Surface properties of membrane vesicles prepared from muscle cells of Ascaris suum.
To facilitate biochemical, pharmacological, and biophysical studies on the membrane of the body muscle of Ascaris suum, a method for preparing intact vesicles was developed. Vesicles were prepared by incubating a muscle flap preparation with 1 mg/ml collagenase in a saline solution and then washing in saline without enzyme. The vesicles then formed gradually over the next hour as outgrowths of the original surface membrane from the bag region of the muscle. The vesicles were harvested readily by suction using a Pasteur pipette. The structure of the vesicles was examined with the transmission electron microscope. The whole-cell patch-clamp technique showed that the vesicles had a high input resistance and that the membrane was complete. The vesicle membrane was shown to contain Ca-activated Cl channels and gamma-aminobutyric acid-activated Cl channels. The vesicles also were shown to be suitable for fluorescence recovery after photobleaching studies designed to examine lateral and vertical movement of a lipid probe (5-N [octadecanoyl]-aminofluorescein) in the membrane. This probe had a mean lateral diffusion coefficient (DL) of 8.1 x 10(-9) cm2/sec, but only a proportion (68.4%) of the probe was mobile. The latter observation illustrated the nonuniform nature of the membrane. Ivermectin (10(-7) M) had no effect on DL or percent recovery. Trypan blue quenching experiments showed that the lipid probe remained in the outer monolayer of the membrane. These observations illustrate the experimental value of the vesicles; they are potentially useful in discerning anthelmintic mode of action and in drug screening.
Department of Preclinical Veterinary Sciences, Summerhall, R.(D.)S.V.S. University of Edinburgh, United Kingdom.
R J
RJ
Martin
J R
JR
Kusel
A J
AJ
Pennington
D000818:Animals; D001200:Ascaris / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D004058:Diffusion; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D007559:Ivermectin / Q000494:pharmacology; D008563:Membrane Lipids / Q000378:metabolism; D008564:Membrane Potentials; D008854:Microscopy, Electron; D013499:Surface Properties; D014343:Trypan Blue / Q000493:pharmacokinetics; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D002712:Chlorides; D007473:Ion Channels; D008563:Membrane Lipids; D005680:gamma-Aminobutyric Acid; D007559:Ivermectin; D014343:Trypan Blue; D002118:Calcium