Effect of cell volume on Acridine Orange fluorescence in hepatocytes

Abstract

Hepatic proteolysis is inhibited by cell swelling following a variety of experimental manoeuvres, such as reduction of extracellular osmolarity, concentrative uptake of amino acids, or blockade of K+ channels by barium. On the other hand, proteolysis is known to be accomplished by pH-sensitive lysosomal proteases. Accordingly, NH3/NH4+ inhibits proteolysis by intralysosomal alkalinization. The present study has been performed to test for an effect of cell volume on the pH of acidic intracellular compartments, as assessed by Acridine Orange fluorescence at > 520 nm (F > 520). F > 520 is enhanced by NH3/NH4+ (2 and 20 mmol/l respectively), by glutamine (2 mmol/l), by the K(+)-channel blocker barium (10 mmol/l) and by reduction of extracellular osmolarity (by 20 and 80 mosmol/l respectively). The observations point to release of Acridine Orange from acidic cellular compartments, which is indicative of alkalinization of these compartments during cell swelling. This effect may contribute to the regulation of proteolysis.

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

“…The present observations confirm previous studies demonstrating that osmotic cell swelling increases the apparent vesicular pH as calculated using FITC-dextran and triggers release of acridine orange from acidic vesicles (3,17,18). Alkalinization of acidic vesicles is expected to increase the fluorescence ratio of FITC-dextran, which remains trapped within vesicular compartments, including endosomes and lysosomes, after being endocytosed (14).…”

“…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.…”

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.…”

The Src family kinases: distinct functions of c-Src, Yes, and Fyn in the liver

“…Evidence has been presented for a direct activation of the vesicular proton pump by Cl - (58) , independent of the role of chloride channels in shunting the endosomal membrane potential, which is generated by the proton pump. A hyperosmolarity-induced increase of the cytosolic chloride concentration may thus indirectly augment vesicular acidification, whereas a decrease in cytosolic chloride could explain the vesicular alkalinization, which is observed in response to cell swelling (64) . Regardless, the mechanisms underlying the osmo-sensitivity of endosomal pH, modulation of endosomal pH must be seen as one site of hepatocellular osmo-sensing through the modulation of acidic sphingomyelinase (ASM) activity and subsequent ceramide formation, which have been described as a downstream consequence of endosomal acidification in this experimental setting (56) .…”