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1.
Cell Physiol Biochem ; 54(5): 899-916, 2020 Sep 19.
Article in English | MEDLINE | ID: mdl-32946686

ABSTRACT

BACKGROUND/AIMS: Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation. METHODS: Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and KV-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR. RESULTS: Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact. CONCLUSION: Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K+ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of KV to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.


Subject(s)
Membrane Potentials/physiology , Photoreceptor Cells/metabolism , Animals , Cell Membrane/metabolism , Membrane Potentials/drug effects , PC12 Cells , Patch-Clamp Techniques/methods , Potassium/metabolism , Potassium Channel Blockers/pharmacology , Potassium Channels/metabolism , Rats
2.
ChemMedChem ; 13(23): 2546-2557, 2018 12 06.
Article in English | MEDLINE | ID: mdl-30430750

ABSTRACT

The renin angiotensin aldosterone system (RAAS) is a hormonal cascade involved in the regulation of blood pressure and electrolyte balance, and represents a common target for the treatment of various diseases including hypertension, heart failure, and diabetes. Herein we present a novel 18 F-labeled derivative of the drug irbesartan, one of the most prescribed angiotensin II type 1 receptor (AT1 R) antagonists, for in vivo positron emission tomography (PET). This allows the in vivo measurement of AT1 R expression, and thus the evaluation of functional changes in its expression under pathophysiological conditions. We followed various synthetic approaches optimized for the introduction of fluorine into different positions of the aliphatic side chain of irbesartan. Radioligand binding studies revealed that fluorine atoms at specified positions (α-position (IC50 =6.6 nm) and δ-position (IC50 =8.5 nm) of the aliphatic side chain) do not alter the binding properties of irbesartan (IC50 =1.6 nm). After successful radiolabeling with fluorine-18 in a radiochemical yield of 11 %, we observed high renal uptake in healthy rats and pigs, which could be decreased by pretreatment with the parent compound irbesartan.


Subject(s)
Angiotensin II Type 1 Receptor Blockers/chemistry , Fluorine Radioisotopes/chemistry , Irbesartan/analogs & derivatives , Positron-Emission Tomography/methods , Radiopharmaceuticals/chemistry , Receptors, Angiotensin/analysis , Angiotensin II Type 1 Receptor Blockers/pharmacokinetics , Animals , Female , Fluorine Radioisotopes/pharmacokinetics , Irbesartan/pharmacokinetics , Kidney/chemistry , Kidney/diagnostic imaging , Kidney/metabolism , Radiopharmaceuticals/pharmacokinetics , Swine
3.
ACS Omega ; 3(9): 10460-10470, 2018 Sep 30.
Article in English | MEDLINE | ID: mdl-30288456

ABSTRACT

Renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure and hormonal balance. Using positron emission tomography (PET) technology, it is possible to monitor the physiological and pathological distribution of angiotensin II type 1 receptors (AT1), which reflects the functionality of RAS. A new 18F-labeled PET tracer derived from the clinically used AT1 antagonist valsartan showing the least possible chemical alteration from the valsartan structure has been designed and synthesized with several strategies, which can be applied for the syntheses of further derivatives. Radioligand binding study showed that the cold reference FV45 (K i 14.6 nM) has almost equivalent binding affinity as its lead valsartan (K i 11.8 nM) and angiotensin II (K i 1.7 nM). Successful radiolabeling of FV45 in a one-pot radiofluorination followed by the deprotection procedure with 21.8 ± 8.5% radiochemical yield and >99% radiochemical purity (n = 5) enabled a distribution study in rats and opened a path to straightforward large-scale production. A fast and clear kidney uptake could be observed, and this renal uptake could be selectively blocked by pretreatment with AT1-selective antagonist valsartan. Overall, as the first 18F-labeled PET tracer based on a derivation from clinically used drug valsartan with almost identical chemical structure, [18F]FV45 will be a new tool for assessing the RAS function by visualizing AT1 receptor distributions and providing further information regarding cardiovascular system malfunction as well as possible applications in inflammation research and cancer diagnosis.

4.
Clin Transl Imaging ; 6(4): 293-303, 2018.
Article in English | MEDLINE | ID: mdl-30148120

ABSTRACT

PURPOSE: We aim to provide an overview of the conventional single photon emission computed tomography (SPECT) and emerging positron emission tomography (PET) catecholamine analogue tracers for assessing myocardial nerve integrity, in particular focusing on 18F-labeled tracers. RESULTS: Increasingly, the cardiac sympathetic nervous system (SNS) is being studied by non-invasive molecular imaging approaches. Forming the backbone of myocardial SNS imaging, the norepinephrine (NE) transporter at the sympathetic nerve terminal plays a crucial role for visualizing denervated myocardium: in particular, the single-photon-emitting NE analogue 123I-meta-Iodobenzylguanidine (123I-mIBG) has demonstrated favorable results in the identification of patients at a high risk for cardiac death. However, cardiac neuronal PET agents offer several advantages including improved spatio-temporal resolution and intrinsic quantifiability. Compared to their 11C-labeled counterparts with a short half-life (20.4 min), novel 18F-labeled PET imaging agents to assess myocardial nerve integrity have the potential to revolutionize the field of SNS molecular imaging. The longer half-life of 18F (109.8 min) allows for more flexibility in the study design and delivery from central cyclotron facilities to smaller hospitals may lead to further cost reduction. A great deal of progress has been made by the first in-human studies of such 18F-labeled SNS imaging agents. Moreover, dedicated animal platforms open avenues for further insights into the handling of radiolabeled catecholamine analogues at the sympathetic nerve terminal. CONCLUSIONS: 18F-labeled imaging agents demonstrate key properties for mapping cardiac sympathetic nerve integrity and might outperform current SPECT-based or 11C-labeled tracers in the long run.

5.
Sci Rep ; 8(1): 11120, 2018 07 24.
Article in English | MEDLINE | ID: mdl-30042495

ABSTRACT

We aimed to explore the impact of ageing on 11C-hydroxyephedrine (11C-HED) uptake in the healthy rat heart in a longitudinal setting. To investigate a potential cold mass effect, the influence of specific activity on cardiac 11C-HED uptake was evaluated: 11C-HED was synthesized by N-methylation of (-)-metaraminol as the free base (radiochemical purity >95%) and a wide range of specific activities (0.2-141.9 GBq/µmol) were prepared. 11C-HED (48.7 ± 9.7MBq, ranged 0.2-60.4 µg/kg cold mass) was injected in healthy Wistar Rats. Dynamic 23-frame PET images were obtained over 30 min. Time activity curves were generated for the blood input function and myocardial tissue. Cardiac 11C-HED retention index (%/min) was calculated as myocardial tissue activity at 20-30 min divided by the integral of the blood activity curves. Additionally, the impact of ageing on myocardial 11C-HED uptake was investigated longitudinally by PET studies at different ages of healthy Wistar Rats. A dose-dependent reduction of cardiac 11C-HED uptake was observed: The estimated retention index as a marker of norepinephrine function decreased at a lower specific activity (higher amount of cold mass). This observed high affinity of 11C-HED to the neural norepinephrine transporter triggered a subsequent study: In a longitudinal setting, the 11C-HED retention index decreased with increasing age. An age-related decline of cardiac sympathetic innervation could be demonstrated. The herein observed cold mass effect might increase in succeeding scans and therefore, 11C-HED microPET studies should be planned with extreme caution if one single radiosynthesis is scheduled for multiple animals.


Subject(s)
Aging/metabolism , Ephedrine/analogs & derivatives , Heart/diagnostic imaging , Myocardium/metabolism , Aging/pathology , Animals , Carbon Radioisotopes/administration & dosage , Ephedrine/administration & dosage , Heart/innervation , Heart/physiology , Humans , Metaraminol/chemistry , Positron-Emission Tomography , Radionuclide Imaging/methods , Rats , Rats, Wistar , Sympathetic Nervous System , Tissue Distribution
6.
EJNMMI Res ; 8(1): 12, 2018 Feb 06.
Article in English | MEDLINE | ID: mdl-29411169

ABSTRACT

BACKGROUND: 18F-N-[3-bromo-4-(3-fluoro-propoxy)-benzyl]-guanidine (18F-LMI1195) is a new class of PET tracer designed for sympathetic nervous imaging of the heart. The favorable image quality with high and specific neural uptake has been previously demonstrated in animals and humans, but intracellular behavior is not yet fully understood. The aim of the present study is to verify whether it is taken up in storage vesicles and released in company with vesicle turnover. RESULTS: Both vesicle-rich (PC12) and vesicle-poor (SK-N-SH) norepinephrine-expressing cell lines were used for in vitro tracer uptake studies. After 2 h of 18F-LMI1195 preloading into both cell lines, effects of stimulants for storage vesicle turnover (high concentration KCl (100 mM) or reserpine treatment) were measured at 10, 20, and 30 min. 131I-meta-iodobenzylguanidine (131I-MIBG) served as a reference. Both high concentration KCl and reserpine enhanced 18F-LMI1195 washout from PC12 cells, while tracer retention remained stable in the SK-N-SH cells. After 30 min of treatment, 18F-LMI1195 releasing index (percentage of tracer released from cells) from vesicle-rich PC12 cells achieved significant differences compared to cells without treatment condition. In contrast, such effect could not be observed using vesicle-poor SK-N-SH cell lines. Similar tracer kinetics after KCl or reserpine treatment were also observed using 131I-MIBG. In case of KCl exposure, Ca2+-free buffer with the calcium chelator, ethylenediaminetetracetic acid (EDTA), could suppress the tracer washout from PC12 cells. This finding is consistent with the tracer release being mediated by Ca2+ influx resulting from membrane depolarization. CONCLUSIONS: Analogous to 131I-MIBG, the current in vitro tracer uptake study confirmed that 18F-LMI1195 is also stored in vesicles in PC12 cells and released along with vesicle turnover. Understanding the basic kinetics of 18F-LMI1195 at a subcellular level is important for the design of clinical imaging protocols and imaging interpretation.

7.
J Nucl Med ; 59(5): 828-832, 2018 05.
Article in English | MEDLINE | ID: mdl-29242399

ABSTRACT

Precise regional quantitative assessment of renal function is limited with conventional 99mTc-labeled renal radiotracers. A recent study reported that the PET radiotracer 2-deoxy-2-18F-fluorosorbitol (18F-FDS) has ideal pharmacokinetics for functional renal imaging. Furthermore, 18F-FDS is available via simple reduction from routinely used 18F-FDG. We aimed to further investigate the potential of 18F-FDS PET as a functional renal imaging agent using rat models of kidney disease. Methods: Two different rat models of renal impairment were investigated: induction of acute renal failure by intramuscular administration of glycerol in the hind legs, and induction of unilateral ureteral obstruction by ligation of the left ureter. At 24 h after these procedures, dynamic 30-min 18F-FDS PET data were acquired using a dedicated small-animal PET system. Urine 18F-FDS radioactivity 30 min after radiotracer injection was measured together with coinjected 99mTc-diethylenetriaminepentaacetic acid urine activity. Results: Dynamic PET imaging demonstrated rapid 18F-FDS accumulation in the renal cortex and rapid radiotracer excretion via the kidneys in healthy control rats. On the other hand, significantly delayed renal radiotracer uptake (continuous slow uptake) was observed in acute renal failure rats and unilateral ureteral obstruction kidneys. Measured urine radiotracer concentrations of 18F-FDS and 99mTc-diethylenetriaminepentaacetic acid correlated well with each other (R = 0.84, P < 0.05). Conclusion:18F-FDS PET demonstrated favorable kinetics for functional renal imaging in rat models of kidney diseases. 18F-FDS PET imaging, with its advantages of high spatiotemporal resolution and simple tracer production, could potentially complement or replace conventional renal scintigraphy in select cases and significantly improve the diagnostic performance of renal functional imaging.


Subject(s)
Fluorine Radioisotopes/chemistry , Kidney Diseases/diagnostic imaging , Kidney/diagnostic imaging , Positron-Emission Tomography , Radiopharmaceuticals/chemistry , Sorbitol/analogs & derivatives , Animals , Disease Models, Animal , Glomerular Filtration Rate , Kidney Function Tests , Kinetics , Male , Rats , Rats, Wistar , Sorbitol/chemistry , Ureteral Obstruction
8.
J Biol Chem ; 292(22): 9365-9381, 2017 06 02.
Article in English | MEDLINE | ID: mdl-28377503

ABSTRACT

Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and ß subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.


Subject(s)
Calcium Channels, N-Type/metabolism , Calcium Channels/metabolism , GTP-Binding Proteins/metabolism , Nerve Tissue Proteins/metabolism , Animals , Calcium Channels/genetics , Calcium Channels, N-Type/genetics , GTP-Binding Proteins/genetics , HEK293 Cells , Humans , Mice , Nerve Tissue Proteins/genetics , Protein Domains
9.
Mol Pharmacol ; 89(3): 348-63, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26733543

ABSTRACT

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.


Subject(s)
Nerve Growth Factors/metabolism , Piperazines/metabolism , TRPC Cation Channels/metabolism , Animals , Calcium Signaling/drug effects , Calcium Signaling/physiology , Drug Evaluation, Preclinical/methods , Female , HEK293 Cells , Humans , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Nerve Growth Factors/chemistry , Nerve Growth Factors/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Rabbits , Rats , Rats, Wistar , TRPC Cation Channels/agonists
10.
Cell Calcium ; 58(3): 296-306, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26142343

ABSTRACT

Autism is a neurodevelopmental psychiatric disorder characterized by impaired reciprocal social interaction, disrupted communication, and restricted and stereotyped patterns of interests. Autism is known to have a strong genetic component. Although mutations in several genes account for only a small proportion of individuals with autism, they provide insight into potential biological mechanisms that underlie autism, such as dysfunction in Ca(2+) signaling, synaptic dysfunction, and abnormal brain connectivity. In autism patients, two mutations have been reported in the Rab3 interacting molecule 3 (RIM3) gene. We have previously demonstrated that RIM3 physically and functionally interacts with voltage-dependent Ca(2+) channels (VDCCs) expressed in neurons via the ß subunits, and increases neurotransmitter release. Here, by introducing corresponding autism-associated mutations that replace glutamic acid residue 176 with alanine (E176A) and methionine residue 259 with valine (M259V) into the C2B domain of mouse RIM3, we demonstrate that both mutations partly cancel the suppressive RIM3 effect on voltage-dependent inactivation of Ba(2+) currents through P/Q-type CaV2.1 recombinantly expressed in HEK293 cells. In recombinant N-type CaV2.2 VDCCs, the attenuation of the suppressive RIM3 effect on voltage-dependent inactivation is conserved for M259V but not E176A. Slowing of activation speed of P/Q-type CaV2.1 currents by RIM3 is abolished in E176A, while the physical interaction between RIM3 and ß subunits is significantly attenuated in M259V. Moreover, increases by RIM3 in depolarization-induced Ca(2+) influx and acetylcholine release are significantly attenuated by E176A in rat pheochromocytoma PC12 cells. Thus, our data raise the interesting possibility that autism phenotypes are elicited by synaptic dysfunction via altered regulation of presynaptic VDCC function and neurotransmitter release.


Subject(s)
Autistic Disorder/genetics , Calcium Channels/metabolism , Calcium Signaling , Mutation , Animals , Autistic Disorder/metabolism , Calcium Signaling/genetics , Guanine Nucleotide Exchange Factors , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Neurons/physiology , PC12 Cells , Rats
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