Involvement of microtubules in the link between cell volume and pH of acidic cellular compartments in rat and human hepatocytes.

Abstract

Cell swelling is shown to induce an increase in acridine orange fluorescence intensity, an effect pointing to the alkalinization of acidic vesicles. Since autophagic hepatic proteolysis is accomplished by pH-sensitive proteinases within acidic lysosomes, this effect may contribute to the well-known inhibitory effect of cell swelling on proteolysis. In the present study, the role ofmicrotubules in volume-dependent alterations of pH in acidic vesicles of rat and human hepatocytes was studied. Colcemid and colchicine were used to depolymerize microtubules and vesicular pH was monitored using two dif- Amino acids elicit cell swelling by their concentrative uptake and cellular accumulation, while insulin exerts its effect by activation of ion uptake via Na+, K+, 2C-cotransport and Na+/H+ exchange (1, 2). The antiproteolytic action of insulin and glutamine is fully mimicked by the respective osmotic alterations of cell volume and is abolished if alterations of cell volume are reversed by appropriate alterations of extracellular osmolarity. Further, inhibition of Na+, K+, 2C-cotransport by furosemide or bumetanide not only blunts the swelling effect of insulin but also leads to a proportional inhibition of proteolysis. In the presence of both furosemide and amiloride, insulin-induced cell swelling and its antiproteolytic action are completely abolished. The mechanism linking cell volume alterations to proteolysis remained elusive, however, until recently when it was observed that cell swelling leads to an increase in acridine orange fluorescence intensity. This effect indicated an alkalinization of acidic intracellular compartments (3). Since proteolysis resides largely within acidic lysosomes and is accomplished by pH-sensitive lysosomal proteinases (4), the alkalinization of acidic intracellular compartments could indeed couple cell swelling to the inhibition of proteolysis. The mechanism by which cell volume changes are communicated to the acidic cellular compartments remained, however, unknown. In view of the described interaction between microtubules and intracellular vesicles (5, 6), the present study has been performed to examine a possible involvement of the microtubule network. To this end, the effect of cell swelling on fluorescein isothiocyanate (FITC)-dextran or acridine orange fluorescence has been studied both in intact cells and in cells treated with either colchicine or colcemid, drugs known to depolymerize microtubules (7,8). In addition, a possible effect of y--lumicolchicine, a stereoisomer of colchicine without an inhibitory effect on microtubules (9), was investigated. METHODSCell Culture. Human hepatocytes were prepared from pieces ofliver not appropriate for transplantation. The pieces were perfused through polyethylene catheters inserted into the main portal veins of the cut surface. The subsequent isolation procedure was according to the method ofBerry and Friend (10) with some modifications (11). Rat hepatocytes were prepared by collagenase treatment according to a method previously de...

Endosomal Acidification and Activation of NADPH Oxidase Isoforms Are Upstream Events in Hyperosmolarity-induced Hepatocyte Apoptosis

“…As reported earlier in studies on FITC-dextran or acridine orange-loaded hepatocytes (27,(43)(44)(45), hypo-osmotic exposure of rat hepatocytes leads to an alkalinization of endocytotic vesicles, whereas hyperosmotic hepatocyte shrinkage induced acidification. The functional significance, however, remained unknown.…”

Influence of Acanthamoeba castellanii on Intracellular Growth of Different Legionella Species in Human Monocytes

“…A decrease of extracellular osmolarity leads to osmotic cell swelling, which modifies a variety of cellular functions, such as transport, metabolism, cell proliferation, and cell death (for a review, see reference 27). Most notably, it is well established that cell swelling results in alkalinization whereas cell shrinkage results in acidification of endosomal pH (10)(11)(12)(13)(14)(15)(42)(43)(44). Previous investigations have shown that, due to inhibition of the fusion of the phagosome and lysosome, the phagosome of L. pneumophila does not become acidified (25,26,38) and therefore enables the intracellular multiplication of bacteria.…”

Untitled

“…Intracellular Ca 2 has been shown to increase upon cell swelling (Hoffmann & Simonsen, 1989;Baquet et al, 1991b;contrast Schreiber et al, 1996, who found no change), but this cannot explain the antiproteolytic effect of cell swelling because an increase in Ca 2 results in activation rather than in inhibition of macroautophagy (Caro, 1989;Gordon et al, 1993). Recent work by Häussinger and colleagues (Schreiber & Häussinger, 1995;Schreiber et al, 1996) with fluorescein isothiocyanate (FITC)-dextran indicates that cell swelling causes alkalinization of an early endocytic compartment rather than alkalinization of the lysosomes, as was originally thought (Busch et al, 1994). The latter finding is in line with recent data of Luiken et al (1996): in order to monitor changes in lysosomal pH in hepatocytes, we used the pH-dependent conversion of lysylalanyl-4-methoxy-2-naphthylamide to lysylalanine and fluorescent 4-methoxy-2-naphthylamine by the lysosomal enzyme dipeptidylpeptidase II, and also failed to observe changes in lysosomal pH after hypo-osmotic swelling of the cells.…”