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Angiotensin II and endothelin-1 signalling in aortic vascular smooth muscle cells.
thesisposted on 2015-11-19, 08:44 authored by Clare. Morton
The rationale behind this thesis was based around the observation that AII and ET-1 were seen to be mitogenic in vascular smooth muscle cells derived from SPIR but not in those derived from WKY rats. This was then used as a model to investigate the AII and ET-1 stimulated mitogenic pathways in vascular smooth muscle cells. AII and ET-1 both stimulated [3H]-thymidine incorporation in SHR derived cells in a dose dependent manner. This data produced a log EC50 of -8.23 0.1 M for AII and -9.24 0.06 M for ET-1. However, no stimulations were observed in AII and ET-1 stimulated WKY derived cells. This increase in [3H]-thymidine incorporation correlated with an increase in cell counts and therefore, indicated a mitogenic response. In addition, time course studies indicated that no stimulations had been missed either side of 24 hours in WKY derived cells. All stimulated [3H]-thymidine incorporation in SHR cells was challenged with an increasing concentration of n- butanol producing a 40% inhibition, which was maximal at a 30mM concentration. However, the ET-l stimulation of [3H]-thymidine incorporation was not affected by n-butanol even at the highest concentrations used. Tertiary butanol did not act as a nucleophile in the transphosphatidylation assay and was therefore, deemed a suitable negative control. It did not have any significant effect on AII stimulated [3H]-thymidine incorporation in SHR cells which would suggest that n-butanol was not mediating non specific effects. AII and ET-1 stimulated PLC activity in SHR and WKY cells with an enhanced PLC activity observed in SHR cells with both agonists. This was seen to be dose dependent and therefore, a receptor activated event with an EC50 of -7.31 0.06 M for AII. AII also stimulated PLD activity in SHR and WKY cells in a dose dependent manner, with an elevated PLD activity present in SHR cells. However, ET-1 only stimulated PLD activity in WKY cells with no significant stimulation in SHR cells. The PKC inhibitor Ro-318220 was shown to completely block AII stimulated PLD activity in SHR and WKY cells. This was also the case in ET-1 stimulated PLD activity in WKY cells. In addition, TPA stimulated PLD activity in SHR and WKY cells on its own. This data indicates that PLD activity is at least partially regulated by PKC activation and therefore, downstream of PLC activation. AII and ET-1 stimulated tyrosine phosphorylation in both SHR and WKY cells. This stimulation was, however, significantly elevated in SHR compared to WKY cells. In addition, the stimulated tyrosine phosphorylation was attenuated by the tyrosine kinase inhibitors, genistein and methyl 2,5 dihydroxycinnamate. However, the two inhibitors did appear to exhibit differential specificity for certain phosphorylated tyrosine residues. Tyrosine kinase inhibition with genistein, methyl 2,5 dihydroxycinnamate and tyrphostin also produced inhibition of AII stimulated PLD activity in SHR cells. This suggested a tyrosine kinase regulation of PLD activation as well as PKC. Genistein (10-100?M) was also seen to prevent AII stimulated [3H]-thymidine incorporation in SHR cells which implicated both tyrosine kinases and PLD activity in AII stimulated mitogenesis. Tyrosine kinase inhibition had no effect on AII stimulated PLC activation providing evidence that tyrosine kinase regulation of PLD activity occurs more directly at the level of PLD activation. In summary the data presented reveals a differential mitogenic response of AII and ET-1 in SHR compared to WKY cells. This may be due to the enhanced activation of PLC and subsequent PLD activation. However, the ET-1 stimulated PLD activation in SHR cells may not be as critical as for AII. In addition, stimulated tyrosine phosphorylation may also be a requirement for a mitogenic response for both AII and ET-1.
Date of award1996-01-01
Author affiliationCell Physiology and Pharmacology
Awarding institutionUniversity of Leicester