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

The norepinephrine transporter (NET) is the carrier that drives the neuronal norepinephrine uptake mechanism (uptake1) in mammalian hearts. The radioligand [3H]mazindol binds with high affinity to NET. In this study, the kinetics of [3H]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 (Bmax) and competitive inhibition assays designed to measure inhibitor binding affinities (KI) 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 [3H]mazindol binding to NET was studied using membranes from rat and canine hearts. Specificity of [3H]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 KI values for several NET inhibitors and substrates. In kinetic studies using rat heart membranes, [3H]mazindol exhibited a dissociation rate constant koff=0.0123+/-0.0007 min(-1) and an association rate constant kon=0.0249+/-0.0019 nM(-1)min(-1). In saturation binding assays, [3H]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 Bmax. Injections of 6-OHDA in rats decreased measured cardiac NET Bmax values in a dose-dependent manner, verifying that [3H]mazindol binds specifically to NET from sympathetic nerve terminals. Competitive inhibition studies provided NET inhibitor and substrate KI values consistent with previously reported values. These studies demonstrate the high selectivity of [3H]mazindol binding for the norepinephrine transporter in membrane preparations from mammalian hearts.