Radiotracers for cardiac sympathetic innervation: Transport kinetics and binding affinities for the human norepinephrine transporter

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Abstract

Introduction

Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [11C]-(−)-meta-hydroxyephedrine, [11C]-(−)-epinephrine, and a series of [11C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [3H]-labeled biogenic amines were also determined.

Methods

Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis–Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [3H]mazindol provided estimates of binding affinities (KI) for NET.

Results

Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for ‘tracer concentrations’ of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r2 = 0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics.

Conclusion

The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density.

Introduction

[11C]-(−)-meta-Hydroxyephedrine (HED) and [11C]-(−)-epinephrine (EPI) are two of the current generation of positron emission tomography (PET) radiotracers used to assess the integrity of cardiac sympathetic nerve terminals [1]. As structural analogs of the endogenous neurotransmitter norepinephrine, HED and EPI are transported into cardiac sympathetic nerve varicosities by norepinephrine transporters (NET) localized in the outer membranes of terminal sympathetic nerve axons [2], [3]. Once inside neurons, they are transported into norepinephrine storage vesicles by the second isoform of the vesicular monoamine transporter (VMAT2).

While HED and EPI both accumulate in cardiac sympathetic neurons by the same transport pathways as norepinephrine, there are differences in the kinetics of their neuronal uptake and retention. For example, kinetic studies in isolated rat hearts have shown that HED is transported into neurons by NET at a rate that is more than four times faster than EPI (2.66 vs. 0.60 ml/min/g wet, respectively) [2], [3]. Also, studies have shown that HED is lipophilic enough (log P = 0.3) to diffuse fairly quickly out of vesicles and neurons [2]. After leaking from neurons, HED is usually taken back up by neurons, setting up a dynamic recycling of HED molecules during PET imaging. On the other hand, EPI is a very polar catecholamine (log P =  1.30) that remains tightly sequestered inside storage vesicles [3]. Isolated rat heart studies of the kinetics and retention mechanisms of cardiac sympathetic nerve radiotracers have provided invaluable insights into differences in the neuronal uptake and storage of these imaging agents [4]. Such insights are critical to interpreting changes observed in the kinetics and myocardial retention of a specific tracer in clinical PET studies of heart diseases [1].

In this study, we sought to better characterize the kinetic properties of HED and EPI by measuring their Michaelis–Menten transport constants Km and Vmax for NET transport. To accomplish this goal, we performed transport kinetics assays using a rat C6 glioma cell line stably transfected with the cloned human NET (C6-hNET cells), a gift from Dr. Amy Eshleman, Oregon Health Sciences University, Portland, OR. For comparison, we also measured Km and Vmax values for the biogenic amines norepinephrine and dopamine. In addition, we performed competitive inhibition binding assays using membranes from homogenized C6-hNET cells to determine the binding affinities (KI) of each compound for hNET.

Km, Vmax and KI values were also measured for five [11C]-labeled phenethylguanidines. Our laboratory is currently investigating radiolabeled phenethylguanidines in an effort to develop a new PET sympathetic nerve imaging agent with more optimal kinetics for quantifying regional nerve density using tracer kinetic analyses [5]. Assessment of the kinetic profiles of these new compounds not only provides valuable structure–activity relation data but also allows comparisons with the existing radiotracers and biogenic amines.

Section snippets

Radiochemistry

[11C]-(−)-meta-Hydroxyephedrine (HED) and [11C]-(−)-epinephrine (EPI) were prepared using previously published methods [6], [7]. The five different [11C]-labeled phenethylguanidines studied were synthesized using methods previously reported [5].

Chemicals

[4′-3H]Mazindol (NET-816; specific activity 24.5 Ci/mmol), levo-[ring-2,5,6-3H]norepinephrine (NET-678; specific activity 74.9 Ci/mmol) and 3,4-[ring-2,5,6-3H]dihydroxyphenylethylamine (NET-673; specific activity 59.3 Ci/mmol) were purchased from Perkin

Binding assays

Saturation binding assays with [3H]mazindol and different batches of C6-hNET membranes were performed to determine the equilibrium dissociation constant KD for [3H]mazindol binding to NET in the C6-hNET cell membrane preparations. Mean binding parameters were KD = 1.20 ± 0.17 nM and Bmax = 457 ± 31 fmol/mg protein (n = 4). In competitive inhibition studies of [3H]mazindol binding to C6-hNET cell membranes, the data from all studies were well-described by a one-site competition model. KI values of the

Discussion

In PET studies of cardiac sympathetic innervation with high specific activity 11C-labeled NET substrates such as [11C]-(−)-meta-hydroxyephedrine (HED) and [11C]-(−)-epinephrine (EPI), tracer-level concentrations of the substrates are used. The substrate concentration [S] outside the neuronal membrane is much lower than the half-saturation transport concentration Km, so that [S]  Km. In this case, the classic Michaelis–Menten equation describing the initial velocity of transport, Vinit = [S]Vmax

Conclusion

In conclusion, the binding affinities (KI) and Michaelis–Menten transport constants (Km, Vmax) of several radiolabeled NET substrates were measured using a cell line stably transfected with the cloned human NET. The results allow for a detailed comparison of the kinetic profiles of several key biogenic amines and some existing PET radiopharmaceuticals for imaging cardiac sympathetic innervation. In addition, the results show that this approach yields valuable structure–activity data, providing

Acknowledgments

We thank the staff of the University of Michigan Cyclotron Facility, especially Lou Tluczek for preparing [11C]-(−)-meta-hydroxyephedrine and [11C]-(−)-epinephrine. This work was supported by PHS grant R01-HL079540 from the National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD USA.

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