Binding of [ 3 H]mazindol to cardiac norepinephrine transporters: kinetic and equilibrium studies

Abstract

The norepinephrine transporter (NET) is the carrier that drives the neuronal norepinephrine uptake mechanism (uptake 1 ) in mammalian hearts. The radioligand [ 3 H]mazindol binds with high affinity to NET. In this study, the kinetics of [ 3 H]mazindol binding to NET were measured using a rat heart membrane preparation. Results from these studies were used to set up saturation binding assays designed to measure cardiac NET densities ( B max ) and competitive inhibition assays designed to measure inhibitor binding affinities ( K I ) for NET. Saturation binding assays measured NET densities in rat, rabbit, and canine hearts. Assay reproducibility was assessed and the effect of NaCl concentration on [ 3 H]mazindol binding to NET was studied using membranes from rat and canine hearts. Specificity of [ 3 H]mazindol binding to NET was determined in experiments in which the neurotoxin 6-hydroxydopamine (6-OHDA) was used to selectively destroy cardiac sympathetic nerve terminals in rats. Competitive inhibition studies measured K I values for several NET inhibitors and substrates. In kinetic studies using rat heart membranes, [ 3 H]mazindol exhibited a dissociation rate constant k off =0.0123±0.0007 min −1 and an association rate constant k on =0.0249±0.0019 nM −1 min −1 . In saturation binding assays, [ 3 H]mazindol binding was monophasic and saturable in all cases. Increasing the concentration of NaCl in the assay buffer increased binding affinity significantly, while only modestly increasing B max . Injections of 6-OHDA in rats decreased measured cardiac NET B max values in a dose-dependent manner, verifying that [ 3 H]mazindol binds specifically to NET from sympathetic nerve terminals. Competitive inhibition studies provided NET inhibitor and substrate K I values consistent with previously reported values. These studies demonstrate the high selectivity of [ 3 H]mazindol binding for the norepinephrine transporter in membrane preparations from mammalian hearts.