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Updated: Aug 7, 2026
A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
Ascorbate enhancement of H1 histamine receptor sensitivity coincides with ascorbate oxidation inhibition by histamine
Patrick F Dillon1, Robert S Root-Bernstein, Charles M Lieder
1Dept. of Physiology, Michigan State University, East Lansing, MI 48824, USA. dillon@msu.edu
Abstract:
Ascorbate has previously been shown to enhance both alpha(1)- and beta(2)-adrenergic activity. This activity is mediated by ascorbate binding to the extracellular domain of the adrenergic receptor, which also decreases the oxidation rate of ascorbate. H1 histamine receptors have extracellular agonist or ascorbate binding sites with strong similarities to alpha(1-) and beta(2)-adrenergic receptors. Physiological concentrations of ascorbate (50 microM) significantly enhanced histamine contractions of rabbit aorta on the lower half of the histamine dose-response curve, increasing contractions of 0.1, 0.2, and 0.3 microM histamine by two- to threefold. Increases in ascorbate concentration significantly enhanced 0.2 microM histamine (5-500 microM ascorbate) and 0.3 microM histamine (15-500 microM ascorbate) in a dose-dependent manner. Histamine does not measurably oxidize over 20 h in oxygenated PSS at 37 degrees C. Thus the ascorbate enhancement is independent of ascorbate's antioxidant effects. Ascorbate in solution oxidizes rapidly. Transfected histamine receptor membrane suspension with protein concentration at >3.1 microg/ml (56 nM maximum histamine receptor) decreases the oxidation rate of 392 microM ascorbate, and virtually no ascorbate oxidation occurs at >0.0004 mol histamine receptor/mol ascorbate. Histamine receptor membrane had an initial ascorbate oxidation inhibition rate of 0.094 min.microg protein(-1).ml(-1), compared with rates for transfected ANG II membrane (0.055 min.microg protein(-1).ml(-1)), untransfected membrane (0.052 min.microg protein(-1).ml(-1)), creatine kinase (0.0082 min.microg protein(-1).ml(-1)), keyhole limpet hemocyanin (0.00092 min.microg protein(-1).ml(-1)), and osmotically lysed aortic rings (0.00057 min.microg wet weight(-1).ml(-1)). Ascorbate enhancement of seven-transmembrane-spanning membrane receptor activity occurs in both adrenergic and histaminergic receptors. These receptors may play a significant role in maintaining extracellular ascorbate in a reduced state.
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