Activation of a NADH Dehydrogenase in the Human Erythrocyte by Beta-Adrenergic Agonists: Possible Involvement of a G Protein in Enzyme Activation

Abstract

NADH dehydrogenase in the plasma membrane transfers electrons from NADH to external oxidants like ferricyanide, through pathways which are linked to metabolic processes in the cell. Hormone binding to specific sites (receptors) can modify the enzyme activity, suggesting a direct or indirect coupling between the redox system and the hormone receptors. Reduction of external ferricyanide to ferrocyanide by human erythrocytes was stimulated by β-adrenergic agonists (adrenaline, ritodrine and isoxsuprine), this effect being dependent upon concentration and pH. The agonist-stimulatory effect was attenuated in the presence of metoprolol (10^–4M), a β-adrenergic antagonist, and was not modified in the presence of prazosin, an α-adrenergic antagonist, suggesting that modification of the redox activity is mediated by binding of the agonists to β-adrenergic receptors present in the human erythrocytes. Basal and agonist-dependent activities were inhibited in the presence of sulfhydryl reagents p-chloromercuriben-zoate (PCMB, 10^–5M) and N-ethylmaleimide (NEM, 10^–3M), indicating the involvement of –SH groups. Inactivation by NEM was reversed by washing the cells with GTP (10^–3M) and GTPγS (10^–4M), suggesting that the specific alkylated –SH group(s) is located on a G protein in the hormone-receptor-G-protein complex. The human erythrocytes contain G proteins, displaying both guanine-nucleotide-binding properties and GTPase activity. Fluoride (10^–2 M) and fluoroaluminate AlF^–₄ (F^–, 10^–2M + Al³⁺, 10^–5M), G protein activators, enhanced the basal and agonist-dependent activities, suggesting the involvement of G proteins in this system. The overall results indicated that one of the coupling components between the hormonal receptors and the redox system is probably a G protein, and the mechanism of enzyme activation after hormone binding to the receptor is based on the redox state of cysteine residues probably within the receptor-G-protein complex.

Novel epinephrine and cyclic AMP-mediated activation of BCAM/Lu-dependent sickle (SS) RBC adhesion

“…Previous studies suggested that the ␤-adrenergic receptor (␤-AR) is the primary mediator of the physiologic effects of epinephrine on RBCs. 9,26,27 As a result, we asked if ␤-AR inhibition could block the epinephrine-stimulated adhesion. The nonselective ␤-AR antagonist propranolol inhibited epinephrine-stimulated SS RBC adhesion by 77% (P Ͻ .01), whereas the ␤2-specific antagonist butoxamine inhibited adhesion by 62% (P Ͻ .001) ( Figure 5B).…”

Protein Kinase A-dependent Phosphorylation of Lutheran/Basal Cell Adhesion Molecule Glycoprotein Regulates Cell Adhesion to Laminin α5

“…Previous studies indicated that the physiologic stress mediator epinephrine acts largely through the ␤2-adrenergic receptor on sickle red blood cells (33)(34)(35)(36)(37). Therefore, we tested whether the ␤2-selective antagonist butoxamine could inhibit epinephrine-stimulated K562-Lu adhesion to laminin.…”

CNS neurons express two distinct plasma membrane electron transport systems implicated in neuronal viability

“…Trans-plasma membrane reductases identified in other cell types are sensitive to modification by extracellular membrane-impermeant sulfhydryl reagents (Crane et al 1985;Vaillant et al 1996;Marques and Bicho 1997). In our experiments, cultured forebrain neurons were pre-incubated with or without 1 mM N-ethylmaleimide (NEM), a sulfhydryl alkylating reagent, for 10 min prior to measuring DCIP reduction.…”