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Activation by calcium of erythrocyte Na+/H+ exchange in men
1Department of Cardiovascular and Molecular Medicine, Catholic University of Leuven, Belgium. paul lijnen@med.kuleuven.ac.be
Insights
Protein kinase C is essential for calcium-mediated activation of sodium-hydrogen exchange in human red blood cells. Depleting protein kinase C prevents calcium from stimulating this vital ion transport.
Area of Science:
- Cellular Physiology
- Biochemistry
- Ion Transport Mechanisms
Background:
- The sodium-hydrogen exchanger (Na+/H+ exchange) plays a crucial role in regulating intracellular pH and cell volume.
- Calcium ions are known modulators of various cellular processes, including ion transport.
Purpose of the Study:
- To investigate the necessity of protein kinase C (PKC) in the calcium-dependent activation of Na+/H+ exchange in human erythrocytes.
- To elucidate the role of PKC in mediating calcium's effects on erythrocyte ion transport.
Main Methods:
- Human erythrocytes were subjected to Na+/H+ exchange measurements using initial rates of sodium influx.
- Protein kinase C was downregulated using phorbol-12-myristate-13-acetate (PMA).
- The effects of varying calcium chloride (CaCl2) concentrations and the calpain inhibitor E-64d on Na+/H+ exchange were assessed in control and treated cells.
Main Results:
- Calcium (1 mmol/l CaCl2) significantly stimulated erythrocyte Na+/H+ exchange by 74% (P<0.001).
- Calcium increased the maximum rate of Na+/H+ exchange but did not alter its affinity for intracellular pH or external sodium.
- Crucially, calcium failed to activate Na+/H+ exchange in erythrocytes with downregulated protein kinase C, and E-64d did not restore this activation.
Conclusions:
- Protein kinase C is indispensable for the calcium-induced activation of Na+/H+ exchange in human erythrocytes.
- These findings highlight a specific signaling pathway where PKC acts as a key mediator for calcium's regulatory effects on this critical ion transporter.
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.
Conclusion:
Our data indicate that protein kinase C is necessary for the activation by calcium of the erythrocyte Na+/H+ exchange.