Lipopolysaccharide Exposure Sensitises TRPV4 Activity In Cultured Bovine Aortic Endothelial Cells

C.A. Sand, M. Nandi, A.D. Grant. King's College London, London, UK

The cation channel Transient Receptor Potential Vanilloid 4 (TRPV4) is expressed in vascular endothelial and smooth muscle cells. It causes vasodilatation in response to acetylcholine or shear stress through stimulation of nitric oxide release or direct hyperpolarisation of vascular smooth muscle (1). Excessive TRPV4 activation leads to profound hypotension and circulatory collapse in mice (2). We have previously observed an enhanced TRPV4 response in keratinocytes exposed to lipopolysaccharide (LPS). We hypothesised that LPS-induced potentiation of TRPV4 activity contributes to endothelial cell dysfunction and death.

Functional expression of TRPV4 was assessed by RT-PCR and calcium fluorimetry in human umbilical vein endothelial cells (HUVECs), bovine aortic endothelial cells (bAECs), a murine dermal microvascular endothelial cell line (sEND1) and murine aortic lysate. Cell viability was determined with the MTT assay.

The selective TRPV4 agonist GSK1016790A (1nM – 10µM) caused a concentration-dependent increase in internal [Ca2+] in bAECs (pEC50 = 7.28 ± 0.23). Pre-incubation with the TRPV4 antagonist HC067047 (1µM) decreased the pEC50 to 5.94 ± 0.24 (p<0.01, unpaired t test; n=4). Incubation with LPS (1-100ng/ml; 1 or 24hr) caused a concentration-dependent enhancement at 24hr of normalised Ca2+ increase in response to GSK1016790A (100nM) (p<0.01, 1-way ANOVA followed by Bonferroni’s post-hoc test). Incubation with LPS (1ng – 1µg/ml; 24hr) caused a concentration-dependent decrease in cell viability that was unaffected by co-incubation with HC067047 (10µM).

We conclude that functional TRPV4 is expressed in vascular endothelial and smooth muscle cells. Exposure to LPS enhanced the response to the TRPV4 agonist GSK1016790A, suggesting a sensitisation of the channel. However, the decrease in cell viability was not prevented by a TRPV4 antagonist, indicating that it is not caused by an increase in tonic TRPV4 activity. Thus, potentiation of TRPV4 activity during sepsis could contribute to altered endothelial signalling and vascular dysfunction.

1. Sonkusare, S. K. et al. (2012). Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function. Science, 336: 597-601.

2. Willette, R. N. et al. (2008). Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2. J. Pharmacol. Exp. Ther., 326: 443-52.