Elsevier

Cell Calcium

Volume 51, Issue 2, February 2012, Pages 194-206
Cell Calcium

Novel pharmacological TRPC inhibitors block hypoxia-induced vasoconstriction

https://doi.org/10.1016/j.ceca.2012.01.001Get rights and content

Abstract

The Ca2+-permeable, nonselective cation channel TRPC6 is gated via phospholipase C-activating receptors and has recently been implicated in hypoxia-induced pulmonary vasoconstriction (HPV), idiopathic pulmonary hypertension and focal segmental glomerulosclerosis (FSGS). Therefore, TRPC6 is a promising target for pharmacological interference. To identify and develop TRPC6-blocking compounds, we screened the Chembionet library, a collection of 16,671 chemically diverse drug-like compounds, for biological activity to prevent the 1-oleoyl-2-acetyl-sn-glycerol-triggered Ca2+ influx in a stably transfected HEKTRPC6-YFP cell line. Hits were validated and characterised by fluorometric and electrophysiological methods. Six compounds displayed inhibitory potency at low micromolar concentrations, lack of cytotoxicity and blocked the receptor-dependent mode of TRPC6 activation. The specificity was tested towards closely (TRPC3 and TRPC7) and more distantly related TRP channels. One of the compounds, 8009-5364, displayed a 2.5-fold TRPC6-selectivity compared to TRPC3, and almost no inhibition of TRPC7 or the other TRP channels tested. Block of native TRPC3/6-like responses was confirmed in dissociated pulmonary artery smooth muscle cells. Two non-polar blockers effectively suppressed the HPV responses in the perfused mouse lung model. We conclude that pharmacological targeting of TRPC6 is feasible and provide a promising concept to treat pulmonary diseases that are characterised by excessive hypoxic vasoconstriction.

Introduction

To ensure an efficient blood oxygenation under resting conditions with low tidal volumes, perfusion of poorly ventilated areas in the lung is limited by hypoxia-induced vasoconstriction (HPV), also referred to as Euler-Lilijestrand mechanism [1]. The HPV reduces shunting of poorly oxygenated blood and increases perfusion in well-ventilated areas of the lung. This effect is especially remarkable since, in other tissues, hypoxia typically triggers a vasodilation rather than vasoconstriction in order to prevent end organ damage. TRPC6, a non-selective cation channel of the transient receptor potential (TRP) channel family [2], has been shown to critically contribute to the early phase of acutely applied HPV (aHPV) [3]. Hence, the molecular pathways that are underlying the Euler–Liljestrand mechanism are now beginning to emerge [4]. Under pathophysiological conditions, HPV may increase the global pulmonary resistance. Consequently, it increases the work burden of the right ventricle and lowers the left atrial filling pressure, eventually leading to right heart failure. In addition, a constitutive dysregulation of pulmonary vasoconstriction or vascular remodelling may cause pulmonary hypertension. The physiological relevance of TRPC6 for regulating pulmonary hemodynamics is fostered by association of the −254(C- > G) allele with idiopathic pulmonary arterial hypertension [5]. Pulmonary artery smooth muscle cells (PASMC) isolated from patients that carry the −254G allele display an increased and NFκB-dependent TRPC6 expression and function. In line with these observations, siRNA-mediated knock-down of TRPC6 expression has been shown to attenuate proliferation of PASMC [6].

Besides the pulmonary phenotypes, mutations in the TRPC6 gene are linked with focal segmental glomerulosclerosis (FSGS), a kidney disease that is characterised by proteinuria and frequently leads to nephrotic syndrome and end-stage renal disease [7]. Although expression of TRPC6 has been shown in podocytes, the cells that form the filtration slits in the Bowman's capsule, and some of the TRPC6 mutations are reported to increase the channel activity, the exact mechanisms of the disease are not yet understood. Since FSGS accounts for about 16% of cases of nephrotic syndrome, and often results in kidney failure, pharmacological inhibition of TRPC6 may be helpful for understanding of and interfering with this severe disease.

The group of TRPC channels contributes to a plethora of physiological functions that are triggered via phospholipase C-coupled receptors, including G-protein-coupled receptors (GPCR) or receptor tyrosine kinases (RTK). Depending on the tissue distribution of individual TRPC channel subunits and the respective co-expression of phospholipase C-activating receptors, homo- or heteromeric TRPC ion channel complexes form a versatile signalling network in vivo by triggering membrane depolarisation and Ca2+ entry. The members of the TRPC3/6/7 subfamily share the unique property of being directly activated by diacylglycerols [8], [9]. The compounds 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 1,2-dioctanoyl-sn-glycerol are membrane-permeable diacylglycerol mimics that open these channels, while bypassing receptor and phospholipase C stimulation. Biophysical properties of TRPC3/6/7 are very similar. They display reversal potentials close to 0 mV in physiological ion concentrations, very short mean open times (<1 ms) and a dually rectifying current–voltage relationship with a prominent outward rectification at potentials higher than +30 mV. Until now, no TRPC6-selective blockers are available [10]. Moreover, most recently described TRPC blockers exhibit a low potency and poorly select between the families. Actually, the frequently applied organic blockers 2-aminoethoxydiphenyl-borate (2-APB) or SKF-96365 as well as the assessment of concentration-dependent effects of La3+ or Gd3+ do not fulfil the criteria that would be desirable for a reliable cell-biological tool and, owing to their toxicity, are not applicable in more complex systems such as ex vivo or in vivo models of diseases.

By screening the Chembionet collection of chemically diverse drug-like molecules [11], we identified and subsequently characterised compounds that block TRPC6 activation in both OAG- and receptor-operated modes of action. The compounds displayed biological activity in TRPC6-expressing PASMC and in a murine model of hypoxia-induced vasoconstriction, indicating that the newly identified chemical entities may provide a valid pharmacological strategy to interfere with disease states such as pulmonary hypertension.

Section snippets

Cell culture and transfections

HEK293 cells were grown in Earle's Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum, 2 mM l-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. Cells were seeded in 35-mm culture dishes, grown for 24 h, and transfected at 80% confluence with 2 μg of plasmid DNA encoding a C-terminally YFP-tagged human TRPC6 and 4 μl of Fugene HD reagent (Roche) in 100 μl serum-free medium. To select stably transfected HEKhTRPC6-YFP clones, 1 mg/ml geneticin (G418) was added to the

Characterisation of the HEKTRPC6-YFP cell line and primary screening

To ensure a consistent functional expression of human TRPC6, we generated and clonally selected a HEK293 cell line (HEKhTRPC6-YFP), upon transfection with a C-terminally YFP-tagged human TRPC6 construct. As expected from earlier results from transiently transfected cells [15], confocal imaging of living HEKhTRPC6-YFP cells revealed expression of a fluorescent protein, which was efficiently inserted into the plasma membrane (Fig. 1A). The cells robustly and homogenously responded to stimulation

Discussion

TRP channel-mediated pathophysiological mechanisms and familiar disorders that involve TRP channel mutations are still emerging [19], and pharmacological targeting of the increasing number of TRP channel-mediated channelopathies is an upcoming and demanding task. The recent finding that activating mutations in TRPC6 or promoter mutations that lead to TRPC6 overexpression are linked with pulmonary hypertension and focal segmental glomerulosclerosis strongly point to a therapeutic potential of

Acknowledgments

The work with the Chembionet compound collection was supported by Ronald Kühne and Jens-Peter von Kries, both Leibniz-Institute of Molecular Pharmacology, FMP, Berlin-Buch, Germany. The cDNA of human TRPA1 was kindly provided by David Julius, UCSF, San Francisco, CA, USA. This work was supported by grants of the Deutsche Forschungsgemeinschaft to M.S. within the framework of the Research Groups FOR748 and FOR806.

References (44)

  • N. Sommer et al.

    Regulation of hypoxic pulmonary vasoconstriction: basic mechanisms

    Eur. Respir. J.

    (2008)
  • L.J. Wu et al.

    International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family

    Pharmacol. Rev.

    (2010)
  • N. Weissmann et al.

    Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

    Proc. Natl. Acad. Sci. U. S. A.

    (2006)
  • Y. Yu et al.

    A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension

    Circulation

    (2009)
  • Y. Yu et al.

    PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression

    Am. J. Physiol. Cell Physiol.

    (2003)
  • J. Reiser et al.

    TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function

    Nat. Genet.

    (2005)
  • T. Hofmann et al.

    Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol

    Nature

    (1999)
  • R. Inoue et al.

    The transient receptor potential protein homologue TRP6 is the essential component of vascular α1-adrenoceptor-activated Ca2+-permeable cation channel

    Circ. Res.

    (2001)
  • C. Harteneck et al.

    Pharmacological modulation of diacylglycerol-sensitive TRPC3/6/7 channels

    Curr. Pharm. Biotechnol.

    (2011)
  • M. Lisurek et al.

    Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept

    Mol. Divers.

    (2010)
  • J.C. Lenz et al.

    Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells

    J. Cell Biol.

    (2002)
  • F. Spöhr et al.

    4-Aminopyridine restores impaired hypoxic pulmonary vasoconstriction in endotoxemic mice

    Anesthesiology

    (2007)
  • Cited by (64)

    • Ca<sup>2+</sup> transport via TRPV6 is regulated by rapid internalization of the channel

      2022, Cell Calcium
      Citation Excerpt :

      After loading the coverslips were mounted in a bath chamber and HBS buffer containing the indicated [Ca2+]i was added. Measurements were performed on an inverted epifluorescence microscope with a Fluar 10 × 0.5 objective (Zeiss) and calibrated as described [21,22]. Coverslips with living cells were mounted on a self-made total internal reflection fluorescence microscope.

    • Valdecoxib blocks rat TRPV2 channels

      2022, European Journal of Pharmacology
      Citation Excerpt :

      Furthermore, we find that the combined addition of two TRPV2 activators, probenecid and 2-APB, is having highly synergistic effects on heterologously and endogenously expressed rat TRPV2 channels, enabling the more selective activation of rat TRPV2 channels in native tissues. HEK293 cells were stably transfected with CFP-tagged rat TRPV2 (HEKTRPV2), CFP-tagged rat TRPV1, CFP-tagged mouse TRPV3, and YFP-tagged mouse TRPV4 using a limiting dilution protocol as described before (Urban et al., 2012). Cells were grown in Earle's minimum essential medium (MEM; Sigma, Munich, Germany) supplemented with 10% fetal calf serum (Gibco Thermo Fisher Scientific, Darmstadt, Germany), 2 mM l-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.6 mg/ml geneticin.

    • TRPC channels: Structure, function, regulation and recent advances in small molecular probes

      2020, Pharmacology and Therapeutics
      Citation Excerpt :

      Because of the prominent expression of TRPC6 in lung tissues and its involvement in lung function and diseases, including hypoxic vasoconstriction, lung ischemia-reperfusion edema (LIRE) and idiopathic PAH (IPAH) (Weissmann et al., 2006; Weissmann et al., 2012; Yu et al., 2004), it would be highly desirable to verify the therapeutic value of inhibiting TRPC6 channels in lung diseases using pharmacological tools. A number of compounds, such as SKF-96365, econazole, W7, compound 8009-5364, norgestimate, and sildenafil, have been shown to inhibit TRPC6 through either direct or indirect mechanisms (Bon & Beech, 2013; Harteneck & Gollasch, 2011; Lu et al., 2010; Miehe et al., 2012; Urban, Hill, Wang, Kuebler, & Schaefer, 2012; Wang et al., 2013). However, limitations exist due to their low potency and poor selectivity.

    • TRPC channels: Regulation, dysregulation and contributions to chronic kidney disease

      2019, Biochimica et Biophysica Acta - Molecular Basis of Disease
    • Pyrazolo[1,5-a]pyrimidine TRPC6 antagonists for the treatment of gastric cancer

      2018, Cancer Letters
      Citation Excerpt :

      Thus, identification of new molecular targets of gastric cancer and development of effective treatment approaches based on these targets are major focus areas of clinical or translational cancer research. Transient receptor potential canonical 6 (TRPC6) proteins form receptor-operated Ca2+-permeable channels, which have been implicated in kidney [11–13], pulmonary [14–18] and cardiac diseases [19,20]. Therefore, drugs that target TRPC6 are thought to bring benefit to the treatment of diseases, including cancers [21–26].

    View all citing articles on Scopus
    View full text