Abstract
Objectives: [11C]-Phenethylguanidines ([11C]-PG) are a novel class of radiotracers for PET studies of cardiac sympathetic innervation. As analogs of the neurotransmitter norepinephrine, [11C]-PG are transported into sympathetic neurons by the norepinephrine transporter (NET). To better characterize the kinetics of [11C]-PG, their Michaelis-Menten constants Km and Vmax for NET transport were measured in vitro using rat C6 glial cells that stably express the human NET (C6-hNET cells). Binding affinities of PG for hNET (KD) were also measured.
Methods: Five [11C]-PGs were studied: [11C]-para-hydroxy-PG (PHPG); [11C]-meta-hydroxy-PG (MHPG); [11C]-4-fluoro-MHPG (4F-MHPG); [11C]-6-fluoro-MHPG (6F-MHPG); and [11C]-N-[guanyl]-meta-octopamine (GMO). For transport studies, C6-hNET cells were incubated for 5 min at 37°C with varying [11C]-PG concentrations. Km and Vmax were estimated by fitting specific uptake vs. [11C]-PG concentration to a one-site transport model. For competitive binding assays, homogenized and purified C6-hNET cell membranes were incubated with 1.5 nM [3H]mazindol at 22 °C for 3 hr in the absence (control binding) and presence of varying PG concentrations. KD was estimated by fitting % control binding vs. PG concentration to a monophasic inhibition process.
Results: KD, Km and Vmax values are given below, along with Vmax/Km ratios, which reflect each substrate’s NET transport rate. Comparable values for [11C]-meta-hydroxyephedrine (HED), [11C]-epinephrine (EPI), [3H]-norepinephrine (NE) and [3H]-dopamine (DA) are also shown. No correlation was found between KD, Km and Vmax, indicating that each constant is independently influenced by structural changes of NET substrates. C6-hNET transport rates (Vmax/Km) were highly correlated with neuronal transport rates measured previously in isolated rat heart (r = 0.96), suggesting that the measured transport constants Km and Vmax accurately reflect in vivo transport kinetics.
|
|
KD (uM) |
Km (uM) |
Vmax (pmol/min/mg
protein) |
Vmax/Km (uL/min/mg protein) |
|
PHPG |
1.93 ± 0.20 |
0.52 ± 0.06 |
3.31 ± 0.40 |
6.42±1.12 |
|
MHPG |
4.87 ± 0.47 |
0.73 ± 0.20 |
7.83 ± 1.73 |
11.2 ± 2.8 |
|
4F-MHPG |
5.60 ± 0.49 |
2.57 ± 0.35 |
7.46± 0.64 |
2.96 ± 0.54 |
|
6F-MHPG |
17.7 ± 0.5 |
2.86 ± 0.43 |
4.06± 0.66 |
1.44±0.31 |
|
GMO |
20.3 ± 2.5 |
4.43 ± 0.31 |
5.57± 0.30 |
1.26±0.13 |
|
HED |
43.2 ± 1.8 |
0.49 ± 0.11 |
5.20 ± 0.90 |
10.7 ± 0.9 |
|
NE |
63.9 ± 2.3 |
0.28 ± 0.03 |
5.83 ± 0.49 |
21.3 ± 2.4 |
|
EPI |
68.4 ± 7.6 |
3.16 ± 0.78 |
6.09 ± 1.04 |
1.97 ± 0.24 |
|
DA |
8.12 ± 0.47 |
0.24 ± 0.04 |
2.91 ± 0.47 |
12.2 ± 1.8 |
|
Values are mean ± SD, N = 5 determinations |
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