Activation by calcium of erythrocyte Na+/H+ exchange in men.
OBJECTIVE
To determine whether protein kinase C is necessary for the calcium activation of the Na+/H+ exchange in human erythrocytes by studying activation by calcium of erythrocyte Na+/H+ exchange in control cells, in protein kinase C-depleted cells after downregulation of protein kinase C with phorbol-12-myristate-13-acetate and in cells that had been treated beforehand with phorbol-12-myristate-13-acetate with and without the calpain inhibitor E-64d.


METHODS
Erythrocyte Na+/H+ exchange was measured by determining the initial rates of the influx of Na+ into Na+-depleted, acid loaded cells. The effects of various concentrations (0-1 mmol/l) of CaCl2 and the effects of 1 mmol/l CaCl2 on activation of the intracellular pH and on the external Na+ activation of Na+/H+ exchange were studied. The effects of 1 mmol/l CaCl2 on Na+/H+ exchange in control cells and cells that had been incubated beforehand with and without 1 micromol/l phorbol-12-myristate-13-acetate and with E-64d and 1 micromol/l phorbol-12-myristate-13-acetate for 1, 2, 3 and 24 h were also investigated.


RESULTS
Addition of Ca2+ to a concentration in the range 0-1 mmol/l in the presence of calcimycin resulted in stimulation of Na+/H+ exchange: 1 mmol/l CaCl2 increased (P< 0.001) the erythrocyte Na+/H+ exchange by 74%. Calcium increased the maximum rate for activations by intracellular pH and by external Na+ of Na+/H+ exchange, whereas it did not affect the Michaelis-Menten constants for activation by intracellular H+ and external Na+. However, calcium did not activate the Na+/H+ exchange in protein kinase C downregulated erythrocytes and administration of the calpain inhibitor E-64d could not prevent this inactivation.


CONCLUSIONS
Our data indicate that protein kinase C is necessary for the activation by calcium of the erythrocyte Na+/H+ exchange.
Lijnen
P
P
Department of Cardiovascular and Molecular Medicine, Catholic University of Leuven, Belgium. paul lijnen@med.kuleuven.ac.be
Fagard
R
R
Petrov
V
V
D002118:Calcium / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D011493:Protein Kinase C / Q000097:blood; D012964:Sodium / Q000097:blood* / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015853:Cysteine Proteinase Inhibitors; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D011493:Protein Kinase C; D002154:Calpain; D007930:Leucine; C108192:aloxistatin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium