Ion channel drug discovery

Ion channel drug discovery is a rapidly evolving field fuelled by recent, but significant, advances in our understanding of ion channel function combined with enabling technologies such as automated electrophysiology. The resurgent interest in this target class by both pharmaceutical and academic scientists was clearly highlighted by the over-subscribed RSC/BPS 'Ion Channels as Therapeutic Targets' symposium in February 2009. This book builds on the platform created by that meeting, covering themes including advances in screening technology, ion channel structure and modelling and up-to-date case histories of the discovery of modulators of a range of channels, both voltage-gated and non-voltage-gated channels. The editors have built an extensive network of contacts in the field through their first-hand scientific experience, collaborations and conference participation and the organisation of the meeting at Novartis, Horsham, increased the network enabling the editors to draw on the experience of eminent researchers in the field. Interest and investment in ion channel modulation in both industrial and academic settings continues to grow as new therapeutic opportunities are identified and realised for ion channel modulation. This book provides a reference text by covering a combination of recent advances in the field, from technological and medicinal chemistry perspectives, as well as providing an introduction to the new 'ion channel drug discoverer'. The book has contributions from highly respected academic researchers, industrial researchers at the cutting edge of drug discovery and experts in enabling technology. This combination provides a complete picture of the field of interest to a wide range of readers

Molecular and cellular changes in vein grafts: influence of pulsatile stretch

Adaptation of saphenous vein, with its intrinsic myogenic tone, from the low-pressure, minimally pulsatile flow of the venous system to the pulsatile flow of the arterial circulation is a minor miracle. Changes in gene expression caused by the pulsatile circumferential deformation (cyclic strain) initiate changes in gene expression, which lead to both vein graft adaptation and pathology. Removal of circumferential deformation by external stenting attenuates the early changes in gene expression and the later development of intimal hyperplasia. Pathways for the transduction of cyclic strain into cellular events have been elucidated in cultured vascular cells: The important second messengers include calcium and reactive oxygen species

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. © Springer-Verlag 2005

Regulation of airway mucosal hydration

Ion channels control the hydration status of the airway epithelium through apical anion secretion and cation absorption, which is accompanied by osmotically obligated water. The key channels in this process are the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), which is principally responsible for Cl- secretion by airway epithelial cells, and the epithelial Na+ channel (ENaC), which is responsible for the absorption of Na ions. In CF, defective CFTR-mediated Cl- secretion and an accompanying upregulation in ENaC-mediated Na absorption results in a reduction in airway surface liquid volume, leading to poorly hydrated mucus and impaired mucociliary clearance. Restoration of normal airway hydration by modulation of ion channel activity represents an important therapeutic strategy for CF. CFTR corrector and potentiator compounds are being developed with the aim of recovering normal Cl- secretion. Ca2+-activated Cl- channels (CaCCs) are expressed by the respiratory epithelia and are reported to be functionally upregulated in CF and offer a 'surrogate pathway for Cl- secretion. TMEM16A has recently been described as a CaCC in the airway epithelium and, as such, represents an alternative target for restoring Cl- secretion in CF. An alternative therapeutic strategy for CF is to inhibit ENaC, thereby blocking excessive Na absorption. This can be achieved by direct blockade of ENaC or inhibition of the channel-activating proteases (CAPs), whose activity regulates ENaC function. This review will describe the regulation of airway mucosal hydration by ion channels and the efforts currently underway to restore normal mucosal hydration in disease patients by modulating the function of these channels. © 2010 Expert Reviews Ltd

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. © Springer-Verlag 2005

Shear stress-induced shedding of soluble intercellular adhesion molecule-1 from saphenous vein endothelium

Within 6 h, shear stress upregulated intercellular adhesion molecule-1 (ICAM-1) (two- to four-fold, P<0.001) and induced matrix metalloproteinase-2 (MMP-2) in cultured human saphenous vein endothelial cells. By 8 h endothelial ICAM-1 levels returned to baseline, with concomitant increase in soluble ICAM-1 (sICAM-1) (P<0.001) and MMP-9 had been induced. Inclusion of a hydroxamate metalloproteinase inhibitor partially reversed the effects on ICAM-1 and sICAM-1 at 8 h, whereas TIMP-1, -2 or -3 had no effect. MMP-9, but not MMP-2, co-immunoprecipitated with ICAM-1. sICAM-1 was processed distal to Arg441, indicating that MMP-9, docking to ICAM-1, contributes to sICAM-1 shedding and attenuation of the shear stress-induced upregulation of ICAM-1. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

Arterial flow conditions downregulate thrombomodulin on saphenous vein endothelium

Background - The antithrombogenic properties of venous endothelium may be attenuated when vein is implanted in the arterial circulation. Such changes may facilitate thrombosis, which is the final common pathway for saphenous vein arterial bypass graft occlusion. Methods and Results - Using human saphenous vein in a validated ex vivo flow circuit, we investigated (1) the possibility that arterial flow conditions (mean pressure, 100 mm Hg, 90 cpm, ?200 mL/min) alter the concentration of proteins involved in regulating thrombosis at the vessel wall and (2) the influence of ion channel blockade on such effects. Concentrations of thrombomodulin and tissue factor were quantified by Western blotting (ratio of yon Willebrand factor staining) and immunohistochemistry (as a percentage of CD31-staining area). Thrombomodulin concentrations after 90 minutes of venous and arterial flow conditions were quantified by immunostaining (68.9±4.8% and 41.0±3.0% CD31, respectively; P<0.01) and by Western blotting (1.35±0.20 and 0.15±0.03 ratio of yon Willebrand factor, respectively; P<0.01). The ability of endothelial cells to generate activated protein C also decreased from 62±14 to 19±10 ng · min- 1 · 1000 cells-1 (P=0.01). The significant reduction in thrombomodulin was attenuated if calcium was removed from the perfusate but not by external vein stenting. Inclusion in the vein perfusate of drugs that reduce calcium entry (including Gd3+, to block stretch-activated ion channels, and nifedipine) abolished the reduction in thrombomodulin concentration observed after arterial flow conditions. In freshly excised vein, negligible concentrations of tissue factor were detected on the endothelium and concentrations did not increase after 90 minutes of arterial flow conditions, although the inclusion of nifedipine caused the immunostaining to increase from 3.0±0.4% to 8.5±0.7% CD31 (P<0.02). Conclusions - In saphenous vein endothelium exposed to arterial flow conditions, there is rapid downregulation of thrombomodulin, sufficient to limit protein C activation, by a calcium-dependent mechanism

Rapid changes in the coagulant proteins on saphenous vein endothelium in response to arterial flow

Healthy endothelium provides a nonthrombogenic surface. In this study the authors investigated the effect of arterial flow on the saphenous vein endothelial expression of proteins controlling thrombosis. Human saphenous vein segments, freshly excised from patients, were placed in a validated in vitro circuit with flow conditions shown to simulate arterial or venous circulations. In separate experiments, placement of an external polytetrafluoroethylene (PTFE) stent was used to differentiate the effects of pulsatile wall deformation and shear stress, while addition of drugs to the vein perfusate allowed study of the role of ion channels in transducing the response of the vein to arterial flow. Endothelial concentrations of thrombomodulin, nitric oxide synthase, tissue factor, and tissue plasminogen activator were assessed by quantitative immunohistochemistry and Western blotting of endothelial cell lysates, in paired vein samples, in comparison to control proteins. Arterial flow conditions caused a rapid and significant reduction in the endothelial concentration of thrombomodulin: The immunostaining area decreased from 80.1 ± 7.0 to 48.3 ± 5.0 and 32.9 ± 3.0% at 45 and 90 minutes respectively, p = 0.01. These findings were confirmed by Western blotting. The reduction in thrombomodulin concentration was unaffected by eliminating vein wall deformation by placement of an external PTFE stent or by including the K+ channel blocker tetraethylammonium (TEA) in the vein perfusate. In contrast, thrombomodulin concentrations remained high when blockers of stretch-activated cation and calcium channels were included in the vein perfusate. The endothelial concentration of nitric oxide synthase increased after 90 minutes of arterial flow and this change was abolished when TEA was included in the vein perfusate. Arterial flow induced rapid changes in saphenous vein antithrombotic proteins. Different cation channels mediated the flow-induced changes in thrombomodulin and nitric oxide synthase

The effects of potassium channel openers on saphenous vein exposed to arterial flow

Objectives: To assess the sensitivity of saphenous vein to potassium channel opening drugs (KCOs). Methods: Saphenous vein, harvested at bypass surgery or high ligation for correction of varicose veins, was exposed to an in vitro flow circuit and vasomotor responses assessed by organ bath pharmacology. Outcome measures: Effective drug concentrations for 50% reduction in vein ring tension (IC50). Results: Vein rings pre-contracted with phenylephrine showed a concentration-dependent relaxation to all the KCOs tested with a potency ranking of HOE 234 > cromakalim > pinacidil > diazoxide. The relaxation to cromakalim was endothelium-independent and was inhibited by glibenclamide (an ATP-sensitive K+ channel blocker). The sensitivity of vein rings to cromakalim increased after exposure to arterial flow conditions for 90 minutes (IC50 before 1.7 ± 0.25 µM and after 0.25 ± 0.08 µM, p > 0.001). This effect was not evident after 90 min of venous flow conditions, 2.19 ± 0.49 µM. When the workload on vein, exposed to arterial flow conditions, was reduced mechanically by external stenting with PTFE the increased sensitivity to cromakalim was abolished. Conclusion: Saphenous vein has ATP-sensitive K+ channels responsive to KCOs. The increased sensitivity to cromakalim, induced by arterial flow conditions, may represent and endogenous protective mechanism limiting ischaemic damage resulting from the higher workload imposed on grafted vein

Maternal and postnatal vitamin D ingestion influences rat aortic structure, function and elastin content

Objectives: Subtle impairment of fetal nutrition appears to predict hypertension and atherosclerosis in adults. It has been hypothesised that impaired aortic elastogenesis is the initiating step in adult hypertension and aortic aneurysms. Vitamin D has been shown to inhibit elastin synthesis by cultured smooth muscle cells. Here we have investigated, in rats, the hypothesis that increased exposure to vitamin D during gestation and in the postnatal period alters aortic elastin content and aortic function. Methods: Nine breeding pairs of Sprague-Dawley rats were allocated to one of three diets containing 3000 (control group), 6000 (low dose) or 12,000 (high dose) IU/kg vitamin D during pregnancy and lactation. Male pups were continued on the same diet until 6 weeks of age. Aortic elastin content was assessed by measuring desmosine+isodesmosine content using capillary zone electrophoresis. Transverse aortic sections were used for quantification of elastic lamellae and morphometric analysis. The contractility of aortic rings was assessed in an organ bath preparation. Results: The desmosine+isodesmosine content of the abdominal aorta of 6-week-old male pups, was 14.1, 10.0 and 10.1 nmol/mg dry weight in the control (n=20), low- (n=23) and high-dose (n=15) groups, respectively (P=0.007). The median number of elastic lamellae of the distal thoracic aorta was 8.25, 7.13 and 6.88 in the control, low-dose and high-dose groups, respectively (P<0.001). There were no significant differences in aortic cross-sectional areas or media:adventitia ratios. The mean peak tension of aortic rings, in response to phenylephrine, was 1.3 g, 1.12 g and 0.87 g in the control, low- and high-dose groups respectively (P=0.002). Conclusion: In rats, exposure to increased amounts of vitamin D during gestation and early life results in a reduction of aortic elastin content, number of elastic lamellae in the aorta and force generation in aortic rings. © 2002 Elsevier Science B.V. All rights reserved