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We have previously reported increased activity of Na+/H+ and Na+/Li+ exchanges in red blood cells (RBC) of patients with hypertension and diabetic nephropathy. The presence in human red blood cells (RBC) of insulin receptors has led us to examine the effects of this hormone on the kinetic parameters of Na+/H+ exchange as a first approach to define its mechanism of action. The antiporter activity was measured as net Na+ influx driven by an outward H+ gradient in acid-loaded, Na-depleted RBCs preincubated with or without (w/wo) insulin (0 to 100 microU/ml) for different time periods. The effects of insulin on the H+ and Na+ activation kinetics of Na+/H+ exchange were examined in RBCs of normal subjects fasted for 12 hours. Insulin (50 microU/ml for 1 hr) increased the Vmax from 28 +/- 6 to 49 +/- 8 mmol/liter cell x hr (N = 10, P < 0.0005) and the Km for Na+ from 72 +/- 10 to 142 +/- 19 mM (N = 4, P < 0.05) but did not change the Km for intracellular H+. Insulin also increased the Vmax of Na+/Li+ exchange at pHi 7.4 (0.34 +/- 0.03 to 0.45 +/- 0.04 mmol/liter cell x hr, N = 9, P < 0.005) as well as the Km for Na+ (31 +/- 3 to 6 +/- 10 mM, P < 0.0003). Therefore, insulin can modulate Na+ sites of Na+/Li+ or Na+/H+ exchanges independent of the occupancy of H+ sites to favor the release of bound Na+ into the cytoplasm. Insulin stimulation of Na+/H+ exchange required endogenous cytosolic Ca2+ levels. The kinetic effects of insulin on Na+/H+ and Na+/Li+ exchanges were imitated by okadaic acid (300 microM), an inhibitor of protein phosphatases which dephosphorylate serine-threonine residues. Okadaic acid increased the Vmax of Na+/H+ and Na+/Li+ exchanges and the Km for Na+ as insulin did. In conclusion, insulin stimulation of the Na+/H+ antiporter occurs by a novel kinetic mechanism leading to a decreased affinity for external Na+ without changes in the affinity for Hi. On the basis that insulin effects were imitated by okadaic acid, we hypothesize that this hormone may increase the phosphorylated state of serine-threonine residues of this antiporter protein.
R Pontremoli, and
G Zerbini, and
A Rivera, and
M Canessa
March 1997,
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R Pontremoli, and
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A Rivera, and
M Canessa
May 2002,
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R Pontremoli, and
G Zerbini, and
A Rivera, and
M Canessa
August 1996,
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R Pontremoli, and
G Zerbini, and
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M Canessa
January 1987,
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R Pontremoli, and
G Zerbini, and
A Rivera, and
M Canessa
December 1989,
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R Pontremoli, and
G Zerbini, and
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M Canessa
June 1994,
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R Pontremoli, and
G Zerbini, and
A Rivera, and
M Canessa
January 1993,
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R Pontremoli, and
G Zerbini, and
A Rivera, and
M Canessa
August 1992,
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R Pontremoli, and
G Zerbini, and
A Rivera, and
M Canessa
July 2003,
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R Pontremoli, and
G Zerbini, and
A Rivera, and
M Canessa
September 1990,
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