Efficient entrapment of large and small compounds during vesiculation of intestinal microvilli.
An efficient method is described permitting the encapsulation of membrane-impermeable compounds at the interior of intestinal microvilli during vesicle formation. Rat intestinal epithelial cells were isolated by high-frequency vibration and exposed transiently to iso-osmotic medium containing 5 mM-EDTA. Vesiculation of microvilli was effected by freeze-thawing instead of mechanical fragmentation or hypo-osmotic lysis. Solutes to be entrapped were mixed with the extracellular medium before freezing in liquid N2. Microvillous vesicles were isolated from thawed cell suspensions by Ca2+- or Mg2+-aggregation of contaminants and differential centrifugation. The yield, purity, orientation and transport properties of the vesicles were similar, or superior, to preparations described in the literature. A high loading efficiency was demonstrated for small impermeants (cyclic GMP, ATP, Arsenazo III) as well as proteins (albumin); in contrast, loading of isolated vesicles by hypo-osmotic shock was only partially effective (cyclic GMP, ATP) or ineffective (albumin). Entrapment of an ATP-regenerating system could partially block a Mg2+-dependent conversion of intravesicular ATP into ADP. No evidence was obtained for the contribution of a proton pump to the intrinsic Mg2+-ATPase of the vesicle. Potential applications of the vesicle-loading technique in studies of brush-border transport regulation by intramicrovillar factors are discussed.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002458:Cell Fractionation / Q000379:methods; D006152:Cyclic GMP / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D005615:Freezing; D007421:Intestine, Small / Q000378:metabolism*; D008297:Male; D008871:Microvilli / Q000378:metabolism*; D008970:Molecular Weight; D009997:Osmotic Pressure; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D011506:Proteins; D000255:Adenosine Triphosphate; D006152:Cyclic GMP