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

作者：Raffel DM^*; Chen W

来源：Naunyn-Schmiedeberg's Archives of Pharmacology, 2004, 370(1): 9-16.

DOI：10.1007/s00210-004-0949-y

• sympathetic nervous system
• norepinephrine transporter
• positron emission tomography
• meta-iodobenzylguanidine
• meta-hydroxyephedrine

The norepinephrine transporter (NET) is the carrier that drives the neuronal norepinephrine uptake mechanism (uptake(1)) in mammalian hearts. The radioligand [H-3]mazindol binds with high affinity to NET. In this study, the kinetics of [H-3]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 [H-3]mazindol binding to NET was studied using membranes from rat and canine hearts. Specificity of [H-3]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, [H-3]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, [H-3]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 [H-3]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 [H-3]mazindol binding for the norepinephrine transporter in membrane preparations from mammalian hearts.

• 出版日期2004-7