Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Functional analysis of anionic phospholipids in cell metabolism in {\it Escherichia coli\/}

The major goal of this work was to understand the function of anionic phospholipid in E. coli cell metabolism. One important finding from this work is the requirement of anionic phospholipid for the DnaA protein-dependent initiation of DNA replication. An rnhA mutation, which bypasses the need for the DnaA protein through induction of constitutive stable DNA replication, suppressed the growth arrest phenotype of a $pgsA$ mutant in which the synthesis of anionic phospholipid was blocked. The maintenance of plasmids dependent on an $oriC$ site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein. In addition, structural and functional requirements of two major anionic phospholipids, phosphatidylglycerol and cardiolipin, were examined. Introduction into cells of the ability to make phosphatidylinositol did not suppress the need for the naturally occurring phosphatidylglycerol. The requirement for phosphatidylglycerol was concluded to be more than maintenance of the proper membrane surface charge. Examination of the role of cardiolipin revealed its ability to replace the zwitterionic phospholipid, phosphatidylethanolamine, in maintaining an optimal membrane lipid organization. This work also reported the DNA sequence of the cls gene, which encodes the CL synthase responsible for the synthesis of cardiolipin. ^

DETERMINANTS OF THE SELECTIVE PHAGOCYTOSIS OF CARCINOGENIC PARTICULATE NICKEL COMPOUNDS

Certain inorganic nickel compounds such as crystalline NiS and Ni(,3)S(,2) are potent inducers of carcinogenesis and in vitro cell transformation, while several closely-related compounds such as amorphous NiS are essentially devoid of genotoxic activity. The phenomenon of selectivity of phagocytosis among such particulate nickel compounds has been hypothesized to account for their widely varying toxicological potency, yet the determinants of this selectivity have not been well characterized. Extracellular medium composition, particle dissolution, and particle surface charge were examined as potential determinants of selective phagocytosis for the carcinogenic crystalline and noncarcinogenic amorphous modifications of NiS. Selectivity and avidity of uptake of crystalline NiS by CHO cells was not dependent upon serum: phagocytosis of crystalline, but not amorphous NiS proceeded readily in a minimal salts/glucose medium at 37(DEGREES)C. The evolution of phagocytosis-inhibiting Ni(II) from the surface of amorphous NiS particles did not demonstrably contribute to the lower uptake of these noncarcinogenic particles despite their somewhat greater dissolution rate than the readily phagocytosed crystalline NiS particles. Significant differences in surface charge were noted between crystalline and amorphous NiS, the former being more negative in charge in distilled water suspension. Exposure of amorphous NiS particles to the vigorously reducing environment of a LiAlH(,4) solution under an inert atmosphere resulted in the particles' acquisition of a more negative surface charge. Amorphous NiS particles thus treated were phagocytosed by CHO cells to an extent similar to that of untreated crystalline NiS particles and likewise were shown to induce morphological transformation of primary Syrian hamster embryo cells with a similar potency. The potentiation of uptake characteristic of LiAlH(,4)-treated amorphous NiS was lost gradually upon storage of particles in ambient oxygenated atmosphere and was lost rapidly by apparent particle surface oxidation in aerated distilled water suspensions aged for up to 7 days. Concomitant with this loss of uptake there occurred a loss of negative surface charge. These results suggest the predominant role of particle surface charge rather than adsorbed serum components or particle dissolution as a determinant of selective phagocytosis among particulate nickel compounds. ^

AN APPROACH TO THE CHARACTERIZATION OF FIBRONECTIN-INTERACTION WITH MAMMALIAN CELLS

Cell adhesion is a fundamentally important process which has been implicated in morphogenesis, metastasis and wound healing. Fibronectin (Fn), a large glycoprotein present in body fluids, the extracellular matrix, and on the cell surface, mediates adhesion of fibroblastic cells. To study the interaction of Fn with Chinese Hamster Cell (CHO) cell membranes, latex beads coated with H('3)-Fn (Fn-beads) were used as surface probes. Binding of Fn-beads was independent of temperature, divalent cations, and metabolic activity. Identification of fibronectin-receptors has been problematical. To study Fn binding components, Fn-beads were pre-incubated with purified glycosaminoglycans (GAGs) and glycolipids. Among the GAGs tested, heparin and heparan sulfate blocked bead binding. Only sialylated glycolipids, GT(,1) and GD(,1) were inhibitory; however, neuraminidase treatment of cells had no effect. It was further shown that Fn-bead binding could be blocked by pre-treating cells with papain. Furthermore, papain digestion releases cellular material which blocks Fn-bead-cell binding. Beads coated with a fragment of Fn which binds to cells but not heparin (F105) were also blocked by soluble papain digests. It was observed that the ability of F105-beads to bind to CHO cells was dependent on surface charge as F105 on uncharged beads did not bind to cells; whereas, F105 on positive or negative beads displayed cell binding activity. The active component in the papain digests was apparently macromolecular (i.e. non-dialysable) and heat stable (i.e. 100(DEGREES)C for 15 min.). This suggested the inhibitory factor is more likely a glycopeptide, rather than a GAG or glycolipid. The findings of this research can be summarized as follows: (1) the expression of cell binding of Fn and Fn fragments can be modulated by the chemical nature of the surface used for adsorption; (2) factors can be released by proteolytic digestion which block Fn and Fn-fragment bead binding; and (3) since bead binding can be done under conditions which reflect initial Fn-cell interaction, it seems likely that the component(s) identified in this way may play a direct role in the recognition phases of cell adhesion to Fn. ^

cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^