-specifically in discrete adenylyl cyclases. Distinction between these sites is essential, or quite spurious inferences may be drawn on the nature or location of high affinity binding sites in the Ca(2+)-inhibitable adenylyl cyclases.     

Permissive role of calcium in the inhibition of T cell mitogenesis by calmodulin antagonists

The importance of Ca++ in the initiation of lymphocyte activation and mitogenesis has been supported by several studies. Because calmodulin functions as the intracellular mediator of the effects of Ca++, it likely plays a major role in the regulation of lymphocyte function. We have examined the effects of known calmodulin antagonists, the phenothiazines, on lectin-induced T cell mitogenesis and have shown a central role for Ca++ uptake in the expression of a phenothiazine-sensitive stage after lectin activation. The drug effects were observed only if the cells were previously activated by PHA or the ionophore A23187, and only in the presence of Ca++. These effects were restricted to a defined time period (5 hr) after lectin activation. The data support the concept that calmodulin is the target for the phenothiazine effects and demonstrate the permissive role of Ca++ in the mediation of these events.