Determination of extracellular space and intracellular electrolytes in rat liver <i>in vivo</i>*

Ja, Williams; Woodbury, Dixon M.

doi:10.1113/jphysiol.1971.sp009311

Cited by 61 publications

(12 citation statements)

References 31 publications

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“…This is less than previously reported values ranging between 16 and 22 mM (16)(17)(18). These disparities may be explained in part by differences in technique.…”

Section: Resultscontrasting

confidence: 68%

Transport of sodium, chloride, and taurocholate by cultured rat hepatocytes

Scharschmidt

Stephens

1981

Proc. Natl. Acad. Sci. U.S.A.

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Transport of sodium, chloride, and taurocholate was studied in primary cultures-ofadult rat hepatocytes incubated in a balanced electrolyte solution containing 150 mM NaCI, various concentrations oftaurocholate, and 'Na, 31C1, [3H] Bile acids are the predominant organic solute in bile and their importance in bile formation is attested to by the observation that the rate of canalicular bile formation is linearly related to bile acid output in all species thus far examined (1). Previous investigations (2-8) have suggested that bile acid uptake is saturable, concentrative, and partially sodium dependent. While these observations are consistent with a sodium-facilitated or sodium-cotransport mechanism, no simultaneous measurement of bile acid and sodium fluxes has been reported, and information regarding the nature of the dependence of concentrative bile acid uptake on extracellular sodium and the sodium gradient is not available. Moreover, little is known regarding the transport by hepatocytes ofeither sodium or chloride, which together account for more than half oftotal bile osmolality.The experiments described in this paper concern three principal areas. First, we report a technique that permits measurement of uptake of sodium and chloride as well as of other inorganic and organic solutes by cultured rat hepatocytes. Second, uptake kinetics and apparent steady-state intracellular concentration of sodium and chloride in cultured rat hepatocytes have been determined. Third, the relationship of uptake and intracellular accumulation of taurocholate to extracellular sodium concentration, sodium uptake, and the transmembrane sodium gradient have been defined. The (14); or ouabain (1 mM), taurocholate, or both were added.Technique. Coverslips with adherent hepatocytes were preincubated 10 min in medium identical to that used in the uptake study but without radioisotope or added tauroctholate. A 1-hr preincubation in ouabain-containing medium was utilized in studies ofthe effect ofouabain on taurocholate transport. The coverslips were then transferred to 26 x 33 mm incubation wells (Flow Laboratories) containing 3 ml of appropriate medium and 1-2 puCi of radioisotope (1 Ci -= 3.7 x 10"°becque-rels). At time intervals varying between 10 sec and 2 hr, the cellcoated coverslips were washed by dipping them for 30 sec in each of eight 30-ml beakers containing 25 ml of ice-cold incubation medium without isotope. The cells were then scraped into Lowry's solution (0.1 M NaOH/0. 189 M Na2CO3) with a rubber spatula. Radioactivity and protein in the Lowry's solution and radioactivity in the incubation medium were measured 986The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…This is less than previously reported values ranging between 16 and 22 mM (16)(17)(18). These disparities may be explained in part by differences in technique.…”

Section: Resultscontrasting

confidence: 68%

Transport of sodium, chloride, and taurocholate by cultured rat hepatocytes

Scharschmidt

Stephens

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…For these drugs, the proportion of drug ionized at physiological pH (7.4) will be between 95.2 and 99.8 based on their pK a values. If the hepatocellular cytosolic pH is assumed to be 7.2 (Williams and Woodbury, 1971;Nilsson et al, 2004), the ratio between the unbound ionized drug inside and outside the cell will be approximately 1.6 for all these compounds. This ratio is close to that of total (ionized and un-ionized) drug because of the degree of ionization (eq.…”

Section: Resultsmentioning

confidence: 99%

“…The concentration of drug in hepatocytes will be influenced by the difference in pH between the hepatocyte cytosol (pH ϭ 7.1-7.2, Williams and Woodbury, 1971;Nilsson et al, 2004) and the medium (pH ϭ 7.4). The relative concentration of ionized drug molecules (for bases) between the cytosol and the medium is given by eq.…”

Section: Methodsmentioning

confidence: 99%

Saturable Uptake of Lipophilic Amine Drugs into Isolated Hepatocytes: Mechanisms and Consequences for Quantitative Clearance Prediction

Hallifax

Houston

2007

Drug Metabolism and Disposition

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ABSTRACT:The hepatic uptake of quinine, fluvoxamine, and fluoxetine (0.1-10 M) was investigated with freshly isolated rat hepatocytes. The cell-to-medium concentration ratios (K p ) were concentration-dependent: the mean maximum K p values (at 0.1 M) were 150 (quinine), 500 (fluvoxamine), and 2000 (fluoxetine). There was also a large capacity site that was not saturable over the concentration range used (possibly partition into the phospholipid component of membranes); representing this site, the mean minimum K p values (at 10 M) were 30 (quinine), 200 (fluvoxamine), and 500 (fluoxetine). To eliminate concomitant metabolism, cells were pretreated with the irreversible P450 inhibitor, aminobenzotriazole. The saturable uptake was substantially eliminated after exposure to carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (ATP inhibitor). The difference between the maximum and minimum K p for these three amine drugs, as well as for dextromethorphan, propranolol, and imipramine, was within a limited range of 3-fold, indicating a common magnitude of saturable uptake. Basic, permeable drugs are expected to be sequestered into lysosomes, which actively maintain their low internal pH (ϳ5) using ATP, and this process is predictable from the combined effects of pH-driven ion accumulation and unsaturable binding representing partition into membranes. The resultant predicted maximum K p correlated strongly with the observed maximum K p . Thus, at low substrate concentrations, the fraction of drug unbound in the hepatocyte incubation (critical for assessing drug clearance and drug-drug interaction potential) may be dependent upon saturable as well as unsaturable binding, and for lipophilic, basic drugs, this can be readily estimated assuming a common degree of uptake into lysosomes.

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“…(iii) an additional quantity of blood that is retained when animals, as in this study, are not exhaustively exsanguinated (10 Fig. 1 by separately measured densities, which were, respectively: 1.126 ± 0.007, 1.068 ± 0.024,1.145 ± 0.009, and 1.135 + 0.019 g/ml (n = 6-8).…”

mentioning

confidence: 99%

“…1 and Table 1). Protein concentrations (mg/g) in the four groups were (left to right) 230.3 ± 6.5, 248.7 ± 4.0, 157.8 ± 2.7, and 204.0 ± 3.1 (8)(9)(10)(11)(12) livers per group). Line B.then was obtained by subtracting from A the following sources of nonhepatocyte protein: (i) sinusoidal cell protein, estimated as -45 mg/ml of parenchyma (17,18), and (ii) extracellular protein, computed from the content of erythrocytes and combined plasma and nonvascular spaces (see Experimental Procedures).…”

mentioning

confidence: 99%

Quantitative correlation between proteolysis and macro- and microautophagy in mouse hepatocytes during starvation and refeeding.

Mortimore¹,

Hutson²,

Surmacz³

1983

Proc. Natl. Acad. Sci. U.S.A.

164

102

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Cytoplasmic protein in hepatocytes is sequestered and degraded by two general classes of lysosomes, overt autophagic vacuoles (macroautophagy) and dense bodies (microautophagy). Volumes of the apparent space in each class that contain the internalized protein, together with estimates of cytoplasmic protein concentration, were used as a basis for predicting rates of protein degradation by the lysosomal system in livers of fed, 48-hr starved, and starved-refed mice. Assuming that the turnover of all sequestered protein is equal to that previously determined in overt autophagic vacuoles (0.087 min'), we obtained close agreement between predicted and observed rates in the three conditions studied. The two autophagic components, though, exhibited different patterns of regulation. Microautophagy followed a downward course through starvation and into refeeding, a trend that explained fully the fall in absolute rates of protein degradation during starvation. By contrast, macroautophagy remained constant throughout starvation but was virtually abolished with refeeding. Whereas regulation of the latter can be explained largely by immediate responses to the supply of amino acids, present evidence together with results of others indicate that microsequestration could be linked to functional and quantitative alterations in the smooth endoplasmic reticulum. Both types of regulation contributed equally to the marked suppression of proteolysis during cytoplasmic regrowth.Protein and other cytoplasmic constituents in hepatocytes are internalized within the lysosomal system in two general ways: (i) by the formation of overt autophagic vacuoles, a highly visible mechanism involving the sequestration of organelles within smooth-surfaced membranes (1)(2)(3)(4)(5), and (ii) by the engulfment of small bits of cytoplasm that subsequently appear in dense bodies (1, 3-5), a microautophagic process that we believe accounts for basal protein turnover (5). Although both are ongoing functions, we have shown in perfused livers of fed animals that the addition or deletion of amino acids can regulate overt autophagy rapidly over a wide range without appreciably affecting microautophagy (3, 5). On the other hand, the induction of cytoplasmic growth, such as that produced by starvation and refeeding, is associated with decreases in rates of protein breakdown (6, 7) and sequestered protein (7) to levels that can be explained only by the suppression of both autophagic functions. Nothing is presently known, however, of the way microinternalization is regulated.The present investigation is an attempt to quantify by stereologic methods the contributions of each process to protein breakdown in livers of fed, starved, and starved-refed mice and to compare degradation rates predicted by this approach with experimentally determined rates. Close agreement was found under all conditions, although activities of the individual autophagic components were affected in distinctly independent ways. Rates of overt autophagy were interpretable from the gene...

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Determination of extracellular space and intracellular electrolytes in rat liver *in vivo**

Cited by 61 publications

References 31 publications

Transport of sodium, chloride, and taurocholate by cultured rat hepatocytes

Transport of sodium, chloride, and taurocholate by cultured rat hepatocytes

Saturable Uptake of Lipophilic Amine Drugs into Isolated Hepatocytes: Mechanisms and Consequences for Quantitative Clearance Prediction

Quantitative correlation between proteolysis and macro- and microautophagy in mouse hepatocytes during starvation and refeeding.

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Determination of extracellular space and intracellular electrolytes in rat liver in vivo*

Cited by 61 publications

References 31 publications

Transport of sodium, chloride, and taurocholate by cultured rat hepatocytes

Transport of sodium, chloride, and taurocholate by cultured rat hepatocytes

Saturable Uptake of Lipophilic Amine Drugs into Isolated Hepatocytes: Mechanisms and Consequences for Quantitative Clearance Prediction

Quantitative correlation between proteolysis and macro- and microautophagy in mouse hepatocytes during starvation and refeeding.

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Determination of extracellular space and intracellular electrolytes in rat liver *in vivo**