Proceedings of The Physiological Society

University of Liverpool (2002) J Physiol 543P, S167


Extraction of cholesterol abolishes phasic contraction of rat and guinea-pig ureter

E. Babiychuk*†, A. Draeger†, T. Burdyga* and Susan Wray*

*Department of Physiology, University of Liverpool, Liverpool L69 3BX, UK and †Institute of Anatomy, University of Bern, Bern 9, CH 3000, Switzerland

The sarcolemma of smooth muscle cells is divided into spatially distinct microcompartments of sphingomyelin- and cholesterol-enriched rafts and glycerophospholipid-containing non-raft regions (Babiychuk & Draeger, 2000). Since the association of various signalling molecules with particular lipid microcompartments is essential for their physiological functioning (Simmons & Toomre, 2000), knowledge of the mechanisms regulating membrane compartmentalization is of paramount importance. In the present study we have investigated in smooth muscle, whether extraction of membrane cholesterol, (1) causes destabilization of membrane rafts and (2) has functional consequences, and the possible mechanisms involved.

Thin-layer chromatography, Western blotting, purification of porcine stomach smooth muscle microsomes and their extraction with non-ionic detergent were performed as described previously (Babiychuk & Draeger, 2000). Ureters were excised from rats or guinea-pigs humanely killed by cervical dislocation after CO2 anaesthesia, muscle strips dissected and tension recorded. Cholesterol was extracted from the ureteric strips by perfusion with Krebs solution containing 2 % methyl-β-cyclodextrin.

In the smooth muscle microscomes, methyl-β-cyclodextrin resulted in preferential solubilization of cholesterol, leaving other lipid constituents of the sarcolemma unaffected. However, such extraction resulted in destabilization of membrane rafts as judged from the loss of their resistance to non-ionic detergents (n = 3). Cholesterol extraction from rat (n = 8) or guinea-pig (n = 3) ureter resulted in the abolition of mechanical activity in response to electrical stimulation. This activity was restored in the presence of 5 mM TEA, an inhibitor of potassium channels (n = 6). The phasic but not tonic contractions of ureter caused by application of 10 µM carbachol were also completely inhibited (n = 3). Extraction of cholesterol had no effect on the amplitude of mechanical responses to depolarisation with 120 mM KCl (n = 12).

In conclusion, our data suggest that extraction of membrane cholesterol results in destabilization of membrane rafts. As a consequence of this there is a specific inhibition of the phasic contractions of rat and guinea-pig ureter, possibly due to a stimulation of K+ channels. This in turn suggests a selective effect of rafts on K+, but not Ca2+, channels, in the smooth muscle membrane.

This work was supported by the Medical Research Council (UK), Swiss National Science Foundation and the Novartis Foundation.

All procedures accord with current UK legislation.

Where applicable, experiments conform with Society ethical requirements