In rat hepatocytes, the hypertonic activation of Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is mediated by protein kinase C.
1. The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an import of extracellular Na(+) via Na(+) conductance, Na(+)-K(+)-2Cl(-) symport, and Na(+)-H(+) antiport. 2. Here, the protein kinase inhibitors staurosporine (100 nmol l(-1)) and bis-indolyl-maleimide I (400 nmol l(-1)) were used to test for a possible contribution of protein kinase C (PKC) to the hypertonic activation of these transporters in confluent primary cultures. 3. Stimulation of Na(+) conductance was monitored: (i) by use of a differential approach based on Na(+) fluxes, (ii) by means of cable analysis, and (iii) in experiments with low Na(+) pulses. All three experimental protocols in concert demonstrated a block of the activation of Na(+) conductance by staurosporine and bis-indolyl-maleimide I. 4. In addition, both compounds significantly reduced the hypertonic activation of Na(+)-K(+)-2Cl(-) symport (quantified on the basis of furosemide-sensitive (86)Rb(+) uptake) to approximately 30 %. 5. In contrast, neither staurosporine nor bis-indolyl-maleimide I had any detectable effect on the hypertonicity-induced alkalinization of cell pH via Na(+)-H(+) antiport (determined fluorometrically). 6. Staurosporine and bis-indolyl-maleimide I completely blocked the RVI of rat hepatocytes (quantified by means of confocal laser-scanning microscopy). The high efficiency of the block suggests an additional inhibitory effect of both compounds on the activity of Na(+)/K(+)-ATPase (determined as ouabain-sensitive (86)Rb(+) uptake). 7. It is concluded that the hypertonic activation of rat hepatocyte Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is probably mediated by PKC.
Heinzinger
H
H
Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Dortmund, Germany.
van den Boom
F
F
Tinel
H
H
Wehner
F
F
D000465:Algorithms; D000818:Animals; D048429:Cell Size; D002478:Cells, Cultured; D004594:Electrophysiology; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012414:Rubidium Radioisotopes; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*
D006982:Hypertonic Solutions; D012414:Rubidium Radioisotopes; D015222:Sodium Channels; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D012964:Sodium; D011493:Protein Kinase C