Osmolyte and Na⁺ transport balances of rat hepatocytes as a function of hypertonic stress

Abstract.

The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an influx of Na⁺ that is then exchanged for K⁺ via stimulation of Na⁺/K⁺-adenosine triphosphatase (ATPase). In this study, we analysed the activation pattern of the Na⁺ transporters underlying RVI as a function of the degree of hypertonic stress. In confluent primary cultures, four hypertonic conditions were tested (changes from 300 to 327, 360, 400 or 450 mosmol/l) and the activities of Na⁺ conductance, Na⁺/H⁺ antiport, Na⁺-K⁺-2Cl^– symport and Na⁺/K⁺-ATPase were quantified using intracellular microelectrodes, microfluorometry and time-dependent, furosemide- or ouabain-sensitive ⁸⁶Rb⁺ uptake, respectively. Neither Na⁺ conductance nor Na⁺-K⁺-2Cl^– symport responded to 327 mosmol/l. At 360, 400 and 450 mosmol/l, uptake via these transporters would lead to increases of cell Na⁺ by 33.0, 49.0 and 49.0 and by 4.5, 10.4 and 9.2 mmol/l per 10 min, respectively. In contrast, Na⁺/H⁺ antiport exhibited 65% of its maximal activation already at 327 mosmol/l. At the four osmolarities tested, this transporter would augment cell Na⁺ by 6.9, 8.9, 9.8 and 10.6 mmol/l per 10 min. The sums of Na⁺ import were consistent with the amounts of Na⁺ exported via Na⁺/K⁺-ATPase plus the actual increases of cell Na⁺ (21.2, 58.5, 63.6 and 68.3 mmol/l per 10 min and 2.2, 4.0, 6.3 and 8.2 mmol/l, respectively). In addition, these elevations of cell Na⁺ plus the increases of cell K⁺ (via Na⁺/K⁺-ATPase) that amounted to 5.0, 6.5, 17.5 and 18.4 mmol/l were consistent with the increases of intracellular osmotic (cationic) activity of 2.5, 11.5, 21.0 and 28.5 mmol/l, respectively, computed from RVI data. It is concluded that the principle of rat hepatocyte RVI, i.e. an initial uptake of Na⁺ that is then exchanged for K⁺ via Na⁺/K⁺-ATPase, is realized over the entire range of 9-50% hypertonicity tested. The set-point for the activation of RVI clearly lies below 327 mosmol/l. Na⁺/H⁺ antiport is the most sensitive Na⁺ importer involved in RVI, whereas Na⁺ conductance plays the prominent role from 360 mosmol/l upwards.