On the distribution of a fluorescent ivermectin probe (4" 5,7 dimethyl-bodipy proprionylivermectin) in Ascaris membranes.
The distribution of the fluorescent ivermectin derivative (4" 5,7 dimethyl bodipy proprionylivermectin, referred to here as bodipy ivermectin) was studied in muscle vesicle membranes from the parasite Ascaris suum. Incubation in bodipy ivermectin, at concentrations greater than 1 ng/ml, produced a dose-dependent fluorescence in the vesicles. The level of fluorescence was not depressed (but increased) by prior incubation with 1 microM unlabelled ivermectin. This observation suggested that most of the bodipy ivermectin was free in the lipid phase of the membrane and not associated with a receptor. The trypan blue quenching technique showed that the bodipy ivermectin did not cross the membrane bilayer but remained in the outer monolayer. Lateral mobility of the probe in the membrane was studied using the fluorescence recovery after photobleaching (FRAP) technique. The lateral diffusion coefficient (DL) was 14.0 x 10(-10) cm2/s which was nearly three times faster than the lipid probe octadecanoyl-aminofluorescein in membranes from the same preparations; only 46% of the probe was mobile, showing that within a 1.2 microns radius the distribution of the bodipy ivermectin in the membrane was not homogeneous. These observations are used to suggest that: the site of action of ivermectin is located in the outer monolayer of the membrane (possibly on the Cl- channel); and that the speed of onset of action in isolated membrane preparations may be limited by diffusion in the membrane.
Martin
R J
RJ
Department of Pre-clinical Veterinary Sciences, University of Edinburgh.
Kusel
J R
JR
D000818:Animals; D001200:Ascaris / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D007559:Ivermectin / Q000378:metabolism*; D014343:Trypan Blue
D005456:Fluorescent Dyes; D007559:Ivermectin; D014343:Trypan Blue