Novel pharmacological TRPC inhibitors block hypoxia-induced 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.
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