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The effects of short-term administration of dexamethasone on sodium/proton antiporter (Na+/H+ antiporter) in human lymphocytes were investigated in vitro.Cytosolic pHi in lymphocytes was measured spectrophotometrically using the pH-sensitive fluorescent dye 2′-7′-bis-carboxyethyl-5(6)-carboxyfluorescein at 530 nm with excitation wavelengths of 440 and 530 nm. The Na+/H+ antiporter activity was determined after intracellular acidification using 100 mmol/l propionic acid.The addition of 1 μmol/l dexamethasone significantly reduced cytosolic pHi from 7.44 ± 0.03 to 7.25 ± 0.05 (mean ± SEM; P < 0.01). Incubation with dexamethasone for 40 min significantly reduced the Na+/H+ antiporter activity from (9.19 ± 0.61) × 10−3 pHi/s (n = 22) to (7.23 ± 0.49) × 10−3 pHi/s (n = 22; P < 0.01). The effect of dexamethasone was time and concentration dependent. The apparent affinity of the Na+/H+ antiporter was not significantly different in the absence or presence of dexamethasone. The inhibition of the Na+/H+ antiporter by dexamethasone was abolished in the presence of the glucocorticoid receptor blocker, mifepristone.Dexamethasone directly inhibits the Na+/H+ antiporter using a receptor-dependent pathway. This effect may be important for pharmacological side effects such as glucocorticoid-induced hypertension.