The mechanical properties of sarcolemmal vesicles from rabbit muscle: The effects of internal calcium and membrane active molecules

Biomechanics

Nichol, James A

University:University of Glasgow

关键词: Biomechanics;   

Others  :  http://theses.gla.ac.uk/71557/1/10391185.pdf

来源: University of Glasgow

【 摘 要 】

The technique of pipette aspiration was first developed by a biophysicist to study the mechanical properties of the red blood cells swollen into spherical form. In light of the discovery that skeletal muscle fibres can be induced to shed membrane vesicles, the technique has here been adapted to examine the mechanical properties of the muscle surface membrane and to provide novel evidence in the investigation of calcium-induced cell damage. For these experiments, sarcolemmal vesicles (mean diameter 71 mum) were prepared from rabbit psoas muscle by treatment with collagenase in a fibre-swelling buffer solution of 140 mM KCl and 5 mM HDEPES at pH 7.4. Vesicles prepared in this way had previously been examined by electronmicroscopic, biochemical and electrophysiological methods which confirmed their outside-out configuration and that they contained diluted muscle cytosol of similar composition to that found in intact muscle fibres. As far as is known, the preparation has retained the normal functional characteristics of the sarcolemma in situ, and represents a valid model for studying otherwise inaccessible properties of the muscle surface membrane. Vesicles were stressed by partial aspiration into parallel bore micropipettes (mean diameter 19.3 mum) pulled from borosilicate glass capillaries. As the aspiration pressure was gradually increased, the membrane projection inside the pipette lengthened as the membrane expanded in response to the increased isotropic membrane tension until a critical point was reached and the vesicle ruptured. A precise value of the isotropic tension was calculated by applying the Law of Laplace to the hemispherical portions of the vesicle inside and outside the pipette. By measuring also the increase in projection length, the area elasticity of the membrane was determined. The stress-strain plot of membrane tension against corresponding area expansion was typically linear right up to the lysis point, indicating that vesicles behaved as perfectly elastic structures. The maximum tension that vesicles could withstand was 12.43 mN m-1 (median) with 95% confidence limits of 12.29 to 12.67 mN m-1, and the corresponding fractional increase in membrane area was 0.027 (median) with 95% confidence limits 0.0265 and 0.0274. The elastic modulus of area expansion, K, was 490 +/- 88 (mean +/- S.D.). In accordance with previous biophysical studies on natural and artificial membranes, which were similarly conducted in nominally Ca2+-free buffers, the tensile strength and elastic modulus of sarcolemma are considered properties of the cohesive forces between membrane lipids and the extent to which membrane proteins perturb the lipid matrix. That the addition of 500 muM Ca2+ to the bathing solution had no effect on the strength or elasticity of vesicles suggested that external Ca2+ does not influence membrane mechanical properties. In studies on cell damage generally, much evidence pointed to the involvement of elevated intracellular Ca2+, an effect that was often demonstrated by using the calcium ionophore A23187. The pipette aspiration technique has revealed for the first time, information on the mechanical properties of the cell membrane which may help to explain how raised intracellular Ca2+ ultimately leads to cell death. It has also been demonstrated that this technique can provide precise, quantitative information on membrane stability and the perturbing effects of intercalating molecules.(Abstract shortened by ProQuest.).