Chris Poll

TRP channels: Emerging targets for respiratory disease

The mammalian transient receptor potential (TRP) superfamily of cation channels is divided into six subfamilies based on sequence homology TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin) and TRPML (mucolipin). The expression of these channels is especially abundant in sensory nerves, and there is increasing evidence demonstrating their existence in a broad range of cell types which are thought to play a key role in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). These ion channels can be activated by a diverse range of chemical and physical stimuli. Physical stimuli include temperature, membrane potential changes and osmotic stress, and some of the more well known chemical stimuli include capsaicin (TRPV1), menthol (TRPM8) and acrolein (TRPA1). There is increasing evidence in this rapidly moving field to suggest that selective blockers of these channels may represent attractive novel strategies to treat characteristic features of respiratory diseases such as asthma and COPD. This review focuses on summarising the evidence that modulation of selected TRP channels may have beneficial effects at targeting key features of these respiratory diseases including airways inflammation, airways hyper-reactivity, mucus secretion and cough.

Activation of Human TRPC6 Channels by Receptor Stimulation

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K0.5 of ∼10 μm and a Hill coefficient of 3–4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30–300 μm) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 μm) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.

Receptor-operated Ca2+ influx channels in leukocytes: a therapeutic target?

Receptor-mediated activation of leukocytes by inflammatory stimuli elicits Ca2+ ion influx as a common and important activation mechanism that has been well established in the literature for over a decade. Inhibiting such receptor-operated Ca2+ influx channels is a potentially attractive strategy for developing anti-inflammatory drugs to attenuate leukocyte activation. Until very recently, the molecular identity of these channels has been unknown, which has hampered drug development in this area. However, the recent explosion of molecular information about one particular family of non-voltage-activated Ca2+ channels, the transient receptor potential (TRP) channels, together with emerging knowledge of their distribution, function and regulation, suggests that they represent a key subgroup of these channels and are therefore potentially attractive drug targets.

Transient receptor potential (TRP) channels as potential drug targets in respiratory disease

Calcium-permeable channels have traditionally been thought of as therapeutic targets in excitable cells. For instance, voltage-operated Ca2+ channels in neurones and smooth muscle cells for neurological and cardiovascular diseases although calcium-permeable channels are also functionally important in electrically non-excitable cells. In the lung, calcium channels play a pivotal role in the activation of all the cell types present, whether resident cells such as airway smooth muscle cells and macrophages or migratory cells such as neutrophils or lymphocytes.Previously, research in this area has been hindered by the lack of obvious molecular identity. More recently, the emergence of the transient receptor potential (TRP) cation family has yielded promising candidates which may underpin the different receptor-operated calcium influx pathways. The challenge now, is to ascribe function to the TRP channels expressed in each cell type as a first step in identifying which TRP channels may be potential drug targets for asthma and chronic obstructive pulmonary disease (COPD) (Fig. 1).

TRP channels in airway smooth muscle as therapeutic targets

Cation channels are of fundamental importance in regulating the function of airway smooth cells especially bronchoconstriction in response to spasmogens, and are therefore key players in the pathogenesis of asthma. To date, the identity of these cation channels remains a mystery. However, the recently emerged transient receptor potential (TRP) cation channel family has provided several promising channel candidates. The identification of the key TRP channels involved in regulating airway smooth muscle contractility, and therefore airway tone, could provide new and exciting prospects for the development of novel therapies for the treatment of airway diseases such as asthma.

Highlights of a workshop to discuss targeting inflammation in cystic fibrosis

A workshop to discuss anti-inflammatory approaches in the treatment of CF was held at Novartis Institutes for Biomedical Research (NIBR, Horsham, UK) in March 2008. Key opinion leaders in the field (Hugo De Jonge, Stuart Elborn, Erich Gulbins, Mike Konstan, Rick Moss, Scott Randell and Adriano Rossi), and NIBR scientists were brought together to collectively address three main aims: (i) to identify anti-inflammatory targets in CF, (ii) to evaluate the pros and cons of targeting specific cell types and (iii) to discuss model systems to profile potential therapeutic agents. The highlights of the workshop are captured in this review.

Therapeutic scope of modulation of non-voltage-gated cation channels.

Although widely regarded as attractive drug targets, less than a tenth of known ion channels are currently commercially exploited as therapeutic targets. Historically, drug discovery efforts on ion channel targets have been encumbered by a lack of molecular and structural information, sub-optimal screening technologies and a paucity of discriminating pharmacological tools. Although challenges remain, recent scientific and technological advances in the area of ion channel research and screening offer the exciting prospect of a new, more-predictive era of ion channel drug discovery. In this article, focusing primarily on non voltage gated cation channels, we describe the continuing evolution of approaches to ion channel drug discovery, highlight recent developments in the ion channel field and consider their potential impact on discovering and ascribing function to ion channel targets. We discuss the renaissance of known ion channel targets, such as nicotinic acetylcholine receptors and calcium-activated potassium channels, as well as the emergence of the transient receptor potential (TRP) channels as a gene family of cation channels with broad therapeutic potential.

Determining the functional role of TRPC channels in primary cells

Although the TRPC members of the mammalian transient receptor potential TRP cation channel family were the first to be described in 1995, the depth of knowledge of TRPC channels has fallen behind that of their counterparts in the TRPV and TRPM subfamilies in the intervening years. The complexities and controversies of TRPC channel composition and regulation have hindered their progress as therapeutic targets in the drug discovery environment to date, however embracing these challenges as opportunities may bring TRPC channels to the forefront of the discovery of novel therapies for many diseases. These challenges and opportunities of exploring TRPC channels as therapeutic targets are highlighted and discussed in this review with respect to respiratory diseases.

Characterisation of a refined rat model of respiratory infection with Pseudomonas aeruginosa and the effect of ciprofloxacin.

BACKGROUND We sought to characterise a refined rat model of respiratory infection with P. aeruginosa over an acute time course and test the antibiotic ciprofloxacin. METHODS Agar beads were prepared ± SPAN(®)80. Rats were inoculated with sterile agar beads or those containing 10(5) colony forming units (cfu) P. aeruginosa via intra-tracheal dosing. Bacterial load and inflammatory parameters were measured. RESULTS Differing concentrations of SPAN(®) 80 modified median agar bead diameter and reduced particle size distribution. Beads prepared with 0.01% v/v SPAN(®)80 were evaluated in vivo. A stable lung infection up to 7 days post infection was achieved and induced BALF neutrophilia 2 and 5 days post infection. Ciprofloxacin (50mg/kg) significantly attenuated infection without affecting the inflammatory parameters measured. CONCLUSION SPAN(®) 80 can control the particle size and lung distribution of agar beads and P. aeruginosa-embedded beads prepared with 0.01%v/v SPAN(®)80 can induce infection and inflammation over 7 days.

Neutral sphingomyelinase-2, acid sphingomyelinase, and ceramide levels in COPD patients compared to controls

Background Increased pulmonary ceramide levels are suggested to play a causative role in lung diseases including COPD. Neutral sphingomyelinase-2 (nSMase-2) and acid SMase (aSMase), which hydrolyze sphingomyelin to produce ceramide, are activated by a range of cellular stresses, including inflammatory cytokines and pathogens, but notably cigarette smoke appears to only activate nSMase-2. Our primary objective was to investigate nSMase-2 and aSMase protein localization and quantification in lung tissue from nonsmokers (NS), smokers (S), and COPD patients. In addition, various ceramide species (C16, C18, and C20) were measured in alveolar macrophages from COPD patients versus controls. Materials and methods Patients undergoing surgical resection for suspected or confirmed lung cancer were recruited, and nSMase-2 and aSMase protein was investigated in different areas of lung tissue (small airways, alveolar walls, subepithelium, and alveolar macrophages) by immunohistochemistry. Ceramide species were measured in alveolar macrophages from COPD patients and controls by mass spectrometry. Results nSMase-2 and aSMase were detected in the majority of small airways. There was a significant increase in nSMase-2 immunoreactivity in alveolar macrophages from COPD patients (54%) compared with NS (31.7%) (P<0.05), and in aSMase immunoreactivity in COPD (68.2%) and S (69.5%) alveolar macrophages compared with NS (52.4%) (P<0.05). aSMase labeling was also increased in the subepithelium and alveolar walls of S compared with NS. Ceramide (C20) was significantly increased in alveolar macrophages from COPD patients compared with controls. Conclusion nSMase-2 and aSMase are both increased in COPD alveolar macrophages at the protein level; this may contribute toward the elevated ceramide (C20) detected in alveolar macrophages from COPD patients.