From early unicellular organisms that formed in salty water environments to complex organisms that live on land away from water, cells have had to protect a homeostatic internal environment favorable to the biochemical reactions necessary for life. In this chapter, we will outline what steps were necessary to conserve the water within our cells and how mechanisms have evolved to maintain and regulate our cellular and organismal volume. We will first examine whole body water homeostasis and the relationship between kidney function, regulation of blood pressure, and blood filtration in the process of producing urine. We will then discuss how the composition of the lipid-rich bilayer affects its permeability to water and salts, and how the cell uses this differential to drive physiological and biochemical cellular functions. The capacity to maintain cell volume is vital to epithelial transport, neurotransmission, cell cycle, apoptosis, and cell migration. Finally, we will wrap up the chapter by discussing in some detail specific channels, cotransporters, and exchangers that have evolved to facilitate the movement of cations and anions otherwise unable to cross the lipid-rich bilayer and that are involved in maintaining or regulating cell volume.

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K+ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba2+ (BaCl2, 5 mmol/l) and the blocker of the Na+/K+ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B4 receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 micromol/l). ATPgammaS (10 micromol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 micromol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPgammaS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.

Two peripheral signaling routes have been proposed to account for the ability of peripheral substances such as glucose to modulate memory processing in the brain. One possible signaling route is by crossing the blood-brain barrier to act directly on brain. A second route involves activation of peripheral nerves with resulting changes in neural activity carried by peripheral nerves to the brain. Because the vagus nerve is a major neural pathway between the periphery and brain, peripherally acting modulators of memory modulators may act via vagal afferents to the brain to enhance memory processing. In the present experiments, systemic injections of either D-glucose or L-glucose, a metabolically inactive enantiomer, facilitated performance of rats on a four-arm alternation task, but at very different doses (D-glucose, 250 mg/kg; L-glucose, 3,000 mg/kg). The enhanced performance seen with L-glucose, but not that seen with D-glucose, was attenuated by vagotomy. These findings suggest that the mechanisms by which these enantiomers act to enhance memory are quite different, with L-glucose acting via vagal afferents but D-glucose acting by other means, including direct modulation of central nervous system (CNS) processes by D-glucose.

The crucial role of the liver as the only organ to produce glucose used by skeletal muscle during exercise is well known. Since hepatic glucose production is central to blood glucose homeostasis during exercise, it has been postulated that the liver may inform the central nervous system and other organs of its diminishing capacity to produce glucose from glycogen, before blood glucose falls. The sensory role of the liver during exercise would be similar to its role in the control of food intake. As a consequence, the experimental approaches used to test the hypothesis that afferent signals from the liver contribute to metabolic regulation during exercise are inspired by those used to test the same hypothesis in the regulation of food intake. In the present review, two questions are addressed. The existing evidence for the liver's sensory influence on metabolic adjustments to exercise is first reviewed; the nature of the initiating stimuli for the afferent contribution of the liver to physical exercise is discussed thereafter. The hypothetical construct upon which rests the contribution of the liver's afferent signals to metabolic regulation during exercise is that a decrease in liver glycogen or a related metabolic intermediate is sensed by the liver, and the signal is transduced to the central nervous system, most likely through the afferent activity of the hepatic vagus nerve, where it contributes to the orchestration of the metabolic and hormonal responses to exercise. Support in favour of this construct comes mainly from the demonstration that sectioning of the hepatic vagus nerve attenuates the normal hormonal response to exercise. It seems that the liver-glucagon axis is particularly responsive to this reflex activation. In other respects, the hepatic mechanism responsible for linking the metabolic activity in the liver to an afferent signal capable of regulating the metabolic response to exercise remains speculative. Substrates or derivatives of substrate oxidation, energy-related compounds (ATP and Pi), or changes in cell volume may all be related to changes in transmembrane potential in the liver cell, which according to the "potentiostatic" theory would determine the afferent vagal activity.

A non-transformed mouse liver cell line (AML12) was used to show that blocking swelling-activated membrane Cl- current inhibits hepatocyte proliferation. Two morphologically distinguishable cell populations exhibited distinctly different responses to hypotonic stress. Hypotonic stress (from 280 to 221 mosmol kg(-1)) to rounded, dividing cells activated an ATP-dependent, outwardly rectifying, whole-cell Cl- current, which took 10 min to reach maximum conductance. A similar anionic current was present spontaneously in 20 % of the dividing cells. Hypotonic stress to flattened, non-dividing cells activated no additional current. The Eisenman halide permeability sequence of swelling-activated anionic current in the dividing cells was SCN(-) > I(-) > Br(-) > Cl(-) > gluconate. Addition of either 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), tamoxifen or mibefradil inhibited swelling-activated anionic current. Hyperosmolarity by added sucrose inhibited the spontaneous anionic current in dividing cells. Added Cl- channel blockers NPPB (IC50 = 40 microM), DIDS (IC50 = 31 microM), tamoxifen (IC50 = 1.3 microM) and mibefradil (IC50 = 7 microM) inhibited proliferative growth of AML12 as determined by cell counts over 4 days or by protein accumulation over 2 days. Only the inhibitory effects of NPPB and mibefradil reversed with the drug washout. Hyperosmolarity by added sucrose (50 and 100 mM) also inhibited cell proliferation. Of the hydrophobic inhibitors neither NPPB at 40 microM nor tamoxifen at 1.3 microM, added for 48 h, reduced cellular ATP; however, DIDS at 31 microM significantly reduced cellular ATP with an equivalent increase in cellular ADP. We conclude that those membrane Cl- currents that can be activated by hypotonic stress are involved in mechanisms controlling liver cell growth, and that NPPB, tamoxifen and mibefradil at their IC50 for growth do not suppress the metabolism of mouse hepatocytes.

The present study was conducted to evaluate the influence of a hepatic portal infusion of hypertonic saline on the metabolic and hormonal responses to exercise. Adrenodemedullated male rats were studied at rest or after 30 min of treadmill exercise (26 m/min, 0% grade). Three groups of rats were infused continuously at a rate of 52 microL/min with one of the following randomly assigned conditions: hypertonic 3.6% NaCl (P3.6% NaCl) or 1.8% NaCl (P1.8% NaCl) infused into the hepatic portal vein, and hypertonic 3.6% NaCl (J3.6% NaCl) infused into the jugular vein. One group of rats received no infusion (SHAM). The infusions of hypertonic NaCl into the portal or the jugular site resulted in a significant (p < 0.05) increase in peripheral concentration of Na+, Cl-, and osmolality at rest and after exercise. The antidiuretic hormone (ADH) concentration was significantly (p < 0.05) increased by the P3.6% NaCl and J3.6% NaCl infusions at rest and after exercise. Exercise caused a significant (p < 0.05). decrease in liver glycogen content, peripheral and portal plasma glycemia, and insulinemia regardless of the different types and sites of infusions. However, the peripheral glucagon response to exercise was significantly (p < 0.05) increased only when hypertonic saline (1.8 or 3.6%) was infused into the portal vein. Portal and peripheral lactate concentrations at rest and after exercise were significantly (p < 0.01) higher in P3.6% NaCl than in all other groups. It is concluded that a 30-min hypertonic saline infusion into the hepatic portal vein does not specifically influence the insulin response at rest and after exercise, but that glucagon response to exercise is increased by such an infusion.

The goal of the present experiment was to measure the volume of the different compartments in liver of exercised rats and to get some insights into the appropriate working of the hepatic function following exercise. Hence, livers from male rats were isolated and perfused after treadmill exercise or rest. This procedure was performed on rats that were overnight semifasted (50% food restriction) or well fed. To evaluate the hepatocyte cell volume, the multiple-indicator dilution curve technique was used after 40 min of perfusion. Radioactive tracers for red blood cells, sucrose, and water were used to measure liver vascular space, liver interstitial space, and water cellular space, respectively. The hepatocyte function was assessed by taurocholate and propanolol clearance. Oxygen consumption, intrahepatic resistance, bile secretion, and lactate dehydrogenase release estimated liver viability. Liver viability and hepatocyte function were not changed following exercise either in the fed or in the semifasted animals. As expected, liver glycogen levels were significantly (P < 0.01) reduced in the food-restricted rats. Consequently, liver glycogen levels following exercise were decreased significantly (P < 0.01) only in the fed rats. Despite this, exercise decreased the hepatocyte water space in both food-restricted and fed groups ( approximately 15%; P < 0.01) without altering the sinusoidal and interstitial space. The present data show that acute exercise decreased the hepatocyte volume and that this volume change is not entirely linked to a decrease in hepatic glycogen level.

Intracellular Na+ accumulation has been shown to contribute to hepatocyte death caused by anoxia or oxidative stress. In this study we have investigated the mechanism by which Na+ overload can contribute to the development of cytotoxicity. ATP depletion in isolated hepatocytes exposed to menadione-induced oxidative stress or to KCN was followed by Na+ accumulation, loss of intracellular K+, and cell swelling. Hepatocyte swelling occurred in two phases: a small amplitude swelling (about 15% of the initial size) with preservation of plasma membrane integrity and a terminal large amplitude swelling associated with cell death. Inhibition of Na+ accumulation by the use of a Na+-free medium prevented K+ loss, cell swelling, and cytotoxicity. Conversely, blocking K+ efflux by the addition of BaCl2 did not influence Na+ increase and small amplitude swelling, but greatly stimulated large amplitude swelling and cytotoxicity. Menadione or KCN killing of hepatocytes was also enhanced by inducing cell swelling in an hypotonic medium. However, increasing the osmolarity of the incubation medium did not protect against large amplitude swelling and cytotoxicity, since stimulated Na+ accumulation and K+ efflux. Altogether these results indicate that the impairment of volume regulation in response to the osmotic load caused by Na+ accumulation is critical for the development of cell necrosis induced by mitochondrial inhibition or oxidative stress.

Using superfused mouse liver slices combined with a conventional microelectrode technique, we investigated: (1) the ionic mechanisms involved in the hyperpolarization of the hepatocyte membrane induced by lactate and other gluconeogenic substrates; (2) whether these mechanisms are similar to those underlying the hyperpolarization induced by cell swelling in hypo-osmotic medium; and (3) whether the hyperpolarizing effect of lactate on the hepatocyte membrane is related to gluconeogenesis. Lactate (5 mmol/l) hyperpolarized the hepatocyte membrane after an exposure of 10-20 min, and the hyperpolarization was still present after 70 min. The hyperpolarization induced by lactate, pyruvate (5 mmol/l) and fructose (10 mmol/l), and by exposure to hypo-osmotic medium (250 mosmol/l) was antagonized by ouabain, tetraethylammonium (TEA), and cetiedil (lactate; hypo-osmotic medium). Hyperpolarization induced by lactate was eliminated or attenuated by agents impairing activation of Ca2+-dependent K+ channels, by amiloride, and by a blockade of non-selective cation channels with flufenamic acid and gadolinium. Thapsigargin, increasing cytosolic Ca2+, mimicked lactate's hyperpolarizing effect. Lactate's effect was dependent on extracellular Ca2+. Finally, lactate's hyperpolarizing effect was reduced by inhibiting gluconeogenesis. These findings suggest that metabolism of lactate hyperpolarizes hepatocytes by mechanisms analogous to those underlying the hyperpolarization induced by cell swelling in hypo-osmotic medium. Gluconeogenesis from lactate may cause cell swelling, subsequent activation of Ca2+-dependent K+ channels and of the Na+/K+-ATPase, and thus hyperpolarize the hepatocyte membrane.

Cells involved in the retrieval and metabolic conversion of amino acids undergo significant increases in size in response to amino acid uptake. The resultant adaptive responses to cell swelling are thought to include increases in membrane K+ and Cl- permeability through activation of volume-sensitive ion channels. This viewpoint is largely based on experimental models of hypotonic swelling, but few mammalian cells experience hypotonic challenge in vivo. Here we have examined volume regulatory responses in a physiological model of cell-swelling alanine uptake in immortalized hepatocytes. Alanine-induced cell swelling was followed by a decrease in cell volume that was temporally associated with an increase in membrane Cl- currents. These currents were dependent both on alanine concentration and Na+, suggesting that currents were stimulated by Na+-coupled alanine uptake. Cl- currents were outwardly rectifying, exhibited an anion permeability sequence of I- > Br- > Cl-, and were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid, features similar to those reported for a widely distributed class of volume-sensitive anion channels evoked by experimental hypotonic stress. These findings suggest that volume-sensitive anion channels participate in adaptive responses to amino acid uptake and provide such channels with a new physiological context.

A large-conductance, Ca2+-activated K+ channel was identified and characterized in embryonic chick hepatocytes using the patch-electrode voltage-clamp technique. The channel conductance was 213 pS in excised patches bathed in symmetrical 145 mM KCI and 1 mM Ca2+. Current-voltage relationships were linear with high K+ on both sides of the membrane but showed constant field rectification as the K+ gradient was increased. The reversal potential shifted 58 mV per 10-fold change in the ratio of external to internal K+. Channel openings occurred at potentials higher than +50 mV in cell-attached patches. The open probability X voltage relationship shifted to more negative potentials in excised, inside-out patches exposed to a solution containing high Ca2+. The voltage sensitivity of the channel was not significantly affected by changes in internal Ca2+ concentration. Conversely, channel gating, reflected in the half-activation potential, shifted 118 mV per 10-fold change in internal Ca2+ at concentrations less than approximately 2 microM, although at higher Ca2+, this parameter was Ca2+ insensitive. Channel open probability in cell-attached patches increased significantly following exposure of the cells to either the Ca2+ ionophore A-23187 or L-alanine, a cell-volume modulator. Channel density increased with time spent in culture from no observations in 10-hr cells, through 13 and 80% of patches in 24-and 48-hr cultured cells, respectively. The implications of delayed functional expression for ion channel studies in acutely dissociated cells is discussed.

These studies of a model liver cell line evaluate the mechanisms responsible for regulated release of K+ ions during metabolic stress. Metabolic inhibition of HTC hepatoma cells by exposure to 2, 4-dinitrophenol (50 microM) and 2-deoxy-D-glucose (10 mM) stimulated outward currents carried by K+ of 974 +/- 75 pA at 0 mV (n = 20, p < 0.001). Currents were inhibited by chelation of intracellular Ca2+ or exposure to apamin (50 nM), an inhibitor of SKCa channels. In cell-attached recordings from intact cells, removal of metabolic substrates (25/28 cells) or exposure to metabolic inhibitors (32/40 cells) opened K+-selective channels with a conductance of 6.5 +/- 0. 2 pS. Channels had an open probability of 0.31 +/- 0.08 and opened in bursts averaging 3.55 +/- 0.27 ms in duration (n = 6). Metabolic stress was associated with rapid translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosol to membrane; and down-regulation of PKCalpha by phorbol esters or exposure to the PKC inhibitor chelerythrine (10 microM) each inhibited currents. Moreover, intracellular perfusion with purified PKCalpha activated currents in a Ca2+- and concentration-dependent manner. These findings indicate that metabolic stress leads to opening of apamin-sensitive SKCa channels in hepatoma cells through a Ca2+- and PKC-dependent mechanism and suggest that PKCalpha may be selectively involved in the response. This mechanism functionally couples the metabolic state of cells to membrane K+ permeability and represents a potential target for modification of liver injury associated with ischemia and preservation.

We studied the ionic mechanisms underlying the regulatory volume increase of rat hepatocytes in primary culture by use of confocal laser scanning microscopy, conventional and ion-sensitive microelectrodes, cable analysis, microfluorometry, and measurements of 86Rb+ uptake. Increasing osmolarity from 300 to 400 mosm/liter by addition of sucrose decreased cell volumes to 88.6% within 1 min; thereafter, cell volumes increased to 94.1% of control within 10 min, equivalent to a regulatory volume increase (RVI) by 44.5%. This RVI was paralleled by a decrease in cell input resistance and in specific cell membrane resistance to 88 and 60%, respectively. Ion substitution experiments (high K+, low Na+, low Cl-) revealed that these membrane effects are due to an increase in hepatocyte Na+ conductance. During RVI, ouabain-sensitive 86Rb+ uptake was augmented to 141% of control, and cell Na+ and cell K+ increased to 148 and 180%, respectively. The RVI, the increases in Na+ conductance and cell Na+, as well as the activation of Na+/K(+)-ATPase were completely blocked by 10(-5) mol/liter amiloride. At this concentration, amiloride had no effect on osmotically induced cell alkalinization via Na+/H+ exchange. When osmolarity was increased from 220 to 300 mosm/liter (by readdition of sucrose after a preperiod of 15 min in which the cells underwent a regulatory volume decrease, RVD) cell volumes initially decreased to 81.5%; thereafter cell volumes increased to 90.8% of control. This post-RVD-RVI of 55.0% is also mediated by an increase in Na+ conductance. We conclude that rat hepatocytes in confluent primary culture are capable of RVI as well as of post-RVD-RVI. In this system, hypertonic stress leads to a considerable increase in cell membrane Na+ conductance. In concert with conductive Na+ influx, cell K+ is then increased via activation of Na+/K(+)-ATPase. An additional role of Na+/H+ exchange in the volume regulation of rat hepatocytes remains to be defined.

The effect of palmitate and metabolizable and nonmetabolizable monosacharides (D-glucose, D-fructose and 2-deoxy-D-glucose = 2-DG) on the membrane potential (Vm) of mouse hepatocytes was investigated employing a superfused mouse liver slice technique. Palmitate hyperpolarized the liver cell membrane in a concentration dependent manner whereas the monosaccharides tested did not. When mice were fed a fat-rich diet, the hyperpolarisation was greater in comparison to mice fed a low fat diet. The hyperpolarization was reversed by ouabain, an inhibitor of the Na+/K(+)-ATPase, by the K(+)-channel blockers tetra-ethyl-ammonium (TEA) and cetiedil and by three inhibitors of fatty acid oxidation (2-bromopalmitate, 2-bromooctanoate and 4-pentenoate). The results suggest that hyperpolarization of the liver cell membrane is due to fatty acid oxidation and that both activation of Na+/K(+)-ATPase and opening of K(+)-channels are involved. The implications of these findings with regard to control of food intake by fatty acid oxidation are discussed. The results are consistent with a role of the hepatic membrane potential in control of food intake by fatty acid oxidation.

Mouse hepatocytes respond to osmotic stress with adaptive changes in transmembrane potential, Vm, such that hypotonic stress hyperpolarizes cells and hypertonic stress depolarizes them. These changes in Vm provide electromotive force for redistribution of ions such as Cl-, and this comprises part of the mechanism of hepatocyte volume regulation. We conducted the present study to determine whether ethanol administered in vitro to mouse liver slices increases hepatocyte water volume, and whether this swelling triggers adaptive changes in the Vm. Cells in mouse liver slices were loaded with tetramethylammonium ion (TMA). Changes in hepatocyte water volume were computed from measurements with ion sensitive microelectrodes of changes in intracellular activity of TMA (a1TMA) that resulted from water fluxes. Ethanol (70 mM) increased hepatocyte water volume immediately, and this peaked at 17% by 7 to 8 min, by which time a plateau was reached. Liver slices also were obtained from mice treated 12 hr prior with 4-methylpyrazole (4 mM). The effect of ethanol on their hepatocyte water volume was identical to that from untreated mice, except that the onset and peak were delayed 2 min. Hepatocyte Vm showed no differences between control or ethanol-treated cells during the course of volume changes. In contrast, hyposmotic stress, created by dropping external osmolality 50 mosm, increased Vm from -30 mV to -46 mV. Ethanol did not inhibit this osmotic stress-induced hyperpolarization, except partially at high concentrations of 257 mM or greater. We infer that ethanol-induced swelling of hepatocytes differs from that resulting from hyposmotic stress.(ABSTRACT TRUNCATED AT 250 WORDS)

The lipophilic cation tetraphenylphosphonium (TPP+) has been extensively utilized as the probe for the membrane potential (Vm) in various cells. For application to mammalian cells, however, two serious problems require resolution: (1), correction of TPP+ binding to intracellular constituents and (2), estimation of the considerable TPP+ accumulation in mitochondria. We propose here a simple corrective method for the TPP+ binding and its accumulation. TPP+ distribution is assumed as: (1), two compartments (a cytosolic and a mitochondrial space); (2), a proportional relationship between TPP+ bound amount and its unbound concentration in each compartment. We theoretically derived the simple equation: Vm = - RT/F ln(C/Mphys ratio/C/Mabol ratio) where R, T and F have their usual thermodynamic significance. Here, the C/M ratio is defined as the ratio of TPP+ concentration of apparent intracellular to extracellular space. The suffixes phys and abol, respectively, mean the physiological and solely Vm-abolished conditions. This equation was checked with hepatocytes, because estimating hepatocytes Vm with TPP+ distribution is not considered possible because of the relatively high mitochondrial content. The selective Vm abolition was achieved by permeabilization with 20 microM of amphotericin B. The Vm value was, thus, estimated to be -38.6 +/- 0.3 mV, compatible with those obtained with microelectrodes in other laboratories. Vm in hepatocytes is composed of transmembrane K+ diffusion potential (-20.6 +/- 0.3 mV) and electrogenic Na+/K(+)-ATPase (-19.6 +/- 0.4 mV). Addition of rheogenic L-alanine caused a transient but significant depolarization (from control to -34 +/- 0.3 mV). These results taken together indicate that hepatocyte Vm can be accurately determined with the present simple method, so that it may possibly be applicable to the evaluation of Vm in other mammalian cells.

1. Whole-cell voltage clamp and intracellular recording techniques were used to study the increase in K+ conductance that accompanies swelling in isolated guinea-pig and rat hepatocytes in short-term culture at 37 degrees C. 2. Swelling was induced (i) by the application of pressure (15 cmH2O) to the shank of the patch pipette, (ii) by exposing the cells to hypotonic solutions and (iii) as a consequence of leakage of electrolyte from an intracellular microelectrode. 3. Applying pressure to the patch pipette caused a large outward current (approximately 600 pA) to develop in guinea-pig hepatocytes voltage clamped to 0 mV. This current reversed direction at -86 mV, close to the reversal potential for K+, EK (-93 mV), and is attributable to the activation of a K+ conductance. 4. Spectral analysis of current noise during this response suggested a single-channel conductance of 7 pS, though this may well be an underestimate. The power spectrum could be fitted by the sum of two Lorentzian components, with half-power frequencies of 7 and 152 Hz. Seventy per cent of the variance was associated with the lower frequency component. 5. The steady-state current-voltage relationship for guinea-pig hepatocytes, as determined by whole-cell recording, was linear over the range -70 to +40 mV both before and during the increase in K+ conductance induced by swelling. 6. Confirming earlier work, intracellular recording using microelectrodes filled with 1 M-potassium citrate sometimes resulted in a slow hyperpolarization and a large rise in input conductance. These changes are also attributable to an increase in K+ conductance as the cell swelled because of leakage from the electrode. 7. Application of hypotonic external solutions during intracellular recording caused hyperpolarization and an increase in conductance. Conversely, hypertonic solution evoked depolarization and a fall in conductance in partly swollen cells. 8. The volume-activated K+ conductance was reversibly blocked by cetiedil, which caused half-maximal inhibition at 2.3 microM. Bepridil, quinine and barium were also effective, with IC50s (concentrations giving 50% maximal inhibition) of 2.7, 12 and 67 microM respectively. 9. Much greater concentrations of cetiedil and bepridil (IC50 approximately 1 mM and 77 microM respectively) were required to inhibit the loss of K+ which follows the application of angiotensin II (100 nM) to guinea-pig hepatocytes, and which occurs via Ca(2+)-activated K+ channels. Our evidence suggests that the activation of K+ channels by cell swelling is Ca2+ independent.(ABSTRACT TRUNCATED AT 400 WORDS)

Swelling-activated K+ and Cl- channels, which mediate RVD, are found in most cell types. Prominent exceptions to this rule include red cells, which together with some types of epithelia, utilize electroneutral [K(+)-Cl-] cotransport for down-regulation of volume. Shrinkage-activated Na+/H+ exchange and [Na(+)-K(+)-2 Cl-] cotransport mediate RVI in many cell types, although the activation of these systems may require special conditions, such as previous RVD. Swelling-activated K+/H+ exchange and Ca2+/Na+ exchange seem to be restricted to certain species of red cells. Swelling-activated calcium channels, although not carrying sufficient ion flux to contribute to volume changes may play an important role in the activation of transport pathways. In this review of volume-activated ion transport pathways we have concentrated on regulatory phenomena. We have listed known secondary messenger pathways that modulate volume-activated transporters, although the evidence that volume signals are transduced via these systems is preliminary. We have focused on several mechanisms that might function as volume sensors. In our view, the most important candidates for this role are the structures which detect deformation or stretching of the membrane and the skeletal filaments attached to it, and the extraordinary effects that small changes in concentration of cytoplasmic macromolecules may exert on the activities of cytoplasmic and membrane enzymes (macromolecular crowding). It is noteworthy that volume-activated ion transporters are intercalated into the cellular signaling network as receptors, messengers and effectors. Stretch-activated ion channels may serve as receptors for cell volume itself. Cell swelling or shrinkage may serve a messenger function in the communication between opposing surfaces of epithelia, or in the regulation of metabolic pathways in the liver. Finally, these transporters may act as effector systems when they perform regulatory volume increase or decrease. This review discusses several examples in which relatively simple methods of examining volume regulation led to the discovery of transporters ultimately found to play key roles in the transmission of information within the cell. So, why volume? Because it's functionally important, it's relatively cheap (if you happened to have everything else, you only need some distilled water or concentrated salt solution), and since it involves many disciplines of experimental biology, it's fun to do.

Hepatocyte transmembrane potential (Vm) behaves as an osmometer and varies with changes in extracellular osmotic pressure created by altering the NaCl concentration in the external medium (Howard, L.D. and Wondergem, R. (1987) J. Membr. Biol. 100, 53). We now have demonstrated similar effects on Vm by increasing external osmolality with added sucrose and not altering ionic strength. We also have demonstrated that hyperosmotic stress-induced depolarization of Vm results from changes in membrane K+ conductance, gK, rather than from changes in the K+ equilibrium potential. Vm and aKi of hepatocytes in liver slices were measured by conventional and ion-sensitive microelectrodes, respectively. Cell water vols. were estimated by differences in wet and dry weights of liver slices after 10-min incubations. Effect of hyperosmotic medium on membrane transference number for K+, tK, was measured by effects on Vm of step-changes in external [K+]. Hepatocyte Vm decreased 34, 52 and 54% when tissue was superfused with medium made hyperosmotic with added sucrose (50, 100 and 150 mM). Correspondingly, aKi increased 10, 18 and 29% with this hyperosmotic stress of added sucrose. Tissue water of 2.92 +/- 0.10 kg H2O/kg dry weight in control solution decreased to 2.60 +/- 0.05, 2.25 +/- 0.06 and 2.22 +/- 0.05 kg H2O/kg dry weight with additions to medium of 50, 100 and 150 mM sucrose, respectively. Adding 50 mM sucrose to medium decreased tK from 0.20 +/- 0.01 to 0.05 +/- 0.01. Depolarization by 50% with hyperosmotic stress (100 mM sucrose) also occurred in Cl-free medium where Cl- was substituted with gluconate. We conclude that hepatocytes shrink during hyperosmotic stress, and the aKi increases. The accompanying decrease in Vm is opposite to that expected by an increase in aKi, and at least in part results from a concomitant decrease in gK. Changes in membrane Cl- conductance most likely do not contribute to osmotic stress-induced depolarization, since equivalent decreases in Vm occurred with added sucrose in cells depleted of Cl- by superfusing tissue with Cl-free medium.

The effect of metabolizable (D-glucose, D-fructose) and nonmetabolizable (2-deoxy-D-glucose, L-glucose) monosaccharides on the membrane potential (Vm) of mouse hepatocytes was investigated employing a superfused liver slice technique. D-Cellobiose was used as an osmotic control. All monosaccharides tested hyperpolarized the liver cell membrane. The short-term effects of D-glucose, D-fructose and 2-deoxy-D-glucose were similar, whereas the effect of L-glucose was less pronounced. The K+ channel blocker quinine reversed the effects of glucose and 2-deoxy-D-glucose on the Vm, suggesting that opening of K+ channels is involved in the hyperpolarizing effect of monosaccharides. The bearing of these findings with regard to hepatic control of food intake is discussed. The findings argue against a role of hepatocytes as glucoreceptors sensing glucose metabolism.

The state of intracellular water has been a matter of controversy for a long time for two reasons. First, experiments have often given conflicting results. Second, hitherto, there have been no plausible grounds for assuming that intracellular water should be significantly different from bulk water. A collective behavior of water molecules is suggested here as a thermodynamically inevitable mechanism for generation of appreciable zones of abnormal water. At a highly charged surface, water molecules move together, generating a zone of water perhaps 6 nm thick, which is weakly hydrogen bonded, fluid, and reactive and selectively accumulates small cations, multivalent anions, and hydrophobic solutes. At a hydrophobic surface, molecules move apart and local water becomes strongly bonded, inert, and viscous and accumulates large cations, univalent anions, and compatible solutes. Proteins and many other biopolymers have patchy surfaces which therefore induce, by the two mechanisms described, patchy interfacial water structures, which extended appreciable distances from the surface. The reason for many conflicting experimental results now becomes apparent. Average values of properties of water measured in gels, cells, or solutions of proteins are often not very different from the same properties of normal water, giving no indication that they are averages of extreme values. To detect the operation of this phenomenon, it is necessary to probe selectively a single abnormal population. Examples of such experiments are given. It is shown that this collective behavior of water molecules amounts to a considerable biological force, which can be equivalent to a pressure of 1,000 atm (1.013 x 10(5) kPa). It is suggested that cells selectively accumulate K+ ions and compatible solutes to avoid extremes of water structure in their aqueous compartments, but that cation pumps and other enzymes exploit the different solvent properties and reactivities of water to perform work of transport or synthesis.

1. Urea synthesis was studied in isolated perfused rat liver during cell volume regulatory ion fluxes following exposure of the liver to anisotonic perfusion media. Lowering of the osmolarity in influent perfusate from 305 mOsm/l to 225 mOsm/l (by decreasing influent [NaCl] by 40 mmol/l) led to an inhibition of urea synthesis from NH4Cl (0.5 mmol/l) by about 60% and a decrease of hepatic oxygen uptake by 0.43 +/- 0.03 mumol g-1 min-1 [from 3.09 +/- 0.13 mumol g-1 min-1 to 2.66 +/- 0.12 mumol g-1 min-1 (n = 9)]. The effects on urea synthesis and oxygen uptake were observed throughout hypotonic exposure (225 mOsm/l). They persisted although volume regulatory K+ efflux from the liver was complete within 8 min and were fully reversible upon reexposure to normotonic perfusion media (305 mOsm/l). A 42% inhibition of urea synthesis from NH4Cl (0.5 mmol/l) during hypotonicity was also observed when the perfusion medium was supplemented with glucose (5 mmol/l). Urea synthesis was inhibited by only 10-20% in livers from fed rats, and was even stimulated in those from starved rats when an amino acid mixture (twice the physiological concentration) plus NH4Cl (0.2 mmol/l) was infused. 2. The inhibition of urea synthesis from NH4Cl (0.5 mmol/l) during hypotonicity was accompanied by a threefold increase of citrulline tissue levels, a 50-70% decrease of the tissue contents of glutamate, aspartate, citrate and malate, whereas 2-oxoglutarate, ATP and ornithine tissue levels, and the [3H]inulin extracellular space remained almost unaltered. Further, hypotonic exposure stimulated hepatic glutathione (GSH) release with a time course roughly paralleling volume regulatory K+ efflux. NH4Cl stimulated lactate release from the liver during hypotonic but not during normotonic perfusion. In the absence of NH4Cl, hypotonicity did not significantly affect the lactate/pyruvate ratio in effluent perfusate. With NH4Cl (0.5 mmol/l) present, the lactate/pyruvate ratio increased from 4.3 to 8.2 in hypotonicity, whereas simultaneously the 3-hydroxybutyrate/acetoacetate ratio slightly, but significantly decreased. 3. Addition of lactate (2.1 mmol/l) and pyruvate (0.3 mmol/l) to influent perfusate did not affect urea synthesis in normotonic perfusions, but completely prevented the inhibition of urea synthesis from NH4Cl (0.5 mmol/l) induced by hypotonicity. Restoration of urea production in hypotonic perfusions by addition of lactate and pyruvate was largely abolished in the presence of 2-cyanocinnamate (0.5 mmol/l). Addition of 3-hydroxybutyrate (0.5 mmol/l), but not of acetoacetate (0.5 mmol/l) largely reversed the hypotonicity-induced inhibition of urea synthesis from NH4Cl.(ABSTRACT TRUNCATED AT 400 WORDS)

We have applied an electrophysiologic technique (Reuss, L. (1985) Proc. Natl. Acad. Sci. USA 82, 6014) to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA+) during transient exposure of cells to nystatin. Intracellular TMA+ activity (alpha 1TMA) was measured with TMA(+)-sensitive, double-barrelled microelectrodes. Loading hepatocytes with TMA+ did not change their membrane potential (Vm), and under steady-state conditions alpha iTMA remained constant over 4 min in a single impalement. Hyperosmotic solutions (50, 100 and 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 and 100 mM) increased and decreased alpha iTMA, respectively, which demonstrated transmembrane water movements. The slope of the plot of change in steady-state cell water volume, [(alpha iTMA)0/(alpha iTMA)4min] -1, on the relative osmolality of media, (experimental mosmol/control mosmol) -1, was less predicted for a perfect osmometer. Corresponding measurements of Vm showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba2+ (2 mM) was present during hyposmotic stress of 0.66 X 286 mosmol (control), cell water volume increased by a factor of 1.44 +/- 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 +/- 0.02, P less than 0.001. Ba2+ also decreased the hyperpolarization of hyposmotic stress from a factor of 1.62 +/- 0.04 to 1.24 +/- 0.09, P less than 0.01. We conclude that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of Vm in the presence of K(+)-channel blocker, Ba2+. This shows that variation in Vm during osmotic stress provides an intercurrent, electromotive force for hepatocyte volume regulation.

1) Addition of glutamine, glycine, alanine, serine, phenylalanine, proline at a concentration of 3mM, each, or of an amino-acid mixture resembling the physiological amino-acid composition of portal venous blood, to influent perfusate of isolated perfused rat liver led to a 4-6% increase of liver mass without increase of the [3H]inulin space, and biphasic K+ movements across the plasma membrane. These K+ movements consisted of an initial net K+ uptake (0.4-0.9 mumol X g-1 liver) for about 2 min, being followed by a net K+ release (1.0-2.8 mumol X g-1 liver) during the next 10 min. Withdrawal of the amino acids from influent perfusate caused a slow net K+ reuptake by the liver and restored the initial liver mass. No effects on liver mass and K+ fluxes were observed following addition of glutamate or glucose at a concentration of 3mM, each. 2) Aminooxyacetate did not affect the alanine (3 mM) induced increase in liver mass. However, in presence of aminooxyacetate the alanine-induced net K+ release from the liver (i.e. K+ release from 2-10 min minus initial K+ uptake) increased from 0.1 to 2.2 mumol X g-1 liver, whereby simultaneously the alanine tissue level rose from 6.8 to 13.3 mumol X g-1 (corresponding to an increase of the intracellular alanine concentration from about 12 to 25 mM) in presence of aminooxyacetate. 3) When livers were perfused with different glutamine concentrations, a maximal increase in liver mass of 5-6% was observed at glutamine concentrations above 1.5-2mM. A halfmaximal increase in liver mass was observed at 0.6-1.0mM glutamine in influent, i.e. at the physiological portal glutamine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholestasis induced by perfusion of the liver with hypocalcemic media has been ascribed to several defects in bile secretion including increased biliary permeability. To investigate this model of cholestasis further, livers perfused with hypo- and normocalcemic media were examined stereologically using thin sections and freeze-fracture replicas. Organization of tight junctions was not altered by hypocalcemia; neither the number of strands nor the junctional depth were significantly affected. By contrast, the volume of hepatocytes decreased by 11% (p less than 0.001), compensated for by an increase in the space of Dissé and of the sinusoids. The canalicular length decreased by 25% (p less than 0.01), while the canalicular membrane surface was not altered. Multiple indicator dilution studies confirmed a decrease in hepatocellular volume, measured as the water space by 14% (p less than 0.03). This was compensated for by an increase in the extravascular sucrose, but not the albumin space. Immediately after switching from normo- to hypocalcemic perfusate a K+ efflux of 62 mumol/g liver was observed corresponding to approx. 8% of the hepatocellular water space. Our results suggest that hypocalcemia-induced cholestasis is due, at least in part, to a disturbance of the osmotic equilibrium, possibly caused by impairment of an ion transport system involved in hepatocellular volume control.

1. In the presence of near-physiological glutamine concentrations, exposure of perfused rat liver to hypotonic perfusion media switched glutamine balance across the liver from net release to net uptake. This was due to both stimulation of flux through glutaminase and inhibition of flux through glutamine synthetase. Conversely, during exposure to hypertonic media, net glutamine release from the liver increased due to inhibition of glutaminase flux and slight stimulation of flux through glutamine synthetase. The effect of perfusate osmolarity on glutaminase flux was observed at an NH4Cl concentration (0.5 mM) sufficient for near-maximal ammonia stimulation of glutaminase. This indicates the involvement of different mechanisms of glutaminase flux control by extracellular osmolarity changes and ammonia. The effects of anisotonicity on flux through glutamine-metabolizing enzymes were fully reversible. Glutamine (0.6 mM) stimulated urea synthesis from NH4Cl (0.5 mM) during hypotonic and normotonic conditions. 2. Exposure to hypotonic and hypertonic media led, after initial liver-cell swelling and shrinkage, respectively to volume-regulatory K+ fluxes which largely restored the initial liver-cell volume despite the continuing osmotic challenge. Even after completion of cell-volume regulatory K+ fluxes, the effects of perfusate osmolarity on hepatic glutamine metabolism persisted. This indicates that in anisotonicity the liver cell is left in an altered metabolic state, even after completion of volume-regulatory responses. 3. During perfusion with isotonic media, addition of glutamine (3 mM) led to an increase of liver mass by about 4% within 2 min, which was accompanied by a net K+ uptake by the liver. Thereafter, the new steady state of increased liver mass was maintained throughout glutamine infusion. When the liver mass had reached this new steady state, a net release of K+ from the liver of about 3 mumol/g liver was observed during the following 10 min. Withdrawal of glutamine was followed by a slow reuptake of K+ and the liver mass returned to its initial value. Following exposure to glutamine (3 mM), the intracellular glutamine concentration (as calculated from glutamine tissue levels, taking into account the extracellular space determined with the [3H]inulin technique) rose from about 1 mM to 30-35 mM within about 12 min, indicating a 10-12-fold concentrative uptake of glutamine into the liver cells and an osmotic challenge for the hepatocyte. When intracellular glutamine had reached its steady-state concentration, net K+ efflux from the liver was also terminated.(ABSTRACT TRUNCATED AT 400 WORDS)

The electrophysiological properties of isolated rat liver cells were studied using the patch clamp method in whole-cell configuration. The membrane potential in isolated hepatocytes was -42 +/- 7 mV (n = 20). The input resistance (Rin) and the time constant (tau m) were 51 +/- 17 M (the range of 34 to 180 M omega) (n = 20) and 4.2 +/- 1.0 msec (the range of 3 to 16.5 ms) (n = 20). Assuming that the specific membrane capacitance is 1 microF/cm2, the membrane resistance and membrane capacitance were 42. +/- 9.0 K omega cm2 and 87 +/- 27 pF. These values indicate that isolated rat hepatocytes are not abnormally permeable or leaky. The current-voltage relationship was linear with no rectification. The depolarizing pulse from the resting potential did not induce fast or slow inward currents even when norepinephrine or high Ca2 (3.6 mM) were applied. This indicates that there is no voltage-sensitive Ca2+ channel in the isolated hepatocytes.

Different calcium-dependent mechanisms may be involved in mediating stimulus-induced aldosterone secretion. Using isolated perfused canine adrenal glands, we determined the effect of reductions in extracellular [Ca2+] and of infusion of a voltage-dependent calcium channel antagonist, nifedipine, on aldosterone secretion induced by decreases in osmolality, by increases in [K+], or by infusion of angiotensin II (ANG II). Aldosterone secretion was stimulated to a similar level by reducing osmolality, by increasing [K+], or by infusing ANG II. After 50 min of stimulation, lowering [Ca2+] from 1.25 to 0.10 mM caused a marked and similar inhibition of osmolality- and [K+]-induced aldosterone secretion that was significantly greater than inhibition of ANG II-induced aldosterone secretion. Similarly, nifedipine at 3.3 X 10(-8) M caused marked and similar inhibition of osmolality- and [K+]-induced aldosterone secretion that was significantly greater than the inhibition of ANG II-induced aldosterone secretion. These data demonstrate that calcium-dependent processes are involved in osmolality-, [K+]-, and ANG II-induced aldosterone secretion. However, the calcium-dependent process(es) evoked by reductions in osmolality or increases in [K+] are considerably different from that evoked by ANG II. Osmolality and potassium appear to induce aldosterone secretion primarily by activating voltage-dependent calcium channels.