Dynamic monitoring of single-terminal norepinephrine transporter rate in the rodent cardiovascular system: A novel fluorescence imaging method.

Here, we validate the use of a novel fluorescent norepinephrine transporter (NET) substrate for dynamic measurements of transporter function in rodent cardiovascular tissue; this technique avoids the use of radiotracers and provides single-terminal resolution. Rodent (Wistar rats and C57BL/6 mice) hearts and mesenteric arteries (MA) were isolated, loaded with NET substrate Neurotransmitter Transporter Uptake Assay (NTUA) ex vivo and imaged with confocal microscopy. NTUA labelled noradrenergic nerve terminals in all four chambers of the heart and on the surface of MA. In all tissues, a temperature-dependent, stable linear increase in intra-terminal fluorescence upon NTUA exposure was observed; this was abolished by NET inhibitor desipramine (1 µM) and reversed by indirectly-acting sympathomimetic amine tyramine (10 µM). NET reuptake rates were similar across the mouse cardiac chambers. In both species, cardiac NET activity was significantly greater than in MA (by 62 ± 29% (mouse) and 21 ± 16% (rat)). We also show that mouse NET reuptake rate was twice as fast as that in the rat (for example, in the heart, by 94 ± 30%). Finally, NET reuptake rate in the mouse heart was attenuated with muscarinic agonist carbachol (10 µM) thus demonstrating the potential for parasympathetic regulation of norepinephrine clearance. Our data provide the first demonstration of monitoring intra-terminal NET function in rodent cardiovascular tissue. This straightforward method allows dynamic measurements of transporter rate in response to varying physiological conditions and drug treatments; this offers the potential to study new mechanisms of sympathetic dysfunction associated with cardiovascular disease.

Pharmacokinetic profiles contribute to the differences in behavioral pharmacology of 071031B enantiomers as novel serotonin and norepinephrine reuptake inhibitors.

Our previous study indicated that a chiral compound 071031B was a novel serotonin and noradrenaline reuptake inhibitor with superior antidepressant activity compared to duloxetine. The present study aimed to investigate chiral pharmacology differences of 071031B enantiomers, S-071031B and R-071031B, and disclose mechanisms underlying the behavioral differences based on target profiles and pharmacokinetic profiles. In vivo behavioral tests indicated that S-071031B was more potent than R-071031B in two depression models (the forced swimming test in mice and rats) and two pain models (the acetic acid-induced writhing and formalin tests in mice). In vitro assays revealed that both S-071031B and R-071031B showed high affinity for human serotonin transporters and norepinephrine transporters with equal potency, and showed consistently equipotent inhibitory effects on serotonin and norepinephrine uptake. Pharmacokinetic studies demonstrated that oral availability and hepatic metabolism, rather than pH stability, intestinal transport, and plasma binding, contributed to enantiomers' behavioral differences. Based on these findings, it is suggested that S-071031B is a more active enantiomer, and the differential pharmacokinetic profiles, but not target affinity, contribute to differences of S-071031B and R-071031B in behavioral pharmacology. Moreover, current PK-PD study may provide positive exploration for chiral antidepressants development.