Rebecca W. Van Dyke

Mechanisms of Hepatic Electrolyte Transport

Why Electrolytes? General Considerations Overview and Definition of Terms Special Considerations With Reference to Liver Sodium-Coupled Transport Regulation of Hepatic Na + ,K+ -Adenosine Triphosphatase Regulation of Cation Permeability Summary Canalicular Bile Formation Bile Acid-Dependent Bile Formation Choleretic Potency of Bile Acids Effects of Ion Substitution on Bile Acid-Dependent Bile Formation Bile Acid-Independent Bile Formation Sodium-Coupled Chloride Transport Bicarbonate Transport Energetics of Canalicular Bile Formation Persisting Uncertainties Other Hepatocellular Functions Gluconeogenesis and Glycolysis Biotransformation Intracellular pH Mitogenesis Cellular Calcium Homeostasis

Acidic Vesicles in Cultured Rat Hepatocytes: Identification and. Characterization of Their Relationship to Lysosomes and Other Storage Vesicles

We and others recently have demonstrated adenosine triphosphate-dependent acidification in a variety of prelysosomal organelles isolated from liver including clathrin-coated vesicles, multivesicular bodies, and Golgi. Little is known, however, regarding the number or distribution of acidic compartments in intact hepatocytes. We therefore have utilized acridine orange, a fluorescent weak base, to study the number and distribution of acidic vesicles of rat hepatocytes in primary culture and compared these with the number and distribution of lysosomes and other storage vesicles. Hepatocytes were found to contain about 170 acidic compartments per cell by fluorescence microscopy. These vesicles were diffusely distributed throughout the cell cytoplasm, with about 50% in the perinuclear area by modified morphometry. The acridine orange staining of these vesicles was reversibly dissipated by monensin, NH4Cl, chloroquine, and primaquine, indicating these vesicles exhibit an acidic interior established by active proton transport. In addition, the cholestatic agent chlorpromazine reversibly inhibited, in a dose-dependent fashion, the redevelopment of a pH gradient in the acidic vesicles after dissipation by monensin. The number and distribution of these acidic vesicles were not significantly different from the number and distribution of vesicles involved in the storage (up to 6 h after internalization) of the fluid phase marker fluorescein-dextran. By contrast, histochemically identifiable lysosomes were fewer in number and significantly more restricted in their distribution to the perinuclear area (89%) than either dextran-storing or acidic vesicles. Electron microscopic studies confirmed that endocytosed dextran as well as another fluid phase marker, colloidal gold, were found predominantly in acid phosphatase- and arylsulfatase-negative vesicles for up to 6 h after internalization. These studies indicate that hepatocytes contain numerous intracellular vesicles acidified by an active H+ transport mechanism. Based on their comparative number and distribution, acidic vesicles probably include vesicles involved in fluid-phase endocytosis but only a minority are lysosomes. The findings also indicate that fluid-phase markers are stored predominantly in vesicles other than histochemically identifiable lysosomes for up to 6 h after internalization. Finally, this technique also affords the opportunity for studying the movement of such vesicles in a vital preparation.

ATP-dependent proton transport by isolated brain clathrin-coated vesicles. Role of clathrin and other determinants of acidification

We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H+ transport specific activity was shown by column chromatograpy to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H+ transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (Cl- greater than Br- much greater than NO3- much greater than gluconate, SO2-(4), HPO2-(4), mannitol; Km for Cl- congruent to 15 mM), but was relatively insensitive to cation replacement as long as Cl- was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC50 less than 10 microM), dicyclohexylcarbodiimide (DCCD) (IC50 congruent to 10 microM), chlorpromazine (IC50 congruent to 15 microM), and oligomycin (IC50 congruent to 3 microM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate much greater than NO3- greater than Cl-), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol greater than potassium gluconate greater than KCl) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H+ transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport (r = 0.95) in the presence of various Cl- salts and KNO3. Finally, vesicles prepared from bovine brain synaptic membranes exhibited H+ transport activity similar to that of the coated vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat pancreatic zymogen granules: An actively acidified compartment

In this study, we looked for acidification in pancreatic zymogen granules as recently reported for other secretory vesicles. In intact dispersed acinar cells, acidic intracellular compartments identified by fluorescence microscopy using acridine orange corresponded exactly to the distribution of zymogen granules visualized by light microscopy. Acridine orange fluorescence in zymogen granules was reversibly dissipated by protonophores (carbonyl cyanide m-chlorophenylhydrazone, monensin) and NH4Cl; and the percentages of cytoplasmic area occupied by the acidic compartments and by zymogen granules were identical under fasting conditions and decreased in parallel after in vivo cholinergic stimulation. Zymogen granules released acutely from hypotonically disrupted cells without homogenization also accumulated acridine orange. Red-orange fluorescence in released granules was also abolished by protonophores and NH4Cl; and it reappeared after washout of protonophores in the presence, but not absence of adenosine triphosphate. Dicyclohexylcarbodiimide, which inhibits all proton pumps, and N-ethylmaleimide, which inhibits the proton pump of endocytic vesicles and lysosomes, but not mitochondria, prevented this adenosine triphosphate-dependent reappearance of acridine orange fluorescence, whereas vanadate did not. In contrast to these observations with zymogen granules in situ or acutely released from disrupted cells, granules isolated by conventional multistep homogenization/centrifugation procedures did not exhibit adenosine triphosphate-dependent acidification or development of a positive membrane potential as measured by quenching of acridine orange or Oxonol V, respectively. The latter findings may indicate release of inhibitors or granule damage during isolation. Collectively, the present results provide direct evidence that zymogen granules contain an active acidification mechanism which appears similar to that of other secretory vesicles and endosomes. This acidification process may have important implications for the storage, stabilization, and secretion of intragranular proteins including proenzymes.

Rat pancreatic zymogen granules

Abstract In this study, we looked for acidification in pancreatic zymogen granules as recently reported for other secretory vesicles. In intact dispersed acinar cells, acidic intracellular compartments identified by fluorescence microscopy using acridine orange corresponded exactly to the distribution of zymogen granules visualized by light microscopy. Acridine orange fluorescence in zymogen granules was reversibly dissipated by protonophores (carbonyl cyanide m-chlorophenylhydrazone, monensin) and NH 4 Cl; and the percentages of cytoplasmic area occupied by the acidic compartments and by zymogen granules were identical under fasting conditions and decreased in parallel after in vivo cholinergic stimulation. Zymogeh granules released acutely from hypotonically disrupted cells without homogenization also accumulated acridine orange. Red-orange fluorescence in released granules was also abolished by protonophores and NH 4 Cl; and it reappeared after washout of protonophores in the presence, but not absence of adenosine triphosphate. Dicyclohexylcarbodiimide, which inhibits all proton pumps, and N -ethylmaleimide, which inhibits the proton pump of endocytic vesicles and lysosomes, but not mitochondria, prevented this adenosine triphosphate-dependent reappearance of acridine orange fluorescence, whereas vanadate did not. In contrast to these observations with zymogen granules in situ or acutely released from disrupted cells, granules isolated by conventional multistep homogenization/centrifugation procedures did not exhibit adenosine triphosphate-dependent acidification or development of a positive membrane potential as measured by quenching of acridine orange or Oxonol V, respectively. The latter findings may indicate release of inhibitors or granule damage during isolation. Collectively, the present results provide direct evidence that zymogen granules contain an active acidification mechanism which appears similar to that of other secretory vesicles and endosomes. This acidification process may have important implications for the storage, stabilization, and secretion of intragranular proteins including proenzymes.

Characterization of the ouabain-insensitive ATPase activity of rat liver plasma membranes.

Very little is currently known about the ouabain-insensitive ATPase activity of the liver plasma membrane; we have therefore characterized it in plasma membranes from rat liver prepared using two different isolation techniques. Greater than 85% of ATPase activity in both preparations was ouabain-insensitive. Based on the effects of multiple inhibitors, including dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide (NEM), vanadate, and oligomycin, the ouabain-insensitive ATPase activity of rat liver plasma membranes consists of a family of ATP-hydrolysing enzymes. Ouabain-insensitive ATPase activity was stimulated by HCO3-in both plasma membranes (13%) and mitochondria (69%) over the range of 7.5 to 9.0, and HCO3--stimulation was similarly inhibited by oligomycin in both preparations. A fraction of the ouabain-insensitive ATPase of liver plasma membranes is inhibited by DCCD and is resistent to inhibition by oligomycin; these characteristics are similar to those of the non-mitochondrial H+-ATPases recently described in lysosomes, endosomes, clathrin-coated vesicles, and Golgi from liver and other cell types.