Studies on Cell Adhesion and Activation

Gasmi, Lakhdar (1990) Studies on Cell Adhesion and Activation. PhD thesis, University of Glasgow.

Abstract

The role of plasmalemmal lipids in the adhesion of Baby Hamster Kidney (BHK) fibroblasts to polystyrene tissue culture grade dishes in serum and serum-free medium was investigated using various fatty acids that differ in their hydrocarbon chain length and saturation. A number of biologically active agents which include arachidonic acid metabolites, as well as some of the factors which affect the calcium/protein kinase C pathway were also considered in the present study. Fatty acids were the first to be tested. The saturated fatty acid derivative stearoyl-CoA (18:0) had two different effects on BHK cell adhesion in serum-free Ham's F-10. Concentrations, ranged between 1.75 and 7 muM, enhanced the adhesion in almost all cases, while higher concentrations such as 14 and 28 uM reduced the adhesion of BHK cells to polystyrene tissue culture dishes by more than 50% of the control values. BHK cells in the presence of long chain unsaturated fatty acids, (oleoyl-CoA (18:1), linoleoyl-CoA (18:2), linolenoyl-CoA (18:3), and arachidonoyl-CoA (18:4), responded in different ways depending on the type of the fatty acid added. When oleoyl-CoA was used at concentrations lower than 7 muM the adhesion was maintained at the control level or slightly enhanced. However,the use of concentrations higher than that reduced the attachment sharply. Linoleoyl-CoA, linolenoyl-CoA and arachidonoyl-CoA generally maintained the adhesion at the control level, though linoleoyl-CoA enhanced the adhesion at fairly low concentrations (lower than 14 muM) . Both, stearoyl-CoA and oleoyl-CoA (1.75 muM and higher) decreased the spreading area of BHK cells by 30-50% of control samples in serum-free medium. The above fatty acyl-CoAs did not however alter either the adhesion or the spreading of BHK fibroblasts in serum-containing medium. In contrast to that, linoleic and arachidonic acid used as free acid form (unbound to the coenzyme A) at concentrations ranged between 2 and 20 microg/ml in combination with 1.25x10e-5 M ATP and 5x10 6 M Coenzyme A, decreased significantly (P< 0.001) the adhesion in both 3%serum and serum-free Ham's F-10 medium. A noticeable loss of viability was found at concentrations higher than 5microg/ml. The effect on cell viability was less pronounced in serum-containing medium. Incorporation studies in serum-free conditions revealed that approximately 40% of the total labeled oleoyl-CoA was taken up by the cells in a period of 20 mins incubation. 21% of the total incorporated is present in the plasma membrane. This results suggest that the observed effect on cell adhesion might be due to changes in plasmalemmal lipids. Hence, the effect on the adhesion and spreading could be explained either in terms of the action of membrane electrodynamic forces or membrane fluidity. It should be noted however, that the total oleoyl-CoA incorporated was reduced by 43% when the experiment was carried out in serum-containing medium. This may explain the lack of effect of acyl-Coenzyme A on BHK cell adhesion as well as other functions in serum-containing medium. The fatty acid metabolites, Prostacyclin (PGI2), prostaglandin E2, E1 and leukotrienes B4 (LTB4) exerted different effects. At the time where 1.25 and 0.125 muM LTB4 increased BHK and slightly endothelial cell-cell attachment and PGE1 (1-50muM) enhanced BHK cell-polystyrene adhesion, prostacyclin and prostaglandin E2 at concentrations up to 5 mug/ml did not have any clear effect on BHK cell-substratum adhesion. Inhibitors of arachidonic acid release, Bromophenacyl Bromide and mepacrine (quinacrine), significantly reduced BHK fibroblast adhesion to polystyrene surfaces. There was an irreversible inhibition of adhesion. Viability tests revealed that approximately 95% of the cells were in a viable state. Tests were also made on calcium and protein kinase C modulators. A diacylglycerol kinase inhibitor (R59022), reduced BHK fibroblast as well as endothelial cell adhesion to polystyrene tissue culture dishes in both serum and serum-free conditions. Endothelial cell adhesion in 3% serum Ham's F-10 was reduced by approximately 90% of the control samples when a concentration of 18.4 mug/ml was used. In contrast, a concentration of 30 mug/ml was needed to get the same reduction with BHK cells under the same conditions. The above concentration (30microg/ml) however, decreased BHK cell adhesion by 40% of the control samples in serum-free conditions.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Cellular biology
• Date of Award: 1990
• Depositing User: Enlighten Team
• Unique ID: glathesis:1990-78215
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 30 Jan 2020 15:36
• Last Modified: 30 Jan 2020 15:36
• URI: https://theses.gla.ac.uk/id/eprint/78215