Ascorbate enhancement of H1 histamine receptor sensitivity coincides with ascorbate oxidation inhibition by histamine receptors

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Abstract
Ascorbate has previously been shown to enhance both α1- and β2-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 α1- and β2-adrenergic receptors. Physiological concentrations of ascorbate (50 μM) 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 μM histamine by two- to threefold. Increases in ascorbate concentration significantly enhanced 0.2 μM histamine (5–500 μM ascorbate) and 0.3 μM histamine (15–500 μM ascorbate) in a dose-dependent manner. Histamine does not measurably oxidize over 20 h in oxygenated PSS at 37°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 μg/ml (56 nM maximum histamine receptor) decreases the oxidation rate of 392 μM 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·μg protein−1·ml−1, compared with rates for transfected ANG II membrane (0.055 min·μg protein−1·ml−1), untransfected membrane (0.052 min·μg protein−1·ml−1), creatine kinase (0.0082 min·μg protein−1·ml−1), keyhole limpet hemocyanin (0.00092 min·μg protein−1·ml−1), and osmotically lysed aortic rings (0.00057 min·μg 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.