Proton gradient formation in early endosomes from proximal tubules.
Heavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (delta pH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (K(m) = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by amiloride derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H(+)-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl(-)-channel, Na+/H(+)-exchanger) present in early endosomes isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient.
Marshansky
V
V
Nephrology Laboratory, Centre de Recherche Louis-Charles Simard, Hôpital Notre-Dame de Montréal, Québec, Canada. marshanv@ere.umontreal.ca
Vinay
P
P
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000165:Acridine Orange; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002458:Cell Fractionation; D018118:Chloride Channels / Q000502:physiology; D002712:Chlorides / Q000494:pharmacology; D004285:Dogs; D011992:Endosomes / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D007672:Kidney Cortex / Q000502:physiology; D007687:Kidney Tubules, Proximal / Q000502:physiology* / Q000648:ultrastructure; D008871:Microvilli / Q000502:physiology* / Q000648:ultrastructure; D008985:Monensin / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology; D025262:Vacuolar Proton-Translocating ATPases
D018118:Chloride Channels; D002712:Chlorides; D009579:Nitrobenzoates; D017923:Sodium-Hydrogen Exchangers; C048793:methylisopropylamiloride; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D000584:Amiloride; D000255:Adenosine Triphosphate; D008985:Monensin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D000165:Acridine Orange; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid