Effect of ATP and UTP on cytokine release, cell viability and collagen synthesis in cardiac fibroblasts

Amarnath Talasila, Renee Germack and John M. Dickenson, School of Biomedical and Natural Sciences, Nottingham Trent University, Nottingham, UK.

Cardiac fibroblasts (CFs) play a vital role in wound healing, hypertrophy and fibrosis observed in cardiovascular diseases. Angiotensin-II (ANG-II) is also associated with the development of hypertrophy and pro-inflammatory properties in heart failure. We have previously reported that rat neonatal CFs functionally express P2Y1,11,13 (ATP), P2Y2 (ATP/UTP) and P2Y6 (UDP/UTP) receptors (Talasila et al., 2005). Since ATP and UTP are released during cardiac ischemia the aim of this study was to determine the effect of these nucleotides on cytokine release (IL-1β , IL-6, TNF- a and TGF- β1), cell viability and collagen synthesis in CFs exposed hypoxia (HX, 0.5% O2) and ANG II (50nM) as model of ischemic heart disease.

Neonatal rat CFs were isolated from 1-3 day-old Wistar rats. Cytokine release was measured by ELISA at 1,2,4,8,18 h of exposure to normoxia (NX), NX/ANG-II, HX, and HX/ANG-II in the presence or absence of ATP-γS (32μM; stable analogue of ATP) and UTP (10μM). Similarly, CF cell viability was assessed by LDH assay and insoluble collagen was evaluated using [3H]-proline at 4 and 18 h exposure.

ATP-γS increased IL-6 release under NX (2h: 110±2% of NX control, 4h: 112±4, 8h: 119±3, 18h: 116±3%; P<0.001; n=9) and HX (2h: 118±3, 4h: 118±5%, 8h: 117±5%; P<0.001; n=8) conditions. Furthermore ATP-γS potentiated ANG-II-induced IL-6 release both in NX (2h: 133±10, 4h: 126±9, 8h: 140±15, 18h: 150±10%; P<0.001; n=5) and HX (2h: 131±11, 4h: 129±7, 8h: 135±17, 18h: 136±13%; P<0.01; n=5) treatments. UTP did not stimulate the production of IL-6. ATP-γS and UTP did not affect the production of IL-1β and TNF- a in NX and HX. However ATP-γS and UTP potentiated ANG-II-induced IL-1β release at 4h (ATP-γS: 113±7, P<0.01, n=5; UTP: 114±3%, P<0.01, n=5). At 18h in HX, UTP inhibited ANG-II-induced IL-1β release (87±6%, P<0.01, n=6) whereas ATP-γS had no effect (97±2%, n=6). UTP augmented TNF- a release at 4h: (117±3%, P<0.001, n=4) in NX/ANG-II. TGF- β1 production was only increased by UTP at 4h: (119±6%, P<0.05, n=4) in NX. ATP-γS had no effect on TNF- a and TGF- β1 in all groups. Since cytokine production was affected by ATP-γS and UTP at 4 and 18h cell viability and collagen synthesis experiments were carried at these time points. Cell viability was not significantly modified by 4h HX exposure whereas 18h HX induced cell death by 252 ± 38% (n=3, p<0.001). ANG-II and the P2Y agonists had no effect on HX-induced cell death. ANG-II induced insoluble collagen production in cells exposed to NX and HX for 4h and 18h. ATP and UTP inhibited ANG-II induced collagen synthesis in cells exposed to 18h HX.

In conclusion the nucleotides ATP and UTP stimulate the production of the hypertrophic cytokine IL-6 and the pro-inflammatory cytokine IL-1β , whereas only UTP induced anti-inflammatory cytokine TGF-β1 release in CFs. In addition, P2Y receptors seem to be involved in hypertrophy development rather than in cardiac protection. Future work will identify which P2Y receptors (P2Y 1,2,6,11,13) are responsible for the modulation of cytokine release and collagen synthesis in CFs.

 Talasila, A. et al (2005) 3 rd James Black Conference, Oxford, UK