Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2015/10/23/mol.115.100792.DC1

1521-0111/89/1/197–213$25.00 http://dx.doi.org/10.1124/mol.115.100792 MOLECULAR PHARMACOLOGY Mol Pharmacol 89:197–213, January 2016 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Identification and Validation of Larixyl Acetate as a Potent TRPC6 Inhibitor s

Nicole Urban, Liming Wang, Sandra Kwiek, Jörg Rademann, Wolfgang M. Kuebler, and Michael Schaefer Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Leipzig, Germany (N.U., M.S.); Institut für Pharmazie, Freie Universität Berlin, Berlin, Germany (S.K., J.R.); and The Keenan Research Centre of St. Michael’s Hospital, Toronto, Canada (L.W., W.M.K.)

Received July 10, 2015; accepted October 21, 2015 Downloaded from

ABSTRACT Classical or canonical transient receptor potential 6 (TRPC6), a in displaying a marked TRPC6-prevalent mode of action. By nonselective and Ca21-permeable cation channel, mediates testing the main constituents of larch resin, we identified larixol pathophysiological responses within pulmonary and renal and larixyl acetate as blockers of Ca21 entry and ionic currents diseases that are still poorly controlled by current medication. through diacylglycerol- or receptor-activated recombinant molpharm.aspetjournals.org Thus, controlling TRPC6 activity may provide a promising and TRPC6 channels, exhibiting approximately 12- and 5-fold challenging pharmacological approach. Recently identified selectivity compared with its closest relatives TRPC3 and chemical entities have demonstrated that TRPC6 is pharma- TRPC7, respectively. No significant inhibition of more distantly cologically targetable. However, isotype-selectivity with regard related TRPV or TRPM channels was seen. The potent inhibition to its closest relative, TRPC3, is difficult to achieve. Reasoning of recombinant TRPC6 by larixyl acetate (IC50 5 0.1–0.6 mM) 21 that balsams, essential oils, or materials that are was confirmed for native TRPC6-like [Ca ]i signals in traditionally used for inhalation may contain biologic activities diacylglycerol-stimulated rat pulmonary artery smooth muscle to block TRPC6 activity, we embarked on a natural compound cells. In isolated mouse lungs, larix-6-yl monoacetate (CAS strategy to identify new TRPC6-blocking chemical entities. 4608-49-5; larixyl acetate; 5 mM) prevented acute hypoxia- Within several preparations of plant extracts, a strong TRPC6- induced vasoconstriction. We conclude that larch-derived at ASPET Journals on September 28, 2021 inhibitory activity was found in conifer balsams. The biologic labdane-type diterpenes are TRPC6-selective inhibitors and activity was associated with nonvolatile resins, but not with may represent a starting point for pharmacological TRPC6 essential oils. Of various conifers, the larch balsam was unique modulation within experimental therapies.

Introduction , but also to activate the DAG-sensitive TRPC3/-6/-7 subgroup of TRPC channels (Hofmann et al., 1999; Okada Classic or canonical transient receptor potential (TRPC) 21 et al., 1999; Gudermann et al., 2004). Membrane-permeable channels are second messenger-gated, Ca -permeable cation DAG analogs, such as 1-oleoyl-2-acetyl-sn-glycerol (OAG) and channels that are activated in a receptor- and phospholipase C 1,2-dioctanoylglycerol can substitute for the endogenously – (PLC) dependent mechanism (Ong et al., 2014). A closely formed DAG species in triggering Na1,K1, and Ca21 currents related subgroup consisting of TRPC3, TRPC6, and TRPC7 through TRPC6. shares the unique property of being directly activated by The expression of TRPC channels is widespread, but diacylglycerols (DAGs) (Hofmann et al., 1999). Within the markedly differs between isoforms. Therefore, selective block- context of ligand-induced signaling events, DAG is mainly ers that modulate the activity of distinct isoforms or sub- formed by phosphatidylinositol-specific phospholipases that groups of TRPC isoforms are expected to exert a tissue-specific cleave phosphatidylinositol-4,5-bisphosphate to generate inhibition of receptor-triggered cellular Ca21 influx and DAG and inositol-1,4,5-trisphosphate. The endogenously plasma membrane depolarization. TRPC6 is expressed in – formed DAG serves as a plasma membrane localized lipid vascular and airway smooth muscle cells (Welsh et al., 2002; second messenger to recruit conventional and novel Dietrich et al., 2006; Wang and Zheng, 2011) and contributes kinase C (PKC) or other C1 domain-bearing signaling to the vasoconstrictor response either by mediating receptor- and second messenger-triggered Ca21 influx, or by depolarizing dx.doi.org/10.1124/mol.115.100792. s This article has supplemental material available at molpharm. the smooth muscle cell, resulting in the opening of voltage- 21 aspetjournals.org. gated Ca channels. In the pulmonary vascular system,

ABBREVIATIONS: BSA, bovine serum albumin; DAG, diacylglycerol; DMSO, dimethylsulfoxide; FRET, Förster resonance energy transfer; GPCR, G protein–coupled receptor; HBS, HEPES-buffered saline; HEK, embryonic kidney; HPV, hypoxia-induced pulmonary vasoconstriction; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); OAG, 1-oleoyl-2-acetyl-sn-glycerol; PASMC, pulmonary artery smooth muscle cell; PBS, phosphate-buffered saline; PKC, protein kinase C; PLC, phospholipase C; TRP, transient receptor potential; TRPC, classical or canonical transient receptor potential.

197 198 Urban et al.

TRPC6 has gained considerable attention from several studies Cell Culture and Transfections. Parental and stably trans- that demonstrated its involvement in hypoxia-induced pulmo- fected human embryonic kidney (HEK) 293 cells were cultured in nary vasoconstriction (HPV) (Weissmann et al., 2006; Fuchs Earle’s minimum essential medium, supplemented with 10% fetal calf et al., 2011; Tabeling et al., 2015) and idiopathic pulmonary serum (Biochrom, Berlin, Germany), 2 mM L-glutamine, 100 U/ml hypertension (Yu et al., 2004, 2009), and also its role in penicillin, and 0.1 mg/ml streptomycin. To generate stably transfected cell lines, HEK293 cells were seeded in 35-mm culture dishes and contributing to lung edema formation (Samapati et al., 2012; transfected with 4 ml Fugene HD (Promega, Mannheim, Germany) Weissmann et al., 2012). In addition, TRPC6 expression in lung and 2 mg of DNA encoding human TRPC3-YFP, mouse TRPC4b-YFP, macrophages (Finney-Hayward et al., 2010) and in bronchial human TRPC6-YFP, human TRPC7-YFP, rat TRPV1-YFP, mTRPV4- smooth muscle cells may provide a link between TRPC6 and YFP, or human TRPM8-CFP. Human TRPC7 (a kind gift from M. pathophysiological mechanisms that induce or aggravate Mori, Fukuoka, Japan) was subcloned into a custom-made pcDNA3- chronic or allergic obstructive airway diseases (Sel et al., 2008). YFP vector with the stop codon replaced by an in-frame ligatable Not-I In the kidney, TRPC6 is expressed in podocytes that restriction site. Stably transfected colonies were obtained in the constitute the glomerular filtration membranes (Reiser presence of 1 mg/ml geneticin with a limiting dilution method. et al., 2005). Since nonsynonymous mutations in the TRPC6 Inducible HEK293mTRPC5-YFP cell lines were maintained and induced are associated with focal segmental glomerulosclerosis, as recently described (Richter et al., 2014). The HEK293mTRPM3a2 cell line was kindly provided by J. Oberwinkler, Marburg, Germany. genetic and functional evidence points to a seminal contribu-

For PKC« translocation assays, a stable HEK293YFP-rPKC« cell Downloaded from tion in controlling the integrity of the filtration slit (Reiser line (Lenz et al., 2002) was seeded on 25-mm glass coverslips for et al., 2005; Winn et al., 2005). Hence, pharmacological subsequent confocal (LSM 510, Carl Zeiss, Jena, Germany) imaging. modulators are intensely sought after that potently and Voltage-gated channels (CaV1.2) were heterologously efficiently block TRPC6 and may provide a biologic tool to expressed as described (Romanin et al., 2000) by cotransfecting validate the role of TRPC6 in pulmonary and renal diseases in HEK293 cells in 35-mm dishes with the following plasmids for a m b m order to establish experimental therapies. visualization: pcDNA3- 1c77 (0.5 g/well), pcDNA3- 2a (0.8 g/well),

a m m molpharm.aspetjournals.org Recently, Urban et al. (2012) demonstrated the general pcDNA3- 2d (0.7 g/well), and pEYFP-C1 (0.2 g/well). Cells were feasibility of identifying inhibitors of human TRPC6 by maintained at 37°C and in a 5% CO2-aerated humidified atmosphere. 21 applying academic high-throughput screening of a chemically Ca Measurements in Multiwell Plates. Unless otherwise 21 diverse library. However, the resulting modulators displayed stated, Ca assays were performed in HEPES-buffered saline (HBS), containing 132 mM NaCl, 6 mM KCl, 1 mM MgCl , 1mM CaCl , only a limited preference to inhibit TRPC6 compared with its 2 2 5.5 mM D-, and 10 mM HEPES adjusted to pH 7.4 with NaOH. closest relatives TRPC3 and TRPC7. Similarly, the biologic When Ca21 influx was induced by OAG, the buffer was supplemented activity of norgestimate to block DAG-sensitive TRPC chan- with 0.03% bovine serum albumin (BSA). HEK293TRP cell lines were nels does not markedly discriminate between single members grown to confluence in 75-cm2 flasks, detached with 0.25% trypsin, of the TRPC3/6/7 subfamily (Miehe et al., 2012). Therefore, we and loaded in culture medium with 4 mM fluo-4/AM (Invitrogen, embarked on another approach, in which we applied natural Darmstadt, Germany) for 30 minutes at 37°C. After washing (100g; at ASPET Journals on September 28, 2021 extracts to identify compound mixtures that exert TRPC6- 3 minutes), cells were resuspended in HBS, and dispensed into pig- prevalent biologic activity, followed by narrowing down the mented clear-bottom 384-well plates (Corning, Kaiserslautern, Germany; fractions and candidate compounds in order to finally pinpoint 36 ml/well). In the case of G protein–coupled receptor (GPCR) m the small molecules larixol and larix-6-yl monoacetate (CAS stimulation the HBS contained 2 M thapsigargin to deplete internal inositol-1,4,5-trisphosphate–sensitive Ca21 stores. Measurements were 4608-49-5, referred to herein as larixyl acetate) as potent and carried out in a two-step protocol using a custom-made fluorescence selective TRPC6 blockers. Validation of the labdane-type imaging plate reader built into a robotic liquid handling station diterpenes included the assessment of their selectivity, cyto- (Freedom Evo 150, Tecan, Männedorf, Switzerland). Fluo 4 was excited toxicity, electrophysiological properties, and confirmation of at 450–470 nm with a light-emitting diode array, filtered with a 475-nm the biologic activity in endogenously TRPC6-expressing cells short-pass filter (DT-blue; Optics Balzers, Balzers, Liechtenstein), and and tissues. the emitted fluorescence was imaged with a cooled charge-coupled device camera (Coolsnap FX, Photometrics, Tucson, AZ), equipped with a Xenon 0.95/25 C-mount lens (Schneider Kreuznach, Bad Kreuznach, Materials and Methods Germany) and a 515-nm long-pass filter (Y515-Di; Fujifilm, Tokyo). Compounds were added at the indicated final concentrations during the Materials. Essential oils were ordered from Neumond (Raisting, recording, and incubated for 5 minutes. In a second time-lapse analysis, Germany); larch oil was purchased from Bauer & Cie. (Munich, cells were stimulated with the respective agonist. During injections, Germany). All resins, turpentines, and balsams were obtained from fluorescence images were continuously monitored under control of the Kremer Pigmente (Aichstetten, Germany) and dissolved in dimethyl- Micromanager software (Edelstein et al., 2010). Fluorescence intensities sulfoxide (DMSO) in due consideration of their density. Abietic acid, in single wells were calculated with ImageJ software (Abramoff et al., neoabietic acid, and isopimaric acid were purchased from Sigma 2004), corrected for the respective background signals and normalized to

Aldrich (Munich, Germany), and pimaric acid was purchased from MP the initial intensities (F/F0). Concentration response curves were Biomedicals (Illkirch, France). For secondary screening, 13-epimanool generated by Hill equation fits. The activators were 50 mMOAGfor and larixyl acetate were purchased from Seqchem Sequoia Research TRPC3, TRPC6, and TRPC7; 2 mM for TRPV1; 10 nM

(Pangbourne, United Kingdom). All of the subsequent experiments GSK1016790A for TRPV4; 1mM H2O2 for TRPM2; 30 mM pregnenolone were conducted with larixol isolated and crystallized from Larix sulfate for TRPM3a2; and 300 mM for TRPM8. GPCR-mediated decidua turpentine following treatment with potassium hydroxide to activation of TRPC4b, TRPC5, and TRPC6 was achieved with a mix of saponify all of the contained esters. Larix-6-yl monoacetate was 1 mM carbachol, 300 mM ATP, and 0.5 U/ml thrombin. Activation of obtained from larixol by acetylation with acetic anhydride and transient receptor potential (TRP) channels was measured as increasing chromatographic purification. The identity and purity of both larixol intracellular calcium concentrations in the form of rising fluo-4 fluores- and larix-6-yl monoacetate were confirmed by 1H- and 13C-NMR cence intensities or as loss of coexpressed YFP fluorescence by spectroscopy and by high-resolution mass spectrometry (Supplemen- acidification in HEK293rTRPV1:YFP cells. If detachment of cells resulted in tal Figs. 1 and 2). impaired apparent channel activity, adherent cells (20,000 cells per well) Larixyl Acetate Is a TRPC6 Inhibitor 199 were grown and loaded with Fluo-4 in tissue culture–treated 384-well injection of Triton X-100 (0.2%) to obtain maximal intensities for plates (Greiner, Frickenhausen, Germany), and experiments were normalization. carried out with monolayers. To assess the cell viability, a (3-(4,5-dimethylthiazol-2-yl)-2,5- PKC« Translocation. Coverslips with adherently growing diphenyltetrazolium bromide) (MTT) assay was executed. HEK293

HEK293PKC«-YFP cells (Schaefer et al., 2001) were placed in a chamber cells were seeded in poly-L-lysine-coated 96-well cell culture plates at a containing HBS with or without 10 mM larixyl actetate and mounted density of 104 cells per well. The next day, medium was exchanged, onto the stage of an inverted LSM 510 META confocal microscope and test compounds were incubated for another 24 hours. After (Carl Zeiss). Imaging was performed with excitation at 514 nm and removal of the medium, cells were incubated for 3 hours in fresh emission filtered with a 530-nm long-pass filter. A Plan-Apochromat medium, containing 0.5 mg/ml MTT. Subsequently, supernatants 100Â/1.46 objective (Carl Zeiss) was used, and pinholes were set to were discarded, cells were lysed, and formed formazan crystals were yield optical slices with a thickness of 0.6–0.8 mm. After an incubation completely dissolved in 100% DMSO. Absorbance was detected at 560 of 5 minutes, visual fields with 5–12 cells were selected, and the PKC«- and 670 nm (reference wavelengths). Finally, the differences of both YFP distribution was imaged over time. Activation of PKC«-YFP was extinctions were calculated. All measurements were executed with a triggered by adding 1 mM carbachol, 300 mM ATP, and 0.5 U/ml multiwell plate reader (Polarstar Omega, BMG Labtech, Ortenberg, thrombin to the bath solution. For statistical analysis, regions of Germany). interest were defined in cytoplasmic areas of single cells, and PKC«- Compound Stability in Whole Blood. Venous blood from YFP translocation was examined in consideration of background and healthy, adult donors was drawn and anticoagulated with trisodium bleaching controls. Finally, data were normalized to initial cytosolic citrate (1:10 v/v, 110 mM). After adding 100 mM larixyl actetate or 1% Downloaded from PKC«-YFP intensity. DMSO, samples were kept at 37°C, aliquots were taken and centri- Electrophysiological Procedures. Whole-cell and single- fuged (2400g; 10 minutes), and citrate plasma was immediately frozen channel patch-clamp analyses were performed at room temperature at 220°C. The biologic activity to inhibit TRPC6 was assessed by 21 using a Multiclamp 700B amplifier with a Digidata 1440A digitizer intracellular Ca measurements in HEK293hTRPC6-YFP cells as de- (Axon CNS, Molecular Devices, Sunnyvale, CA) controlled with scribed previously in the presence of 10% of the conditioned plasma PClamp 10 software (Molecular Devices). HEK293hTRPC6-YFP, samples (corresponding to a final larixyl acetate concentration of

HEK293hTRPC3-YFP, and pulmonary artery smooth muscle cells 10 mM). Due to an inactivating protein binding of OAG, stimulation molpharm.aspetjournals.org (PASMCs) were seeded at low density on glass coverslips 6–24 hours with a GPCR agonist mix was performed (1 mM carbachol, 300 mM before the experiments. Coverslips were mounted in a perfused ATP, and 0.5 U/ml thrombin). chamber (Warner Instruments, Hamden, CT) placed on the stage of Smooth Muscle Cell Isolation, Immunofluorescence, and an inverted microscope. The standard extracellular solution contained Western Blot. After thoracotomy of 3-week-old rats, pulmonary 140 mM NaCl, 5 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, and 10 mM arteries were dissected from generation one to four. The blood vessels HEPES adjusted to pH 7.4 with NaOH. In the case of stimulation with were washed and mechanically cut in small pieces in a cold buffer

OAG, the bath solution additionally contained 0.1% BSA as well as containing 127 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 2.4 mM CaCl2, 1 mM nifedipine and 1 mM bisindolylmaleimide if PASMCs were 1.8 mM glucose, and 10 mM HEPES adjusted to pH 7.4. Enzymatic examined. Patch pipettes were made from borosilicate glass (Science digestion was done in a mix of one volume Dulbecco’s modified Eagle’s

Products, Hofheim, Germany) and had a resistance of 4–8MV when medium/Hams F12-solved papain (0.5 mg/ml, Sigma Aldrich Munich, at ASPET Journals on September 28, 2021 filled with the standard pipette solution: 140 mM CsCl, 4 mM MgCl2, Germany) and one volume of buffer without calcium but with 10 mM EGTA, and 10 mM HEPES adjusted to pH 7.2 with CsOH. In additional 32 mM DTT and 0.5% bovine albumin (Sigma Aldrich) for all whole-cell experiments, serial resistance was lower than 12 MV, 30 minutes at 37°C. Upon short centrifugation, supernatants were and was compensated by 70%. A modified pipette solution with 5 mM discarded, and pulmonary artery pieces were digested with a buffer 21 5 EGTA and 1.1 mM CaCl2 (calculated free Ca 100 nM) was used containing 0.75 mg/ml collagenase (Sigma Aldrich) in the presence of 2 when AlF4 infusion, inside-out, and outside-out experiments were 0.5% BSA for 10 minutes at 37°C. The enzyme was removed by m 2 m carried out. For TRPC6 activation by 30 M AlF4 , 500 l of this centrifugation, and smooth muscle cells were resuspended with cell pipette solution was freshly mixed with 10 ml of 0.5 M NaF and 2.5 ml culture medium (Dulbecco’s modified Eagle’s medium/Ham’s F12 1:1) of 6 mM AlCl3, and filled in the pipettes. To record current-voltage supplemented with 10% fetal calf serum, 2 mM L-glutamine, 2.7 g/l 2 1 21 curves, voltage ramps from 100 to 100 mV with a slope of 400 mV/s D-glucose, 25 mM HEPES, 100 U/ml penicillin, and 0.1 mg/ml were applied. All whole-cell currents were filtered at 3 kHz (four-pole streptomycin, and seeded on glass coverslips for immunofluorescence 21 Bessel filter) and sampled at 10 kHz. and single-cell [Ca ]i analysis. Electrophysiological Measurements of Recombinant For smooth muscle-specific a-actin staining, PASMCs were fixed CaV1.2. The bath solution contained 82 mM NaCl, 20 mM with 4% paraformaldehyde, blocked and permeabilized with triethanolamine-Cl, 30 mM BaCl2, 5 mM CsCl, 1 mM MgCl2,0.1mM phosphate-buffered saline (PBS) including 3% BSA and 0.1% Triton- EGTA, 10 mM glucose, and 5 mM HEPES adjusted to pH 7.4 with X100. Coverslips were incubated with monoclonal mouse antibodies NaOH. The intracellular solution contained 102 mM CsCl, 10 mM against smooth muscle a-actin (Sigma Aldrich; 1:200) overnight. After triethanolamine-Cl, 1 mM MgCl2, 10 mM EGTA, 3 mM Na2ATP, and washing three times for 5 minutes in PBS, alexa fluor 488-conjugated 5 mM HEPES adjusted to pH 7.4 with CsOH. The voltage step protocol donkey anti-mouse antibody (Invitrogen; 10 mg/ml) was added for consisted of 300 millisecond pulses, stepping from a holding potential 2 hours. Nuclei were stained with Hoechst 33258 (Sigma Aldrich, of 270 to 250 to 180 mV in increments of 10 mV. Inhibition of Cav1.2 10 mg/ml), coverslips were washed and mounted with Mowiol 4-88 was examined after incubation with the respective compounds for (Roth, Karlsruhe, Germany). All antibodies were diluted and in- 200 seconds by applying a 300 millisecond test pulse to 120 mV, cubated in PBS with 3% BSA. Confocal imaging was performed with followed by a second test pulse 160 seconds after washout of the a40Â water immersion objective. modulators. Maximum current amplitudes were determined in the To prepare PASMCs for western blotting, cells were harvested with presence of the modulators and after washout. a cell scraper, washed with PBS and homogenized with a lysis buffer, Detection of Cytotoxicity and Cell Viability. Membrane in- containing 150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% NP-40, 0.25% tegrity was assessed with a propidium iodide exclusion assay upon natrium deoxycholate, 1 mM NaF, 1 mM natrium orthovanadate, short-term (10 minutes) and long-term (24 hours) incubation with the 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinine, 1 mg/ml test compounds in HEK293 cell suspensions and adherent HEK293 leupeptine, and 1 mg/ml pepstatin. After assessment of protein yield cell cultures, respectively. After addition of 2 mg/ml propidium iodide, using a BCA Protein Assay Kit (Pierce Thermo Scientific, Darmstadt, fluorescence signals were detected with excitation at 544 nm and Germany), 30 mg was lysed in Laemmli buffer, and loaded to 8% SDS- emission at 620/10 nm in a plate reader device, followed by acute PAGE. Separated proteins were electroblotted onto polyvinylidene 200 Urban et al. difluoride membranes, and membranes were blocked for 1 hour in 5% day, the membrane was washed, followed by incubation with second- dried milk Tris-buffered saline/Tween-20 (20 mM Tris pH 7.4, 137 mM ary anti-rabbit (TRPC6) or anti-mouse (b-actin) peroxidase- NaCl, 0.1% Tween-20). Anti-TRPC6 antibody (Alomone, Jerusalem, conjugated antibodies (Sigma Aldrich; Germany; 1:5000) for 3 hours Israel; 1:200) or anti b-actin (Sigma Aldrich; 1:5000) was incubated in and detection using a chemiluminescence reagent Novex ECL 1% milk Tris-buffered saline and 0.5% Tween-20 overnight. The next (Invitrogen). Downloaded from

Fig. 1. Conifer turpentines or resins molpharm.aspetjournals.org concentration-dependently block Ca2+ entry through TRPC6 and TRPC3. HEK293 cell lines that stably express TRPC6 (HEKhTRPC6-YFP; black symbols and lines) or TRPC3 (HEKhTRPC3-YFP; gray symbols and lines) were loaded with fluo-4/AM in suspension and dispensed (TRPC6) into 384-well plates, or loaded as adherent cell layers (TRPC3). (A) True turpentines or resins of Picea alba (Bur- gundy resin); (B) Abies balsamea (Canada balsam); (C) Larix decidua (larch turpentine);

(D) Pinus spp. (pine resin); (E) propolis; (F) at ASPET Journals on September 28, 2021 Abies alba (Strassburg turpentine); (G) turpen- tine resin oil from Pinus spp.; or (H) a mixture of pine resin and Larix occidentalis turpentine (Venice turpentine) were serially diluted to the indicated final concentrations and incubated for 5 minutes. Fluorescence intensities of fluo-4 were recorded during injection of OAG (50 mM), applying a plate reader device. Relative in- creases in fluorescence intensities (F/F0)were obtained, and peak intensities after OAG stim- ulation were determined and normalized to the peak levels that were reached without treat- ment with turpentines (controls). Shown are mean and S.E. of 2–4 independent experiments, each performed in duplicate. Lines represent the results of nonlinear regression analyses, applying a four-parameter Hill equation, or with the sum of two Hill equations (C). Note the larger difference between TRPC6- and TRPC3-blocking properties achieved with the larch turpentine. Larixyl Acetate Is a TRPC6 Inhibitor 201

21 21 21 21 Smooth Muscle Cell [Ca ]i Imaging and Mn Quench. between 25, 50, 75, and 100 ml×kg/b.wt. ×min to generate a four- PASMCs were loaded with 5 mM fura-2/AM (Invitrogen) in HBS point pressure-flow curve from which the intrinsic vascular resistance, containing 0.5% BSA for 30 minutes at 30 minutes. Coverslips were R0 (i.e., the resistance that would exist if the lung vessels were at their washed, mounted in a bath chamber and perfused with HBS contain- respective diameter at zero vascular pressure), was calculated using a ing 0.1% BSA. Measurements were performed on an inverted nonlinear regression model (Wang et al., 2012). epifluorescence microscope (Carl Zeiss) equipped with a 10Â/0.5 Fluar objective, and a monochromator (Polychrome V, Till Vision, Gräfelfing, Germany) at alternating excitation wavelengths of 340, 358, and Results 380 nm. Digital video imaging was performed with a cooled charge- coupled device camera (PCO Sensicam, Kelheim, Germany) through a Identification of a TRPC6-Blocking Compound in 510-nm long-pass filter. To calibrate Ca21 signals, a spectral unmixing Larch Resin. Essential oils, resins, turpentines, or balsams method was used (Lenz et al., 2002). Activation of TRPC6 was induced of Pinophyta (conifers) are frequently applied as homespun with 100 mM OAG after a preincubation with 5 mM bisindolylmalei- remedies to treat or alleviate symptoms of respiratory disor- mide to exclude PKC interference. To evaluate the activity of store- 21 ders. To assess possible TRPC3- and TRPC6-inhibitory activ- operated calcium channels, PASMCs were perfused with Ca -free ity of oils or resins, we applied several plant extracts, HBS and pretreated with 5 mM larixyl acetate or 10 mM lanthanum(III) turpentines, resins, and essential oils to stably transfected acetate as a positive control. After a stable baseline, 2 mM 21 HEK293 cell lines that either overexpress human TRPC6 thapsigargin was injected for 5 minutes to deplete internal Ca Downloaded from (HEK ) or human TRPC3 (HEK ), and we stores. Then, 300 mMMnCl2 was added and store-operated TRPC6-YFP TRPC3-YFP activity was followed by measuring decreases in measured the effect of serially diluted extracts on the OAG- 1 fluorescence intensity at 358 nm, corresponding to the isosbestic induced Ca2 responses in a fluorescence imaging plate reader wavelength of fura-2/AM. In each experiment signals were device. Among them, Strasbourg turpentine (silver fir; Abies assessedandaveragedfrom100to200cells. alba), Canada balsam (balsam fir, Abies balsamea), Burgundy Hypoxia-Induced Pulmonary Vasoconstriction. Acute HPV resin (spruce; Picea alba), Pine resin (pine colophony; var. Pine was induced in isolated mouse lungs and assayed as recently described spp.), balsam turpentine oil (var. Pine spp.), Venice turpentine molpharm.aspetjournals.org (Urban et al., 2012). In brief, isolated lungs were prepared from male (a mixture of larch turpentine and colophony), and pure larch C57BL/6 mice (Charles River Laboratories, St. Constant, Quebec, turpentine (European larch; Larix decidua), or propolis (a Canada) with a body weight of 20–30 g and perfused with Hanks’ balanced salt solution containing 5% BSA and 5% dextran at a rate of resiniferous mixture collected by honey bees) were tested in 2 2 50 ml×kg/b.wt. 1×min 1. Pulmonary artery pressure was measured HEKTRPC6-YFP or HEKTRPC3-YFP cells. Both channels were continuously during ventilation with a normoxic gas mixture (21% O2, C-terminally tagged with a yellow fluorescent protein to identify 5% CO2, balance N2) and after switching to hypoxia (1% O2, 5% CO2, clones and later to ascertain the homogeneity of the expres- balance N2). At both normoxia and hypoxia, flow rates were varied sion of the respective channel transgene. Fluo-4/AM-loaded at ASPET Journals on September 28, 2021 TABLE 1 Half-maximally effective concentrations of various conifer turpentines and their constituents to inhibit TRPC6 or TRPC3

Half-maximally effective concentrations (IC50) of the indicated natural products or compounds were obtained by monitoring OAG (50 mM)–induced increases in Ca2+ as shown in Figs. 1–3, and fitting the data to a four-parameter Hill equation. If half-maximal inhibition was not achieved at the highest test concentration, the IC50 value was assumed to be higher than the indicated concentration. If responses were augmented, the effect is indicated as positive modulation of Ca2+ signals. Dashes indicate that half-maximal inhibition was not reached at concentrations up to 20 ml/l or that a ratio of potencies could not be calculated. The ratio of potencies is calculated as IC50(hTRPC3-YFP)/IC50(hTRPC6). Note that high concentrations of essential oils are cytotoxic, and effects may be unrelated to TRPC6 or TRPC3.

Ratio of hTRPC6-YFP hTRPC3-YFP Potencies Resins and turpentines Burgundy resin 0.002 g/l 0.007 g/l 3.5-fold Canada balsam 0.1 g/l 0.2 g/l 2.0-fold Larch turpentine 0.013 g/l 0.3 g/l 23.1-fold Pine resin 0.018 g/l 0.1 g/l 5.6-fold Propolis 0.014 g/l 0.045 g/l 3.2-fold Strassburg turpentine 0.2 g/l 0.68 g/l 3.4-fold Turpentine resin oil 0.03 g/l 0.11 g/l 3.7-fold Venice turpentine 0.14 g/l 0.61 g/l 4.4-fold Essential oils Abies alba 5.9 ml/l 10.7 ml/l 1.8-fold Abies sibirica — 15.8 ml/l — Cedrus atlantica — 14 ml/l — Cupressus sempervirens 5.0 ml/l 14.2 ml/l 2.8-fold Larix decidua Positive modulator . 20 ml/l — Pinus cembra 19 ml/l 6.3 ml/l 0.33-fold Pinus mugo Positive modulator 19.2 ml/l — Pinus sylvestris — . 20 ml/l — Resin acids and neutral compounds Abietic acid 21 mM30mM 1.4-fold Neoabietic acid 24 mM60mM 2.5-fold Pimaric acid 25 mM71mM 2.8-fold Isopimaric acid 33 mM48mM 1.5-fold 13-Epimanool 10 mM 28.6 mM 2.9-fold Larixol 2.04 mM 8.32 mM 4.1-fold Larixyl acetate 0.58 mM 6.83 mM 11.8-fold 202 Urban et al.

HEKhTRPC6-YFP cell suspensions or adherent cultures of HEKhTRPC3-YFP cells as described for the turpentines (data not HEKhTRPC3-YFP cells were incubated with the respective shown). None of the conifer oils were capable of blocking serially diluted turpentines (dissolved in DMSO) for 5 minutes, TRPC6 at concentrations typically used for inhalation, as and then challenged with 50 mM OAG to activate Ca21 entry bathing additives, or in ointments. In some cases, the highest in a receptor- and PLC-independent fashion. As depicted in concentrations applied in the assay (5–20 ml/l) exerted both 21 Fig. 1 and Table 1, various conifer turpentines or propolis increases in [Ca ]i and signs of cellular toxicity, such as exerted an inhibitory activity, with Burgundy resin and rounding and swelling of adherent HEK293 cell cultures. Strasbourg turpentine having the highest (IC50 5 2mg/l) Since these signals were unrelated to the expression of the and lowest (IC50 5 200 mg/l) TRPC6-blocking potencies, channels, they presumably reflect nonspecific signals. Nota- respectively. At saturating concentrations, the TRPC6 activ- bly, the TRPC6-prevalent block induced by larch turpentine ity was completely abrogated by either coniferous turpentine was not reproduced with the essential oil of Larix decidua. or resin tested. The preference to block TRPC6 but not TRPC3 Therefore, we conclude that the biologic activity of larch was in general poor, with the exception of larch turpentine. turpentine is contained in its nonvolatile resiniferous Larch turpentine exerted a relatively high potency (about constituents. 13 mg/l) to block the OAG-induced activation of TRPC6, while The nonvolatile conifer resins contain large amounts of half-maximal block of TRPC3 was obtained only with about carboxylic acids, such as abietic acid, pimaric acid, or iso- 20-fold higher concentrations of the turpentine (see Fig. 1C; pimaric acid. When applied individually, these resin acids Downloaded from Table 1). contained in any conifer turpentine were poorly potent and did We also asked whether inhibitory activity is found in not exert a marked TRPC6 prevalence in their blocking essential oils that are frequently inhaled to relieve respiratory activity (Fig. 2). When compared with other genus, larch symptoms such as cough, bronchospasms, or congestion. To resins contain a fraction of neutral diterpenes that feature a test this hypothesis, we applied serial dilutions (20–0.02 ml/l) bicyclic, labdane-type structure. The most prominent ones are of the essential oils of various Pinophyta spp.,includ- larixol and larixyl acetate. Larixol exerted a moderate potency molpharm.aspetjournals.org ing European larch (Larix decidua), and preformed (IC50 5 2.04 mM) in blocking TRPC6, but showed a marked concentration-response analysis on HEKhTRPC6-YFP and preference to block TRPC6 compared with TRPC3 (Fig. 3, A, C, at ASPET Journals on September 28, 2021

Fig. 2. (A–F) Resiniferous compounds that are con- tained in various conifer turpentines inhibit both TRPC6 and TRPC3. Experiments were performed es- sentially as described in Fig. 1, but after preincubating cells for 5 minutes with the indicated nonvolatile resin constituents that are generally contained in conifer turpentines, or with the corresponding concentrations of the solvent DMSO. Note the moderate potency and TRPC6 prevalence of inhibition of OAG-induced Ca2+ entry. Larixyl Acetate Is a TRPC6 Inhibitor 203 Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 28, 2021

Fig. 3. Isoform-selective inhibition of Ca2+ entry through TRPC6 by larixol and larixyl acetate. The impact of larixol and larixyl acetate on OAG- 2+ stimulated Ca entry was assessed in fluo-4 loaded HEKhTRPC6-YFP cells or in HEKhTRPC3-YFP cells (gray symbols and lines in D and E). Multiwell plates containing fluo-4-loaded HEKhTRPC6-YFP cells were placed in a fluorescence plate imaging device, and monitored during the sequential application of various concentrations of larixol (A) or larixyl acetate (B), followed by the addition of 50 mM OAG. (C) Representative images of wells, containing fluo-4- loaded HEKhTRPC6 cell suspensions imaged before (1) and 30 second after (2) stimulation with OAG (time points are indicated in A and B). The respective concentrations of the modulator or the solvent DMSO in the wells (double determinations) are indicated. (D and E) Concentration response curves were obtained from n =10–13 independent experiments with HEKhTRPC6-YFP (black lines) and HEKhTRPC3-YFP lines (gray lines) and for each modulator, performed as shown in (A–C).

and D). With an IC50 value of about 0.58 mM, larixyl acetate presumably accounts for the TRPC6-selective inhibitory effect displayed higher potency in blocking the OAG-induced Ca21 of the complete larch turpentine. signals in HEKhTRPC6-YFP cells (Fig. 3, B, C, and E). The IC50 Larixol and Larixyl Acetate-Induced Inhibition of value of 6.38 mM to block TRPC3 demonstrates that the Receptor-Induced Ca21 Entry through TRPC6. The TRPC6-selective activity is maintained in the acetylated initial identification of conifer resin constituents as TRPC6- derivative. Together, these data suggest that the efficient selective inhibitors was achieved by activating TRPC6 or and selective block of TRPC6 by larixol and larixyl acetate TRPC3 with the membrane-permeable DAG analog OAG. We 204 Urban et al. next assessed the biologic activity of larixol and larixyl acetate 300 mM ATP, 1 mM carbachol, or 0.5 U/ml thrombin without to block heterologously expressed TRPC6 channels in a GPCR- thapsigargin pretreatment. The resulting transient increases 21 triggered activation regimen that includes activation of in [Ca ]i were essentially identical irrespective of the pres- endogenous phospholipases C in HEK293 cells. The apparent ence or absence of 10 mM larixyl acetate, a concentration that IC50 values of larixol and larixyl acetate were 1.57 and blocks TRPC6 activity by more than 90% (Fig. 4, A and B). The 0.65 mM when observing the GPCR-induced Ca21 entry PLC-mediated formation of endogenous DAG species and in thapsigargin-pretreated HEKhTRPC6-YFP cells (2 mM for possible effects on DAG availability or degradation were more 5 minutes) (Supplemental Fig. 3). Again, the isotype selectivity directly assessed by monitoring the plasma membrane re- appeared considerably high, since GPCR-induced activation of cruitment of a fluorescent fusion protein of the C1 domain- Ca21 entry through TRPC3 was half-maximally inhibited by bearing PKC« (PKC«-YFP) upon receptor activation in the 19.0 6 0.9 mM larixol or 7.3 6 0.7 mM larixyl acetate (data not absence and presence of 10 mM larixyl acetate. Applying shown). Thus, the about 12-fold preference of larixyl acetate to confocal life cell microscopy, the plasma membrane associa- block TRPC6 compared with TRPC3 in the OAG-activated tion can be quantified by following the background-corrected mode was confirmed by an about 11-fold higher potency of the fluorescence intensity of PKC«-YFP in the cytosol before compound in the GPCR-activated mode of TRPC6 and TRPC3. [Icyt(0)] and during [Icyt(t)] GPCR activation. Since PKC«- Larixyl Acetate Does Not Interfere with GPCR- YFP is not enriched in the plasma membrane of resting cells Induced PLC Activation. To exclude that the most potent and PKC«-YFP is selectively recruited to the plasma mem- Downloaded from TRPC6 blocker larixyl acetate uncouples the GPCR-induced brane (Fig. 4C), the fraction of plasma membrane–bound PKC« activation of PLC, we measured the Ca21 mobilization re- is reflected by the fluorescence disappearance in the cytosol by 21 sponse by stimulating parental HEK293 cells in a Ca -free calculating IPM(t) 5 [Icyt(0 seconds) 2 Icyt(t)]/Icyt(0 seconds). HBS (1 mM Ca21 replaced by 200 mM EGTA, pH 7.4) with The results showed that 10 mM larixyl acetate did not cause a molpharm.aspetjournals.org

Fig. 4. Larixyl acetate does not interfere with the GPCR-induced Ca2+ mobiliza- tion and PKC« translocation. (A and B) at ASPET Journals on September 28, 2021 To control for possible inhibition of the GPCR-induced and PLC-mediated Ca2+ mo- bilization from inositol-1,4,5-trisphosphate– sensitive stores, parental HEK293 cells were loaded with fluo-4, stimulated with the indicated concentrations of agonists or activators that act on endogenously expressed PLC-activating GPCRs, and Ca2+ responses were followed in the pres- ence of 10 mM larixyl acetate (filled sym- bols) or in solvent controls (open symbols). Shown are mean and S.E. of eight deter- minations. (C–E) The GPCR-dependent DAG formation was monitored in liv- ing HEK293 cells stably expressing a C-terminally YFP-tagged rat PKC« (PKC«-YFP) as a translocating fluo- rescent biosensor. (C) Confocal micro- graphs were taken before (15 seconds), and during (120 and 280 seconds) com- bined stimulation with ATP 300 mM, carbachol 1 mM, and thrombin 0.5 U/ ml. Bars: 20 mm. (D and E) A quanti- tative analysis of PKC«-YFP translo- cation was obtained in single cells by determining the percentage of disap- pearance of fluorescence in regions of interest defined over cytosolic areas. Data represent mean and S.E. of 6–8 independent experiments, comprising data of 5–15 cells per imaging experiment. Larixyl Acetate Is a TRPC6 Inhibitor 205

21 significant decrease in the GPCR-triggered translocation single-cell [Ca ]i analyses, or in electrophysiological experi- efficiency of PKC«-YFP (Fig. 4, D and E). We conclude that ments when acutely applying up to 15 mM larixyl acetate (data larixyl acetate does not interfere with the GPCR-induced not shown). activation of PLC or accelerate DAG breakdown, but pre- To assess the activity of larixyl acetate on the CaV1.2 sumably acts by direct TRPC6 channel inhibition. subtype of voltage-gated Ca21 channels, heterologously Isoform Selectivity of TRP Channel Block by Larch- expressed CaV1.2 was analyzed electrophysiologically by Derived Labdanes. The selectivity of the identified TRPC6 applying voltage step protocols. Robust CaV1.2 currents were inhibitors was further profiled with TRPC7, and TRPC4 and obtained by stepping the membrane potential from 270 to TRPC5, representing the more distantly related TRPC family 120 mV (Supplemental Fig. 4). In contrast to 1 mM nifedipine, members. In line with the phylogenetic distance, OAG- 5–10 mM larixyl acetate exerted only partial and statistically activated TRPC7 was inhibited with an IC50 value of insignificant inhibition of CaV1.2-mediated peak current 2.9 mM, whereas half-maximal block of GPCR-induced activa- densities. Similarly, the inactivation time constants remained tion of TRPC4b and TRPC5 required 18.0 and 19.3 mM unchanged in the presence of 5 or 10 mM larixyl acetate. concentrations of the compound, respectively (Fig. 5A). Of Although rat PASMCs almost exclusively expressed note, a steeper concentration response curve was seen for TRPC6, but not TRPC3, these channel subunits may be TRPC7. This effect may be caused by a distinct and more coexpressed in vascular smooth muscle cells of other tissues cooperative mode of inhibition or by changes of the channel and species. To assess the impact of larixyl acetate in cells that Downloaded from inactivation properties. Extending the range of TRP channels are assumed to form heteromeric TRPC6:TRPC3 channel to TRPV1, TRPV4, TRPM2, and TRPM8 revealed no discern- complexes, we generated a stable HEKTRPC6:YFP:TRPC3:CFP cell ible inhibition at 15 mM concentrations that almost completely line, which coexpresses both channel subunits. The stoichi- block TRPC6 (Fig. 5B). In fluorescence imaging plate reader ometry of TRPC6:TRPC3 expression was estimated by com- analyses, TRPM3 was weakly inhibited by 15 mM larixyl paring CFP and YFP fluorescence intensities to those in cells acetate, but no statistically significant inhibition was seen in that expressed an intramolecularly fused CFP-YFP fusion molpharm.aspetjournals.org at ASPET Journals on September 28, 2021

2+ Fig. 5. Isoform-selective block of TRPC, TRPV, and TRPM channels by larixol and larixyl acetate. The impact of larixyl acetate on [Ca ]i signals elicited via stimulation of various TRP channel isoforms were assessed in fluo-4 loaded suspensions or adherent cultures of HEK293 cells, stably expressing the following TRP channels: mouse TRPC4b (mTRPC4) or mouse TRPC5 (mTRPC5) stimulated by ATP 300 mM, carbachol 1 mM, and thrombin 0.5 U/ml after depleting intracellular Ca2+ storage organelles with thapsigargin (2 mM for 5 minutes); human TRPC7 (hTRPC7) stimulated with 50 mM OAG; rat TRPV1 (rTRPV1) stimulated with 2 mM capasaicin; mouse TRPV4 (mTRPV4) stimulated with 10 nM GSK1016790A, human TRPM2 (hTRPM2) stimulated with 1 mM H2O2; mouse TRPM3a2 (mTRPM3) stimulated with 50 mM ; or mouse TRPM8 (mTRPM8) stimulated with 300 mM menthol. Effects of TRPC6 inhibitors were depicted as concentration response curves to inhibit TRPC channels (A). For better comparison, the HEKhTRPC6-YFP and HEKhTRPC3-YFP cell data are redrawn. (B) The inhibition of TRPV or TRPM channels by 0.15% DMSO (open bars) or 15 mM larixyl acetate (filled bars) is depicted as percentage of peak Ca2+ responses to the respective activators achieved in a buffer control. With the exception of mTRPM3, none of the differences reached statistical significance. (C) Expression ratios of and FRET efficiencies between TRPC6-YFP and TRPC3-CFP 2+ transiently or stably coexpressed in HEK293 cells. (D) Concentration response curve of larixyl acetate effects on OAG (50 mM)–induced [Ca ]i signals detected in the stably transfected HEKTRPC6-YFP:TRPC3-CFP cell line. Data represent mean and S.E. of 2–7 independent experiments, each performed in duplicate. 206 Urban et al. protein, with CFP intensities detected after eliminating cell line amount to 19.2% and 1.3%, respectively. Hence, most Förster resonance energy transfer (FRET) by photobleaching TRPC6-YFP channel subunits would be contained in hetero- YFP. The stable cell line expresses TRPC6-YFP and TRPC3- meric complexes, along with one (10.2%), two (30.0%), or three CFP at a 1:2 ratio (Fig. 5C). As reported previously, TRPC6 (39.2%) TRPC3-CFP subunits. In these cells, larixyl acetate 21 channel assembly can be monitored by FRET assays (Hofmann inhibited the OAG-induced [Ca ]i signal with an IC50 value of et al., 2002). In HEKTRPC6:YFP:TRPC3:CFP cells subjected to the about 3.74 mM (Fig. 5D), hinting to a TRPC6 homomer- method described therein, the FRET efficiency was about 35% prevalent inhibitory property of larixyl acetate. lower than in transiently transfected HEK293 cells, express- Electrophysiological Characterization of the TRPC6 ing both subunits at a 1:1 stoichiometry (see Fig. 5C). The Block. The electrophysiological properties of larixyl acetate slight reduction of FRET efficiencies in the stable cell line were determined in HEK293 cells that stably expressed expressing the partners with a molar excess of the FRET TRPC6 (HEKhTRPC6-YFP) or TRPC3 (HEKhTRPC3-YFP). In donor (TRPC3-CFP) is in good agreement with a maintained HEKhTRPC6-YFP cells, stimulation with 50 mM OAG elicited a heteromer formation, but with a fraction of homomeric characteristic nonselective cation conductance with a reversal TRPC3-CFP and a lower abundance of FRET acceptors potential close to 0 mV and a dually rectifying current-voltage (TRPC6-YFP) in individual channel complexes. Assuming a relationship. In the presence of 0.1–5 mM larixyl acetate, channel subunit assembly with no preference for momomeric inward and outward currents through TRPC6 were versus heteromeric association the expected percentages of concentration-dependently blocked (Fig. 6, A–C). Of note, Downloaded from homomeric TRPC3 and homomeric TRPC6 channels in this the IC50 values of larixyl acetate to block TRPC6 inward and molpharm.aspetjournals.org

Fig. 6. Electrophysiological properties of TRPC6 and TRPC3 inhibition by larixyl acetate. Ionic currents in HEKhTRPC6-YFP

and HEKhTRPC3-YFP cells were recorded in at ASPET Journals on September 28, 2021 the whole-cell mode by path-clamp analy- sis. (A) Characteristic TRPC6 currents were elicited by adding 50 mM OAG to the bath solution. Currents were recorded during application of slow voltage ramps from 2100 to +100 mV (400 mV/s21)as shown in (B). Larixyl acetate was added to the bath solution at the indicated concen- trations; outward (+100 mV; open bars) and inward (2100 mV; black bars) cur- rents through TRPC6 were determined in n =5–20 cells and normalized to the cell capacity to obtain current densities (pA/pF21). (B) Examples of leak-corrected current- voltage ramps in OAG-stimulated HEKhTRPC6-YFP cells in the absence (black line) and presence of the indicated concen- trations of larixyl acetate. (C and D) Statistical analysis of OAG-induced peak outward (open bars) and inward (filled bars) currents measured as shown in (A) in HEKhTRPC6 (C) or HEKhTRPC3 (D) cells (n =6–16 patched cells). Note the higher test concentrations of larixyl acetate added to HEKhTRPC3-YFP cells. (E and F) Shown are the corresponding four- parameter Hill equations of current den- sities of (C and D) with calculated IC50 values. Larixyl Acetate Is a TRPC6 Inhibitor 207 outward currents were 0.12–0.13 mM (Fig. 6E), which is current-voltage relationships in current-voltage curves pro- markedly lower than the concentrations that were required vided no evidence for a voltage dependence of the TRPC6 block to half-maximally suppress increases in fluo-4 fluorescence. In (see Fig. 6B). However, it should be mentioned that upon 21 agreement with the selectivity observed in [Ca ]i assays, addition of larixyl acetate the physiologically relevant inward TRPC3 current inhibition required substantial higher larixyl currents (at 2100 mV) decayed slightly faster than the acetate concentrations with half-maximal effects seen at outward currents measured at 1100 mV. Since both TRPC6 concentrations of 1.4 mM (Fig. 6, D and F). The analysis of channel opening and channel inactivation/closure typically Downloaded from Fig. 7. Larixyl acetate acts via an extra- cellularly accessible binding site in a re- versible and repeatable fashion. (A) HEKhTRPC6-YFP cells were patched with a pipette solution supplemented with 30 2 mMAlF4 (30 mMAlCl3 and 10 mM NaF) and either no (black line) or 1 mMof

larixyl acetate (gray line). Shortly after molpharm.aspetjournals.org reaching maximal inward currents (Vclamp = 260 mV), 1 mM larixyl acetate was added to the bath solution (outside) as indicated by the bar. Note the main- tained response to the extracellularly applied modulator in cells that are in- tracellularly perfused with larixyl ace- tate. (B) Statistical analysis of TRPC6 currents measured as shown in (A), or TRPC6 currents that were elicited by adding 50 mM OAG to the bath solution

in cells without intracellular larixyl ace- at ASPET Journals on September 28, 2021 tate perfusion (open bars) or in cells that were patched with pipette solutions, con- taining 1 mM larixyl acetate (gray bars). Filled bars: effect of extracellularly ap- plied larixyl acetate. Mean and S.E. of 8–10 experiments are shown. (C) Repeated application and washout of 1 mMlarixyl acetate to a HEKhTRPC6 cell. (D) Magnifi- cation of the current decay during acute extracellular addition of 1 mMlarixyl acetate and monoexponential fit of the decay kinetic. (E–H) Effects of 1 mM larixyl acetate on OAG-activated TRPC6 currents were tested in excised mem- brane patches during application to the intracellular surface in inside-out patches (E and F) or to the extracellular surface in outside-out patches (G and H). Shown are representative current traces and open probability (nPo) analyses, covering con- secutive 2-second bins. Magnified current traces represent time points of basal activity, OAG stimulation, and larixyl acetate as indicated by the numbers in the upper left panels. (F and H) Statistical analysis (mean and S.E.) of nPo from 8 to 10 cells under each condition. Inhibition of OAG-induced increases in nPo was tested for significance (P , 0.05) using the Student’s t test for unpaired data sets. 208 Urban et al. develop in a voltage-dependent fashion with outward currents of larixyl acetate appear less frequently, but can still reach preceding the development and lagging behind of the decay of amplitudes of more than 4 pA (see the insets in Fig. 7, E and inward currents, this partially voltage-sensitive behavior of G), a slow or flickery mode of channel inhibition is assumed. the blocker is not unexpected. Assessment of Cytotoxic Activity and Stability in To assess the reversibility of the inhibition, intracellularly Human Blood. The application of pharmacological TRPC6 perfused fluoroaluminate was used to obtain a persistent G modulators in more complex systems ex vivo or in vivo protein activation and more long-lasting TRPC6 currents. requires the absence of cytotoxic effects of the respective Inhibition by 1 mM larixyl acetate was again effective, and the modulator. To follow possible acute and subacute cytotoxic block was reversible upon washout of the compound (Fig. 7, A effects of larixol or larixyl acetate, we tested the membrane and B). A second addition of the blockers was again effective, integrity after 10-minute or 24-hour exposure of HEK293 cells indicating a slowly developing but fully reversible and repeat- to various compound concentrations with a propidium iodide able mode of action (Fig. 7, C and D). Of note, larixyl acetate assay. Under these conditions, no discernible membrane added to the pipette solution used for recordings in the whole- instability was elicited by larixol or larixyl acetate (Fig. 8, A cell mode did not prevent the activation of TRPC6 by in- and B). Likewise, cellular viability and proliferation appeared tracellularly perfused fluoroaluminate or extracellularly unaffected by both compounds as tested with a colorimetric applied OAG (gray trace and gray bars in Fig. 7, A and B, MTT conversion test (Fig. 8C). To gain first insight into the respectively). Under these conditions, TRPC6 activation was stability of larixyl acetate in human blood samples, the Downloaded from still blocked by adding larixyl acetate to the bath solution. To compound was added to citrate-supplemented whole-blood further substantiate the observation that larixyl acetate samples for up to 24 hours, and the remaining bioactivity was preferentially gets access to its binding site from the extra- tested in HEKhTRPC6-YFP cells by applying an appropriate cellular side, we compared the effectiveness of bath solution– dilution of cleared supernatants containing an initial concen- applied larixyl acetate in excised inside-out and outside-out tration of 10 mM larixyl acetate. The block of TRPC6 activity membrane patches. Indeed, TRPC6 current inside-out was not impaired by prolonged exposure to human blood molpharm.aspetjournals.org patches were less efficiently inhibited by 1 mM larixyl acetate obtained from three different donors (see Fig. 8D). (Fig. 7, E and F) than those measured in the outside-out Biologic Activity of Larixyl Acetate to Block Native configuration (Fig. 7, G and H). Owing to the very short TRPC6-Like Channels. TRPC6 is expressed in PASMCs. In (,1 millisecond) mean open time of TRPC6, the kinetics of the primary cultures of rat PASMCs, TRPC6 expression was block is hardly resolvable using electrophysiological patch- confirmed by western blot analysis, and immunofluorescence clamp methods. Since single-channel openings in the presence detection of smooth muscle-specific a-actin confirmed the at ASPET Journals on September 28, 2021

Fig. 8. Effects of larixyl acetate on cell viability, cell proliferation, and mainte- nance of biologic activity after prolonged incubation in full blood. (A–C) HEK293 cells were incubated with the indicated concentrations of larixyl acetate, buffer or solvent controls, and positive test com- pounds. Membrane integrity (A and B) and cellular metabolic activity (C) were tested by propidium iodide staining and MTT assays, respectively. To induce apo- ptosis or directly permeabilize the plasma membrane, 2 mM staurosporine or 50 mM digitonin were applied as controls. Data represent mean and S.E. of three to four experiments; #, significant differences to solvent (DMSO)-treated cells as tested by a Student’s t test for unpaired data. (D) Larixyl acetate was incubated with anti- coagulated (11 mM trisodium citrate) human full blood at 37°C for the indicated times. Aliquots were cleared (superna- tants of 2400g centrifugation), diluted to a final concentration of 10 mM in HBS and added to fluo-4-loaded and thapsigargin- pretreated (2 mM for 5 minutes) HEKhTRPC6-YFP cells. A solvent control (0.1% DMSO) was treated and diluted in a similar fashion. TRPC6 activation was induced by adding ATP (300 mM), carba- chol (1 mM), and thrombin 0.5 U/ml to stimulate PLC-coupled receptors. Depicted are increases in fluo-4-fluores- cence, normalized to values observed in the presence of the solvent. Larixyl Acetate Is a TRPC6 Inhibitor 209

21 purity of the PASMC cultures and ongoing expression of decline of [Ca ]i (see the gray line in Fig. 9B) compared with contractile filaments (Fig. 9A). In these cells, TRPC6-like the untreated controls. Upon preincubation with 1 or 5 mM 21 responses were elicited by stimulation with OAG (100 mM), larixyl acetate, the OAG-stimulated increase in [Ca ]i was which caused a significant rise of the intracellular Ca21 strongly or completely suppressed (see Fig. 9, C and D), concentrations in about 70%–100% of PASMCs, reaching an indicating efficient inhibition of native TRPC6 channel com- 21 average peak concentration of about 300 nM [Ca ]i (Fig. 9B). plexes. Inclusion of 5 mM bisindolylmaleimide-1 served to The acute addition of 5 mM larixyl acetate resulted in a rapid ascertain that these effects are independent of PKC activation Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 28, 2021

2+ 2+ Fig. 9. Larixyl acetate blocks OAG-induced increases in [Ca ]i but not stored depletion-associated Mn entry in primary cultures of PASMCs. Primary cultures of rat PASMCs were obtained and maintained without passaging for 5–7 days prior to the experiments. (A) Western blot analysis of TRPC6 and b-actin expression, and immunofluorescence staining of smooth muscle–specific a-actin in cultured PASMCs. Nuclei are counterstained with Hoechst 33258. A micrograph of a representative visual field taken by confocal laser-scanning microscopy is shown. (B) Adherent PASMCs were loaded with fura- 2+ 2+ 2/AM, and subjected to single-cell [Ca ]i determination using a digital fluorescence video imaging system. The [Ca ]i in PASMCs was recorded during sequential addition of the PKC blocker bisindolylmaleimide-1 (BIM), 100 mM OAG, and 5 mM larixyl acetate (gray line) or no TRPC6 modulator (black line). (C) Experiments as in (B), but with larixyl acetate (1 mM, light gray; 5 mM, dark gray) or no larixyl acetate (black line) preincubated for 5 minutes prior to OAG application. Shown are mean and S.E. of 7–15 independent experiments. (D) Statistical analysis of the basal and respective OAG-stimulated 2+ peak [Ca ]i from experiments as shown in (C); #, significant (P , 0.05) differences between OAG-stimulated control cells. (E) Manganese quench of fura-2 fluorescence was followed at the isosbestic wavelength of fura-2 (358 nm in our system), after incubating PASMCs with 5 mM larixyl acetate (dark gray), 10 mM lanthanum acetate (La3+; light gray), or the solvent control (0.1% DMSO; black line). Thapsigargin was added to deplete intracellular Ca2+ stores 3+ and to activate store-operated channels. Note that addition of 300 mM MnCl2 results in fura-2 quenching, which is inhibited by La but not by larixyl acetate. Data represent mean and S.E. of four independent determinations, each representing 90–160 cells. (F) Statistical analysis of four independent experiments performed as shown in (E). Depicted are mean fura-2 fluorescence intensities 350 seconds after the addition of 300 mM MnCl2 to the bath solution; #, statistically significant (P , 0.05) differences to thapsigargin-treated control cells in the absence of La3+. 210 Urban et al. by OAG. By contrast, the store/depletion-associated Mn21 Discussion entry in thapsigargin-treated PASMCs was unaffected by In this study, larch-derived diterpenes have been identified 5 mM larixyl acetate but completely abrogated by 10 mM as a new class of natural compounds that exhibit TRPC6- La31 (Fig. 9, E and F). Electrophysiological recordings of OAG- inhibitory biologic activity. In contrast to the recently reported stimulated whole-cell currents in PASMCs revealed TRPC6- subfamily spanning inhibitor norgestimate (Miehe et al., like transient inward and outward currents, reversing close to 2012) or a slightly more TRPC6-selective inhibitor 8009- 0 mV and showing the typical n-shaped current-voltage 5364 (Urban et al., 2012), larixol and larixyl acetate display relationship (Fig. 10). Acute addition of 5 mM larixyl acetate a marked selectivity for a single isotype of the closely related at a time point close to the maximal activation levels led to TRPC3/6/7 subfamily. Since electrophysiological analyses di- immediate decay of both outward and inward currents (Fig. rectly monitor the channel activity, the estimated IC value of 10B). Together, these data strongly suggest a reliable block of 50 larixyl acetate to block inward currents through TRPC6 is TRPC6-like channels but not store-operated channels by the about 0.1 mM. The selectivity of TRPC6 inhibition compared larixol derivative in rat PASMCs. with TRPC3 is about 12-fold. Thus, together with the recently Larixyl Acetate Prevents HPV. To test the impact of disclosed compound SAR7334, a substituted aminoindanole larixyl acetate on a physiologically relevant response that that also displays about 20- to 30-fold selectivity for TRPC6 involves TRPC6 activation, we measured the HPV, also over TRPC3 or TRPC7 (Maier et al., 2015), larixyl acetate can Downloaded from referred to as the von Euler-Lilljestrand mechanism. In the be considered a TRPC6-selective blocker. The inhibition of isolated mouse lung, a perfusion pressure of 5.7 6 0.3 mmHg TRPC6 activity is reversible, largely voltage-independent, and was required to achieve and maintain a baseline flow rate of affects both Ca21 and Na1 entry through TRPC6, while 2 2 50 ml/×kg/b.wt. 1×min 1 during normoxic ventilation. After leaving the GPCR-induced activation of PLC and PKC trans- switching to hypoxic ventilation the perfusion pressure in- location unaffected. Similar to the recombinantly expressed 1 creased by 2.5 6 0.2 mmHg, reaching peak values about 10 human TRPC6, native TRPC6-related Ca2 entry in rat molpharm.aspetjournals.org minutes after the onset of hypoxic ventilation. In the presence PASMCs was efficiently suppressed by low micromolar con- of 5 mM larixyl acetate applied via the perfusate, the increase centrations of larixyl acetate. Low cytotoxicity of the com- in the perfusion pressure upon hypoxic ventilation was sup- pound allowed the application of the inhibitor in an ex vivo pressed by 80% (P 5 0.008; n 5 5) (Fig. 11, A and B). Before model of HPV in mouse lungs, mimicking the effect of TRPC6 and 10 minutes after switching to hypoxic ventilation, the deficiency to abrogate the early vasoconstrictive response to vascular resistance was determined by applying a discontin- hypoxia known as the von Euler-Liljestrand mechanism. 2 2 uous flow ramp (25–100 ml/min 1×kg 1) and calculating the Previous publications have highlighted a prominent in- intrinsic vascular resistance R0, in which R0 increased from teraction of various natural compounds with TRP channels 2 2 2 0.25 6 0.03 to 0.53 6 0.13 mmHg×ml 1×kg/b.wt. 1×min 1 to activate, positively modulate, or inhibit the channel at ASPET Journals on September 28, 2021 during hypoxic ventilation; however, this effect was strongly activity. Our initial finding that conifer turpentines contain attenuated in mouse lungs that were perfused with 5 mM TRPC6- and TRPC3-inhibitory activity was a starting point larixyl acetate (Fig. 11C). for further screening of different conifer species and extracts.

Fig. 10. TRPC6-like ionic currents in primary PASMCs are inhibited by larixyl acetate. Rat PASMCs were obtained as in Fig. 9, but subjected to electrophysiologi- cal patch-clamp experiments to measure ionic currents in the whole-cell mode as in Fig. 6. (A and B) Inward and outward currents were measured by repeated ap- plication of voltage ramps as depicted in (C and D) during application of 50 mM OAG to the bath solution. Recordings from two representative cells with higher or lower current amplitudes are shown. Time points of voltage ramps shown in (C and D) are indicated in (A and B). (B and D) Larixyl acetate was added to the bath in the continued presence of OAG. Larixyl Acetate Is a TRPC6 Inhibitor 211

Fig. 11. Larixyl acetate blocks acute HPV in mouse lungs. Mouse lungs were cannu- lated and perfused with a modified Hanks’ balanced salt solution containing no (black line) or 5 mM larixyl acetate (gray line) at a constant flow rate of 50 ml/kg21×min21.(A) The pulmonary artery perfusion pressure (PAP) was measured before (normoxia) Downloaded from and after switching the ventilation from 21% O2 to a gas mixture containing 1% O2 (hypoxia). To measure the vascular resis- tance, the perfusion rate was briefly changed before and after hypoxic ventila- tion as shown in the lower chart. (B and C) Analysis of perfusion pressure (B) and vascular resistance (C) during experiments molpharm.aspetjournals.org performed as shown in (A). Data represent mean and S.E. of five independent exper- iments, each with and without larixyl acetate. Differences were considered sig- nificant (P , 0.05) after testing with the Mann-Whitney U test for unpaired (#) and Wilcoxon matched pairs test for paired (*) comparisons. at ASPET Journals on September 28, 2021

Since the turpentine of Larix decidua (European larch) promiscuous activity of larixyl acetate or other labdane- or displayed noticeable TRPC6-prevalent biologic activity, we labdene-type diterpenes in potently inhibiting Ca21-permeable focused our interest on identifying its active ingredient. Larch ion channels or affecting other modulators of cellular Ca21 turpentine consists of 15% (w/w) essential oils, which were not homeostasis. acting as TRPC6 inhibitors. The nondistillable resin contains Until the end of the twentieth century, Venetian turpentine, resin acids that are common for various Pinaceae and a mixture of larch turpentine and pine resin (colophony), was displayed inhibitory activity toward both TRPC6 and TRPC3. available in pharmacy and externally applied for wound In the genus Larix, the neutral fraction contains substantial disinfection. In addition, larch resin was slowly burned, and amounts of larixyl acetate (Sandermann and Bruns, 1965). By vapors were inhaled for relaxation and invigoration of re- testing subfractions, and finally the larch-specific resin com- spiratory organs. The larch resin was also used as a gum by ponents larixol and larixyl acetate, the selective inhibitory Native Americans, and in Siberia to treat toothaches. Larch activity was identified in the two chemically defined com- sawdust, a cheap by-product of the wood industry, has been pounds. In contrast to the biologic activity of some monoter- proposed as feed supplement for pigs and ruminants with anti- penes on cation channels, including TRP channels (Oz et al., inflammatory activity (Bauer et al., 2009). In vitro, labdane- 2015), a channel-modulating activity of larch-derived bicyclic type diterpenes have been implicated with inhibition of COX-2 diterpenes has not yet been described. Calcium channel and leukotriene LTB4 formation (Pferschy-Wenzig et al., inhibition by other labdane diterpenes has been occasionally 2008). The European Medical Agency lists larch turpentine reported (El-Bardai et al., 2003; de Oliveira et al., 2006); as Terebinthinae laricina for topical application as an anti- however, the inhibition of CaV1.2 currents required concen- septic and wound healing-promoting drug in veterinary trations that are more than two orders of magnitude higher medicinal products (EMEA/MRL/398/98). Likewise, larch than the inhibitory activity of larixyl acetate toward TRPC6 turpentine is contained in some commercially available currents. Thus, to our knowledge, there is no evidence of balsams and ointments. 212 Urban et al.

Orally ingested conifer turpentine has been reported to (Winn et al., 2005; Krall et al., 2010). On the other hand, exert acute toxicity with an LD50 of 5760 mg/kg in rats TRPC6 seems to protect podocytes from complement-induced (Skramlik 1959). The highly lipophilic and volatile essential injury (Kistler et al., 2013). Hence, further work is required to oils of conifer trees, such as pinenes and other bicyclic dissect out potential fields of use for pharmacological modu- monoterpenes, may cause toxicity upon inhalation, cutaneous lators of TRPC6. Of note, the marked TRPC6 expression in the exposure, or massive oral ingestion (Mercier et al., 2009). By dentate gyrus of the hippocampus (Nagy et al., 2013), contrast, the toxicity of the resiniferous ingredients in larch contributing to the regulation of neurite outgrowth (Heiser turpentines, including larixol or larixyl acetate has not been et al., 2013), may either give rise to additional fields of use or— reported in detail. To gain first insight into cytotoxicity and if inhibition would be detrimental—pose a need to avoid blood- possible interference with cell proliferation in the presence of brain-barrier permeability of pharmacological TRPC6 larixol and larixyl acetate, we performed a propidium iodide modulators. In addition, the acute modulation of TRPC6 staining and MTT assay in HEK293 cells. Only prolonged may circumvent problems that can arise from compensatory incubation with high micromolar concentrations of larixyl upregulation of other TRPC channels, an effect that has been acetate exerted a marked reduction of metabolic activity, observed in vascular smooth muscle cells of TRPC6-deficient indicative of a cytotoxic effect. animals (Dietrich et al., 2005). Therefore, the development of The biologic activity of larixyl acetate on native, TRPC6-like selectively acting pharmacological TRPC6 inhibitors is signals in PASMCs, and in abrogating acute HPV in mouse expected to fuel future studies that may contribute to a more Downloaded from lungs suggests that the compound is effective on native, precise understanding of physiologic and pathophysiological TRPC6-bearing complexes. The complex signaling functions of this member of the TRP channel family. mechanism behind HPV has not yet been fully elucidated; however, the available data suggest that several intermedi- Acknowledgments ates are critical. Besides TRPC6, other ion channels have been This work was supported by the Deutsche Forschungsgemeinschaft implicated in mediating HPV, including TRPV4 (Goldenberg (TRR 152). molpharm.aspetjournals.org et al., 2015), voltage-gated potassium channels, and obviously voltage-gated calcium channels in vascular smooth muscle Authorship Contributions cells (Veit et al., 2015). Therefore, selectivity tests were Participated in research design: Rademann, Kuebler, Schaefer. extended to cover the Ca21-permeable TRPV4 and CaV . Conducted experiments: Urban, Wang, Kwiek. 1.2 Contributed new reagents or analytic tools: Kwiek, Rademann. Although these channels are not (TRPV4) or are only poorly Performed data analysis: Urban, Kuebler, Schaefer. inhibited by larixyl acetate (CaV1.2), we cannot exclude that Wrote or contributed to the writing of the manuscript: Urban, other ion channels or nonchannel targets may contribute to Kuebler, Schaefer. the inhibition of HPV observed in isolated mouse lungs.

Owing to the ester bond, the stability of the natural References at ASPET Journals on September 28, 2021 compound may be compromised by unspecific serum ester- Abramoff MD, Magalhaes PJ, and Ram SJ (2004) Image processing with ImageJ. Biophotonics International 11:36–42. ases. Since the activity was maintained upon prolonged Bauer R, Wenzig EM, Franz C, Stefanon B, Tzika E, Kyriakis S, Farinacci M, and incubation in whole blood, the acetate ester seems to with- Sgorlon S (2 July 2009) inventors, Use larch wood material for treating in- flammation. WO 2009079680 A1. University of Graz, University of Udine, Aristotle stand these plasma esterases. However, at present the University Of Thessaloniki, University of Veterinary Medicine Vienna, Austria. pharmacokinetic properties of larixyl acetate, including oral de Oliveira AP, Furtado FF, da Silva MS, Tavares JF, Mafra RA, Araújo DA, Cruz JS, and de Medeiros IA (2006) Calcium channel blockade as a target for the cardio- bioavailability, distribution volume, metabolism, and elimi- vascular effects induced by the 8 (17), 12E, 14-labdatrien-18-oic acid (labdane-302). nation half-times are not yet known. Thus, long-term treat- Vascul Pharmacol 44:338–344. Dietrich A, Chubanov V, Kalwa H, Rost BR, and Gudermann T (2006) Cation ments, for example, to prevent or delay the progression of channels of the transient receptor potential superfamily: their role in physiological TRPC6-related kidney diseases in preclinical disease models, and pathophysiological processes of smooth muscle cells. Pharmacol Ther 112: 744–760. will require characterization of the absorption, distribution, Dietrich A, Mederos Y Schnitzler M, Gollasch M, Gross V, Storch U, Dubrovska G, metabolism, and excretion profile of larixyl acetate. Since Obst M, Yildirim E, Salanova B, and Kalwa H et al. (2005) Increased vascular smooth muscle contractility in TRPC62/2 mice. Mol Cell Biol 25:6980–6989. larixol is readily available in gram amounts, further com- Edelstein A, Amodaj N, Hoover K, Vale R, and Stuurman N (2010) Computer control pound optimization within the chemical entity may be prom- of microscopes using mManager. Curr Protoc Mol Biol Chapter 14:Unit14.20. doi: 10.1002/0471142727. ising. Other applications, such as protection from pulmonary El-Bardai S, Wibo M, Hamaide MC, Lyoussi B, Quetin-Leclercq J, and Morel N vasoconstriction, bronchoconstriction, and allergic airway (2003) Characterisation of marrubenol, a diterpene extracted from Marrubium disease, may be realized via nebulizer-assisted topic applica- vulgare, as an L-type calcium channel blocker. Br J Pharmacol 140:1211–1216. Finney-Hayward TK, Popa MO, Bahra P, Li S, Poll CT, Gosling M, Nicholson AG, tion, thereby circumventing uncertainties of hepatic first-pass Russell RE, Kon OM, and Jarai G et al. (2010) Expression of transient receptor metabolism and lowering the systemic exposure to the drug. potential C6 channels in human lung macrophages. Am J Respir Cell Mol Biol 43: 296–304. Although the process of validating TRPC6 as a potential Fuchs B, Rupp M, Ghofrani HA, Schermuly RT, Seeger W, Grimminger F, pharmacological target is still ongoing, specific pulmonary Gudermann T, Dietrich A, and Weissmann N (2011) Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6. Respir Res 12:20. and renal diseases are the most advanced examples in which Goldenberg NM, Wang L, Ranke H, Liedtke W, Tabuchi A, and Kuebler WM (2015) TRPC6 inhibition may resolve currently unmet medical needs. TRPV4 is required for hypoxic pulmonary vasoconstriction. Anesthesiology 122: 1338–1348. In the lung, dysregulated TRPC6 activity may contribute to Gudermann T, Hofmann T, Mederos y Schnitzler M, and Dietrich A (2004) Activa- pulmonary hypertension (Yu et al., 2009; Smith et al., 2015), tion, subunit composition and physiological relevance of DAG-sensitive TRPC proteins. Novartis Found Symp 258:103–118 (discussion 118–122, 155–159, 263– allergic airway disease (Sel et al., 2008), and ischemia/ 266). reperfusion-associated edema (Weissmann et al., 2012) or Heiser JH, Schuwald AM, Sillani G, Ye L, Müller WE, and Leuner K (2013) TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons in- permeability-type barrier failure (Samapati et al., 2012). In the volves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling. J Neurochem kidney, familial forms of focal segmental glomerulosclerosis are 127:303–313. Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, and Schultz G a prominent phenotype that can be caused by gain-of-function (1999) Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. mutations in the TRPC6 gene or by TRPC6 overexpression Nature 397:259–263. Larixyl Acetate Is a TRPC6 Inhibitor 213

Hofmann T, Schaefer M, Schultz G, and Gudermann T (2002) Subunit composition of Sel S, Rost BR, Yildirim AO, Sel B, Kalwa H, Fehrenbach H, Renz H, Gudermann T, mammalian transient receptor potential channels in living cells. Proc Natl Acad and Dietrich A (2008) Loss of classical transient receptor potential 6 channel re- Sci USA 99:7461–7466. duces allergic airway response. Clin Exp Allergy 38:1548–1558. Kistler AD, Singh G, Altintas MM, Yu H, Fernandez IC, Gu C, Wilson C, Srivastava Skramlik EV (1959) [On the toxicity and tolerance of ethereal oils]. Pharmazie 14: SK, Dietrich A, and Walz K et al. (2013) Transient receptor potential channel 6 435–445. (TRPC6) protects podocytes during complement-mediated glomerular disease. J Smith KA, Voiriot G, Tang H, Fraidenburg DR, Song S, Yamamura H, Yamamura A, Biol Chem 288:36598–36609. Guo Q, Wan J, and Pohl NM et al. (2015) Notch activation of Ca21 signaling Krall P, Canales CP, Kairath P, Carmona-Mora P, Molina J, Carpio JD, Ruiz P, Mezzano mediates hypoxic pulmonary vasoconstriction and pulmonary hypertension. Am J SA, Li J, and Wei C et al. (2010) Podocyte-specific overexpression of wild type or Respir Cell Mol Biol 53:355–367. mutant trpc6 in mice is sufficient to cause glomerular disease. PLoS One 5:e12859. Tabeling C, Yu H, Wang L, Ranke H, Goldenberg NM, Zabini D, Noe E, Lenz JC, Reusch HP, Albrecht N, Schultz G, and Schaefer M (2002) Ca21-controlled Krauszman A, Gutbier B, and Yin J et al. (2015) CFTR and sphingolipids competitive diacylglycerol binding of protein kinase C isoenzymes in living cells. J mediate hypoxic pulmonary vasoconstriction. Proc Natl Acad Sci USA 112: Cell Biol 159:291–302. E1614–E1623. Maier T, Follmann M, Hessler G, Kleemann HW, Hachtel S, Fuchs B, Weissmann N, Urban N, Hill K, Wang L, Kuebler WM, and Schaefer M (2012) Novel pharmaco- Linz W, Schmidt T, and Löhn M et al. (2015) Discovery and pharmacological logical TRPC inhibitors block hypoxia-induced vasoconstriction. Cell Calcium 51: characterization of a novel potent inhibitor of diacylglycerol-sensitive TRPC cation 194–206. channels. Br J Pharmacol 172:3650–3660. Veit F, Pak O, Brandes RP, and Weissmann N (2015) Hypoxia-dependent reactive Mercier B, Prost J, and Prost M (2009) The essential oil of turpentine and its major oxygen species signaling in the pulmonary circulation: focus on ion channels. volatile fraction (a- and b-pinenes): a review. Int J Occup Med Environ Health 22: Antioxid Redox Signal 22:537–552. 331–342. WangL,YinJ,NicklesHT,RankeH,TabuchiA,HoffmannJ,TabelingC, Miehe S, Crause P, Schmidt T, Löhn M, Kleemann HW, Licher T, Dittrich W, Rütten Barbosa-Sicard E, Chanson M, and Kwak BR et al. (2012) Hypoxic pulmonary H, and Strübing C (2012) Inhibition of diacylglycerol-sensitive TRPC channels by vasoconstriction requires 40-mediated endothelial signal conduction. synthetic and natural steroids. PLoS One 7:e35393. J Clin Invest 122:4218–4230.

Nagy GA, Botond G, Borhegyi Z, Plummer NW, Freund TF, and Hájos N (2013) DAG- Wang YX and Zheng YM (2011) Molecular expression and functional role of canonical Downloaded from sensitive and Ca21 permeable TRPC6 channels are expressed in dentate granule transient receptor potential channels in airway smooth muscle cells. Adv Exp Med cells and interneurons in the hippocampal formation. Hippocampus 23:221–232. Biol 704:731–747. Okada T, Inoue R, Yamazaki K, Maeda A, Kurosaki T, Yamakuni T, Tanaka I, Weissmann N, Dietrich A, Fuchs B, Kalwa H, Ay M, Dumitrascu R, Olschewski A, Shimizu S, Ikenaka K, and Imoto K et al. (1999) Molecular and functional char- Storch U, Mederos y Schnitzler M, and Ghofrani HA et al. (2006) Classical tran- acterization of a novel mouse transient receptor potential protein homologue TRP7. sient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary Ca21-permeable cation channel that is constitutively activated and enhanced by vasoconstriction and alveolar gas exchange. Proc Natl Acad Sci USA 103: stimulation of G protein-coupled receptor. J Biol Chem 274:27359–27370. 19093–19098. Ong HL, de Souza LB, Cheng KT, and Ambudkar IS (2014) Physiological functions Weissmann N, Sydykov A, Kalwa H, Storch U, Fuchs B, Mederos y Schnitzler M,

and regulation of TRPC channels. Handbook Exp Pharmacol 223:1005–1034. Brandes RP, Grimminger F, Meissner M, and Freichel M et al. (2012) Activation of molpharm.aspetjournals.org Oz M, Lozon Y, Sultan A, Yang KH, and Galadari S (2015) Effects of monoterpenes on TRPC6 channels is essential for lung ischaemia-reperfusion induced oedema in ion channels of excitable cells. Pharmacol Ther 152:83–97. mice. Nat Commun 3:649. Pferschy-Wenzig EM, Kunert O, Presser A, and Bauer R (2008) In vitro anti- Welsh DG, Morielli AD, Nelson MT, and Brayden JE (2002) Transient receptor po- inflammatory activity of larch (Larix decidua L.) sawdust. J Agric Food Chem 56: tential channels regulate myogenic tone of resistance arteries. Circ Res 90: 11688–11693. 248–250. Reiser J, Polu KR, Möller CC, Kenlan P, Altintas MM, Wei C, Faul C, Herbert S, Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, Daskalakis Villegas I, and Avila-Casado C et al. (2005) TRPC6 is a glomerular slit diaphragm- N, Kwan SY, Ebersviller S, and Burchette JL et al. (2005) A mutation in the associated channel required for normal renal function. Nat Genet 37:739–744. TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science Richter JM, Schaefer M, and Hill K (2014) Clemizole hydrochloride is a novel and 308:1801–1804. potent inhibitor of transient receptor potential channel TRPC5. Mol Pharmacol 86: Yu Y, Fantozzi I, Remillard CV, Landsberg JW, Kunichika N, Platoshyn O, Tigno 514–521. DD, Thistlethwaite PA, Rubin LJ, and Yuan JX (2004) Enhanced expression of Romanin C, Gamsjaeger R, Kahr H, Schaufler D, Carlson O, Abernethy DR, transient receptor potential channels in idiopathic pulmonary arterial hyperten- and Soldatov NM (2000) Ca21 sensors of L-type Ca21 channel. FEBS Lett 487: sion. Proc Natl Acad Sci USA 101:13861–13866. 301–306. Yu Y, Keller SH, Remillard CV, Safrina O, Nicholson A, Zhang SL, Jiang W, Vangala at ASPET Journals on September 28, 2021 Samapati R, Yang Y, Yin J, Stoerger C, Arenz C, Dietrich A, Gudermann T, Adam D, N, Landsberg JW, and Wang JY et al. (2009) A functional single-nucleotide poly- Wu S, and Freichel M et al. (2012) Lung endothelial Ca21 and permeability re- morphism in the TRPC6 gene promoter associated with idiopathic pulmonary ar- sponse to platelet-activating factor is mediated by acid sphingomyelinase and terial hypertension. Circulation 119:2313–2322. transient receptor potential classical 6. Am J Respir Crit Care Med 185:160–170. Sandermann W and Bruns K (1965) Über die Konformation des Larixols. Tetra- hedron Lett 42:3757–3760. Address correspondence to: Dr. Michael Schaefer, Universität Leipzig, Schaefer M, Albrecht N, Hofmann T, Gudermann T, and Schultz G (2001) Diffusion- Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Härtelstraße 16- limited translocation mechanism of protein kinase C isotypes. FASEB J 15: 18, 04107 Leipzig, Germany. E-mail: [email protected] 1634–1636.