Effects Of Angiotensin Peptides On Calcium Mobilisation In Fibroblasts From Healthy And Idiopathic Pulmonary Fibrosis Lung Tissue

SM Maitland1, AP Sampson1, JA Warner1, DA Hall2. 1University of Southampton, Southampton, UK, 2GlaxoSmithKline, Stevenage, UK

Fibroblasts are a key cell type responsible for the deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF). Angiotensin (ANG) II increases fibroblast proliferation and ECM deposition (Hafizi et al, 2004), but little is known about other angiotensin peptides, such as ANG(1-7), and their effects on fibroblasts. The aim of this study was to determine the ability of ANG peptides to stimulate intracellular Ca2+ release in healthy and IPF human lung fibroblasts (HLF and ILF respectively).

Intracellular Ca2+ release was measured using a fluorescence imaging plate reader (FLIPR) tetra as previously described (Strachan et al, 2009). In brief, fibroblasts were seeded at 10000 per well in black-walled, clear-bottomed 96 well plates and grown to confluency. Cells were loaded with 2µM FLUO-4AM at 37oC for one hour and the FLIPR tetra used to measure fluorescence upon peptide addition. To allow Ca2+ concentrations to be calculated, MnCl2 (5mM) and ionomycin (5µM) were added to each well at the end of the experiment. Peptides that failed to induce Ca2+ release were retested as antagonists of ANGII induced Ca2+ release.

ANGI, ANGII and ANGIII mobilised calcium with mean pEC50 values of 5.87±0.17, 7.67±0.01 and 7.43±0.02 respectively in HLFs (n≥6), with no difference in the pEC50 values observed between HLFs and ILFs (6.12±0.13, 7.72±0.06 and 7.22±0.19 respectively, n≥6). However, compared to HLFs (Mann-Whitney U, n≥6), ILFs displayed significantly lower maximal Ca+ responses to ANGI (median of 170.4nM, IQR of 105.2-269.1nM versus 57.6nM, IQR of 33.9-134.1nM, p<0.05), ANGII (277.5nM, IQR of 240.3-403.7nM, versus 133.5nM, IQR of 115.7-158.5nM), p<0.0001) and ANGIII (308.8nM, IQR of 157.2-373.0nM, versus 119.5nM, IQR of 77.82-174.3nM, p<0.01). Telmisartan (AT1R antagonist, 10nM), caused complete inhibition of the calcium response to all active peptides in both cell types (n=3), with no effect observed in the presence of PD-123319 (AT2R antagonist) or A-779 (Mas antagonist).

Pretreatment with 10µM ANG(1-7) caused 40.5±20.1 (n=3) and 46.4±7.9% (n=5) inhibition of the Ca2+ response to ANGII in HLFs and ILFs respectively. ANG(1-9) (10µM) caused an inhibition of 63.1±4.7 (n=5) and 63.34±10.9% (n=4) in HLFs and ILFs respectively. Upon further investigation, a pIC50 of 5.71±0.25 for ANG(1-7) was documented against 10µM ANGII in ILFs. For ANG(1-9), pIC50 values of 5.54±0.09 in HLFs (n=3) and 5.49±0.3 in ILFs (n=3) were recorded. Two similar pIC50 values were observed in the presence of lower agonist concentrations (0.1 and 1µM ANGII). Furthermore, the inhibition observed with ANG(1-9) was not reversed by PD-123319, A-779 or D-Pro7-ANG(1-7) (ANG(1-7) receptor antagonist) in HLFs (n=3) or ILFs (n=3). This lack of reversibility was also observed for ANG(1-7) in ILFs (n=3).

Three ANG peptides generate a Ca2+ response in both HLFs and ILFs, via AT1R. Two peptides, ANG(1-7) and ANG(1-9) cause partial inhibition of this Ca2+ release that does not appear to be via simple competitive antagonism, nor is reversed by the addition of PD-123319, A-779 or D-Pro7-ANG(1-7), indicating a novel binding site for these peptides.

1. Hafizi S et al, Peptides 25:1031, 2004

2. Strachan RT et al, J Biol Chem 284:5557, 2009