Effect of nicotine on fibroblast beta 1 integrin expression and distribution in vitro.

BACKGROUND: Integrins are a family of transmembrane cell surface glycoproteins, and those with the beta 1-subunit function in both cell-to-cell and cell-to-substrate adhesion. The purpose of this study was to determine nicotine's effect on the expression and distribution of the beta 1 integrin subunit on the human gingival fibroblast cell surface. METHODS: Pure nicotine was diluted in medium to the following concentrations: 0 (control), 0.025, 0.05, 0.1, 0.2, and 0.4 microM. Human gingival fibroblasts (HFG) were grown for 24 hours in each concentration and fluorescein-labeled with a mouse monoclonal anti-human beta 1 antibody and secondarily incubated with a urease-labeled anti-mouse IgG antibody. After a final wash, the cells were incubated with urea/bromcresol blue substrate for 15 minutes at 37 degrees C and measured in a microplate reader at 570 nm. RESULTS: The integrin beta 1-subunit was detected on the HGF surface membrane by fluorescence labeling, and cell-enzyme-linked immunosorbent assay testing demonstrated its decreased expression with increasing nicotine concentrations that were statistically different at the concentrations of 0.2 and 0.4 microM versus controls (P < 0.05). CONCLUSIONS: Nicotine concentrations of 0.2 and 0.4 microM significantly decrease beta 1 integrin expression in human gingival fibroblasts that may affect cell-cell and cell-substratum adhesion during wound healing.

Exocellular phospholipase C activity from a Propionibacterium acnes strain isolated from a periodontal pocket.

The culture supernatant of a strain of Propionibacterium acnes was investigated for its phospholipase (PL) activity. The microorganism was isolated from a periodontal pocket of a patient with periodontal disease. Supernatants from cultures of this microorganism were used as a source to obtain enzymes. Proteins from the supernatants were concentrated, and their enzymatic activity was partially purified through molecular sieving. The procedure yielded two peaks of activity. This activity was shown to hydrolyze phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) and was effective in a pH range of 5 to 9, with an optimal activity at pH 7.0. Divalent cations were not required for activity of the enzymes. Analysis of the products obtained from the hydrolysis of PC labeled in the choline, phosphoryl, or acyl moieties and PE containing labeled oleic acid indicated that the supernatants' activity was mostly phospholipase C (PL-C). Phospholipase C can act synergistically with other factors to produce tissue damage, and hence may contribute to the pathogenesis of periodontal disease.

Effects of an aminomethacrylate on epithelial cell lipid metabolism.

Methacrylates can affect cell functions by surfactant-like effects or by altering cell lipid composition. Dimethylaminoethyl methacrylate (DMAEMA), an activator widely used in visible-light polymerized dental resins has been shown to elute readily into aqueous environments. The current study examined the metabolism of this material by oral epithelial cells (HCP) and its subsequent effects on cell lipids. Cells were plated in culture medium, then exposed to DMAEMA in the presence of 14C-acetate, a precursor which labeled the cell lipids. Other cultures were prelabeled with radioisotope, then exposed to DMAEMA. After incubation, the cell lipids were extracted and separated by TLC. Radioactive lipids were located and quantitated. Exposure of the cells to DMAEMA resulted in decreased synthesis of cholesterol with a concomitant increase in sterol precursors. Cholesterol esters and triacylglycerides also increased. Among the polar lipids, phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) decreased in response to DMAEMA. However, dimethylphosphatidyl ethanolamine (DMPE), a precursor of PC not detectable in control cultures, accumulated to a significant extent in cells exposed to DMAEMA. Furthermore, changes in PC and DMPE levels persisted in the cells for at least 48 h after removal of the DMAEMA. The results indicate that DMAEMA produces alterations in the relative amounts of several cellular neutral and polar lipids. Such alterations, especially of the normal phospholipid composition, along with an alteration in cellular cholesterol, could result in altered membrane-associated cell functions.

Binding and uptake of N-nitrosonornicotine by oral epithelial cells.

N-nitrosonornicotine (NNN), a significant nitrosamine component of tobacco binds to and is taken up by cells prior to its activation by intracellular enzymes. A variety of factors may affect the binding of substances to a cell including the cell type, medium composition and the cell membrane lipid composition. This study examines the binding and uptake of NNN to various cell types and relates these to the cells' lipid composition. Hamster buccal pouch keratinocytes were exposed to NNN and the amount of bound NNN determined. This was compared with cells pretreated with 12-0-tetradecanoylphorbol-13-acetate (TPA). While the keratinocytes bound significant amounts of NNN, this binding was not specific. Pretreatment of the cells with TPA significantly increased the amount of NNN bound. Cultures of human gingival fibroblasts and a human liver cell line also bound NNN, in quantities greater than that bound to the keratinocytes. The TPA caused changes in long chain unsaturated fatty acids although major changes in lipid metabolism were not detected. The fatty acid composition of the liver cells more closely resembled that of the TPA treated keratinocytes than the untreated ones. The data suggest that NNN binds to the cells non-specifically and the binding may be related to the amount of long-chain, unsaturated fatty acids present.

In vivo effects of Sn-1,2-dioctanoylglycerol, TPA and A23187 on hamster cheek pouch epithelium.

Cheek pouches of male Syrian golden hamsters were topically treated with a single dose of TPA (.5 microgram), calcium ionophore A23187 (75 micrograms) or Sn-1,2-dioctanoylglycerol (DiC8) (500 micrograms) dissolved in 0.25 ml acetone. Acetone-treated animals served as controls. After 48 h the mitotic index for the control group was 1.1 +/- 0.1 per 1 mm of the basement membrane length. All the test congeners exhibited higher mitotic indices than controls: TPA (4.8 +/- 0.4), A23187 (3.9 +/- 0.3), DiC8 (2.1 +/- 0.2). All groups exhibited an increase in the epithelial thickness manifested by cellular hyperplasia. The treatment of the pouches with the anti-inflammatory agent fluocinolone acetonide inhibited the mitogenic and hyperplasiogenic affects on the epithelium induced by the various test chemicals. These studies indicate a possible role of calcium-phospholipid dependent protein kinase (protein kinase C) in the mediation of oral epithelial cell proliferation.

Phorbol ester binding to oral epithelial cells in the presence of retinoic acid or N-nitrosonornicotine.

Although the specific mechanisms by which phorbol ester tumour promoters exert their various effects are not known, their actions are mediated by cell membrane receptors which contain lipids as major components of the receptor complex. Since cell modulators such as retinoic acid (RA) and nitrosonornicotine (NNN) can alter cell lipids, the binding of a phorbol ester to cells was examined at time intervals when lipid changes mediated by these modulators occur. Epithelial cells prepared from hamster cheek pouches were treated with all-trans RA or NNN for varying periods of time, then specific binding of phorbol esters was investigated. Cells treated with RA for intervals up to 24 h showed decreased binding when compared with untreated cells. Those treated with NNN for up to 168 h showed increased specific binding. The results suggest that alterations in the cell lipids may affect the specific binding of phorbol esters to cells.

Oral epithelial cell lipid synthesis in the presence of retinoic acid or nitrosonornicotine.

The current study examines the effects of N-nitrosonornicotine (NNN) and all trans-retinoic acid (RA) on the synthesis and composition of lipids of oral epithelial cells grown in culture. Cells were exposed to NNN, RA or methylene chloride (the vehicle for the NNN and RA) and the lipids labelled with [14C]-acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. Cholesterol labelling was significantly decreased by the RA between 4 and 48 h when compared to the NNN-treated or control cells. After 48 h the incorporation levels in the presence of the two last compounds decreased. Free fatty acid labelling was also significantly less in cells exposed to RA, while labelling of triglycerides and phospholipids was increased. N-nitrosonornicotine seemed to produce a decreased labelling of cholesterol after 96 h continuous exposure. The results of these studies suggest that retinoid rapidly elevates the cell content of lipid formed from the glycerolipid pathway and membrane mechanisms may be modulated by this effect.

A simplified method for culture of oral epithelial cells.

In order to facilitate studies on oral mucosa a simplified method for the culture of oral epithelial cells from adult hamsters was developed. Cheek pouches were excised and epithelial cells isolated by collagenase digestion. These were grown in CM-V medium containing spermine in order to inhibit overgrowth of the epithelial cells by fibroblasts. The epithelial cells were subcultured by routine tissue culture procedures. The cells isolated were examined by light microscopy and scanning and transmission electron microscopy. Morphologically the cells were typical of epithelial cells. Ultrastructural examination showed structures typical of epithelia including filaments, keratohyalin granules and desmosomal junctions. The culture system provides epithelial cells that can be used for a variety of biochemical and morphological studies.

The effects of retinoic acid on [14C]acetate incorporation into lipids of normal and transformed hamster fibroblasts.

This study examined the effects of retinoic acid (RA) on [14C]acetate incorporation and fatty acid composition of hamster embryo fibroblasts (HEF) and two cell lines derived from the same inbred strain but transformed by herpes simplex-2 virus (HSV) or polyoma virus (HFT). Cells were exposed to all trans RA, or dimethylsulfoxide (DMSO), the vehicle for RA, and the lipids labeled with [14C]acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. The distribution of fatty acids in total cell lipids was examined by gas chromatography. HEF cells incorporated more acetate into cholesterol than either transformed cell type. The HFT line incorporated more acetate into triglycerides and less into total phospholipids than either the HSV line or the HEF line. RA caused a significant decrease in incorporation of acetate into cholesterol and sphingomyelin in all three cell lines. HEF and HSV cells had decreased incorporation into phosphatidyl inositol-phosphatidyl serine and increased incorporation into triglycerides, changes not evident in the HFT cell. The control fatty acid profiles of the HEF and HSV cells were similar, while the HFT cells had a larger proportion of C16:0 and 18:1 fatty acids. Following treatment with RA all three cell types showed an increase in palmitic and a decrease in oleic acids. The three related cell types showed different [14C]acetate labeling patterns which did not respond uniformly to RA. On the other hand, exposure elicited some like responses in all cell types.

Effects of 12-0-tetradecanoyl-phorbol-13-acetate on oral epithelium in vitro.

TPA, a tumor promotor whose initial site of action is the cell membrane, was examined for its actions on hamster oral mucosa using light and scanning electron-microscopic procedures. Hamster cheek-pouch explants were cultured in vitro, then treated for 72 h with 1.6 X 10(-8) M TPA. Treated cultures showed a higher mitotic index and more extensive growth than control cultures. Epithelial cells in the control cultures appeared polygonal, with thin, small to medium microvilli. The cells in the treated cultures showed variable shape and surface morphology. Some were interconnected by broad cytoplasmic extensions while others demonstrated long, thin processes that traversed great distances. The unusual surface morphology may be a manifestation of altered phospholipid metabolism that produces a more "fluid" cytoplasmic membrane.

Effects of calcium ions on hamster cheek pouch epithelium grown in vitro.

Epithelial outgrowths from hamster cheek pouch explants were cultured for 14 days in media containing calcium concentrations of 0.05 mM, 0.075 mM, 0.1 mM, 0.25 mM, 0.75 mM or 1.45 mM (control). Compared with controls, the cultures grown at lower calcium concentrations (0.25-0.75 mM), exhibited higher mitotic indices and an increase in outgrowth size. The mitotic index of cultures at 0.25 mM calcium concentration was 30 +/- 2.26, compared to 24 +/- 2.24 for controls. Increase in size of the outgrowths was also observed at 0.25 mM calcium concentration compared with those grown in control medium. The epithelial outgrowths grown in control media exhibited cell stratification not observed at lower calcium concentrations. These studies indicate that lower calcium concentration in the media (0.25-0.75 mM) increased epithelial cell proliferation and inhibited keratinization.

Phospholipase A activity in supernatants from cultures of Bacteroides melaninogenicus.

The phospholipase A activity in culture supernatants of two strains of Bacteroides melaninogenicus is described. The enzyme utilize phosphatidylcholine as substrate and produce mainly lysophosphatidylcholine and free fatty acids. The activities are Ca2+-independent, are not affected by the presence of a chelating agent, have a broad pH range (5-9) and an optimum temperature for activity of approx. 50 degrees C. The activity in a growing bacterial culture increases from the end of the lag phase to the late exponential phase of growth. Analysis of the products resulting from the actions of the enzymes on L-alpha-palmitoyl-beta-oleoyl[1-14C]phosphatidylcholine indicates that the enzymes are phospholipase A1 (EC 3.1.1.32).

Effects of retinoic acid on the differentiation of hamster cheek pouch epithelium grown in vitro.

Recently our laboratories have successfully attained preferential epithelial outgrowths from hamster cheek pouch explants. This has allowed us to systematically analyze oral epithelial cell differentiation as weel las examine the modulatory effects of various physiologic and pharmacologic agents in this epithelial cell system. The objective of the current investigation was to study the effects of various doses of retinoic acid (RA) on the epithelial outgrowths of hamster cheek pouch. Light microscopic observations employing colcemid treatment indicated that 10 IU/ml RA enhanced mitotic activity of the epithelium up to 25%. Scanning electron microscopic observations of control cultures showed flat, polyhedral epithelial cells containing thin long microvilli connecting the adjacent cells. In contrast, cultures treated with 10 IU/ml RA showed round or spherical cells exhibiting short of medium sized thicker microvilli. These were covered with a fuzzy appearing layer probably representing glycocalyx. Many bleb-like formation were also observed. Transmission electron microscopic examination of the control cultures showed the presence of tonofilaments and desmosomes, characteristic components of the keratinizing epithelia. In contrast, the RA treated cultures included cells which apparently were either devoid of tonofilaments or contained few fine filaments and small intercellular functions. In addition many vacuoles as well as variable numbers of electron-dense and electron-lucent granules were observed. Thus, it appears that RA inhibited keratinization and altered the differentiation pattern of keratinization and altered the differentiation pattern of keratinizing epithelium.