A high affinity binding site for [3H]-Dofetilide on human leukocytes

Area of Science:

• Pharmacology
• Immunology
• Cardiovascular Research

Background:

• Class III anti-arrhythmic agents are known to interact with human leukocytes.
• Leukocyte activation by antigenic and mitogenic stimuli is a key immune response.
• Understanding drug interactions with immune cells is crucial for predicting side effects.

Purpose of the Study:

• To investigate the hypothesis that a binding site for the Class III anti-arrhythmic agent dofetilide exists on human leukocytes.
• To characterize the binding kinetics and identify potential molecular targets for dofetilide on leukocytes.
• To assess the functional consequences of dofetilide binding on leukocyte activity.

Main Methods:

• Binding isotherm analysis was performed to determine the affinity (Kd) and density (Bmax) of dofetilide binding sites on mononuclear cells and neutrophils.
• Competitive binding assays were conducted using other Class III drugs, verapamil, charybdotoxin, tetraethylammonium, and apamin to identify the binding site.
• Functional assays evaluated the effect of dofetilide on fMLP-stimulated superoxide production in leukocytes.

Main Results:

• Dofetilide exhibited high-affinity binding to both mononuclear cells (Kd 26±4 nm) and neutrophils (Kd 33±14 nm).
• Other Class III drugs, verapamil, charybdotoxin, and tetraethylammonium inhibited dofetilide binding, suggesting a Ca2+-activated K+ channel as a potential binding site.
• Dofetilide (30 nm) significantly suppressed fMLP-stimulated superoxide production in leukocytes, indicating functional alteration.

Conclusions:

• Dofetilide binds to human leukocytes at physiologically relevant concentrations.
• The binding site may involve a calcium-activated potassium channel, specifically an apamin-insensitive isoform.
• Dofetilide binding alters leukocyte function, impacting superoxide production, which may have implications for immune response.