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

Article abstract of DOI:10.1124/mol.115.102863

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

Full text of DOI:10.1124/mol.115.102863

Molecular Pharmacology Fast Forward. Published on January 5, 2016 as DOI: 10.1124/mol.115.102863
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Screening of TRPC 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
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Kyoto, Japan (S.S., M.H., Y.T., H.H., M.H., R.R., S.K.,
M.M., Y.M.); Department of Technology and Ecology, Hall of Global Environmental
Studies, Kyoto University, Kyoto, Japan (S.K., Y.M.); Sumitomo Dainippon Pharma Co.,
Ltd. (Y.T., K.H., T.K., J.T., H.N., R.N.); Division of Systems Medical Science, Institute
for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan (A.N.);
Division of Cardiocirculatory Signaling, Okazaki Institute for Integrative Bioscience
(National Institute for Physiological Sciences), National Institutes of Natural Sciences,
Japan (M.N.); Department of Physiology, School of Medicine, Fukuoka University
Fukuoka, Japan (Y.H., R.I.)
1

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Running title:
Neurotrophic Action of Novel TRPC Channel Activators
Address for correspondence:
Yasuo Mori, Ph.D.
Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological
Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus,
Nishikyo-ku, Kyoto 615-8510, Japan.
Tel: 81-75-383-2761, Fax: 81-75-383-2765
E-mail: mori@sbchem.kyoto-u.ac.jp
Number of text pages: 56
Tables: 0
Figures: 9
References: 69
Number of words in the Abstract: 247
Number of words in the Introduction: 750
Number of words in the Discussion: 1708
Abbreviations:
BDNF,
brain-derived
neurotrophic
factor;
BAPTA,
2
+
2+
1
,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; [Ca ] , intracellular Ca
i
2
+
concentration; CaMK, Ca /calmodulin-dependent protein kinase; capsazepine,
N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-
carbothioamide; CGNs, cerebellar granule neurons; CNS, central nervous system;
2

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CREB, cAMP response element-binding protein; DAG, diacylglycerol; DIV, days in
vitro; DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhanced green
®
fluorescent protein; FMP, FLIPR membrane potential; HEK, human embryonic
kidney;
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; Norad,
noradrenaline; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PKC, protein kinase C; PLC,
phospholipase C; RT-PCR, reverse transcriptase-polymerase chain reaction; Trk,
tropomyosin-related
kinase;
PD98059,
2
-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; PI, propidium iodide; PPZ1,
[4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone;
PPZ2,
2
1
-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide; SK&F 96365,
-[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;
U73122,
1
-[6-[[(17 )-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione;
U73343,
-[6-[[(17 )-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione
1
3

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Abstract
2
+
Transient receptor potential canonical (TRPC) proteins form Ca -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 neurological deficits, but its therapeutic potential is limited by its poor
pharmacokinetic profile. Elucidation of a framework for designing small molecules,
that 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 the rabbit portal vein. Also,
2
+
PPZ2 evoked cation currents and Ca 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.
2+
Inhibitors of Ca signaling pathways except calcineurin impaired neurite outgrowth
promotion induced by PPZ compounds. PPZ2 increased activation of the
Ca -dependent transcription factor, cAMP response element-binding protein (CREB).
2
+
2+
These findings suggest that Ca signaling mediated by activation of DAG-activated
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TRPC channels underlies neurotrophic effects of PPZ compounds.
piperazine-derived activators of DAG-activated TRPC channels provide important
insights for future development of a new class of synthetic neurotrophic drugs.
Thus,
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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 (TrkB), a receptor tyrosine kinase, to stimulate
various intracellular signaling pathways mediated by mitogen-activated protein kinase
(MAPK), phosphatidylinositol 3-kinase, or phospholipase C (PLC)
(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,
2
012). 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 neurological and
psychiatric disorders.
Drosophila melanogaster transient receptor potential (TRP) protein and its
homologs are putative six-transmembrane polypeptide subunits that form tetrameric
2
+
Ca -permeable non-selective cation channels (Clapham, 2003; Clapham et al., 2005;
Nilius and Szallasi, 2014). Mammalian TRP channels are grouped into six related
subfamilies, TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML. TRP channels are
activated by diverse stimuli including receptor stimulation, heat, osmotic pressure,
mechanical stress and environmental irritants from the extracellular and intracellular
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Molecular Pharmacology Fast Forward. Published on January 5, 2016 as DOI: 10.1124/mol.115.102863
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milieu. “Canonical” 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.
1
A) and inositol 1,4,5-trisphosphate. In addition to their well-known activation of
protein kinase C (PKC), a kinase regulating a plethora of biological functions
Nishizuka, 1995), DAG and its analogues also activate the closely related TRPC
(
subtypes, TRPC3, TRPC6, and TRPC7 (Hofmann et al., 1999).
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 TrkB (Li et al., 1999, Amaral and Pozzo-Miller, 2007;
2+
Jia et al., 2007). This cascade contributes to increased intracellular Ca concentration
2
+
2+
(
[Ca ]
i
) and subsequent Ca -dependent activation of such intracellular signaling
2
+
molecules as MAPKs, Ca /calmodulin-dependent protein kinases (CaMKs) and
2
+
Ca -dependent transcription factor cAMP response element-binding protein (CREB),
all of which play beneficial roles on neuronal functions (Shaywitz and Greenberg, 1999;
Agell et al., 2002; Jia et al., 2007; Wayman et al., 2008). The importance of TRPC6
has been also recognized through investigation of hyperforin (Fig. 1A), an active
ingredient of St. John’s wort, which is a widely used and well-investigated medicinal
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Molecular Pharmacology Fast Forward. Published on January 5, 2016 as DOI: 10.1124/mol.115.102863
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plant (Wolfle et al., 2014). Hyperforin is a multi-target compound that modulates
various molecular functions and exerts various beneficial effects, showing
anti-microbial, anti-inflammatory, anti-cancer and antidepressant activity (Russo et al.,
2
014). It has been previously reported that hyperforin exerts antidepressant and
neurotrophic effects via activation of TRPC6 in neurons (Leuner et al., 2007; Leuner et
al., 2013; Griesi-Oliveira et al., 2014). However, the activity of hyperforin remains
controversial: 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 neurological and psychiatric disorders. However, a
framework for designing small molecule 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
2
+
outgrowth and neuronal survival via downstream Ca -dependent signals. These
findings clearly support the potential of novel small molecule mimics of BDNF that
2+
target TRPC channels-dependent Ca signaling as an important basis to develop leads
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Molecular Pharmacology Fast Forward. Published on January 5, 2016 as DOI: 10.1124/mol.115.102863
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of therapeutic drugs for neurological and psychiatric disorders.
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Materials and Methods
Synthesis of Piperazine Derivatives. The details of the synthesis of piperazine
derivatives are provided in the Supplemental Data.
Reagents. Pyr3 and Pyr4 were synthesized as previously reported (Kiyonaka et al.,
2
009). Dimethyl sulfoxide (DMSO), SK&F 96365, ML-7, 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, U73343, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and
KN-93 were from Calbiochem (Nottingham, UK). Recombinant human BDNF
(248-BD) was from R&D systems (Minneapolis, MN). STO-609 was from Wako Pure
Chemical Industries (Osaka, Japan). PD98059 was from Biomol (Plymouth Meeting,
PA). Carbachol (CCh), fura-2 AM and BAPTA-AM were from Dojindo (Kumamoto,
Japan). FK506 was from Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan).
Animals. Any procedures and protocols used for animal studies using rats were
approved by the Institutional Animal Care and Use Committee of Kyoto University
prior to the start of experiments. Two to six-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)
1
0

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cDNA Cloning and Recombinant Plasmid Construction. Plasmids of the pCI-neo
vector carrying mouse TRPC1 , TRPC2, TRPC3, TRPC4 , 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. Human embryonic kidney (HEK) 293 cells
Boehringer Ingelheim, Ingelheim, Germany) were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum, 30 units/ml penicillin,
and 30 g/ml streptomycin at 37°C under 95% air and 5% CO HEK 293 cells were
(
2
.
transfected with recombinant plasmids using SuperFect transfection reagent (Qiagen,
Valencia, CA), according to the manufacturer’s instruction. For measurements of
2
+
[
Ca ]
i
and TRPC currents, recombinant plasmids were co-transfected with pEGFP-F
(Clontech Laboratories, Mountain View, CA) and pEGFP-N1 (Clontech Laboratories),
respectively. HEK 293 cells harboring green fluorescence were used for analysis.
2
+
Transfected cells were cultured for 36–48 hours prior to [Ca ]
i
measurement 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
1
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®
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% FBS in Minimum Essential Eagle’s Medium supplemented with
G418 (400 g/ml) and Blasticidin (5 g/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
5
cells (Stratagene) were seeded at a density of 2.5 × 10 cells/well in 6-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. Chemical compound library,
comprising 8,000 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,
3
Austria) at a density of 5 × 10 cells/well. After overnight culture, the culture medium
were removed, and then cells were incubated with 20 l of FMP probe diluted in
2
+
Ca -free 20 mM HEPES/Hank's Balanced Salt Solution for 1 hour at room temperature.
Compounds diluted in Hank's Balanced Salt Solution were added at 10 M (10 l/well).
Relative fluorescence intensities (530/595 nm; excitation/emission) were measured in a
®
FLIPR Tetra fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) in
1
second interval for 140 seconds. Following first round of screening, 315 positive
compounds were further tested under the same protocol twice to verify reproducibility.
1
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Molecular Pharmacology Fast Forward. Published on January 5, 2016 as DOI: 10.1124/mol.115.102863
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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
2
93 cells and HEK 293 cells stably expressing TRPC6, each in three independent
experiments.
2
+
[
Ca ]
i
Measurement. Transfected HEK 293 cells (Boehringer) were subjected to
2
+
[
Ca ]
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 MgSO , 2 mM CaCl , 11.5 mM
4
2
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
2
+
pixel-by-pixel basis and were converted to Ca concentrations by in vivo calibration
using 10 M 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
n
to the Hill equation: f (x) = A
0
+ (Amax − A
0
)/[1 + (EC50/x) ], where A
0
is the basal
response, Amax is the maximum response, x is the PPZ1 or PPZ2 concentration, and n is
Hill coefficient, using Kaleidagraph software (Synergy software, Reading, PA).
Preparation of Single Portal Vein Smooth Muscle Cells. Albino rabbits of either
sexes weighing 1.5–2.0 kg (Biotec) were used. Each rabbit was anesthetized with an
1
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intravenous injection of sodium pentobarbital (40 mg/kg) and butorphanol tartate (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 mm × 2 mm in length and width, were made and incubated
2+
successively in nominally Ca -free Krebs solution and one 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
2
+
into small pieces in Ca -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 3 or 4 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
4
2
3
.5-cm glass bottom dish at a density of 5 × 10 cells/cm for reverse
transcriptase-polymerase chain reaction (RT-PCR), neurite outgrowth measurement and
5
2
western blotting and 2.5 × 10 cells/cm for neuronal survival assay. Two to four hours
after plating, the medium was changed to DMEM with 10% newborn calf serum, 26
mM KCl, 30 units/ml penicillin, and 30 g/ml streptomycin. Cultures were maintained
at 37°C in 95% air, 5% CO
2
.
Freshly isolated CGNs were transfected with 300 nM of
®
siRNAs using the Rat Neuron Nucleofector Kit (Lonza, Walkersville, MD) and G-13
1
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program (Lonza) according to the manufacturer’s instructions. pEGFP-N1 (1 g) was
co-transfected 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). Non-targeting, randomized siRNA was synthesized and purified with
(
the Silencer siRNA construction kit (Ambion, Foster City, CA). The oligonucleotide
sequence used for synthesis of non-targeting siRNA is
’-GGGTATACTAGTGAATTAG-3’ (forward), and 5’-CTAATTCACTAGTATACCC-3’
reverse).
5
(
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 M 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 (Heka Elektronik) software was used for command
pulse control, data acquisition, and analysis. Series resistance was compensated (by
5
0–70%) to minimize voltage errors. Solutions had the following compositions, with
2
+
the concentrations in mM unless otherwise indicated. Ca -containing bath solution:
+
+
2+
2+
–
1
20 Na , 5 K , 1.2 Mg , 1 Ca , 129.4 Cl , 10 glucose, and 10 HEPES (pH 7.4 adjusted
2
+
with NaOH, and osmolality adjusted to 300 mOsM with D-mannitol) and Ca -free bath
1
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+
+
2+
–
solution: 120 Na , 5 K , 1.2 Mg , 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
+
2+
2+
–
internal solution for whole-cell recording: 120 Cs , 2 Mg , 4 Ca , 32 Cl , 100
aspartate, 2 Na ATP, 10 BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid), 0.1 GTP, and 5 HEPES (pH 7.2 adjusted with CsOH, and osmolality adjusted to
90 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
2
2
-
1
0
.4 mV ms after a 50-ms step to –100 mV from a holding potential of –60 mV.
For whole-cell recording from CGNs at 1–3 DIV 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 M for CGNs were used in the recordings. Solutions had the
following compositions, with the concentrations in mM unless otherwise indicated.
+
+
2+
2+
–
Bath solution: 140 Na , 5 Tetraethylammonium , 1.2 Mg , 1 Ca , 149.4 Cl , 15
2+
glucose, 10 HEPES, 0.001 tetrodotoxin, 0.1 picrotoxin, 0.01 nifedipine, and 0.03 Cd
(pH 7.4 adjusted with Tris, and osmolality adjusted to 300 mOsM with D-mannitol)
+
+
2+
2+
–
(
Zhang et al., 2008); Cs internal solution: 120 Cs , 2 Mg , 4 Ca , 32 Cl , 100
aspartate, 2 Na
2
ATP, 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 I-V relationship, ramp pulses
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-
1
were applied from –100 to +100 mV at a speed of 0.4 mV ms 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’ DA, AD converter:
Patchmaster v.1.1.2 (Heka Elektronik). The input resistance of electrodes used for
whole-cell cell recording ranged from 8–15 M , and 50–70% of access resistance (5–7
M ) was electronically compensated. For long-term recordings (> 1 minutes), the
current signal was obtained using PowerLab 4/35 (digitized at 1 kHz; AD Instruments,
Bella Vista, NSW) after low-pass filtering at 500 Hz from which the mean current
amplitude were 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
-way electromagnetic valves by DA outputs from the computer. Solutions had the
following compositions, with the concentrations in mM unless otherwise indicated.
2
+
+
2+
2+
–
Bath solution: 135 Na , 5 K , 1.2 Mg , 1 Ca , 151.4 Cl , 5 glucose, and 10 HEPES;
+
+
2+
–
and Cs internal solution for whole-cell recording: 140 Cs , 2 Mg , 24 Cl , 120
2
+
aspartate, 2 Na
2
ATP, 5 phosphocreatine, 5 EGTA or BAPTA/1.5Ca , 10 glucose and 10
2
+
+
+
HEPES. The composition of Ca -free Krebs solution was, in mM; 140 Na , 6 K , 1.2
2
+
–
Mg , 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).
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RT-PCR. Total RNA of cultured rat CGNs was extracted using ISOGEN following
the manufacturer’s instructions (Wako Pure Chemical Industries, Osaka, Japan).
Reverse transcription of total RNA to cDNA was performed using the RNA LA PCR Kit
(TaKaRa-Bio, Shiga, Japan). This cDNA was used as PCR template to detect genes
expression
using
the
following
primers:
(forward)
(reverse);
(forward)
(reverse);
(forward)
(reverse);
(forward)
(reverse);
(forward)
(reverse);
(forward)
(reverse);
TRPC1,
and
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
’-GGTACATCTTCTCCTTAGCGC-3’
’-TGGTCTGTGCTCTGCATCTTC-3’
’-AGAAGCTGGGCAATTTCAACG-3’
’-CGATGAGCATGTTGAGTAGCACA-3’
’-TGAGGTGAACGAAGGTGAACTG-3’
’-CCTGTCCCCCAAGGAACTCT-3’
’-GCTTCGAAGTTCTGGGATTGC-3’
’-CACCACCACCTTCTCCGACTT-3’
’-CTAGCAGCGCTGAATTCTCTC-3’
TRPC2,
and
TRPC3,
and
TRPC4,
and
TRPC5,
and
’-AAGTTTCGAATTTGAGGAGCAGATG-3’
’-GGTTTACGGCAGCAGACCAT-3’
’-CGCATGTGCTACGAACTTCATG-3’
’-GAAGTACGACCACAAGTTCATCG-3’
’-TCAGGTGGTCTTTGTTCAAAT-3’
’-TTCTACAATGAGCTGCGTGTGGC-3’
TRPC6,
and
TRPC7,
and
(forward)
(reverse);
(forward)
-actin,
and
’-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 -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
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lengths of products were 477, 166, 469, 555, 502, 152, and 649 base pairs for
TRPC1–C7 and 445 base pair (bp) for -actin, respectively.
Measurement of Neurite Outgrowth in CGNs. Two to four 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 treating with siRNA, CGNs
were stimulated with DMSO, 50 ng/ml BDNF, PPZ1 or PPZ2 at 24–48 hours after
transfection for 24 hours. Neurons were fixed in phosphate buffer containing 4%
paraformaldehyde/4% sucrose solution. Cells were permeabilized, and blocked with
phosphate buffer containing 1% BSA/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 were 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 cells using NeuronJ software (Meijering et al., 2004).
Only neurites longer than one cell diameter were measured and values were averaged.
Ten to twenty images from independent three experiments were used for the analyses.
Isolation, Culture and Neurite Measurements of Rat Hippocampal Neurons.
Primary hippocampal neurons were prepared from frozen hippocampus of 19-day
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embryo Wistar rat (MBX0402; Sumitomo Bakelite, Tokyo, Japan) using Nerve Cell
Dissociation Medium (MBX0802; Sumitomo Bakelite). The primary hippocampal
neurons were plated onto a poly-D-lysine-coated 8 well chamber slides (BD Bioscience,
4
San Jose, CA) at a density of 5 × 10 cells/well. The cultures were maintained at 37°C
with 95% air and 5% CO
2
.
Four hours after plating, the medium was replaced with
Neuron Assay Medium (MBX0701; Sumitomo Bakelite) containing 0.1% DMSO, 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 with CGNs
described above. Five to ten images from independent three experiments were used
for the analyses.
Western Blotting. Two to four hours after plating, CGNs were stimulated with 0.1%
DMSO or PPZ2 for indicated time. Then, CGNs were washed with PBS buffer and
lysed with 300 l 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) as
previously described (Chung et al, 2011), respectively, using the ECL system
(Amersham Pharmacia Biotech, Piscataway, NJ). For loading normalization, -tubulin
was detected by using an anti- -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,
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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, 50 ng/ml BDNF, PPZ1 or
PPZ2 with or without 1 M Pyr4 for 24 hours at 4 DIV, 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 g/ml) and PI (0.2
2
g/ml) for 5 min at 37°C with 5% CO to visualize 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 nucleus, and shown as % PI
positive cells. Four to ten 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 FMP assay,
2
+
[
i
Ca ] 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 the Student’s t-test and a P value < 0.05 was considered
significant.
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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 a 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 a.u. for
3
0 M compound 1 and 789.4 ± 15.5 a.u. for 30 M 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
(
2+
TRPC3, TRPC6, and TRPC7, another DAG-activated cation channel, in [Ca ]
i
2+
measurements. These compounds, each at 10 M, evoked significant [Ca ]
i
increases
in TRPC3-, TRPC6-, and TRPC7-expressing HEK 293 cells compared to
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
2
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most potent effect on DAG-activated TRPC channels. Notably, compound 6 at 10 M
2
+
i
elicited robust [Ca ] increase in TRPC6-expressing cells.
Characterization of the Compounds PPZ1 and PPZ2
For precise characterization, we focused on compound 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 (compound 1–5) for further investigation. Because
both compounds share phenyl-piperazine structure, we designated them as PPZ1
(compound 1) and PPZ2 (compound 6), respectively. PPZ1 (100 M) and PPZ2 (30
2+
M) elicited significant [Ca ]
i
elevation in HEK 293 cells expressing TRPC3, TRPC6,
and TRPC7 but not in cells expressing TRPC1, TRPC2, TRPC4, and TRPC5 (Fig. 3, A
2
+
and B). The data of PPZ1- and PPZ2-induced Ca responses suggest activation
selectivity of the compounds PPZ1 and PPZ2 to TRPC3, TRPC6, and TRPC7 among
2
+
TRPC subfamily.
PPZ1 and PPZ2 evoked these Ca
signals in
a
concentration-dependent manner (Fig. 3, C and D). To estimate functional expression
2
+
levels of TRPC channels in HEK 293 cells, we have analyzed Ca responses to 100 M
2+
2+
2+
CCh by the Ca add-back protocol, which separates the [Ca ]
i
increase through Ca
2+
release from that through Ca influx (Supplemental Fig. S1, A and B). CCh-induced
2
+
Ca influx is significantly higher in TRPC channel-transfected HEK 293 cells than
2
+
vector-transfected cells. The maximum [Ca ]
i
rises elicited by 100 M CCh are
comparable in TRPC3-, TRPC6-, and TRPC7-expressing HEK 293 cells, suggesting
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that the levels of functional expression of these TRPC channels are similar in our
system. The dose-response data was reanalyzed by normalizing the value of maximum
2
+
[
i
Ca ] rise induced by PPZ compounds to those induced by adding back extracellular
2
+
Ca upon stimulation with 100 M 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, and 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
2+
2+
PPZ2-induced Ca responses were attributable mainly to Ca influx through TRPC
2
+
i
channels, because PPZ1 and PPZ2 failed to evoke significant [Ca ] elevation in
2
+
Ca -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
2
+
2+
[
Ca ]
and TRPC7-expressing HEK 293 cells, but 10
-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DAG-analogue, failed to
i
measurements. Ten M PPZ2 enhanced CCh-induced Ca influx in TRPC6
M
PPZ1 and 30
M
1
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.
PPZ1 and PPZ2 were further examined for their effects on TRPM2, TRPA1, and
2
+
i
TRPV1 using [Ca ] measurements and for TRPV4 by whole-cell patch clamp
recordings (Supplemental Fig. S4). PPZ1 had no significant effects on TRPM2,
TRPA1, or TPRV4, while it activated TRPV1 at 10–100 M. PPZ2 had no significant
effects on TRPM2 and TRPV4, while it activated TRPA1 and TRPV1 at 30 M, a
markedly higher concentration than those needed to activate TRPC3, TRPC6, and
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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 as well at 10 μM.
Both PPZ1 and PPZ2, in the external solution also containing the PLC inhibitor
2+
i
U73122 (Bleasdale et al., 1990), still induced [Ca ] increases in TRPC3-, TRPC6-, and
2+
TRPC7-expressing cells (Fig. 3, E–H). This indicated that the Ca 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). Current-voltage (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
2
+
extracellular Ca -dependence of PPZ1- and PPZ2-induced currents in
TRPC6-expressing HEK 293 cells. TRPC6 currents induced by 100 M PPZ1, 30 M
2
+
PPZ2, and 100 M CCh in the absence of extracellular Ca were suppressed by
2+
addition of 1 mM extracellular Ca (Supplemental Fig. S5). This result suggests that
2
+
extracellular Ca is not an obligatory necessity but can act as a modulator in PPZ1- and
PPZ2-induced TRPC6 activation as in receptor-induced TRPC6 activation.
We further examined PPZ2-induced TRPC6 channel activity by a cell-attached
configuration in TRPC6-transfected HEK 293 cells. Application of PPZ2 from pipette
o
significantly increased ion channel activity with a mean NP (the product of the number
of channels (N) and the channel open probability (P )) value from 0.0093 ± 0.0083
o
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(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 to
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 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, being 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 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.,
2
001). Ten M noradrenaline (Norad) was used as a positive control to induce
non-selective cation currents through TRPC6. Since TRPV1 is also expressed in
smooth muscle cells (Mori et al., 2015), it may contribute to ionic currents induced by
PPZ2 at 30 M in rabbit portal vein smooth muscle cells according to our results
(Supplemental Fig. S4C). However, PPZ2 at 1 and 3 M, which are subthreshold
concentrations to activate TRPV1 channels, enhanced the ionic currents in rabbit portal
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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.
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 cerebellar granule
neurons (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.
We tested the effects of co-treatment of PPZ1 or PPZ2 at various concentrations
(0.01–10 M) with BDNF in neurite outgrowth assay (Supplemental Fig. S8A). PPZ2
at relatively high concentrations ( 1 M) but not PPZ1 (0.01–10 M) suppressed
promotion of neurite outgrowth induced by BDNF. To address the underlying
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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 M) (Supplemental Fig.
2
+
S8B). This was rescued by co-treatment of the membrane-permeable Ca chelator
BAPTA-AM (Supplemental Fig. S8C). These results may suggest that PPZ2 at high
2+
concentrations induces Ca 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 lack promoting effects at relatively high
concentrations 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 M SK&F
2
+
9
6365 (SKF), a non-selective inhibitor for various cation and Ca influx channels
including TRPC channels (Merritt et al., 1990) and 1 M Pyr4, but not by 0.1 M
capsazepine (CPZ), 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 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
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A–D: compare with recombinant currents in Fig. 4), and knockdown of TRPC3, TRPC6,
and TRPC7 using 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.
2+
Neurite Outgrowth is Promoted by Piperazine Compounds via Ca -Dependent
Signaling Pathways
2
+
Ca signaling initiated by TRPC3 and TRPC6 plays multiple roles in
physiological functions in neurons (Abramowitz and Birnbaumer, 2009; Nilius and
2
+
Szallasi, 2014; Feng et al., 2015). We sought to clarify how Ca signaling pathways
are involved in the biological effects of PPZ compounds in neurons. In cultured rat
2
+
2+
CGNs, PPZ2 induced [Ca ]
i
increases (Fig. 8, A–C). These Ca responses were
2
+
2+
predominantly attributable to Ca influx, because, in the absence of extracellular Ca ,
2
+
i
PPZ2 caused only a marginal [Ca ] increases in CGNs. Next, we tested various
2+
pharmacological inhibitors of Ca -dependent signaling, which suppressed
BDNF-induced neurite outgrowth promotion in CGNs (Fig. 8D). SK&F 96365 (SKF,
2
0 M), the myosin light chain kinase (MLCK) inhibitor ML-7 (10 M), the CaMK
kinase (CaMKK) inhibitor STO-609 (STO, 5 M), the CaMKII inhibitor KN-93 (2 M),
and the MAPK/ERK kinase (MEK) inhibitor PD98059 (PD, 50 M) significantly
suppressed the promoting effects of PPZ1 and PPZ2 on neurite outgrowth in CGNs. In
contrast, the calcineurin inhibitors cyclosporine A (CsA, 10 M) and FK506 (1 M)
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
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Molecular Pharmacology Fast Forward. Published on January 5, 2016 as DOI: 10.1124/mol.115.102863
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MOL #102863
(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 M CsA and 1 M 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.
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CaMKK, CaMKII and MEK are known to activate Ca -dependent transcription
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factor CREB in response to [Ca ]
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
i
increases (Shaywitz and Greenberg, 1999). Indeed,
(
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
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motility via inhibition of MLCK (Ruchhoeft and Harris, 1997), the Ca -dependent
actin filament modulator. PPZ compounds therefore may also affect the cytoskeleton
during neurite outgrowth through MLCK. Thus, PPZ1 and PPZ2 promote neurite
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outgrowth of CGNs by triggering Ca -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 above, our data
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