Author:

Keywords:

Calcium, Calpain, Cysteine Proteinase Inhibitors, Down-Regulation, Erythrocytes, Humans, Hydrogen-Ion Concentration, Intracellular Fluid, Ion Transport, Kinetics, Leucine, Male, Protein Kinase C, Sodium, Sodium-Hydrogen Antiporter, Tetradecanoylphorbol Acetate, Science & Technology, Life Sciences & Biomedicine, Peripheral Vascular Disease, Cardiovascular System & Cardiology, Na+/H+ exchange, calcium, protein kinase C, erythrocytes, PROTEIN-KINASE-C, SODIUM-PROTON EXCHANGE, HUMAN RED-CELLS, HUMAN-PLATELETS, CA-2+ MOBILIZATION, PH, HYPERTENSION, SPECIFICITY, INHIBITORS, AMILORIDE, In Vitro Techniques, Sodium-Hydrogen Exchangers, 1102 Cardiorespiratory Medicine and Haematology, 1103 Clinical Sciences, 1116 Medical Physiology, Cardiovascular System & Hematology, 3201 Cardiovascular medicine and haematology, 3202 Clinical sciences

Abstract:

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.