| Summary: | Ion channel permeability and calcium ion (Ca2+) signalling are essential in regulating cellular responses to external and internal stimuli. Changes in the Ca2+ signalling machinery during pathologies of the cardiovascular system are a common occurrence, with an underlying deregulation of Ca2+ signalling a driving force for many pathological processes. This study particularly focusses on the change in Ca2+ regulatory mechanisms under conditions of adversity that are often attributed to pathological environments. An elevation of inflammatory mediators and an acidic extracellular environment can be present in multiple pathologies, including cancer and atherosclerosis. This study uses Ca2+ measurement techniques and patch-clamp electrophysiology to demonstrate that the transient receptor potential canonical (TRPC) class of ion channels are stimulated in an environment where high levels of the inflammatory mediator arachidonic acid (AA) are present alongside extracellular acidosis. This environment was demonstrated in both human umbilical vein endothelial cells (HUVECs) and HT29 colorectal adenocarcinoma cells. Antibody inhibition of individual TRPC subunits could inhibit AA-evoked signalling by greater than 40 % in HUVECs and greater than 50 % in HT29 cells. It is suggested that these signalling pathways are partly mediated by TRPC1, TRPC4 and TRPC5 heteromeric ion channel formations. This hypothesis was supported by the ability to stimulate TRPC5 homomeric channels overexpressed in HEK293 cells with AA in conditions of extracellular acidosis. Tools for investigating endogenous TRPC ion channel function are limited and lack potency and specificity. A search for small molecule TRPC modulators identified two novel TRPC5 inhibitor series. C42a1 was the most potent of those compounds investigated, with an IC50 of 4.28 ± 0.04 μM against Gd3+-evoked TRPC5 activity. The TRPC4β/TRPC5 activator Englerin-A ((-)EA) was utilised to demonstrate a 4.1-fold selectivity for TRPC5 over TRPC4β for C42a1. This is the first study utilising this direct channel agonist in both Ca2+ measurement and patch-clamp assays against these channel types. Modifying the chemical structure of the C42 inhibitor series identified compounds with altered selectivity and potency. This compound series also demonstrated good selectivity for TRPC5 over closely related ion channels that were investigated (TRPC6, TRPM2, TRPM3, and Orai1). TRPC activation by AA and (-)EA could both evoke cell death in a variety of cell types with (-)EA particularly effective in renal cell carcinoma, as has been previously reported. Our ability to modulate cell death using our novel TRPC inhibitors was limited, assumed to be due to our inability to completely block Ca2+ signalling. Protection of cells against (-)EA-evoked cell death using the C42 inhibitor series was more successful compared to that for AA-evoked cell death. This was likely due to the wide-array of activity of AA and its metabolites on protein function, with only a small cell death component likely attributable to TRPC activation. This study identified novel TRPC-mediated activation mechanisms that have relevance in cellular functions during cardiovascular disease and cancer pathology. The newly identified classes of TRPC inhibitors are in the early stage of development though initially demonstrate the potential to be developed to yield highly potent and therapeutically valuable modulators of endogenous TRPCs.
|
|---|
