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Research ArticleArticle

Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds

Seishiro Sawamura, Masahiko Hatano, Yoshinori Takada, Kyosuke Hino, Tetsuya Kawamura, Jun Tanikawa, Hiroshi Nakagawa, Hideharu Hase, Akito Nakao, Mitsuru Hirano, Rachapun Rotrattanadumrong, Shigeki Kiyonaka, Masayuki X. Mori, Motohiro Nishida, Yaopeng Hu, Ryuji Inoue, Ryu Nagata and Yasuo Mori
Molecular Pharmacology March 2016, 89 (3) 348-363; DOI: https://doi.org/10.1124/mol.115.102863

Abstract

Transient receptor potential canonical (TRPC) proteins form Ca2+-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 Ca2+ 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 Ca2+ signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca2+-dependent transcription factor, cAMP response element–binding protein. These findings suggest that Ca2+ 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.

Introduction

Brain-derived neurotrophic factor (BDNF) is the most abundant and widely distributed neurotrophin in the mammalian central nervous system (CNS). BDNF binds to tropomyosin-related kinase B, a receptor tyrosine kinase, to stimulate various intracellular signaling pathways mediated by mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, or phospholipase C (PLC) γ (Huang and Reichardt, 2003; Reichardt, 2006; Zhang et al., 2012). Through these intracellular signaling pathways, BDNF exerts neurotrophic functions, such as promoting neuronal growth and survival or modulating synaptic transmission and plasticity (Huang and Reichardt, 2001; Park and Poo, 2013). BDNF administration showed beneficial effects in models for various diseases, such as Alzheimer’s disease, Parkinson’s disease, depression, and schizophrenia (Nagahara and Tuszynski, 2011; Autry and Monteggia, 2012). However, its pharmacokinetic properties, including short plasma half-life and poor blood-brain barrier penetration, limit its therapeutic applications (Poduslo and Curran, 1996). Therefore, discovery of small molecules that mimic the neurotrophic actions of BDNF would be a promising therapeutic strategy for neurologic and psychiatric disorders.

Drosophila melanogaster transient receptor potential (TRP) protein and its homologs are putative six-transmembrane polypeptide subunits that form tetrameric Ca2+-permeable nonselective cation channels (Clapham, 2003; Clapham et al., 2005; Nilius and Szallasi, 2014). Mammalian TRP channels are grouped into six related subfamilies: TRP “canonical” (TRPC), TRP “vanilloid” (TRPV), TRP “melastatin” (TRPM), TRP “ankyrin” (TRPA), TRP “polycystin”, and TRP “mucolipin. TRP channels are activated by diverse stimuli, including receptor stimulation, heat, osmotic pressure, mechanical stress, and environmental irritants from the extracellular and intracellular milieu. The TRPC subfamily comprises seven members (TRPC1–C7), highly expressed in the mammalian CNS and mediating cation influx induced by activation of PLC-coupled metabotropic receptors, such as G protein–coupled receptors and receptor tyrosine kinases (Vazquez et al., 2004; Abramowitz and Birnbaumer, 2009). PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to form diacylglycerol (DAG) (Fig. 1A) and inositol 1,4,5-trisphosphate. In addition to their well known activation of protein kinase C (PKC), a kinase regulating a plethora of biologic functions (Nishizuka, 1995), DAG and its analogs also activate the closely related TRPC subtypes: TRPC3, TRPC6, and TRPC7 (Hofmann et al., 1999).

Fig. 1.
Fig. 1.

Chemical structures of DAG-activated TRPC channel activators. (A) Chemical structures of DAG, the DAG analog OAG, hyperforin, and the hyperforin analog Hyp9. (B) Chemical structures of piperazine-derived activators (compounds 1–9) of DAG-activated TRPC channels.

In the CNS, TRPC3 and TRPC6 modulate neurotransmission, synaptic plasticity, neuronal development, and neuronal survival (Abramowitz and Birnbaumer, 2009; Nilius and Szallasi, 2014). Previous studies further showed that TRPC3 and TRPC6 are important for growth cone turning, spine formation, and neuronal survival induced by BDNF stimulation in neurons (Li et al., 2005; Amaral and Pozzo-Miller, 2007; Jia et al., 2007). It has been suggested that, with BDNF stimulation, TRPC3 and TRPC6 are activated by the PLC pathway via tropomyosin-related kinase B (Li et al., 1999, Amaral and Pozzo-Miller, 2007; Jia et al., 2007). This cascade contributes to increased intracellular Ca2+ concentration ([Ca2+]i) and subsequent Ca2+-dependent activation of such intracellular signaling molecules as MAPKs, Ca2+/calmodulin-dependent protein kinases (CaMKs), and Ca2+-dependent transcription factor cAMP response element–binding protein (CREB), all of which play beneficial roles in neuronal functions (Shaywitz and Greenberg, 1999; Agell et al., 2002; Jia et al., 2007; Wayman et al., 2008). The importance of TRPC6 has also been recognized through investigation of hyperforin (Fig. 1A), an active ingredient of St. John’s wort, which is a widely used and well investigated medicinal plant (Wolfle et al., 2014). Hyperforin is a multitarget compound that modulates various molecular functions and exerts various beneficial effects, showing antimicrobial, anti-inflammatory, anticancer, and antidepressant activity (Russo et al., 2014). It has been previously reported that hyperforin exerts antidepressant and neurotrophic effects via activation of TRPC6 in neurons (Leuner et al., 2007, 2013; Griesi-Oliveira et al., 2015). However, the activity of hyperforin remains controversial: a recent publication implicates that the protonophore activity of hyperforin contributes to its pharmacological effects (Sell et al., 2014). These insights support the concept that activation of DAG-activated TRPC3 and TRPC6 channels are potential therapeutic approaches for neurologic and psychiatric disorders. However, a framework for designing small molecules that activate DAG-activated TRPC channels, thus inducing neurotrophic effects on CNS, remains to be discovered.

In this study, we identified small-molecule activators of DAG-activated TRPC3, TRPC6, and TRPC7 channels which showed trophic effects on neurons. Using a high-throughput screening assay, we identified nine piperazine derivatives by monitoring depolarizing effects on membrane potential in human embryonic kidney (HEK) cells expressing recombinant TRPC channels (Fig. 1B). Two piperazine compounds designated as PPZ1 ([4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone) and PPZ2 (2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide) potently activated TRPC3, TRPC6, and TRPC7 among TRPC subfamily and promoted neurite outgrowth and neuronal survival via downstream Ca2+-dependent signals. These findings clearly support the potential of novel small-molecule mimics of BDNF that target TRPC channel–dependent Ca2+ signaling as an important basis to develop leads of therapeutic drugs for neurologic and psychiatric disorders.

Materials and Methods

Synthesis of Piperazine Derivatives.

The details of the synthesis of piperazine derivatives are provided in the (Supplemental Material).

Reagents.

Pyr3 and Pyr4 were synthesized as previously reported (Kiyonaka et al., 2009). Dimethylsulfoxide (DMSO), SK&F 96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H]-imidazole hydrochloride), ML-7 (hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride), tetrodotoxin, nifedipine, picrotoxin, capsazepine, and cyclosporine A were purchased from Sigma-Aldrich (St. Louis, MO). FLIPR Membrane Potential (FMP) Assay Kit was from Molecular Devices (Sunnyvale, CA). U73122 (1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), U73343 (1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione), 1-oleoyl-2-acetyl-sn-glycerol (OAG), and KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide) were from Calbiochem (Nottingham, UK). Recombinant human BDNF (248-BD) was from R&D Systems (Minneapolis, MN). STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) was from Wako Pure Chemical Industries (Osaka, Japan). PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one was from Biomol (Plymouth Meeting, PA). Carbachol (CCh), fura-2 acetoxymethyl ester, and 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) acetoxymethyl ester (BAPTA-AM) were from Dojindo (Kumamoto, Japan). FK506 ([3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5, 19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14, 16-dimethoxy-4,10,12, 18-tetramethyl-8-(2-propenyl)-15, 19-epoxy-3H-pyrido[2,1-c][1,4] oxaazacyclotricosine-1,7,20, 21(4H,23H)-tetrone, monohydrate) was from Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan).

Animals.

Any procedures and protocols used for animal studies involving rats were approved by the Institutional Animal Care and Use Committee of Kyoto University prior to the start of experiments. Two- to 6-day-old Wistar rat pups were purchased from Japan SLC (Shizuoka, Japan) with their mother. The rats were group-housed (8–10 pups with their mother) in a standard 12-hour light/12-hour dark cycle environment with water and food provided ad libitum. Any procedures and protocols used for animal studies with rabbits were approved by the Institutional Animal Care and Use Committee of Fukuoka University prior to the start of experiments. Japan white rabbits weighing 1.5–2.0 kg were purchased from Biotec (Tokyo, Japan).

cDNA Cloning and Recombinant Plasmid Construction.

Plasmids of the pCI-neo vector carrying mouse TRPC1α, TRPC2, TRPC3, TRPC4β, TRPC5, TRPC6, and TRPC7 cDNA were used as previously described (Okada et al., 1999; Inoue et al., 2001; Yoshida et al., 2006; Kiyonaka et al., 2009). Plasmids of the pCI-neo vector carrying mouse TRPA1, TRPM2, rat TRPV1, and human TRPV4 cDNA were used as previously described (Hara et al., 2002; Yoshida et al., 2006; Takahashi et al., 2014). A pEF-BOS expression vector encoding human muscarinic type 1 receptor was provided by T. Haga (Gakushuin University, Tokyo, Japan).

Cell Culture and cDNA Expression.

HEK 293 cells (Boehringer Ingelheim, Ingelheim, Germany) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 30 U/ml penicillin, and 30 μg/ml streptomycin at 37°C under 95% air and 5% CO2. HEK 293 cells were transfected with recombinant plasmids using SuperFect transfection reagent (Qiagen, Valencia, CA), according to the manufacturer’s instruction. For measurements of [Ca2+]i and TRPC currents, recombinant plasmids were cotransfected with pEGFP-F and pEGFP-N1 (Clontech Laboratories, Laboratories, Mountain View, CA), respectively. HEK 293 cells harboring green fluorescence were used for analysis. Transfected cells were cultured for 36–48 hours prior to [Ca2+]i and electrophysiological measurements.

Generation of Stably TRPC6-Expressing Cell Clone.

HEK 293 cells (Stratagene, La Jolla, CA) were transfected with a pCI-neo vector carrying mouse TRPC6 using Lipofectamine LTX transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For the selection of stably transfected cell clones, cells were incubated in 10% fetal bovine serum in minimum essential Eagle’s medium supplemented with O-2-amino-2,7-dideoxy-D-glycero-α-D-gluco-heptopyranosyl-(1→4)-O-[3-deoxy-4-C-methyl-3-(methylamino)-β-L-arabinopyranosyl-(1→6)]-2-deoxy-D-streptamine, disulfate (G418) (400 μg/ml) and blasticidin (5 μg/ml). Among 219 cell clones, clone 112 was selected as the best based on its response to CCh.

Transfection of HEK 293 Cells for FLIPR Membrane Potential Assay.

HEK 293 cells (Stratagene) were seeded at a density of 2.5 × 105 cells/well in six-well plates. After overnight culture, HEK 293 cells were transfected with a plasmid of the pCI-neo vector carrying mouse TRPC3 using Lipofectamine LTX for 4–6 hours at 37°C according to the manufacturer’s instructions.

FLIPR Membrane Potential Screening Assay.

A chemical compound library, comprising 8000 compounds purchased from ChemBridge (San Diego, CA), was screened for TRPC6 activators. HEK 293 cells stably expressing TRPC6 (clone #112) were plated in 384-well, black, clear-bottom plates (Greiner Bio-one, Kremsmuenster, Austria) at a density of 5 × 103 cells/well. After overnight culture, the culture medium was removed, and then cells were incubated with 20 μl of FMP probe diluted in Ca2+-free 20 mM HEPES/Hanks' balanced salt solution for 1 hour at room temperature. Compounds diluted in Hanks' balanced salt solution were added at 10 μM (10 μl/well). Relative fluorescence intensities (530/595 nm; excitation/emission) were measured in a FLIPR Tetra fluorometric imaging plate reader (Molecular Devices) at 1-second intervals for 140 seconds. Following the first round of screening, 315 positive compounds were further tested under the same protocol twice to verify reproducibility. Next, 124 positive compounds were subjected to a further round of testing, in vector-transfected HEK 293 cells, designed to eliminate false positives. Finally, nine positive compounds were further characterized in vector- or TRPC3-transfected HEK 293 cells and HEK 293 cells stably expressing TRPC6, each in three independent experiments.

[Ca2+]i Measurement.

Transfected HEK 293 cells (Boehringer) were subjected to [Ca2+]i measurement 3–16 hours after plating onto poly-l-lysine–coated glass coverslips. Fluorescence signals from fura-2 were measured in HEPES-buffered saline containing the following: 107 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 11.5 mM glucose, and 20 mM HEPES (pH adjusted to 7.4 with NaOH). Fluorescence images of the cells were recorded and analyzed with the video image analysis system AQUACOSMOS (Hamamatsu Photonics, Shizuoka, Japan) according to the manufacturer’s instructions. The 340:380-nm ratio images were obtained on a pixel-by-pixel basis and were converted to Ca2+ concentrations by in vivo calibration using 10 μM ionomycin (Calbiochem/EMD Chemicals, San Diego, CA) as described previously (Takahashi et al., 2008). For calculation of the EC50 value of PPZ1 and PPZ2 in TRPC3-, TRPC6-, and TRPC7-expressing HEK 293 cells, the plots were fitted to the Hill equation, f (x) = A0 + (Amax − A0)/[1 + (EC50/x)n], where A0 is the basal response, Amax is the maximum response, x is the PPZ1 or PPZ2 concentration, and n is the Hill coefficient, using KaleidaGraph software (Synergy Software, Reading, PA).

Preparation of Single Portal Vein Smooth Muscle Cells.

Albino rabbits of either sex weighing 1.5–2.0 kg (Biotec) were used. Each rabbit was anesthetized with an intravenous injection of sodium pentobarbital (40 mg/kg) and butorphanol tartrate (0.08 mg/kg) and killed quickly by exsanguination via the femoral artery. The abdominal cavity was opened and an approximately 1-cm-long segment of the portal vein was excised between the hepatic and splenic bifurcations. Connective tissues were removed with fine scissors and forceps, and thin strips of portal vein smooth muscle, approximately 10 × 2 mm in length and width, were made and incubated successively in nominally Ca2+-free Krebs solution and the same solution containing 2 mg/ml collagenase type 3 (Worthington, Lakewood, NJ) at 36°C for 5 and 35–40 minutes, respectively. The collagenase-digested strips were then thoroughly rinsed and minced into small pieces in Ca2+-free Krebs solution, and gently triturated with a large-bore Pasteur pipette to disperse single cells. These cells were used for electrophysiological measurements within 3 hours after the dispersion.

Isolation, Culture, and Transfection of Rat Cerebellar Granule Neurons.

Rat cerebellar granule neurons (CGNs) were prepared from three or four cerebella from 6- to 9-day-old Wistar rat pups (Japan SLC) as previously described (Uriu et al., 2010). Cells were plated on a polyethyleneimine-coated 13-mm circle glass, 3.5-cm dish, or 3.5-cm glass-bottom dish at a density of 5 × 104 cells/cm2 for reverse-transcriptase polymerase chain reaction (RT-PCR), neurite outgrowth measurement, and western blotting, and 2.5 × 105 cells/cm2 for the neuronal survival assay. Two to 4 hours after plating, the medium was changed to DMEM with 10% newborn calf serum, 26 mM KCl, 30 U/ml penicillin, and 30 μg/ml streptomycin. Cultures were maintained at 37°C in 95% air, 5% CO2. Freshly isolated CGNs were transfected with 300 nM small interfering RNAs (siRNAs) using the Rat Neuron Nucleofector Kitand G-13 program (Lonza, Walkersville, MD) according to the manufacturer’s instructions. pEGFP-N1 (1 μg) was cotransfected as a transfection marker in neurite measurements and patch-clamp recordings. The siRNAs targeting rat TRPC3 (ON-TARGETplus SMARTPool, L-088687), TRPC6 (ON-TARGETplus SMARTPool, L-095094), and TRPC7 (ON-TARGETplus SMARTPool, L-090059) were purchased from Dharmacon (Lafayette, IN). Nontargeting, randomized siRNA was synthesized and purified with the Silencer siRNA construction kit (Ambion, Foster City, CA). The oligonucleotide sequence used for synthesis of nontargeting siRNA is 5′-GGGTATACTAGTGAATTAG-3′ (forward) and 5′-CTAATTCACTAGTATACCC-3′ (reverse).

Electrophysiology.

For whole-cell recording from transfected HEK 293 cells (Boehringer), coverslips with cells were placed in a chamber containing bath solutions. Macroscopic currents were recorded at room temperature using the whole-cell mode of the patch-clamp technique with an EPC-10 amplifier (Heka Elektronik, Lambrecht/Pfalz, Germany) as described previously (Okada et al., 1999). The patch electrodes prepared from borosilicate glass capillaries had a resistance of 2–4 MΩ and were used in the recordings. Current signals were filtered at 2.9 kHz with a 4-pole Bessel filter and digitized at 20 kHz. Patchmaster software (Heka Elektronik) was used for command pulse control, data acquisition, and analysis. Series resistance was compensated (by 50–70%) to minimize voltage errors. Solutions had the following compositions, presented in millimolar concentrations unless otherwise indicated: Ca2+-containing bath solution: 120 Na+, 5 K+, 1.2 Mg2+, 1 Ca2+, 129.4 Cl–, 10 glucose, and 10 HEPES (pH 7.4 adjusted with NaOH, and osmolality adjusted to 300 mOsM with d-mannitol), and Ca2+-free bath solution: 120 Na+, 5 K+, 1.2 Mg2+, 129.4 Cl–, 1 EGTA, 10 glucose, and 10 HEPES (pH 7.4 adjusted with NaOH, and osmolality adjusted to 300 mOsM with d-mannitol); Cs+ internal solution for whole-cell recording: 120 Cs+, 2 Mg2+, 4 Ca2+, 32 Cl–, 100 aspartate, 2 Na2ATP, 10 BAPTA, 0.1 GTP, and 5 HEPES (pH 7.2 adjusted with CsOH, and osmolality adjusted to 290 mOsM with d-mannitol). For the recordings of whole-cell currents in HEK 293 cells, ramp pulses were applied every 5 seconds from –100 to +100 mV at a speed of 0.4 mV ms−1 after a 50-ms step to –100 mV from a holding potential of –60 mV.

For whole-cell recording from CGNs at 1–3 days in vitro and CGNs transfected with siRNA for 24–48 hours, macroscopic currents were recorded and analyzed with an EPC-10 amplifier (Heka Elektronik) and Patchmaster (Heka Elektronik) as described for HEK cells. The patch electrodes prepared from borosilicate glass capillaries had a resistance of 5–20 MΩ for CGNs used in the recordings. Solutions had the following compositions, presented in millimolar concentrations unless otherwise indicated: bath solution: 140 Na+, 5 tetraethylammonium+, 1.2 Mg2+, 1 Ca2+, 149.4 Cl–, 15 glucose, 10 HEPES, 0.001 tetrodotoxin, 0.1 picrotoxin, 0.01 nifedipine, and 0.03 Cd2+ (pH 7.4 adjusted with Tris, and osmolality adjusted to 300 mOsM with d-mannitol) (Zhang et al., 2008); Cs+ internal solution: 120 Cs+, 2 Mg2+, 4 Ca2+, 32 Cl–, 100 aspartate, 2 Na2ATP, 10 BAPTA, 0.1 GTP, and 5 HEPES (pH 7.2 adjusted with CsOH, and osmolality adjusted to 290 mOsM with d-mannitol). Bath solutions containing the same concentration of DMSO (0.3%) with PPZ2-containing test solutions were used to record basal currents. The currents were continuously recorded at –60 mV and low-pass filtered at 500 Hz before analyses. To construct a current-voltage (I-V) relationship, ramp pulses were applied from –100 to +100 mV at a speed of 0.4 mV ms−1 after a 50-ms step to –100 mV.

For current recording from rabbit portal vein smooth muscle cells, recording of whole-cell currents and data analysis were performed using a standard patch-clamp system consisting of an EPC-8 patch-clamp amplifier; LIH8+8 digital to analog/ analog to digital converter; and Patchmaster v.1.1.2 (Heka Elektronik). The input resistance of electrodes used for whole-cell recording ranged from 8 to 15 MΩ, and 50–70% of access resistance (5–7 MΩ) was electronically compensated. For long-term recordings (>1 minute), the current signal was obtained using PowerLab 4/35 (digitized at 1 kHz; AD Instruments, Bella Vista, NSW, Australia) after low-pass filtering at 500 Hz, from which the mean current amplitude was calculated over every 1-second segment. The liquid junction potential between pipette and bath solutions (–6 mV) was corrected when constructing current-voltage relationships. Test solutions were topically applied using the so-called “Y-tube” fast solution exchange device, which was controlled by one 3-way and two 2-way electromagnetic valves by digital to analog outputs from the computer. Solutions had the following compositions, presented in millimolar concentrations unless otherwise indicated: bath solution: 135 Na+, 5 K+, 1.2 Mg2+, 1 Ca2+, 151.4 Cl–, 5 glucose, and 10 HEPES; and Cs+ internal solution for whole-cell recording: 140 Cs+, 2 Mg2+, 24 Cl–, 120 aspartate, 2 Na2ATP, 5 phosphocreatine, 5 EGTA or BAPTA/1.5 Ca2+, 10 glucose, and 10 HEPES. The composition of Ca2+-free Krebs solution was (in millimolar concentrations) 140 Na+, 6 K+, 1.2 Mg2+, 148.4 Cl–, 10 glucose, and 10 HEPES (adjusted to pH 7.4 with Tris base). All experiments were performed at 24–26°C, either at ambient temperature or using a commercial warmer unit (TC-344B; Warner Instrument, Hamden, CT).

RT-PCR.

Total RNA of cultured rat CGNs was extracted using ISOGEN following the manufacturer’s instructions (Wako Pure Chemical Industries). Reverse transcription of total RNA to cDNA was performed using the RNA LA PCR Kit (TaKaRa-Bio, Shiga, Japan). This cDNA was used as a PCR template to detect gene expression using the following primers: TRPC1, 5′-GGTACATCTTCTCCTTAGCGC-3′ (forward) and 5′-TGGTCTGTGCTCTGCATCTTC-3′ (reverse); TRPC2, 5′-AGAAGCTGGGCAATTTCAACG-3′ (forward) and 5′-CGATGAGCATGTTGAGTAGCACA-3′ (reverse); TRPC3, 5′-TGAGGTGAACGAAGGTGAACTG-3′ (forward) and 5′-CCTGTCCCCCAAGGAACTCT-3′ (reverse); TRPC4, 5′-GCTTCGAAGTTCTGGGATTGC-3′ (forward) and 5′-CACCACCACCTTCTCCGACTT-3′ (reverse); TRPC5, 5′-CTAGCAGCGCTGAATTCTCTC-3′ (forward) and 5′-AAGTTTCGAATTTGAGGAGCAGATG-3′ (reverse); TRPC6, 5′-GGTTTACGGCAGCAGACCAT-3′ (forward) and 5′-CGCATGTGCTACGAACTTCATG-3′ (reverse); TRPC7, 5′-GAAGTACGACCACAAGTTCATCG-3′ (forward) and 5′-TCAGGTGGTCTTTGTTCAAAT-3′ (reverse); and β-actin, 5′-TTCTACAATGAGCTGCGTGTGGC-3′ (forward) and 5′-CTCATAGCTCTTCTCCAGGGAGGA-3′ (reverse). PCR was conducted under the following conditions: 94°C for 2 minutes followed by 35 cycles for TRPC1–C7 or 20 cycles for β-actin of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute, and finally 72°C for 7 minutes. Predicted lengths of products were 477, 166, 469, 555, 502, 152, and 649 bp for TRPC1–C7 and 445 bp for β-actin, respectively.

Measurement of Neurite Outgrowth in CGNs.

Two to 4 hours after plating, neurons were stimulated with 0.1% DMSO, 50 ng/ml BDNF, PPZ1, or PPZ2 with or without various inhibitors for 24 hours. In experiments with siRNA treatment, CGNs were stimulated for 24 hours with DMSO, 50 ng/ml BDNF, PPZ1, or PPZ2 at 24–48 hours after transfection. Neurons were fixed in phosphate buffer containing 4% paraformaldehyde/4% sucrose solution. Cells were permeabilized and blocked with phosphate buffer containing 1% bovine serum albumin/0.1% Triton X-100. Neurons were incubated overnight at 4°C with an anti-MAP2 antibody (M1406, 1:200; Sigma-Aldrich) in phosphate buffer and staining was detected by incubation with a secondary antibody, Alexa Fluor 488-conjugated goat anti-mouse IgG (A11017, 1:2000; Invitrogen) for 1 hour at room temperature. F-actin was labeled for simultaneous detection by incubation with Alexa Fluor 546-phalloidin (A22283, 1:40; Invitrogen). Fluorescence images were acquired using a confocal laser-scanning microscope (IX71; Olympus, Tokyo, Japan) with FV500 software (Olympus). For analyses of total neurite length/cell, neurite lengths were measured by tracing neurites according to F-actin signals in each MAP2-positive cell using NeuronJ plugin (http://www.imagescience.org/meijering/software/neuronj/) for ImageJ software (NIH, Bethesda, MD) (Meijering et al., 2004). Only neurites longer than one cell diameter were measured, and values were averaged. Ten to 20 images from three independent experiments were used for the analyses.

Isolation, Culture, and Neurite Measurements of Rat Hippocampal Neurons.

Primary hippocampal neurons were prepared from the frozen hippocampus of 19-day embryo Wistar rat (MBX0402; Sumitomo Bakelite, Tokyo, Japan) using Nerve Cell Dissociation Medium (MBX0802; Sumitomo Bakelite). The primary hippocampal neurons were plated onto poly-d-lysine–coated 8-well chamber slides (BD Bioscience, San Jose, CA) at a density of 5 × 104 cells/well. The cultures were maintained at 37°C with 95% air and 5% CO2. Four hours after plating, the medium was replaced with Neuron Assay Medium (MBX0701; Sumitomo Bakelite) containing 0.1% DMSO and 50 ng/ml BDNF, PPZ1, or PPZ2 with or without SK&F 96365, Pyr4, or capsazepine for 48 hours. Total neurite length/cell was determined by the same procedure as CGNs, described earlier. Five to 10 images from three independent experiments were used for the analyses.

Western Blotting.

Two to 4 hours after plating, CGNs were stimulated with 0.1% DMSO or PPZ2 for the indicated time. Then, CGNs were washed with phosphate-buffered saline and lysed with 300 μl of 1 × SDS sample buffer containing 50 mM dithiothreitol. Proteins from each lysate were separated by electrophoresis on a 10% SDS-polyacrylamide gel and then transferred onto immobilon-P membranes (Millipore, Billerica, MA). Phosphorylated and total CREB were detected by western blotting using an anti–phospho-CREB (Ser-133) –specific antibody (#9198, 1:1000; Cell Signaling, Danvers MA) and an anti-CREB antibody (#9197, 1:1000; Cell Signaling), respectively, as previously described (Chung et al., 2011), using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ). For loading normalization, α-tubulin was detected by using an anti–α-tubulin antibody (T6074, 1:2000; Sigma-Aldrich). The chemiluminescence intensities of the bands were measured by Multigauge version 3.0 (Fuji Film, Tokyo, Japan). Ratios of phosphorylated CREB to total CREB, reflecting CREB activation, were calculated from band intensities obtained in at least three independent experiments.

Cell Survival Assay.

Cell survival assays were performed as previously described (Jia et al., 2007) with minor modification. Isolated CGNs were plated on 3.5-cm glass-bottom dishes. CGNs were pretreated with 0.1% DMSO and 50 ng/ml BDNF, PPZ1, or PPZ2 with or without 1 μM Pyr4 for 24 hours at 4 days in vitro, then medium was washed with DMEM containing 5 mM KCl without serum and incubated with this medium containing the pretreated agents for 24 hours to assess cell death. Cell death was evaluated by Hoechst 33342 (Dojindo) and propidium iodide (PI) (Setareh Biotech, Eugene, OR) staining. Cells were incubated with Hoechst 33342 (1 μg/ml) and PI (0.2 μg/ml) for 5 minutes at 37°C with 5% CO2 to visualize the nucleus and dead cells, respectively. Then, the staining was detected by using a fluorescence microscope (IX81; Olympus) with MetaMorph software (Molecular Devices). Cell death ratio was calculated by dividing the number of PI-positive cells by the number of nuclei, and shown as percentage of PI-positive cells. Four to 10 images containing at least 50 cells from at least three independent experiments were analyzed.

Statistical Analyses.

All data are expressed as means ± S.E.M. In the FMP assay, [Ca2+]i measurements, electrophysiology, RT-PCR, real-time PCR, neurite outgrowth measurements, western blot, survival assays, and luciferase assay, we accumulated the data for each condition from at least three independent experiments. The statistical analyses were performed using Student’s t test, and a P value <0.05 was considered significant.

Results

Screening of Novel Small-Molecule Activators of DAG-Activated TRPC Channels.

To search for small molecules that activate DAG-activated TRPC channels, we first carried out a high-throughput screening using an FMP assay in HEK 293 cells stably expressing TRPC6 (Fig. 2A). Fluorescence increases, indicating membrane potential depolarization elicited by stimulation of endogenous muscarinic cholinergic receptors by CCh (Boulay et al., 1997; Kiyonaka et al., 2009), served as a positive control for TRPC6 channel activation (Fig. 2B). From a chemical library of 8000 compounds, we identified nine piperazine compounds, each eliciting a more than 20% increase in fluorescent intensity of the FMP probe in TRPC6-expressing cells (334.8 ± 95.0 arbitrary units for 30 μM compound 1 and 789.4 ± 15.5 arbitrary units for 30 μM compound 6) and not inducing significant changes in vector-transfected control cells. These nine piperazine compounds are categorized into two structural groups: five compounds contain a benzoyl or phenylsulfonyl piperazine structure (compound A group), and four compounds contain a 1-phenyl piperazine-4-acetamide structure (compound B group) (Fig. 1B). Further experiments with compounds 1 and 6 showed a gradual increase in membrane potential in not only TRPC6-expressing, but also TRPC3-expressing cells (Fig. 2, C and D). The compound groups A and B were examined for stimulation of TRPC3, TRPC6, and TRPC7, another DAG-activated cation channel, in [Ca2+]i measurements. These compounds, each at 10 μM, evoked significant [Ca2+]i increases in TRPC3-, TRPC6-, and TRPC7-expressing HEK 293 cells compared with vector-transfected HEK 293 cells (Fig. 2E). These results suggest that the nine identified piperazine compounds are a new series of activators of DAG-activated TRPC3, TRPC6, and TRPC7 channels. Among nine compounds, compound 6 had the most potent effect on DAG-activated TRPC channels. Notably, compound 6 at 10 μM elicited a robust [Ca2+]i increase in TRPC6-expressing cells.

Fig. 2.
Fig. 2.

Screening of small-molecule activators of the TRPC6 channel. (A) The principle of the FMP assay to detect TRPC6 channel activation. TRPC6 channel activators induce influx of cations such as Na+ and Ca2+ from the extracellular solution, leading to membrane potential depolarization. In response to the depolarization, the FMP probe translocates to the cytoplasm from the extracellular space and exhibits increased fluorescence intensity. (B) Time courses of FMP probe fluorescence after addition of CCh to HEK 293 cells stably expressing TRPC6 (a.u., arbitrary units). (C and D) Representative traces of fluorescence changes induced by compound 1 (PPZ1) (C) or compound 6 (PPZ2) (D) in vector-transfected cells and TRPC3- or TRPC6-expressing cells. The compounds were added 1 second after beginning the observation. (E) Maximum increases in [Ca2+]i (Δ[Ca2+]i) induced by addition of the nine identified activators, each at 10 μM, for 8 minutes in HEK 293 cells transfected with TRPC3, TRPC6, TRPC7, or vector (n = 24–80). *P < 0.05; **P < 0.01; ***P < 0.001 compared with vector. Data points show the means ± S.E.M.

Characterization of the Compounds PPZ1 and PPZ2.

For precise characterization, we focused on compounds 1 and 6, which evoked robust and comparatively similar responses of TRPC3/TRPC6/TRPC7 in measurements conducted so far. Compound 3 is similarly potent but has a bulky and lipophilic benzyloxy group, which may limit optimization of the compound toward pharmaceutical drugs. Therefore, considering the smaller and simpler structure, we selected compound 1 from group A (compounds 1–5) for further investigation. Because both compounds share a phenyl-piperazine structure, we designated them as PPZ1 (compound 1) and PPZ2 (compound 6), respectively. PPZ1 (100 μM) and PPZ2 (30 μM) elicited significant [Ca2+]i elevation in HEK 293 cells expressing TRPC3, TRPC6, and TRPC7, but not in cells expressing TRPC1, TRPC2, TRPC4, and TRPC5 (Fig. 3, A and B). The data of PPZ1- and PPZ2-induced Ca2+ responses suggest activation selectivity of the compounds PPZ1 and PPZ2 to TRPC3, TRPC6, and TRPC7 among the TRPC subfamily. PPZ1 and PPZ2 evoked these Ca2+ signals in a concentration-dependent manner (Fig. 3, C and D). To estimate functional expression levels of TRPC channels in HEK 293 cells, we analyzed Ca2+ responses to 100 μM CCh by the Ca2+ add-back protocol, which separates the [Ca2+]i increase through Ca2+ release from that through Ca2+ influx (Supplemental Fig. S1, A and B). CCh-induced Ca2+ influx is significantly higher in TRPC channel-transfected HEK 293 cells than vector-transfected cells. The maximum [Ca2+]i rises elicited by 100 μM CCh are comparable in TRPC3-, TRPC6-, and TRPC7-expressing HEK 293 cells, suggesting that the levels of functional expression of these TRPC channels are similar in our system. The dose-response data were reanalyzed by normalizing the value of maximum [Ca2+]i rise induced by PPZ compounds to those induced by adding back extracellular Ca2+ upon stimulation with 100 μM CCh, and the EC50 values were obtained by fitting the plots to the Hill equation (Supplemental Fig. S1, C and D). For TRPC3, TRPC6, and TRPC7, EC50 values of PPZ1 were 57.0, 67.3, and 45.9 µM, and Hill coefficients were 0.80, 1.59, and 2.14, respectively; EC50 values of PPZ2 were 10.20, 8.37, and 2.90 µM, and Hill coefficients were 0.67, 1.67, and 1.25, respectively. Importantly, PPZ1- and PPZ2-induced Ca2+ responses were attributable mainly to Ca2+ influx through TRPC channels, because PPZ1 and PPZ2 failed to evoke significant [Ca2+]i elevation in Ca2+-free extracellular solutions (Supplemental Fig. S2, A–D). We also examined the effects of PPZ1 and PPZ2 on CCh-induced activity of TRPC3, TRPC6, and TRPC7 in [Ca2+]i measurements. Ten micromolar PPZ2 enhanced CCh-induced Ca2+ influx in TRPC6- and TRPC7-expressing HEK 293 cells, but 10 μM PPZ1 and 30 μM OAG, a membrane-permeable DAG analog, failed to exert such effects (Supplemental Fig. S3, A and B). This result suggests that PPZ2 may activate TRPC6 and TRPC7 in part via a mechanism not induced by receptor stimulation.

Fig. 3.
Fig. 3.

Characterization of TRPC channel activation by PPZ1 and PPZ2 based on [Ca2+]i measurements. (A and B) [Ca2+]i changes induced by PPZ1 (A) and PPZ2 (B) in HEK 293 cells expressing TRPC subtypes TRPC1–C7, respectively. Averaged time courses and maximum increases in [Ca2+]i (Δ[Ca2+]i) (n = 25–99). ***P < 0.001, as compared with vector. (C and D) Dose-response relationships for Δ[Ca2+]i induced by PPZ1 (C) and PPZ2 (D) in HEK 293 cells expressing TRPC3, TRPC5, TRPC6, TRPC7, or vector (n = 17–169). (E–H) Effects of 5 μM U73122 or U73343 on [Ca2+]i increases induced by 100 μM PPZ1 (E and F) and 30 μM PPZ2 (G and H) in HEK 293 cells expressing TRPC3, TRPC6, and TRPC7. Averaged time courses of [Ca2+]i changes in TRPC7-expressing cells and Δ[Ca2+]i in TRPC3-, TRPC6-, and TRPC7-expressing cells (n = 21–48) are shown. n.s., not significant compared with DMSO or U73343. Data points show the means ± S.E.M.

PPZ1 and PPZ2 were further examined for their effects on TRPM2, TRPA1, and TRPV1 using [Ca2+]i measurements, and for TRPV4 by whole-cell patch-clamp recordings (Supplemental Fig. S4). PPZ1 had no significant effects on TRPM2, TRPA1, or TRPV4, whereas it activated TRPV1 at 10–100 μM. PPZ2 had no significant effects on TRPM2 and TRPV4, whereas it activated TRPA1 and TRPV1 at 30 μM, a markedly higher concentration than those needed to activate TRPC3, TRPC6, and TRPC7. Thus, the obtained results suggest that PPZ2 selectively targets DAG-activated TRPC channels at concentrations as high as 10 μM, in contrast to PPZ1, which efficiently activates TRPV1 at 10 μM.

Both PPZ1 and PPZ2, in the external solution also containing the PLC inhibitor U73122 (Bleasdale et al., 1990), still induced [Ca2+]i increases in TRPC3-, TRPC6-, and TRPC7-expressing cells (Fig. 3, E–H). This indicated that the Ca2+ responses induced by PPZ1 and PPZ2 are not due to an indirect activation of these channels via the PLC pathway.

In whole-cell patch-clamp recordings, PPZ1 and PPZ2 increased whole-cell currents in HEK 293 cells expressing TRPC3, TRPC6, or TRPC7, but not in control cells (Fig. 4, A–D). I-V relationships of the currents in TRPC3-, TRPC6-, or TRPC7-expressing cells showed prominent rectification at depolarizing potentials, consistent with previous reports for receptor-operated TRPC3, TRPC6, or TRPC7 currents (Hofmann et al., 1999; Okada et al., 1999). We then tested the extracellular Ca2+ dependence of PPZ1- and PPZ2-induced currents in TRPC6-expressing HEK 293 cells. TRPC6 currents induced by 100 μM PPZ1, 30 μM PPZ2, and 100 μM CCh in the absence of extracellular Ca2+ were suppressed by the addition of 1 mM extracellular Ca2+ (Supplemental Fig. S5). This result suggests that extracellular Ca2+ is not an obligatory necessity but can act as a modulator in PPZ1- and PPZ2-induced TRPC6 activation as in receptor-induced TRPC6 activation.

Fig. 4.
Fig. 4.

Electrophysiological characterization of TRPC3, TRPC6, and TRPC7 channel activation by PPZ1 and PPZ2. (A and B) Whole-cell patch-clamp recordings of ionic currents evoked by 100 μM PPZ1 (A) and 30 μM PPZ2 (B) in HEK 293 cells transfected with TRPC3, TRPC6, TRPC7, or vector. Representative traces of inward and outward currents recorded at –100 and +100 mV, respectively, under ramp clamp (top rows). Corresponding I-V relationships at time points 1 and 2 (bottom rows). (C and D) Peak current densities at –100 and +100 mV during the application of 100 μM PPZ1 (C) and 30 μM PPZ2 (D) (n = 8–19). *P < 0.05; **P < 0.01; ***P < 0.001 compared with vector. Data points show the means ± S.E.M.

We further examined PPZ2-induced TRPC6 channel activity by a cell-attached configuration in TRPC6-transfected HEK 293 cells. Application of PPZ2 from a pipette significantly increased ion channel activity with a mean NPo value [the product of the number of channels (N) and the channel open probability (Po)] from 0.0093 ± 0.0083 (DMSO) to 0.070 ± 0.032 (PPZ2; P < 0.05), which was not observed in vector-transfected HEK 293 cells (0.0055 ± 0.0030; P < 0.05 compared with TRPC6-transfected cells) (Supplemental Fig. S6, A and B). PPZ2 induced TRPC6 currents which opened to multiple unitary current amplitude levels, comparable to the TRPC6 currents observed in mesenteric artery myocytes (Saleh et al., 2006). Other than a unitary current which had a smaller single-channel conductance (15.8 pS), we found a larger unitary current amplitude with a conductance of 30.4 pS (calculated from the inward portion of the I-V relationship) almost identical to that of receptor-activated recombinant TRPC6 currents in previous reports (Hofmann et al., 1999; Shi et al., 2004) (Supplemental Fig. S6C). The amplitude of the unitary current with the conductance of 30.4 pS was 2.1 pA in the presence of PPZ2 at –60 mV, similar to 2.0 pA of the unitary current observed in the absence of PPZ2. These results may suggest that PPZ2 triggers activation gating or stabilizes the open state of the TRPC6 channel without affecting the ion permeability of the opened channel, at least in part through a mechanism shared by physiological receptor stimulation.

PPZ2, in a concentration-dependent manner, also induced whole-cell currents in isolated rabbit portal vein smooth muscle cells (Fig. 5, A–C), in which TRPC6 was reported to play a predominant role in receptor-operated cation influx (Inoue et al., 2001). Ten micromolar noradrenaline was used as a positive control to induce nonselective cation currents through TRPC6. Since TRPV1 is also expressed in smooth muscle cells (Mori et al., 2016), it may contribute to ionic currents induced by PPZ2 at 30 μM in rabbit portal vein smooth muscle cells, according to our results (Supplemental Fig. S4C). However, PPZ2 at 1 and 3 μM, which are subthreshold concentrations to activate TRPV1 channels, enhanced the ionic currents in rabbit portal vein smooth muscle cells. This supports that we were able to isolate native TRPC6 currents activated by PPZ2 in rabbit portal vein smooth muscle cells.

Fig. 5.
Fig. 5.

Whole-cell currents evoked by PPZ2 in rabbit portal vein smooth muscle cells. (A) Representative traces of the currents activated by 10 μM noradrenaline (Norad) and PPZ2 recorded at –66 mV. (B) Corresponding I-V relationships at time points 1, 2, and 3. (C) Peak current densities induced by PPZ2 normalized to that of 10 μM Norad (n = 4–13). Data points show the means ± S.E.M.

Piperazine Compounds PPZ1 and PPZ2 Promote Neurite Outgrowth via TRPC3, TRPC6, and TRPC7 Channels.

TRPC3 and TRPC6 are reportedly involved in neuronal development, such as neurite outgrowth and dendritic morphological changes (Li et al., 2005; Amaral and Pozzo-Miller, 2007; Tai et al., 2008). Therefore, to investigate whether the PPZ compounds have such pharmacological effects, we isolated rat CGNs, which endogenously express TRPC channels as confirmed by RT-PCR and real-time PCR (Fig. 6A; Supplemental Fig. S7). CGNs treated with PPZ1 and PPZ2 (3–300 nM) had significantly greater neurite outgrowth, measured by total neurite length, as compared with control CGNs treated with vehicle (DMSO) (Fig. 6, B and C). Promotion of neurite outgrowth by both compounds was suppressed by Pyr3, the selective TRPC3 inhibitor, or by Pyr4, which inhibits TRPC3, TRPC6, and TRPC7 (Kiyonaka et al., 2009) (Fig. 6D). It was also inhibited by a mixture of specific siRNAs for TRPC3, TRPC6, and TRPC7 (siTRPC3/6/7), each of which efficiently suppressed expression (Fig. 6, E and F). These data suggest that PPZ1 and PPZ2 promote neurite outgrowth through activation of endogenous TRPC3, TRPC6, and TRPC7 in CGNs.

Fig. 6.
Fig. 6.

Neurite outgrowth promotion by PPZ1 and PPZ2 in cultured neurons. (A) RT-PCR analysis for mRNA expression of TRPC channels in CGNs at 1 day in vitro. (B) Representative fluorescence images of CGNs stained with the neuronal marker MAP2 (green) and the morphological indicator F-actin (red). CGNs were treated with 0.1% DMSO, 50 ng/ml BDNF, 10 nM PPZ1, or 10 nM PPZ2 for 24 hours and fixed at 1 day in vitro. (C) Total neurite length relative to that in DMSO-treated control cells in response to 50 ng/ml BDNF or PPZ1 and PPZ2 at various concentrations, as indicated (n = 32–127). *P < 0.05; **P < 0.01; ***P < 0.001 compared with DMSO. (D) Effects of TRPC inhibition by 0.3 μM Pyr3 and 1 μM Pyr4 on PPZ1- or PPZ2-induced promotion of neurite outgrowth in CGNs (n = 44–92). *P < 0.05; **P < 0.01; ***P < 0.001 compared with DMSO. (E) RT-PCR analyses of TRPC3, TRPC6, and TRPC7 expression in CGNs electroporated with a randomized siRNA (siControl) or an siRNA mixture targeting TRPC3, TRPC6, and TRPC7 (siTRPC3/6/7). (F) Effects of TRPC knockdown on 10 nM PPZ1- or PPZ2-induced promotion of neurite outgrowth in CGNs (n = 66–120). *P < 0.05; **P < 0.01; ***P < 0.001 compared with siControl or DMSO. (G) Total neurite length of cultured hippocampal neurons, relative to DMSO-treated control neurons, in response to 50 ng/ml BDNF or PPZ1 and PPZ2 at the indicated concentrations (n = 22–32). *P < 0.05; ***P < 0.001 compared with DMSO. (H) The effects of 1 μM SK&F 96365 (SKF), 1 μM Pyr4, and 0.1 μM capsazepine (CPZ) on neurite outgrowth in hippocampal neurons induced by 50 ng/ml BDNF, 100 nM PPZ1, or 100 nM PPZ2 (n = 38–98). *P < 0.05; **P < 0.01; ***P < 0.001 compared with DMSO. Data points show the means ± S.E.M.

We tested the effects of cotreatment of PPZ1 or PPZ2 at various concentrations (0.01–10 μM) with BDNF in a neurite outgrowth assay (Supplemental Fig. S8A). PPZ2 at relatively high concentrations (≥1 μM), but not PPZ1 (0.01–10 μM), suppressed promotion of neurite outgrowth induced by BDNF. To address the underlying mechanisms of suppressive effects of PPZ2 on BDNF-induced neurite outgrowth, we assessed cell toxicity induced by PPZ1 or PPZ2 by itself. PPZ2, but not PPZ1, increased neuronal apoptosis at high concentrations (10 and 30 μM) (Supplemental Fig. S8B). This was rescued by cotreatment of the membrane-permeable Ca2+ chelator BAPTA-AM (Supplemental Fig. S8C). These results may suggest that PPZ2 at high concentrations induces Ca2+ overload in CGNs, leading to induction of cell death and suppression of BDNF-induced neurite outgrowth. This is consistent with the observation that PPZs applied alone at relatively high concentrations lack promoting effects on neurite growth (Fig. 6C) and, interestingly, contrasts with the promoting effect of PPZs at relatively low concentrations on neurite outgrowth (Fig. 6, C and D) and neuronal survival (see the data in the last part of the Results section).

PPZ1 and PPZ2 also promoted neurite outgrowth in isolated rat hippocampal neurons, in which DAG-activated TRPC channels were endogenously expressed, as shown by real-time PCR, but TRPV1 was poorly expressed (Supplemental Fig. S7). PPZ1- and PPZ2-induced neurite outgrowth was suppressed by 1 or 20 μM SK&F 96365, a nonselective inhibitor for various cation and Ca2+ influx channels, including TRPC channels (Merritt et al., 1990), and by 1 μM Pyr4, but not by 0.1 μM capsazepine, a TRPV1 inhibitor (Dickenson and Dray, 1991) (Fig. 6, G and H; (Supplemental Fig. S9). These results support the growth-promoting effects of the PPZ compounds PPZ1 and PPZ2 in neurons, endogenously expressing TRPC3, TRPC6, or TRPC7.

We examined activation of endogenous DAG-activated TRPC channels by PPZ2 in a whole-cell mode of patch-clamp recordings in CGNs. Application of PPZ2 induced inward currents which showed TRPC3/TRPC6/TRPC7-like I-V relationships (Fig. 7, A–D: compare with recombinant currents in Fig. 4), and knockdown of TRPC3, TRPC6, and TRPC7 using a mixture of specific siRNAs (siTRPC3/6/7: Fig. 6, E and F) nearly abolished PPZ2-induced currents in CGNs (Fig. 7, E and F). These results support that PPZ2 activates endogenous DAG-activated TRPC channels in CGNs.

Fig. 7.
Fig. 7.

(A) A representative trace of whole-cell currents induced by 30 μM PPZ2 in CGNs recorded at –60 mV. (B) Corresponding I-V relationships at time points 1 and 2. (C) The I-V relationship for the PPZ2-induced currents was obtained by subtraction in (B). (D) Averaged peak current densities of CGNs before (control) and after application of PPZ2 at –60 mV (n = 11–14). *P < 0.05 compared with control. (E) Representative traces of whole-cell currents induced by 10 and 30 μM PPZ2 in CGNs transfected with randomized siRNA (siControl) or siRNA mixture targeting TRPC3, TRPC6, and TRPC7 (siTRPC3/6/7) at –60 mV. (F) Averaged peak current densities of CGNs transfected with siControl or siTRPC3/6/7 before and after application of PPZ2 at –60 mV (n = 5–8). *P < 0.05. Data points show the means ± S.E.M.

Neurite Outgrowth Is Promoted by Piperazine Compounds via Ca2+-Dependent Signaling Pathways.

Ca2+ signaling initiated by TRPC3 and TRPC6 plays multiple roles in physiological functions in neurons (Abramowitz and Birnbaumer, 2009; Nilius and Szallasi, 2014; Feng et al., 2015). We sought to clarify how Ca2+ signaling pathways are involved in the biological effects of PPZ compounds in neurons. In cultured rat CGNs, PPZ2 induced [Ca2+]i increases (Fig. 8, A–C). These Ca2+ responses were predominantly attributable to Ca2+ influx, because, in the absence of extracellular Ca2+, PPZ2 caused only a marginal [Ca2+]i increase in CGNs. Next, we tested various pharmacological inhibitors of Ca2+-dependent signaling, which suppressed BDNF-induced neurite outgrowth promotion in CGNs (Fig. 8D). SK&F 96365 (20 μM), the myosin light-chain kinase (MLCK) inhibitor ML-7 (10 μM), the CaMK kinase (CaMKK) inhibitor STO-609 (5 μM), the CaMKII inhibitor KN-93 (2 μM), and the MAPK/ERK kinase (MEK) inhibitor PD98059 (50 μM) significantly suppressed the promoting effects of PPZ1 and PPZ2 on neurite outgrowth in CGNs. In contrast, the calcineurin inhibitors cyclosporine A (10 μM) and FK506 (1 μM) failed to suppress the PPZ1 and PPZ2 effects. To confirm the suppressive activity of these inhibitors on calcineurin, we tested their effects on the activity of the main downstream signaling molecule of calcineurin, nuclear factor of activated T-cells (NFAT), using luciferase reporter vector, which contains NFAT binding sequences in CGNs (Supplemental Fig. S10). BDNF significantly increased NFAT activity in CGNs, as previously reported (Groth and Mermelstein, 2003). By contrast, 10 nM PPZ1 and PPZ2 failed to show this effect. Ten micromolar cyclosporine A and 1 μM FK506 nearly abolished the constitutive NFAT activity, which was slightly rescued by BDNF. These results indicate that the calcineurin/NFAT pathway is induced in our experimental condition, further supporting the notion that the calcineurin/NFAT pathway is not involved in PPZ-induced neurite outgrowth.

Fig. 8.
Fig. 8.

Neurite growth–promoting effects of PPZ1 and PPZ2 via Ca2+-dependent signaling in CGNs. (A–C) 30 μM PPZ2-induced Ca2+ responses in cultured CGNs at 1 day in vitro. Representative averaged time courses in 2 mM Ca2+–containing (A) or Ca2+-free, 0.5 mM EGTA–containing (B) external solutions and averaged maximum increases in [Ca2+]i (Δ[Ca2+]i) (C) (n = 92–248). *P < 0.05; **P < 0.01; ***P < 0.001 compared with DMSO. (D) Total neurite outgrowth of CGNs treated for 24 hours with 0.1% DMSO, 50 ng/ml BDNF, 10 nM PPZ1, or with 10 nM PPZ2 alone or in combination with various inhibitors of Ca2+-dependent signaling, as indicated. Mean values of total neurite length were normalized to those in DMSO-treated control cells without inhibitors. CGNs were fixed at 1 day in vitro (n = 22–137). *P < 0.05; **P < 0.01; ***P < 0.001 compared with DMSO. CsA, cyclosporine A; PD, PD98059; SKF, SK&F 96365; STO, STO-609. (E) PPZ2-induced CREB phosphorylation detected by western blotting (WB) using a Ser-133–phosphorylated CREB-specific antibody and a total CREB antibody. Cultured CGNs at 1 day in vitro were treated with DMSO or 10 μM PPZ2 for the indicated times and lysed. Total cell lysates were subjected to western blotting. (F) CREB phosphorylation was quantified as the ratio of the amount of phosphorylated CREB to the total amount of CREB. Mean ratios were normalized to the ratio of the cells treated with 0.1% DMSO for 15 minutes (n = 3–4). **P < 0.01 compared with DMSO. Data points show the means ± S.E.M.

CaMKK, CaMKII, and MEK are known to activate Ca2+-dependent transcription factor CREB in response to [Ca2+]i increases (Shaywitz and Greenberg, 1999). Indeed, treatment with PPZ2 significantly increased CREB Ser-133 phosphorylation in CGNs (Fig. 8, E and F). This result is consistent with a previous report demonstrating the importance of TRPC3 and TRPC6 in BDNF-induced activation of MAPK, CaMK, and the downstream molecule CREB (Jia et al., 2007). ML-7 decreases growth cone motility via inhibition of MLCK (Ruchhoeft and Harris, 1997), the Ca2+-dependent actin filament modulator. Therefore, PPZ compounds may also affect the cytoskeleton during neurite outgrowth through MLCK. Thus, PPZ1 and PPZ2 promote neurite outgrowth of CGNs by triggering Ca2+-dependent signaling pathways essential for modulation of gene expression and actin dynamics.

Piperazine Compounds PPZ1 and PPZ2 Promote Neuronal Survival through TRPC3, TRPC6, and TRPC7 Channels.

BDNF-induced TRPC3 and TRPC6-dependent CREB activation is known to promote neuronal survival in CGNs (Jia et al., 2007). As described earlier, our data indicate that PPZ2 induces activation of CREB. Therefore, we tested neuroprotective effects of the PPZ compounds in cultured CGNs subjected to serum deprivation–induced cell death. This is also an important experiment for determining the biological activity of PPZ2 at concentrations in the context of neuronal death/survival. When CGNs were pretreated for 24 hours before serum deprivation and incubated during serum deprivation (24 hours) with the compounds PPZ1 and PPZ2, CGNs showed significantly reduced cell death, indicated by the marker PI, as compared with DMSO-treated control CGNs (Fig. 9, A and B). PPZ1 was effective at concentrations of 100 and 1000 nM, whereas PPZ2 was effective at 1 and 10 nM, in contrast to higher concentrations of PPZ2 (10 and 30 μM), required to induce cell death. Coadministration of Pyr4 (1 μM) with BDNF, PPZ1, or PPZ2 reversed the percentage of PI-positive cells to the level seen with the DMSO-treated control (Fig. 9C). These results show that PPZ1 and PPZ2, similar to BDNF, promote survival of CGNs.

Fig. 9.
Fig. 9.

Neuroprotective effects of PPZ1 and PPZ2 on cultured CGNs. (A) Representative images of CGNs treated with 0.1% DMSO, 50 ng/ml BDNF, 100 nM PPZ1, or 10 nM PPZ2 were obtained by staining with the nuclear marker Hoechst 33342 (Hoechst) (blue) and dead cell marker PI (red). Scale bar, 100 μm. (B) Percentages of PI-positive CGNs treated with DMSO, BDNF, PPZ1, or PPZ2 (n = 20–47). *P < 0.05; **P < 0.01; ***P < 0.001 compared with DMSO. Cell death was induced in CGNs by 24-hour serum deprivation. Cells were pretreated with 0.1% DMSO, 50 ng/ml BDNF, PPZ1, or PPZ2 for 24 hours prior to serum deprivation. (C) Effects of TRPC inhibition by 1 μM Pyr4 on neuroprotection induced by 50 ng/ml BDNF, 100 nM PPZ1, and 10 nM PPZ2 (n = 18–40). *P < 0.05; **P < 0.01; ***P < 0.001 compared with DMSO. Data points show the means ± S.E.M.

Taken together, our findings suggest that the PPZ compounds PPZ1 and PPZ2 activate DAG-activated TRPC3, TRPC6, and TRPC7 channels, and stimulate Ca2+-dependent signaling pathways, leading to trophic effects such as promotion of neurite outgrowth and survival in neurons.

Discussion

We have identified novel small-molecule, piperazine-derived activators of DAG-activated TRPC channels as a new class of BDNF mimetics. More specifically, PPZ1 and PPZ2 selectively activate, among TRPC subfamily members, TRPC3/C6/C7, thereby promoting neurite outgrowth and survival in isolated rat neurons. We hypothesize that activation of Ca2+-dependent signaling by Ca2+ influx via TRPC3/C6/C7 mediates the neurotrophic action of the PPZs.

The nine low-molecular-weight small compounds identified by our chemical library screening all have a piperazine scaffold, and fall into two groups. Group A comprises benzoyl-piperazines (compounds 1–4) and sulfonyl piperazines (compound 5), and Group B comprises 1-phenyl-piperazine-4-aceto amide derivatives (compounds 6–9) (Fig. 1B). Although our screening was based on TRPC6 activation, subsequent experiments revealed that the selected compounds also activated the closely related TRPC subtypes, TRPC3 and TRPC7. DAG and its analog OAG (Fig. 1A), originally used as potent activators of PKCs (Nishizuka, 1995), activate TRPC3/C6/C7 (Hofmann et al., 1999; Okada et al., 1999). Although further studies are needed to clarify the mechanism of action, it is important that PPZs selectively activate DAG-activated TRPC channels without directly affecting PKCs. Because PKCs directly phosphorylate various signaling proteins, including TRPC3/C6/C7, to control intracellular signal transduction upon PLC-coupled receptor stimulation or OAG application (Okada et al., 1999; Venkatachalam et al., 2003; Bousquet et al., 2010; Numaga et al., 2010), PPZs would activate particular pathways more selectively than PLC-coupled receptor stimulation or OAG. In [Ca2+]i measurements and patch-clamp recordings, the compounds PPZ1 and PPZ2 failed to activate other TRPCs, TRPM2, or TRPV4. PPZ1 also activated TRPV1 at 10 μM, whereas PPZ2 activated TRPV1 and TRPA1 only at 30 μM, a concentration markedly higher than those needed to activate TRPC3/C6/C7. Based only on the data obtained so far, we cannot exclude the possibility that PPZ1 and PPZ2 affect other channels, receptors, or intracellular molecules. However, PLC inhibition failed to affect PPZ1- and PPZ2-induced TRPC activation, suggesting that involvement of PLC pathways is an unlikely possibility. Moreover, immediate action of the compounds in whole-cell recordings is consistent with a direct action of PPZs on the channels. Although our cell-attached recordings suggest that PPZ2-induced TRPC6 currents share functional properties with receptor-activated TRPC6 currents, [Ca2+]i measurements showed that PPZ2 further enhances TRPC6 and TRPC7 activity elicited under CCh stimulation, suggesting a mechanism different from receptor stimulation in the activation of TRPC6 and TRPC7 by PPZ2. Further evaluations will be required to demonstrate mechanisms of selective action of the PPZs on DAG-activated TRPC channels.

PPZ1 and PPZ2 dose-dependently activate TRPC3/C6/C7. Although we could not obtain complete dose-response relationship data due to poor water solubility, by fitting the plots to the Hill equation, we obtained the EC50 values, which reveal that PPZ2 has higher affinities to DAG-activated TRPC channels than PPZ1. Interestingly, both compounds showed the lowest EC50 values to TRPC7. Moreover, the nine compounds identified in our screening activated TRPC7 at high potencies. Therefore, our compounds are potential new leads to develop selective activators of TRPC7, providing useful tools to investigate the physiological and pharmacological significance of TRPC7, the most elusive TRPC channel.

PPZ1 and PPZ2 promoted neurite outgrowth and survival in CGNs. Inhibition or downregulation of TRPC3/C6/C7 suppressed these beneficial effects. PPZ-induced neurite outgrowth was suppressed by inhibitors of MLCK, CaMKK, CaMKII, and MEK, all of which are activated by [Ca2+]i increases (Kamm and Stull, 2001; Agell et al., 2002; Wayman et al., 2008). This, together with the observations that PPZ2 induced TRPC3/C6/C7-like currents and [Ca2+]i increases in CGNs, suggests that Ca2+-induced signaling pathways via TRPC are of pivotal importance for neurotrophic effects of PPZs.

MLCK is an important regulator of growth cone motility through Ca2+-dependent modulation of actin dynamics (Ruchhoeft and Harris, 1997; Zheng and Poo, 2007). Therefore, PPZ-induced MLCK activation via [Ca2+]i increase may affect growth cone motility. This idea is consistent with essential roles of TRPC3 and TRPC6 in BDNF-induced growth cone turning (Li et al., 2005). In addition to MLCK, PPZ2-induced [Ca2+]i increases stimulate CaMKK, CaMKII, and MEK, components of CaMK and MAPK pathways. Because PPZ2 promoted CREB activation in CGNs, Ca2+-dependent activation of these pathways by PPZ2 is likely to converge at CREB activation, thus leading to the neurotrophic actions of PPZ2 (Shaywitz and Greenberg, 1999). This is supported by the previous findings that TRPC3 and TRPC6 mediate Ca2+-dependent activation of CaMK, MAPK, and CREB in neurons (Jia et al., 2007; Tai et al., 2008; Heiser et al., 2013).

Our data fail to reveal the roles of calcineurin in PPZ-induced neurite outgrowth, although important roles have been previously suggested for calcineurin and its downstream transcription factor, NFAT, in neurite outgrowth and survival induced by neurotrophins (Graef et al., 2003; Benedito et al., 2005). Importantly, in addition to TRPC-dependent pathways, BDNF induces Ca2+ release from endoplasmic reticulum and Ca2+-independent activation of MAPK-ERK and phosphatidylinositol 3-kinase-Akt pathways to induce neurotrophic effects (Huang and Reichardt, 2003). This was apparent in neurite growth, when TRPC3/C6/C7 were fully blocked by SK&F 96365 or Pyr4 in hippocampal neurons (Fig. 6H). In BDNF-induced calcineurin activation, [Ca2+]i increase via Ca2+ release has a primary role (Nguyen and Di Giovanni, 2008). In contrast, PPZ1 and PPZ2 failed to induce Ca2+ release in HEK 293 cells or evoked only marginal Ca2+ release in CGNs (Fig. 8, A–C; Supplemental Fig. S2). Moreover, BDNF, but neither 10 nM PPZ1 nor PPZ2, activated NFAT (Supplemental Fig. S10). Altogether, these results suggest that PPZ1 and PPZ2 activate a part of the pathways of BDNF to partially mimic its neurotrophic effect. Importantly, only “recombinantly overexpressed” TRPC6 has been reported to activate the calcineurin pathway without cellular stimulation in cultured hippocampal neurons (Shen et al., 2013). In the calcineurin-mediated pathway, previous reports have studied roles of endogenous TRPC3/C6/C7 activation upon receptor stimulation, which induces Ca2+ release (Onohara et al., 2006; Kiyonaka et al., 2009), but not roles of endogenous TRPC3/C6/C7 “directly” activated.

An aberrant elevation of calcineurin/NFAT signaling is associated with numerous deleterious processes linked to neurodegenerative disorders, such as synaptic dysfunction, neuritic atrophy, and neuronal death (Wu et al., 2010). Also, TRPC3 and TRPC6 have been implicated in cardiac hypertrophy through activation of calcineurin and NFAT (Onohara et al., 2006; Kiyonaka et al., 2009). Considering the lack of effect of PPZ1 and PPZ2 on the calcineurin/NFAT pathway, PPZ1 and PPZ2 may have a merit to evade undesired side effects induced through activation of calcineurin/NFAT signaling in the brain and the heart. These considerations may suggest that PPZ1 and PPZ2 have the potential to become leading compounds for the development of new drugs for neurodegenerative disorders.

CGNs mainly express TRPC6, whereas hippocampal neurons express TRPC3/C6/C7 at almost the same and much higher level than CGNs, which may allow PPZ1 (0.1 and 1 μM) to show a higher potency of neurite outgrowth higher in hippocampal neurons than in CGNs. By contrast, PPZ2 showed a similar potency in these neurons. Interestingly, at relatively low concentrations (1 μM), PPZ2 preferentially activates TRPC7, whereas PPZ1 activates TRPC3 and TRPC7 (Fig. 3, C and D), suggesting that TRPC3 may contribute to the robust effect of PPZ1 in hippocampal neurons. In hippocampal neurons and CGNs, TRPV1, which is activated by PPZ1, is only marginally expressed, compared with TRPC3/C6/C7. Furthermore, capsazepine failed to affect PPZ1- and PPZ2-induced neurite growth promotion in hippocampal neurons. These results suggest that TRPV1 is not involved in PPZ-induced promotion of neurite outgrowth in CGNs and hippocampal neurons (Fig. 6H). Although we cannot completely exclude the possibility of off-target effects of PPZ1, TRPV1 is unlikely the main pathway in robust PPZ1-induced hippocampal neurite outgrowth.

High concentrations of PPZ2 showed neuronal toxicity which was rescued by BAPTA-AM in CGNs, suggesting that Ca2+ overload due to excessive activation of TRPC channels underlies the suppressive effects of PPZ2 at high concentrations. PPZ1 at 100 μM induced prominent Ca2+ responses and ionic currents (poor solubility precluded us from testing 100 μM PPZ2). However, PPZ2 showed a higher efficacy than PPZ1 up to 30 μM in terms of Ca2+ responses and membrane depolarization (Figs. 2 and 3; Supplemental Fig. S1). Also, PPZ2 (>1 μM), but not PPZ1 (1–30 μM), induced cell death (Supplemental Fig. S8). Taken together, PPZ2 has stronger effects to enhance TRPC activity than PPZ1. This may enable PPZ2 to exert neurotrophic effects at low concentrations, but deleterious effects on neurons via Ca2+ overload and maybe Na+ overload at high concentrations.

PPZ1 and PPZ2 induced neurotrophic effects at concentrations lower than those required to activate recombinant TRPCs. This difference implies enhancements of PPZ-induced signaling in native preparations. It has been reported that TRPC3 and TRPC6 activation leads to membrane depolarization that activates voltage-dependent Ca2+ channels in excitable cells (Reading et al., 2005; Onohara et al., 2006; Qu et al., 2012). In CGNs, 30 μM PPZ2 evoked spike-like [Ca2+]i increases that are abolished by depriving external Ca2+. This robust Ca2+ response may be due to voltage-dependent Ca2+ channel activation following cation influx through TRPCs, a possible explanation for enhancement of the PPZ-induced signaling. Our results also suggest that PPZ-induced Ca2+ influx via TRPCs activates CREB, which regulates expression of BDNF (Tao et al., 1998). It is possible that TRPC3-mediated [Ca2+]i increases promote BDNF release (Vohra et al., 2013) to enhance PPZ-induced neurotrophic effects. Interestingly, hyperforin and its analog 2,4-(1-ketohexyl)phloroglucinol (Hyp9) (Fig. 1A) activate only TRPC6 but still promote neurite outgrowth and dendritic morphological changes (Leuner et al., 2007, 2010; Heiser et al., 2013). However, not only TRPC6 but also TRPC3 plays an important role in neuronal functions, such as chemoattraction, neurotransmitter release, synaptic plasticity, and neuronal survival (Li et al., 2005; Amaral and Pozzo-Miller, 2007; Jia et al., 2007; Hartmann et al., 2008). This leads us to postulate a synergy between PPZ-induced activation of TRPC3 and TRPC6 and their ability to promote neurotrophic effects at nanomolar concentrations.

In conclusion, we have identified novel PPZ activators of TRPC3/C6/C7, thereby identifying a novel pharmacophore for these TRPCs. PPZs exert potent neurotrophic effects by inducing Ca2+-dependent signaling via TRPC3/C6/C7. It is important to note that PPZs, especially PPZ1 and PPZ2, are different in a number of aspects of their activity, including TRP subtype selectivity. This suggests that there is still room for improvement in affinity/selectivity to DAG-activated TRPCs to enhance the BDNF-like effect of PPZs. Further structural optimization and in vivo experiments are necessary to eventually allow PPZ compounds to become leads in the development of BDNF-mimetic TRPC activators.

Acknowledgments

The authors thank T. Numata for confirmation of the stably mouse TRPC6-expressing HEK 293 cell clone and helpful discussion, R. Sakaguchi and B. Heba for critical feedback on the manuscript, N. Takahashi for experimental design and helpful discussion, and H. Sasaki for patch-clamp recordings in TRPC channel-expressing HEK 293 cells.

Authorship Contributions

Participated in research design: Sawamura, Kawamura, Tanikawa, Kiyonaka, M. X. Mori, Nagata, Y. Mori.

Conducted experiments: Sawamura, Hatano, Takada, Hino, Kawamura, Tanikawa, Nakagawa, Hase, Nakao, Hirano, Rotrattanadumrong, Hu, Inoue.

Performed data analysis: Sawamura, Hatano, Takada, Hino, Nakagawa, M. X. Mori, Inoue.

Wrote or contributed to the writing of the manuscript: Sawamura, Takada, Nakagawa, Kiyonaka, M. X. Mori, Nishida, Inoue, Y. Mori.

Footnotes

    • Received December 9, 2015.
    • Accepted January 4, 2016.

  • This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Oxygen Biology: A New Criterion for Integrated Understanding of Life” [Grant 26111004] of The Ministry of Education, Culture, Sports, Science and Technology, Japan; and a Grant-in-Aid for Scientific Research (A) [Grant 24249017] of Japan Society for the Promotion of Science. This work was funded by Sumitomo Dainippon Pharma Co., Ltd. Y.T., K.H., T.K., J.T., H.N., and R.N. were full-time employees of Sumitomo Dainippon Pharma Co., Ltd., when this study was conducted.

  • dx.doi.org/10.1124/mol.115.102863.

  • ↵Embedded ImageThis article has supplemental material available at (molpharm.aspetjournals.org).

Abbreviations

BAPTA
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
BAPTA acetoxymethyl ester
BAPTA-AM
BDNF
brain-derived neurotrophic factor
[Ca2+]i
intracellular Ca2+ concentration
CaMK
Ca2+/calmodulin-dependent protein kinase
CaMKK
CaMK kinase
capsazepine
N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide
CCh
carbachol
CGN
cerebellar granule neuron
CNS
central nervous system
CREB
cAMP response element–binding protein
DAG
diacylglycerol
DMEM
Dulbecco’s modified Eagle’s medium
DMSO
dimethylsulfoxide
FK506
[3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5, 19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14, 16-dimethoxy-4,10,12, 18-tetramethyl-8-(2-propenyl)-15, 19-epoxy-3H-pyrido[2,1-c][1,4] oxaazacyclotricosine-1,7,20, 21(4H,23H)-tetrone, monohydrate
FMP
FLIPR membrane potential
G418
O-2-amino-2,7-dideoxy-D-glycero-α-D-gluco-heptopyranosyl-(1→4)-O-[3-deoxy-4-C-methyl-3-(methylamino)-β-L-arabinopyranosyl-(1→6)]-2-deoxy-D-streptamine, disulfate
HEK
human embryonic kidney
Hyp9
2,4-(1-ketohexyl)phloroglucinol
KN-93
N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide
MAPK
mitogen-activated protein kinase
MEK
MAPK/ERK kinase
ML-7
hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride
MLCK
myosin light-chain kinase
NFAT
nuclear factor of activated T-cells
OAG
1-oleoyl-2-acetyl-sn-glycerol
PD98059
2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
PI
propidium iodide
PKC
protein kinase C
PLC
phospholipase C
PPZ1
[4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone
PPZ2
2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide
RT-PCR
reverse-transcriptase polymerase chain reaction
siRNA
small interfering RNA
SK&F 96365
1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H]-imidazole hydrochloride
STO-609
7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid
TRP
transient receptor potential
TRPA
transient receptor potential ankyrin
TRPC
transient receptor potential canonical
TRPM
transient receptor potential melastatin
TRPV
transient receptor potential vanilloid
U73122
1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione
U73343
1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione

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Neurotrophic Action of Novel TRPC Channel Activators

Seishiro Sawamura, Masahiko Hatano, Yoshinori Takada, Kyosuke Hino, Tetsuya Kawamura, Jun Tanikawa, Hiroshi Nakagawa, Hideharu Hase, Akito Nakao, Mitsuru Hirano, Rachapun Rotrattanadumrong, Shigeki Kiyonaka, Masayuki X. Mori, Motohiro Nishida, Yaopeng Hu, Ryuji Inoue, Ryu Nagata and Yasuo Mori
Molecular Pharmacology March 1, 2016, 89 (3) 348-363; DOI: https://doi.org/10.1124/mol.115.102863
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