Two types of clathrin-coated vesicles isolated from rat brain: analysis of biochemical properties and cellular origin.
Two major fractions rich in clathrin-coated vesicles (CVs) (fraction I, rho = 1.140 g/cm3; fraction II, rho = 1.113 g/cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl- translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85 +/- 9.5 nm in diameter; fraction II, 72 +/- 6.8 nm in diameter). Fraction II contained potent ouabain-sensitive ATPase activity, whereas fraction I contained only a little activity. Immunoblot analysis for the Na+,K(+)-ATPase catalytic subunit, alpha and alpha(+), demonstrated that fraction II exhibited predominantly alpha(+), whose proportion to alpha was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling-chase experiments using 125I-transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface-bound ligand in the fraction with lower density (rho = 1.090 g/cm3), a result suggesting that the involvement of Na+,K(+)-ATPase in fraction II is attributable to endocytic vesicles. Cl- uptake in fraction II was approximately threefold higher than that in fraction I. N-Ethylmaleimide (100 microM) completely inhibited the Cl- uptake in fraction I but partially (approximately 50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types--glial and neuronal cells--and differ in size distribution of CVs, content of Na+,K(+)-ATPase, and mechanism for Cl- uptake.
Uriu
T
T
Department of Pharmacology, Kansai Medical University, Osaka, Japan.
Omori
K
K
Omori
K
K
Yamamoto
A
A
Inoue
M
M
Inagaki
C
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002460:Cell Line; D005591:Chemical Fractionation; D002712:Chlorides / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D009457:Neuroglia / Q000648:ultrastructure; D009474:Neurons / Q000648:ultrastructure; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D002712:Chlorides; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase