G. D. V. van Rossum

Net movements of sodium and potassium, and their relation to respiration, in slices of rat liver incubated in vitro

In adult rat liver the intracellular concentration of potassium is normally higher, and the sodium concentration lower, than the concentration in the extracellular fluid in vivo. The maintenance of the intracellular cation concentrations is dependent upon transport mechanisms which are linked to the metabolic activity of the celis. Thus, when the metabolism of rat-liver slices is inhibited by cooling in a medium having the same sodium and potassium concentration as the extracellular fluid in vivo, the slices lose potassium and gain sodium (Leaf, 1956; Heckmann & Parsons, 1959 a). Upon subsequent incubation at 38° C in an oxygenated medium these changes are reversed, the slices regaining much of the lost potassium and losing sodium (McLean, 1960; Parsons & van Rossum, 1962a). The effects of anaerobiosis suggest that the recovery process at 380 C is to a great extent linked to the respiration of the liver slices (Flink, Hastings & Lowry, 1950), but about 10% of the reaccumulation of potassium by slices prepared from adult rats persists when respiration is inhibited, provided that anaerobic glycolysis can continue (van Rossum, 1963). The object of the work described below was further to characterize the mechanisms which are responsible for the transport of cations by cells of adult rat liver. To this end the time course has been examined of the metabolically linked net movements of sodium and potassium that occur when cold-incubated rat-liver slices are subsequently incubated at 380 C. The effects of the cation composition of the medium and of inhibitors were also studied. The results suggest that there is a close coupling between the inward movement ofpotassium and the outward movement of sodium, and that these cation movements are closely coupled to the respiration of the tissue.

Ouabain-resistant mechanism of volume control and the ultrastructural organization of liver slices recovering from swelling in vitro

SummaryWe have studied the net extrusion of water by liver slices recovering from swelling at 1°C and have attempted to relate this to ultrastructural alterations. Special attention was paid to the ouabain-resistant extrusion of water. The restoration of many details of intracellular architecture was dissociated from the net loss of water, since an osmotic stimulus (produced by 5% inulin) caused a passive withdrawal of water with little recovery of structure. Also, a similar recovery of structure was produced during active extrusion of water in the absence and presence of ouabain, even though ouabain reduced the water extrusion by 50%. The time-course of water extrusion in the presence of ouabain was correlated with the formation of cytoplasmic vesicles. Incubation without K+ in the medium had similar effects to those caused by ouabain. Colchicine had little effect on water extrusion in presence or absence of ouabain except at concentrations which reduced tissue ATP levels and caused much necrosis. Cytochalasin B alone had little effect on water extrusion, but led to the accumulation of many vesicles in the cytoplasm and appeared to abolish the access of such vesicles to the canaliculi. In the presence of ouabain, cytochalasin B had a similar effect on ultrastructure, and totally prevented the ouabain-resistant water extrusion. Ni2+ had rather similar effects to cytochalasin B both in the presence and absence of ouabain, although to a smaller degree. The results support our previous suggestion that the ouabain-resistant water extrusion proceeds by secretion of water into cytoplasmic vesicles, followed by the exocytotic expulsion of the vesicular contents into the bile canaliculi. Microfilaments appear to play an important role in this process.

Properties of the calcium‐extruding mechanism of liver cells.

1. The movements of Ca2+ between liver slices and the suspending medium have been followed with a Ca2+‐specific electrode and with measurements of the efflux of 45Ca. 2. A net entry of Ca2+ into the tissue occurred during anaerobiosis. Reoxygenation of the medium resulted in a net extrusion of Ca2+ which was reversed by the addition of a respiratory inhibitor (Amytal), an uncoupler of oxidative phosphorylation (pentachlorophenol) or the divalent‐cation‐specific ionophore, A23187. 3. The net extrusion of Ca2+ was dependent on the presence of Mg2+ in the medium, but was not prevented by ouabain or by incubation in a Na+‐free medium. Extrusion was not prevented by cytochalasin B or colchicine. 4. The undirectional efflux of 45Ca required Mg2+ to attain its maximal rate, and the magnitude was not affected by the presence of ouabain or the absence of Na+ from the medium. 5. It is concluded that extrusion of Ca2+ from liver cells is a metabolically dependent transport process that occurs independently of the exchange of Na+ between tissue and medium.

Observations on the regulation of cell volume and metabolic control in vitro ; changes in the composition and ultrastructure of liver slices under conditions of varying metabolic and transporting activity

SummaryLiver slices incubated at 1 °C underwent swelling of both cellular and intercellular compartments, as judged by electronmicroscopy. The ultrastructure showed marked changes, including disorganization of the cytocavitary network and plasma membrane and alterations of mitochondria. Restoration of metabolically favorable conditions (oxygenated medium at 38 °C) caused a nearly complete recovery of ultrastructure closely associated with extrusion of water; measurements of inulin space and electronmicroscopy both indicate a recovery of cell volume, with intercellular spaces remaining somewhat expended. The fluid lost was a roughly isotonic solution of Na+ and Cl−, while K+ was reaccumulated in exchange for Na+. Cyanide prevented recovery. Ouabain and oligomycin each partially prevented fluid extrusion, but had little effect on ultrastructural recovery except to induce intracellular vesicles containing particles of thorium dioxide derived from sinusoidal spaces. The vesicles were, however, markedly different in form with each inhibitor. There are, thus ouabain-sensitive and-insensitive components of volume regulation; the former appears to depend on the coupled transport of Na+ and K+ and the latter, we suggest, on a secretion of Na+ and Cl− into vesicles which release their contents into the bile canaliculi by an oligomycin-sensitive mechanism. Mitochondria showed conformational changes between orthodox and condensed forms, but these could not be directly related to tissue energy states; the numbers of mitochondrial dense granules bore a closer relation to tissue ATP.

Role of Cytoplasmic Vesicles in Volume Maintenance

Publisher Summary This chapter considers some instances in which the role of intracellular membranes or vesicular systems in the compartmentalization or movement of water is either well established or strongly suspected. It also considers evidence that intracellular vesicles are implicated in regulation of the volume of vertebrate cells when Na-K transport is inhibited under isosmotic conditions. Vacuoles and vesicles are widespread in living cells. Those with watery contents necessarily contribute to the partition and distribution of cell water and, if they undergo exocytosis, will contribute in some measure to the net expulsion of water from the cell. Such factors play a major role in volume regulation in organisms possessing contractile vacuoles, but in vertebrates, it is the movement of water through plasma membranes, driven by Na-K transport in isosmotic conditions and also by furosemide-sensitive cotransport systems in anisosmotic experiments, that appears to be most important. However, net extrusion of water from several vertebrate tissues continues to a considerable extent under isosmotic conditions even when the Na-K transport system is inhibited to the degree that no net accumulation of K + takes place.

ROLE OF VACUOLAR ADENOSINE TRIPHOSPHATASE IN THE REGULATION OF CYTOSOLIC PH IN HEPATOCYTES

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-ATPases make an important contribution to the regulation of the cytosolic pH of hepatocytes.

Ions and energy metabolism in duck salt‐gland: possible role of furosemide‐sensitive co‐transport of sodium and chloride

1. The effects of methacholine on net ionic movements and energy metabolism of the avian salt‐gland have been studied, using slices of glands taken from salt‐adapted Pekin ducks. The slices were equilibrated with media and drugs for 120 min at 1 °C before the experimental incubation at 38 °C.

Morphological and physiological studies of rat kidney cortex slices undergoing isosmotic swelling and its reversal: a possible mechanism for ouabain-resistant control of cell volume

SummarySlices of rat kidney cortex were induced to swell by preincubation at 1°C in an isotonic Ringers solution, and their capacity to reverse swelling, by net extrusion of cellular water, was studied during subsequent incubation at 25°C. The recovery from swelling was prevented by the respiratory inhibitor, antimycin A. On the other hand, extrusion of water was little affected by ouabain. The extrusion of water continuing in the presence of ouabain (but not that in its absence) was significantly reduced when furosemide was added or when medium Cl− was replaced by NO3−, or I−. There was substantial variability in the morphological appearance of cells within the cortical slices. Different segments of the nephron showed different structural changes during swelling and its reversal, the proximal tubules being most markedly affected. Proximal tubular cells of swollen slices showed disorganization of brush borders and expansion of their apical surfaces, and contained vesicles in their apical cytoplasm. Upon recovery at 25°C, the apical portions of these cells showed reversal of the expansion, but some apical vesicles remained. These vesicles were much more numerous after recovery in the presence of ouabain, but they were much reduced in numbers, or totally absent, when recovery took place in the presence of furosemide or absence of Cl−, with or without ouabain. The vesicles seen in the presence of ouabain alone appeared to fuse with each other and with infoldings of the basolateral plasma membrane. Rather similar results were obtained with distal tubular cells in the slices. We suggest that volume regulation in the proximal and distal tubular cells proceeds by way of two mechanisms. The first consists of extrusion of water coupled to the ouabain-sensitive transport of Na+ and K+. The other proceeds by way of an ouabain-resistant entry of water into apical cytoplasmic vesicles, following furosemide-sensitive movements of Cl− and Na+; the vesicles then expel their contents by exocytosis at the basolateral cell borders.

Observations on the size of the fluid compartments of rat liver slices in vitro.

Previous work (Leaf, 1956; Heckmann & Parsons, 1959 a, b; Parsons & van Rossum, 1962a) has shown that the water content of liver slices prepared from rats of many ages increases (i.e. the tissue swells) when the slices are incubated in an isotonic saline medium in vitro under metabolically unfavourable conditions. The swelling is accompanied by an exchange of ions between the slices and the incubation medium such that the final distribution between the tissue water and the medium is in accordance with the requirements of a Gibbs-Donnan system at ionic equilibrium. These changes can be accounted for in terms of the inhibition of a mechanism which actively excludes sodium ions from the cells. When this mechanism is inhibited, sodium and chloride, the principal ions of the medium, are free to enter the cells towards the equilibrium distribution required by a Gibbs-Donnan system and are accompanied secondarily by water (Leaf, 1956). According to this hypothesis the swelling of the whole slice should be due entirely to an increase in the water content of the cells. We have investigated this point by attempting to compare the absolute sizes of the extracellular and intracellular water compartments of fresh and swollen liver slices. In view of the changes during post-natal growth both in the sizes of the intracellular and extracellular water compartments of fresh liver tissue (Parsons & van Rossum, 1961) and also in the extent of the swelling in vitro (Parsons & van Rossum, 1962a) it seemed to be of interest to undertake this work with liver slices prepared from rats of different ages. A further consequence of the hypothesis described above is that the restoration of the tissue to metabolically favourable conditions should result in an extrusion of sodium from the cells, secondarily accompanied by anions and water, so that the cells will shrink. Potassium, which is lost from the cells during swelling, should be reaccumulated. The results of other workers suggested that swollen liver slices are at least partially able

Relative effects of furosemide and ethacrynic acid on ion transport and energy metabolism in slices of rat kidney-cortex

SummaryThe effects of furosemide and ethacrynic acid have been studied using slices of rat kidney cortex incubated in a Ringer medium. At concentrations from 0.2–2.0 mM, furosemide had no significant effect on the tissue ATP content or on the metabolism-dependent net movements of intracellular Na+, K+ and Ca2+. It did, however, induce an increase in the net, outward movement of Cl−; we suggest that this may have srisen from inhibition of a Cl− accumulating mechanism. In contrast, ethacrynic acid in the same concentration range caused marked reduction of cell respiration and ATP content and virtually total inhibitition of several processes of ion transport (Na+, Cl− and Ca2+ loss, and K+ uptake). Concentrations of furosemide greater than 5 mM caused marked inhibition of energy metabolism and transport of ions, and 10 mM furosemide had quantitatively similar effects to 2 mM ethacrynic acid. Electron micrographs of kidney-cortex slices treated with the diuretics at 2 mM show that the ultrastructure was well maintained in the presence of furosemide but that ethacrynic acid caused severe structural disorganisation and necrosis. The mitochondria were generally in the orthodox configuration in the presence of furosemide, but swollen in ethacrynic acid in accord with the marked effects of 2 mM ethacrynate on mitochondrial energy metabolism. Of the effects we have detected, that of low concentrations of furosemide on Cl− movement appears to be rather specific. Higher concentrations of this agent (5 mM and above), and all concentrations of ethacrynic acid studied (0.1–5.0 mM), have several inhibitory effects which seem to result from primary inhibition of mitochondrial activities and are presumably manifestations of toxicity.