Several tracers sharing similarities in their benzylguanidine structure were designed to compensate for the disadvantages of the clinically used SPECT tracer MIBG. They all represent similarities to MIBG in order to achieve comparable in vitro intracellular retention and in vivo distribution properties [14]. Among them, 18F-LMI1195 is so far the best examined 18F-labeled PET tracer and has successfully proceeded with a clinical phase I trial [5]. In addition to the current literatures [5, 8, 9, 15], our research group has also performed a number of investigations with 18F-LMI1195 using animal models and ex vivo systems [11, 16, 17]. A further understanding of the properties of 18F-LMI1195 and its performance at a subcellular and molecular level is still of importance for its clinical application.
Therefore, we investigated the storage mechanism and depletion kinetics of LMI1195 on both rat pheochromocytoma PC12 and human neuroblastoma SK-N-SH cells, using 131I-MIBG as a comparator. The former cell line is rich of storage vesicles that could retain either the physiological neurotransmitter norepinephrine or radioactive tracers with analogous structures, whereas the SK-N-SH cells are poor of such secretory vesicles, and therefore, the taken-up tracers can only be stored in cytoplasm or mitochondria [18]. All cells were first preloaded with both tracers to reach equilibrium and thereafter were treated with either high concentration KCl buffer or reserpine in order to trigger the depletion of preloaded radiotracers.
As shown in Fig. 1, depolarization of PC12 cells caused by stimulation of high concentration KCl buffer evoked apparent tracer release, with approximately 60–70% depletion of additional 18F-LMI1195 or 131I-MIBG from the cells. By applying high concentration KCl to neuronal cells, Blaustein has proposed that neurotransmitter release from the nerve terminal is caused by Ca2+ influx via voltage-gated calcium channels [19]. Therefore, when using either Ca2+-free KCl buffer with Ca2+ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or calcium channel blocker nifedipine, Araujo et al. further verified the suppression of norepinephrine release [20]. Similar conclusions were also drawn by Mandela et al. yielding that norepinephrine depletion is dependent on extracellular Ca2+ and could be fully suppressed by EDTA [21]. Thus, as expected, the outcome of exposing cells to Ca2+-free high KCl buffer containing EDTA lead to comparable findings in our study with a diminished release effect (Fig. 2).
This result attained from high KCl induction is consistent with the conclusion achieved from our research group using isolated rabbit hearts, in which the electrical provocation evoked enhanced tracer release [11]. Electrical field stimulation is known to induce norepinephrine overflow by releasing storage vesicles [22]. Since we could measure the radioactivity in the whole heart, including neuronal cells and myocytes, it was suggested that 18F-LMI1195 was taken up by the cells and stored within the vesicles [11]. In addition to our previous findings, we further confirmed this distinct uptake, storage, and release characteristics by using an in vitro assay.
As a human neuroblastoma cell line, SK-N-SH also expresses NET on the plasma membrane [23] and they are able to transport either 131I-MIBG or 18F-LMI1195 into cells. However, due to the shortage of storage vesicles, no apparent release of stored tracers could be observed after the application of high KCl buffer compared to controls (Fig. 1b). The response of high KCl-leading tracer release compared with the control group is of utmost importance: Since no statistical difference could be observed between both groups, a robust conclusion can be derived from the setup of our experiment.
Reserpine is known for its potential to release norepinephrine from synaptic nerve cells by triggering the exocytosis of storage vesicles [21]. In this study, reserpine induced significant tracer release after 30 min of its application to vesicle-rich PC12 cells (Fig. 3), whereas such an effect was not observed in SK-N-SH cells, which is in accordance with the conclusion drawn by Smets et al. from a reserpine-induced MIBG depletion study [24]. Due to the deficiency of storage vesicles in SK-N-SH cells, no clear tracer overflow, either with 18F-LMI1195 or 131I-MIBG, could be observed. The efflux of tracers from SK-N-SH cells may be only due to slow passive diffusion. The current study of using either high concentration KCl or reserpine is the opposite way as the results achieved from rabbit heart [16], in which pretreatment of desipramine was followed by tracer injection (Fig. 5). Firstly, this in vivo study provided the first proof of successfully prohibiting the uptake of tracer into storage vesicles by using desipramine. Secondly, the in vitro cell study demonstrated the clear depletion mechanism of an already taken-up tracer in the storage vesicles. By comparing the two methods (high concentration KCl and reserpine), it was revealed that the application of these exogenous radioactive sympathetic nerve tracers apparently mimics the physiological neurotransmitter norepinephrine turnover quite well, including transporter-mediated uptake as well as modes of storage and exocytosis (Fig. 6). Integrating our previous animal study (Fig. 5) and ex vivo results [11, 16, 17] with the present in vitro findings, the intracellular behavior of 18F-LMI1195 is analogous to its SPECT counterpart MIBG and the neurotransmitter norepinephrine.
Similar to high KCl-induced exocytosis, reserpine-mediated 18F-LMI1195 release is also Ca2+ dependent. Mandela et al. have investigated and reported how reserpine influences NET in a non-competitive manner by Ca2+ dependency and how it interferes with the interaction between NET and norepinephrine storage vesicles. Strikingly, it was revealed that reserpine induces a non-competitive inhibition of norepinephrine uptake in PC12 cells [13]. This effect requires the presence of vesicular monoamine transporter (VMAT) and storage/secretory vesicles, which explains the finding for exposure to reserpine alone and reserpine/desipramine-induced tracer release—a demonstration of analogous uptake and efflux mechanisms associated with the benzylguanidine structure common to both tracers (Fig. 4). By contrast, as demonstrated previously, cardiac retention of 11C-hydroxyephedrine (11C-HED) is mediated through a continuous cyclical mode of release (diffusion out) and reuptake via NET from the nerve terminal [11, 16]. 11C-HED showed enhanced washout from both in vivo and isolated perfused rabbit heart after desipramine chase. On the other hand, 18F-LMI1195 and MIBG are not sensitive to a NET inhibitor chase protocol in an in vivo setting, which was imitated in the present in vitro study by adding desipramine while incubating with reserpine (Fig. 4). Therefore, on a subcellular level, a stable vesicle-storing mechanism mimicking physiological norepinephrine turnover was corroborated.
It should be mentioned that in addition to the application of these NET tracers in cardiac diseases, there are many potential applications in tumor diagnosis [25]. 123I-MIBG imaging had been used in the evaluation of neuroblastoma for years [26]. 18F-LMI1195 would also be available because of their structural and property similarities in NET imaging: A previous study of high and specific accumulation of LMI1195 in pheochromocytomas has already made the first attempt in proving this potential [15].