In recent years, it has become evident that the volume of a given cell is an important factor not only in defining its intracellular osmolality and its shape, but also in defining other cellular functions, such as transepithelial transport, cell migration, cell growth, cell death, and the regulation of intracellular metabolism. In addition, besides inorganic osmolytes, the existence of organic osmolytes in cells has been discovered. Osmolyte transport systems-channels and carriers alike-have been identified and characterized at a molecular level and also, to a certain extent, the intracellular signals regulating osmolyte movements across the plasma membrane. The current review reflects these developments and focuses on the contributions of inorganic and organic osmolytes and their transport systems in regulatory volume increase (RVI) and regulatory volume decrease (RVD) in a variety of cells. Furthermore, the current knowledge on signal transduction in volume regulation is compiled, revealing an astonishing diversity in transport systems, as well as of regulatory signals. The information available indicates the existence of intricate spatial and temporal networks that control cell volume and that we are just beginning to be able to investigate and to understand.

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 t, low C1-) revealed that these membrane effects are due to an increase in hepatocyte Na t conductance. During RVI, ouabain-sensitive S6Rb+ uptake was augmented to 141% of control, and cell Na t 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 t conductance. In concert with conductive Na t 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.This work was done with the technical assistance of

In rat hepatocytes under hypertonic stress, the entry of Na+ (which is thereafter exchanged for K+ via Na+‐K+‐ATPase) plays the key role in regulatory volume increase (RVI). In the present study, the contributions of Na+ conductance, Na+‐H+ exchange and Na+‐K+‐2Cl− symport to this process were quantified in confluent primary cultures by means of intracellular microelectrodes and cable analysis, microfluorometric determinations of cell pH and buffer capacity, and measurements of frusemide (furosemide)/bumetanide‐sensitive 86Rb+ uptake, respectively. Osmolarity was increased from 300 to 400 mosmol l−1 by addition of sucrose. The experiments indicate a relative contribution of approximately 4:1:1 to hypertonicity‐induced Na+ entry for the above‐mentioned transporters and the overall Na+ yield equalled 51 mmol l−1 (10 min)−1. This Na+ gain is in good agreement with the stimulation of Na+ extrusion via Na+‐K+‐ATPase plus the actual increase in cell Na+, namely 55 mmol l−1 (10 min)−1, as was determined on the basis of ouabain‐sensitive 86Rb+ uptake and by means of Na+‐sensitive microelectrodes, respectively. The overall increase in Na+ and K+ activity plus the expected concomitant increase in cell Cl− equalled 68 mmol l−1, which fits well with the increase in osmotic activity expected to occur from an initial cell shrinkage to 87.5 % and a RVI to 92.6 % of control, namely 53 mosmol l−1. The prominent role of Na+ conductance in the RVI of rat hepatocytes could be confirmed on the basis of the pharmacological profile of this process, which was characterized by means of confocal laser‐scanning microscopy.

At moderate cell shrinkage, activation of Na⁺ channels is the most prominent mechanism of regulatory cell volume increase in rat hepatocytes. The amiloride sensitivity of these channels suggests a relation to the family of epithelial Na⁺ channels (ENaCs). The present study was performed to determine the pharmacological profile of shrinkage-activated Na⁺ channels and to test for ENaC expression in primary cultures of rat hepatocytes; in addition, the influence of the cell volume regulated serine/threonine kinase hSGK on activity and pharmacological profile of rENaC was examined in Xenopus oocytes. Conventional electrophysiology in hepatocytes reveals that the shrinkage-activated Na⁺ channel is inhibited by amiloride and EIPA with IC₅₀ values of 6.0 and 0.12 μmol/l, respectively. Western blots and RT-PCR demonstrate that rat hepatocytes do express all three subunits (α, β, γ) of ENaC. Coexpression of hSGK with rENaC in Xenopus oocytes reveals that the kinase stimulates ENaC by a factor of 4. Moreover, hSGK decreases the affinity to amiloride (increase of IC₅₀ from 0.12 to 0.26 μmol/l) and increases the affinity to EIPA (decrease of IC₅₀ from 250 to 50 μmol/l). In conclusion, rat hepatocytes express ENaC, which is activated by the cell volume-sensitive kinase hSGK. ENaC may contribute to the Na⁺ channels activated by osmotic cell shrinkage in hepatocytes, whereby the relatively low amiloride and high EIPA sensitivity of the channel could at least be partially due to modification by SGK, which decreases the amiloride and increases the EIPA sensitivity of ENaC.

In whole-cell recordings on single HeLa cells, the hypertonic activation of a cation conductance with a selectivity ratio P Na :P Li :P K :P Cs :P NMDG :P Ca :P Cl of 1.00:0.86:0.84:0.56: 0.10:0.07:0.15 was observed. This (non-selective) cation conductance was reduced to 59 and 30% of maximal stimulation by Gd 3+ and £ufenamate, respectively, but it was insensitive to amiloride (with each compound applied at 100 W Wmol/l). As was determined by the Coulter counter technique, the cation conductance was the main mechanism of regulatory volume increase (RVI) in HeLa cells. Whereas a signi¢cant contribution of Na + / H + antiport was also detectable, Na + -K + -2Cl 3 symport most likely did not contribute to RVI. ß