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1.
Biomed Res Int ; 2015: 487256, 2015.
Article in English | MEDLINE | ID: mdl-25866786

ABSTRACT

Cellular swelling is controlled by an active mechanism of cell volume regulation driven by a Na(+)/K(+)-dependent ATPase and by aquaporins which translocate water along the osmotic gradient. Na(+)/K(+)-pump may be blocked by ouabain, a digitalic derivative, by inhibition of ATP, or by drastic ion alterations of extracellular fluid. However, it has been observed that some tissues are still able to control their volume despite the presence of ouabain, suggesting the existence of other mechanisms of cell volume control. In 1977, by correlating electron microscopy observation with ion and water composition of liver slices incubated in different metabolic conditions in the presence or absence of ouabain, we observed that hepatocytes were able to control their volume extruding water and recovering ion composition in the presence of ouabain. In particular, hepatocytes were able to sequester ions and water in intracellular vesicles and then secrete them at the bile canaliculus pole. We named this "vesicular mechanism of cell volume control." Afterward, this mechanism has been confirmed by us and other laboratories in several mammalian tissues. This review summarizes evidences regarding this mechanism, problems that are still pending, and questions that need to be answered. Finally, we shortly review the importance of cell volume control in some human pathological conditions.


Subject(s)
Cell Size , Cytoplasmic Vesicles/metabolism , Ouabain/metabolism , Water-Electrolyte Balance/physiology , Animals , Humans , Ion Transport/physiology , Sodium-Potassium-Exchanging ATPase/metabolism
2.
J Cell Biochem ; 81(1): 1-8, 2001.
Article in English | MEDLINE | ID: mdl-11180393

ABSTRACT

Heterotrimeric G protein G12 stimulates diverse physiological responses including the activities of Na+/H+ exchangers and Jun kinases. We have observed that the expression of the constitutively activated, GTPase-deficient mutant of Galpha(12) (Galpha(12)QL) accelerates the hyperosmotic response of NIH3T3 cells as monitored by the hyperosmotic stress-stimulated activity of JNK1. The accelerated response appears to be partly due to the increased basal activity of JNK since cell lines-such as NIH3T3 cells expressing JNK1-in which JNK activity is elevated, show a similar response. NIH3T3 cells expressing Galpha(12)QL also display heightened sensitivity to hyperosmotic stress. This is in contrast to JNK1-NIH3T3 cells that failed to enhance sensitivity although they do exhibit an accelerated hyperosmotic response. Reasoning that the increased sensitivity seen in Galpha(12)QL cells is due to a signaling component other than JNK, the effect of dimethyamiloride, an inhibitor of Na+/H+ exchanger in this response, was assessed. Treatment of vector control NIH3T3 cells with 50 microM dimethylamiloride potently inhibited their hyperosmotic response whereas the response was only partially inhibited in Galpha(12)QL-NIH3T3 cells. These results, for the first time, identify that NHEs are upstream of the JNK module in the hyperosmotic stress-signaling pathway and that Galpha(12) can enhance this response by modulating either or both of these components namely, JNKs and NHEs in NIH3T3 cells.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , 3T3 Cells , Animals , Heterotrimeric GTP-Binding Proteins/genetics , JNK Mitogen-Activated Protein Kinases , Mice , Mitogen-Activated Protein Kinases/metabolism , Mutation , Osmotic Pressure
3.
J Membr Biol ; 172(2): 101-11, 1999 Nov 15.
Article in English | MEDLINE | ID: mdl-10556358

ABSTRACT

Although both amiloride- and phloretin-sensitive Na(+)/Li(+) exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li(+) (0.2-2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li(+) by hepatocytes, preloaded with Na(+) and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K(m) 1.2 mm Li(+), V(max) 40 mumol. (kg dry wt. min)(-1), showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H(+) was curvilinear suggesting a modifier role of [H(+)](i). This system persisted in Na(+)-depleted cells, although with apparent K(m) 3.8 mm. (b) A phloretin-sensitive component, with K(m) 1.2 mm, V(max) 21 mumol. (kg. min)(-1), was unaffected by pH but was inactive in Na(+)-depleted cells. Phloretin inhibited Li(+) uptake and Na(+) efflux in parallel. (c) A residual uptake increased linearly with the external Li(+) concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li(+) uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H(+)].


Subject(s)
Lithium/administration & dosage , Lithium/pharmacokinetics , Liver/metabolism , Amiloride/pharmacology , Animals , Antiporters/antagonists & inhibitors , Antiporters/metabolism , Biological Transport/drug effects , Bumetanide/pharmacology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Hydrogen-Ion Concentration , Inhibitory Concentration 50 , Kinetics , Lithium/pharmacology , Liver/cytology , Liver/drug effects , Male , Ouabain/pharmacology , Phloretin/pharmacology , Rats , Rats, Sprague-Dawley , Sodium/metabolism , Thermodynamics
4.
J Membr Biol ; 162(2): 147-56, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9538508

ABSTRACT

The uptake of 3H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 microM and 5.0 microM medium choline; at 5 microM, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 microM), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na+ was replaced by Li+, K+ or Mg2+, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na+ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na+ concentration. Kinetic properties of the uptake of 0.1 microM 3H-choline in the presence and absence of medium Na+ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5-100 microM) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na+; a Na(+)-dependent portion (a mean of 0.52 of the total) had a K(m) for choline of 1.5 microM while K(m) in the absence of Na+ (Li+ substituting) was 18.6 microM. (b) Inhibition by HC-3 (0.3-300 microM) gave Ki values of 1.7 microM and 5.0 microM HC-3 for the Na(+)-dependent and -independent fractions. The apparent K(m) of the Na(+)-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K(m) values reported for high-affinity, Na(+)-dependent choline uptake by neuronal cells.


Subject(s)
Choline/pharmacokinetics , Epithelial Cells/metabolism , Sodium/pharmacology , Animals , Cells, Cultured , Choline/metabolism , Epithelial Cells/drug effects , Hemicholinium 3/pharmacology , Isotope Labeling , Kinetics , Lithium , Lung/metabolism , Magnesium , Male , Potassium , Rats , Time Factors , Tritium
5.
J Biol Chem ; 272(46): 28829-32, 1997 Nov 14.
Article in English | MEDLINE | ID: mdl-9360946

ABSTRACT

Galpha12 and Galpha13 regulate diverse responses through the small GTPases Ras, CDC42, Rac, and Rho. Whereas they activate similar responses in many different cell types, they also activate more specific and critical signaling pathways in other cell types. In COS cells, in which both Galpha12 and Galpha13 stimulate Na+/H+ exchange, they do so by activating different signaling pathways. Here we report that the differential recruitment of specific small GTPases by Galpha12 and Galpha13 defines the molecular basis for their functional differences. We have observed that the stimulation of Na+/H+ exchange by the GTPase-deficient mutant of Galpha12 (Galpha12QL) requires a functional Ras and is independent of Rac/CDC42 and Jun kinase signaling module. By contrast, the stimulation of Na+/H+ exchange by Galpha13QL requires a functional Rac/CDC42 and the Jun kinase signaling module. Our results also indicate that Galpha12QL-Ras stimulation of Na+/H+ exchange involves a D609-sensitive phospholipase and protein kinase C. These studies, for the first time, describe a novel Galpha12-specific signaling pathway involving Ras, phosphatidylcholine hydrolysis, and protein kinase C in the regulation of Na+/H+ exchange.


Subject(s)
GTP Phosphohydrolases/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go , GTP-Binding Proteins/genetics , Proto-Oncogene Proteins/genetics , Signal Transduction/genetics , Animals , COS Cells , Enzyme Activation , GTP-Binding Protein alpha Subunit, Gi2 , Hydrolysis , Phosphatidylcholines/metabolism , Protein Kinase C/metabolism , Sodium-Hydrogen Exchangers/metabolism
6.
Metabolism ; 45(9): 1130-5, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8781301

ABSTRACT

The safety and efficacy of vanadyl sulfate (VS) was tested in a single-blind, placebo-controlled study. Eight patients (four men and four women) with non-insulin-dependent diabetes mellitus (NIDDM) received VS (50 mg twice daily orally) for 4 weeks. Six of these patients (four men and two women) continued in the study and were given a placebo for an additional 4 weeks. Euglycemic-hyperinsulinemic clamps were performed before and after the VS and placebo phases. VS was associated with gastrointestinal side effects in six of eight patients during the first week, but was well tolerated after that. VS administration was associated with a 20% decrease in fasting glucose concentration (from 9.3 +/- 1.8 to 7.4 +/- 1.4 mmol/L, P < .05) and a decrease in hepatic glucose output (HGO) during hyperinsulinemia (from 5.0 +/- 1.0 pre-VS to 3.1 +/- 0.9 micromol/kg x min post-VS, P < .02). The improvement in fasting plasma glucose and HGO that occurred during VS treatment was maintained during the placebo phase. VS had no significant effects on rates of total-body glucose uptake, glycogen synthesis, glycolysis, carbohydrate (CHO) oxidation, or lipolysis during euglycemic-hyperinsulinemic clamps. We conclude that VS at the dose used was well tolerated and resulted in modest reductions of fasting plasma glucose and hepatic insulin resistance. However, the safety of larger doses and use of vanadium salts for longer periods remains uncertain.


Subject(s)
Carbohydrates/blood , Diabetes Mellitus, Type 2/blood , Hypoglycemic Agents/therapeutic use , Lipids/blood , Vanadium Compounds/therapeutic use , Blood Glucose/analysis , Female , Glucose Clamp Technique , Humans , Hypoglycemic Agents/adverse effects , Insulin/blood , Insulin/metabolism , Male , Middle Aged , Placebos , Vanadium Compounds/adverse effects
7.
Arch Biochem Biophys ; 322(2): 453-9, 1995 Oct 01.
Article in English | MEDLINE | ID: mdl-7574721

ABSTRACT

Morris hepatomas 3924A and 9618A have much lower endogenous contents of Mn than normal rat liver. This work studied the uptake of Mn by slices of these three tissues over a range of concentrations from 0.05 to 100 microM. The influx was assessed with 54Mn while atomic absorption measurements determined the total content. At medium Mn from 0.05 to 5 microM, entry of 54Mn in 2 min was taken as the initial rate and within this period the apparent concentration of Mn in the cell water exceeded that in the medium. Liver showed three apparently saturable uptake systems, the medium concentrations of Mn for half-maximal uptake rate being 0.075, approximately 2, and 100 microM. Hepatoma 3924A appeared to have only two systems, the half-maximal concentration for the higher affinity mechanism being, at 0.34 microM, substantially greater than that for liver. At no concentration was the uptake rate of Mn by hepatoma 3924A less than that of liver although there was some indication that Mn uptake by 9618A was somewhat less than that by the other two tissues. It is concluded that liver and hepatoma 3924A have systems for Mn uptake with affinities that enable them to be active at the plasma concentration (approximately 0.1 microM) as well as uptake systems of less affinity. However, differences in these systems between liver and hepatomas do not account for the differences in endogenous Mn content.


Subject(s)
Liver Neoplasms, Experimental/metabolism , Liver/metabolism , Manganese/metabolism , Animals , Biological Transport , Carrier Proteins/metabolism , In Vitro Techniques , Liver/chemistry , Liver Neoplasms, Experimental/chemistry , Manganese/analysis , Manganese/pharmacokinetics , Radioisotopes , Rats , Rats, Sprague-Dawley , Spectrophotometry, Atomic , Water/analysis
8.
J Membr Biol ; 142(1): 21-34, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7707351

ABSTRACT

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of 14C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25-37.5 mM in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO3-, both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A1 caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO2) was sensitive to both 25 mM NO3- and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5-1.0 mM ouabain resulted in an initial alkalinization (0.5-2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mM NO3-, but not by 25 mM gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H+ uptake by V-ATPase activity. We conclude that V-ATPase make an important contribution to the regulation of the cytosolic pH of hepatocytes.


Subject(s)
Hydrogen-Ion Concentration , Intracellular Fluid/chemistry , Liver/metabolism , Macrolides , Proton-Translocating ATPases/physiology , Amiloride/pharmacology , Animals , Anti-Bacterial Agents/pharmacology , Carbon Dioxide/pharmacology , Chloride Channels/drug effects , Chloride Channels/metabolism , Chlorides/metabolism , Chlorides/pharmacology , Endocytosis , Fluorescent Dyes , Intracellular Membranes/metabolism , Male , Membrane Potentials/drug effects , Nitrates/pharmacology , Ouabain/pharmacology , Proton-Translocating ATPases/antagonists & inhibitors , Protons , Rats , Rats, Sprague-Dawley , Sodium-Hydrogen Exchangers/drug effects , Sodium-Hydrogen Exchangers/physiology , Vacuoles/enzymology
9.
Eur J Cell Biol ; 64(2): 229-42, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7813511

ABSTRACT

Rat hepatocytes in primary monolayer culture have been studied by a combination of physiological and morphological approaches under conditions affecting ion transport and cell volume. A concentration of ouabain completely inhibiting the coupled transport of Na+ and K+ had little effect on cell volume, as indicated by cell water content, but induced the formation of many vesicles in the cytoplasm. Apparent fusion of vesicles was often observed. By itself, replacement of medium Cl by NO3- had little effect on cell volume or morphology. However, when NO3- replaced Cl- in the presence of ouabain the cells swelled and the numbers and size of vesicles were much reduced. The vesicles accumulating in the presence of ouabain showed a yellow fluorescence after the cells were loaded with acridine orange, implying that the vesicular contents were acidic. Total fluid-phase endocytosis, determined by uptake of Lucifer yellow, was not affected by ouabain or the absence of Cl-. However, ouabain considerably retarded the subsequent release of Lucifer yellow; this suggests that the dye originally taken into endocytotic vesicles became diluted by mixing with contents of ouabain-induced vesicles, an explanation consistent with the vesicle fusion seen by electron microscopy. The Cl-free medium also retarded Lucifer yellow efflux, to the same extent as ouabain, and the effects of the two treatments were not additive. These observations are consistent with the activity in hepatocytes of an ouabain-resistant, Cl(-)-dependent mechanism for cell volume control. It is suggested that this depends on the accumulation of water into acidic vesicles, which is driven by the Cl(-)-coupled activity of the vacuolar ATPases of the organelles, followed by exocytotic expulsion of their contents.


Subject(s)
Chlorides/physiology , Exocytosis/drug effects , Liver/drug effects , Ouabain/pharmacology , Proton-Translocating ATPases/physiology , Animals , Cell Size/drug effects , Cells, Cultured , Culture Media , Endocytosis/drug effects , Liver/ultrastructure , Microscopy, Electron, Scanning , Osmotic Pressure , Rats , Temperature , Vacuoles/enzymology
10.
Exp Physiol ; 76(5): 745-63, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1742014

ABSTRACT

Slices of lungs from late-fetal (1 day pre-partum) and adult rats lost K+ and gained Na+, Cl-, water and Ca2+ during pre-incubation at 1 degrees C. These changes were reversed upon restoration to 37 degrees C. The recovery of composition at 37 degrees C was completely dependent on cell respiration in adult slices; by contrast, glycolysis could support partial recovery in the fetal slices. Ouabain completely inhibited K+ reaccumulation at both ages but inhibited net extrusion of water by no more than 50%. Replacement of medium Cl- with NO3- prevented the extrusion of water in the presence of ouabain in adult but not fetal slices. Transmission electron microscopy of type II epithelial cells in slices of both ages showed that ouabain induced the formation of many cytoplasmic vesicles, apparently derived from the Golgi apparatus. Regulation of cell ionic and water content is thus generally similar in late-fetal and adult lung tissue, but there are differences in the source of ATP and in some features of ouabain-resistant volume regulation.


Subject(s)
Body Water/metabolism , Lung/metabolism , Animals , Biological Transport, Active , Energy Metabolism , Female , Lung/embryology , Lung/ultrastructure , Male , Ouabain/pharmacology , Potassium/metabolism , Pregnancy , Rats , Rats, Inbred Strains
11.
Hepatology ; 13(3): 523-33, 1991 Mar.
Article in English | MEDLINE | ID: mdl-1847895

ABSTRACT

We have studied the mechanism by which liver Golgi apparatus maintains the acidity of its contents, using a subcellular fraction from rat liver highly enriched in Golgi marker enzymes. Proton accumulation (measured by quenching of acridine-orange fluorescence) and anion-dependent ATPase were characterized and compared. Maximal ATPase and proton accumulation required ATP; GTP and other nucleotides gave 10% to 30% of maximal activity. Among anions, Cl- and Br- approximately doubled the activities; others were much less effective. Half-maximal increase of ATPase and H+ uptake required 55 mmol/L and 27 mmol/L Cl-, respectively. In predominantly chloride media, SCN- and NO3- markedly inhibited H+ uptake. Nitrate competitively inhibited both the chloride-dependent ATPase (apparent Ki 6 mmol/L) and proton uptake (apparent Ki 2 mmol/L). Nitrate and SCN- also inhibited uptake of 36Cl. Replacing K+ with Na+ had no effect on the initial rate of proton uptake but somewhat reduced the steady state attained. Replacement of K+ with NH4+ and choline reduced proton uptake without affecting ATPase. The ATPase and H+ uptake were supported equally well by Mg2+ or Mn2+. The ATPase was competitively inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (apparent Ki 39 mumol/L). Other agents inhibiting both H+ uptake and ATPase were N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn2+, Co2+ and Cu2+. In the Cl- medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin greater than valinomycin greater than carbonyl cyanide mchlorophenylhydrazone; the last of these also reduced ATPase activity. In the absence of Cl-, monensin and valinomycin both stimulated the ATPase. These results show a close association between ATPase activity and acidification of liver Golgi vesicles. They support a role for Cl- that depends on its uptake as a counter ion for H+ and suggest that it may also stimulate proton transport by a more direct effect on a component of the transport system.


Subject(s)
Adenosine Triphosphatases/metabolism , Golgi Apparatus/enzymology , Liver/metabolism , Protons , Animals , Chlorides/pharmacology , Golgi Apparatus/drug effects , Ions , Male , Nitrates/pharmacology , Nucleotides/pharmacology , Rats , Rats, Inbred Strains
12.
Toxicol In Vitro ; 5(4): 311-23, 1991.
Article in English | MEDLINE | ID: mdl-20732032

ABSTRACT

The effects of carbon tetrachloride and 1,2-dichloroethane (1,2DCE) on the recovery of slices of rat liver from cellular swelling in vitro were studied. Slices took up water during pre-incubation at 1 degrees C, then cellular volume and ultrastructure were rapidly restored during subsequent incubation at 38 degrees C. Ouabain (2 mm) inhibited water extrusion by less than 50%, while inducing formation of peri-canalicular vesicles, apparently derived from the Golgi apparatus. Neither CCl(4) nor 1,2DCE (up to 10 mm) affected the initial extrusion of water at 38 degrees C in the absence of ouabain, but renewed swelling occurred after 60 min with either agent; this was associated with loss of membrane selectivity and some histological damage. By contrast, 1,2DCE inhibited water extrusion in the presence of ouabain after less intensive exposure, for example with 5 mm-1,2DCE for 60 min or 10 mm for 15-30 min. With ouabain present, 1,2DCE (10 mm) caused marked swelling of the endoplasmic reticulum, reduced the peri-canalicular vesicles seen with ouabain alone and reduced the formation of canalicular microvilli. Both CCl(4) and 1,2DCE inhibited the ATP-dependent accumulation of Cl(-) by isolated vesicles of the Golgi apparatus, The delayed swelling of hepatocytes at high concentrations of 1,2DCE and CCl(4) in the absence of ouabain is probably a non-specific consequence of membrane damage. By contrast, 1,2DCE specifically inhibits the ouabain-resistant extrusion of water, possibly by interfering with a postulated mechanism for the exocytotic expulsion of water.

13.
J Physiol ; 417: 389-402, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2621601

ABSTRACT

1. We have examined whether the apparently ouabain-resistant fraction of cellular volume regulation in liver slices under isosmotic conditions is due to a failure of ouabain to cause complete inhibition of the coupled transport of Na+ and K+. The ion and water contents of rat and rabbit liver slices were altered by pre-incubation at 1 degree C and then allowed to recover at 38 degrees C, with or without ouabain or other inhibitors. The net movements of ions and water were determined during the recovery. The influx of 86Rb under steady-state conditions was taken as a measure of unidirectional influx of K+. 2. Concentrations of ouabain for half-maximal inhibition of 86Rb influx were 0.15 mM for rat and 0.15 microM for rabbit liver slices, with maximal inhibition at 2 mM and 10 microM respectively. Inhibition of net K+ reaccumulation closely followed inhibition of 86Rb influx. 3. The 86Rb influx persisting in the presence of maximally inhibiting concentrations of ouabain was not reduced by inhibitors of cellular respiration or glycolysis. 4. In rat liver slices, about 50% of net water extrusion was resistant to 2 mM-ouabain; rabbit liver showed a much smaller, but statistically significant, extrusion of water in the presence of 10 microM-ouabain. 5. In rat liver slices, a small, net uptake of K+ continued in the presence of amytal alone, when water extrusion was completely inhibited; by contrast, ouabain gave complete inhibition of K+ uptake while permitting 50% of the control water extrusion. 6. Isolated rat hepatocytes in primary culture were pre-incubated at 4 degrees C for 20 h. They recovered their original K+ content within 60 min of restoration to 37 degrees C. Ouabain, 1-2 mM, completely prevented this recovery. 7. The results imply that ouabain completely inhibits the coupled transport of Na+ and K+ in both rat and rabbit liver slices. Thus, the fraction of total water extrusion continuing in the presence of maximally inhibiting concentrations of ouabain is the consequence of a truly ouabain-resistant mechanism.


Subject(s)
Liver/metabolism , Ouabain/pharmacology , Potassium/pharmacokinetics , Animals , In Vitro Techniques , Liver/cytology , Liver/drug effects , Male , Rabbits , Rats , Rats, Inbred Strains
14.
J Membr Biol ; 108(3): 235-46, 1989 Jun.
Article in English | MEDLINE | ID: mdl-2778798

ABSTRACT

Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38 degrees C with 0.1 microM monensin induced time-dependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 microM, monensin also reduced net, active movements of K+, Na+, Cl- and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1-1.0 microM) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.


Subject(s)
Adenosine Triphosphate/metabolism , Liver/cytology , Lung/cytology , Monensin/pharmacology , Adenosine Triphosphate/analysis , Animals , Female , Fetus/cytology , Fetus/metabolism , Fetus/ultrastructure , Liver/metabolism , Liver/ultrastructure , Lung/metabolism , Lung/ultrastructure , Male , Microscopy, Electron , Pregnancy , Rats , Rats, Inbred Strains
15.
Toxicol In Vitro ; 3(1): 59-68, 1989.
Article in English | MEDLINE | ID: mdl-20702325

ABSTRACT

Carbon tetrachloride and 1,2-dichloroethane (1,2DCE) were added in vitro to freshly prepared slices of rat liver and the time- and concentration-dependence of their toxic effects on several metabolic parameters determined. With each agent, the most sensitive effect was an increase of malondialdehyde production by a microsomal preparation isolated from the treated slices. The next most sensitive parameter was the inhibition of amino acid incorporation into slice proteins, followed by inhibition of net K(+) accumulation and the induction of early necrotic changes, as indicated by loss of histological staining with azure II. Substantially greater exposures were required to reduce cellular ATP and to initiate entry of Ca(2+). This sequence was similar with both agents, but CCl(4) was the more potent in each case. When added in combinations of submaximally effective concentrations, the two agents produced at least additive inhibitions of protein synthesis and K(+) accumulation. We conclude that metabolic effects in liver slices can be a useful in vitro test for potential toxicity of chlorinated hydrocarbons. Amino acid incorporation and K(+) transport are the most convenient indicator systems, combining considerable sensitivity to relatively low levels of exposure with convenience of measurement.

16.
Br J Exp Pathol ; 69(2): 221-34, 1988 Apr.
Article in English | MEDLINE | ID: mdl-3377963

ABSTRACT

The subcellular distribution and ultrastructural effects of lead have been studied in the kidneys and liver of rats given lead acetate (600 ppm Pb2+) in their drinking water for 6 months. Control rats were given sodium acetate. Tissue samples were fixed in glutaraldehyde and examined by electron microscopy with and without staining. Unstained sections of both kidney and liver from lead-treated animals showed small particles (2-5 nm diameter) of very high electron density which appear to represent a deposited form of Pb2+. Ultrastructural changes in the kidneys were largely confined to glomeruli (swelling of endothelial cells, fusion of foot processes, thickening of basement membrane) and proximal tubules (ranging from minimal sub-lethal changes to necrotic disorganization). The electron-dense particles of Pb2+ occurred in large clusters in basement membranes. As individual particles, or small groups, they were numerous in nuclei of proximal epithelium but usually only a few, largely confined to vesicles or inclusion bodies, were present in the cytoplasm. Only when cells were markedly damaged morphologically were particles more generally distributed in the cytoplasm. Liver damage by Pb2+ was largely confined to centrilobular regions. Endothelial and Kupffer cells were the most affected; they often sequestered large numbers of the particles. In parenchymal cells, particles were few and mainly in vesicles, but they were more widely distributed in the cytoplasm when morphological injury was apparent. The free distribution of Pb2+ in liver and kidney seems to be limited by its deposition in basement membranes and sequestration in reticulo-endothelial cells; intracellular distribution in healthy cells is also limited, by deposition in nuclei (in kidney only) or cytoplasmic vesicles.


Subject(s)
Kidney/analysis , Lead/analysis , Liver/analysis , Organometallic Compounds/pharmacokinetics , Animals , Basement Membrane/ultrastructure , Energy Metabolism , Kidney/ultrastructure , Kidney Cortex/analysis , Kidney Glomerulus/ultrastructure , Kidney Tubules, Proximal/ultrastructure , Liver/ultrastructure , Male , Microscopy, Electron , Mitochondria/ultrastructure , Rats , Rats, Inbred Strains
18.
Lab Invest ; 54(6): 695-707, 1986 Jun.
Article in English | MEDLINE | ID: mdl-3713139

ABSTRACT

The development of cellular damage in response to ethacrynic acid and to amytal has been studied in slices of rat liver. After a preincubation at 1 degree C, the slices were incubated with or without the agents at 38 degrees C. Control slices showed a net extrusion of water, Na+, Cl-, and Ca2+, and a net reaccumulation of K+, during incubation at 38 degrees C. Ethacrynic acid (3 mM) reduced the extrusion of water, Na+ and Cl- during the first 5 minutes at 38 degrees C, but Ca2+ extrusion was not affected. Morphological indications of damage to mitochondria were apparent already at 5 minutes, although unaccompanied by a change of tissue ATP content. The mitochondrial damage was more marked at 15 minutes, when signs of early necrosis were also evident in some cells and ATP content started to fall. At longer times with ethacrynate, water and Ca2+ started to re-enter slices and there were indications of a loss of selective permeability of the plasma membranes; no significant uptake of K+ occurred at any time. Electron-dense particles, apparently of Ca2+, were deposited in the mitochondria and cisternae of the endoplasmic reticulum. The morphological appearance rapidly progressed to a marked disorganization. Slices incubated with 4 mM amytal showed greater and earlier loss of ATP than slices with ethacrynate and complete inhibition of water extrusion; other biochemical and morphological effects were generally less marked than with ethacrynate. Some of the morphological and biochemical effects of the two agents after 15 minutes at 38 degrees C could be reversed upon removal of the drugs. However, Ca2+ contents showed a late rise which started 20 minutes after removal of the drugs, indicating that an early, nonreversible damage of the calcium regulating mechanism had occurred. The results suggest that early effects of ethacrynate are on cell volume regulation and mitochondrial structure and function; there follow more general changes of membrane permeability and loss of control of cell Ca2+. A variety of biochemical and morphological markers of cell damage gave different indications of the level of damage in the early phases of treatment with each of the drugs used here. At longer times (e.g., 60 min) there was a greater degree of uniformity in the indications of cell damage.


Subject(s)
Ethacrynic Acid/pharmacology , Liver/drug effects , Adenosine Triphosphate/metabolism , Amobarbital/pharmacology , Animals , Calcium/metabolism , Chlorides/metabolism , In Vitro Techniques , Kinetics , L-Lactate Dehydrogenase/metabolism , Liver/physiology , Liver/ultrastructure , Male , Microscopy, Electron , Mitochondria, Liver/metabolism , Mitochondria, Liver/ultrastructure , Potassium/metabolism , Rats , Rats, Inbred Strains , Sodium/metabolism , Water/metabolism
19.
Biochem Pharmacol ; 34(9): 1439-48, 1985 May 01.
Article in English | MEDLINE | ID: mdl-3994757

ABSTRACT

Slices of rat renal cortex were shown to take up Pb2+ during incubation in vitro; Pb2+ was also shown to enter mitochondria within the slices. The uptake of Pb2+ by isolated mitochondria was inhibited by N-3, La3+ and ruthenium red. A steady state of uptake was attained within 60 sec. The concentration dependence of uptake was complex; maximum uptake was attained at 25 microM and inhibition ensued at higher concentrations. A substantial inhibitor-resistant component of Pb2+ uptake was noted, especially at medium Pb2+ concentrations greater than 25 microM, and these concentrations also inhibited respiration state 3. The effects on respiration were reduced if the mitochondria had been preincubated with ruthenium red. Slices of renal cortex incubated at 1 degree in medium with various concentrations of Pb2+ showed two fractions of uptake, one saturating at 50-100 microM external Pb2+ and the other at 150-200 microM. Subsequent incubation for 60 min at 25 degrees led to further uptake at all concentrations. Upon isolation of mitochondria from incubated slices, significant amounts of Pb2+ were detected in the mitochondria within 5 min of addition of Pb2+ (200 microM), with maximum attained at 30 min. Electron microscopy of slices showed electron-dense particles, apparently of Pb2+, in the cortical cells but the greatest concentration was deposited in the basement membranes. The results indicate the importance of the basement membrane in limiting access of Pb2+ to cortical cells, and of mitochondria in accumulating Pb2+ once it is in the cells. They also illustrate the importance of interactions between Pb2+ and Ca2+.


Subject(s)
Kidney Cortex/metabolism , Lead/metabolism , Mitochondria/metabolism , Animals , In Vitro Techniques , Kidney Cortex/ultrastructure , Male , Rats , Rats, Inbred Strains
20.
Arch Toxicol ; 56(3): 175-81, 1985 Jan.
Article in English | MEDLINE | ID: mdl-3977597

ABSTRACT

The effects of Pb2+ added in vitro to tissue slices, isolated tubules and isolated mitochondria of rat kidney cortex have been studied. Slices were depleted of K+ and loaded with Na+, Cl- and water by pre-incubation at 1 degree C, and reversal of these changes was then induced by incubation under metabolically favourable conditions. The net reaccumulation of K+ was reduced by a maximum of 30% when Pb2+ was present in the medium, the maximal effect being caused by 200 microM Pb2+. Lead also caused a reduction of Na+ extrusion which was approximately equimolar with its effect on K+, but it did not affect the extrusion of Cl- and water. The initial rates of the net, active movements of K+ and Na+ were not altered by Pb2+, divergence from control values only being noted after 15-30 min incubation. The O2 consumption and the ATP content were 25-30% lower in slices incubated with 200 microM Pb2+ than in control slices; the effect on ATP content was not observed until incubation had continued for 30 min. In tubules isolated from the renal cortex, the rate of respiration (50%) and ATP content (30%) were also partly reduced by 200 microM Pb2+. The consumption of O2 by mitochondria isolated from the cortex was much more sensitive to Pb2+ added in vitro than the respiration of intact cells; the rate of respiration in state 3 (presence of phosphate acceptor) and the respiratory control ratio were drastically reduced, with half-maximal inhibition at 30 and 20 microM Pb2+ respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Ion Exchange , Kidney Cortex/metabolism , Lead/pharmacology , Oxygen Consumption/drug effects , Adenosine Triphosphate/metabolism , Animals , Antimycin A/pharmacology , Energy Metabolism/drug effects , In Vitro Techniques , Kidney Cortex/drug effects , Kidney Tubules/metabolism , Male , Mitochondria/metabolism , Ouabain/pharmacology , Rats
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