Vasoconstrictor-stimulated phosphoinositide signalling in caveolae/lipid rafts of intact small arterie

Caveolae/rafts, specialised plasma membrane microdomains, were recently implicated in vascular smooth muscle (VSM) contraction but all work to date has focused on protein signalling pathways. Vasoconstrictors such as noradrenaline (NA) and endothelin-1 (ET-1) signal through phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) producing the second messengers inositol-1,4,5-trisphosphate and diacylglycerol (DAG). DAG is phosphorylated by DAG kinases (DGK) to produce phosphatidic acid (PA) - also a second messenger with distinct cellular targets. Both NA and ET-1-stimulated PIP2 hydrolysis was localised to caveolae/rafts. This involved the PLC-delta1 isoform in an extracellular calcium- dependent manner for NA but not for ET-1 suggesting agonist-specific activation of PLC isoforms. Both NA and ET-1-stimulated DGK activity and DGK-derived PA production was localised to caveolae/rafts although this was not exclusive for ET-1. Whilst DGK activity in response to either agonist was unaffected by extracellular calcium removal, PA levels were reduced suggesting calcium-dependent regulation of PA metabolism. NA-stimulated DGK activity and PA production was partially dependent on phosphoinositide 3-kinase (PI3K) with the PI3K-dependent DGK activity localising to caveolae/rafts. However, ET-1-stimulated DGK activity and PA production were independent of PI3K suggesting agonist-specific activation of DGK isoforms. The PI3K-dependent DGK0 isoform and the downstream PI3K target protein kinase B (PKB/Akt) were both present in caveolae/rafts. Furthermore, PKB/Akt levels in caveolae/rafts were increased by NA but not ET-1. This suggests differential activation of DGK0 and this could be a result of different effects of NA and ET-1 on PKB/Akt levels in caveolae/rafts. Cholesterol depletion is reported to disrupt caveolae/rafts and enhanced the contractile response to NA in intact RMSA. Cholesterol depletion enhanced NA but not ET-1-stimulated PIP2 hydrolysis and had no effect on DGK activity with either agonist. However, PA levels for both NA and ET-1 were increased. Whilst this suggests that caveolae/raft disruption affects vasoconstrictor-stimulated PI signalling in VSM, it was unclear if caveolae/rafts were disrupted in RMSA as evidenced by different effects of cholesterol depletion on caveolae/raft markers. Additionally, cholesterol depletion is now known to have effects other than disruption of caveolae/rafts, which could also account for the differences observed. In conclusion, the data presented in this thesis supports a role for caveolae/rafts in regulation of vascular contractility as sites where agonist-stimulated PI turnover couples to intracellular signalling pathways through localised production of second messengers. The agonist-specific activation of enzyme isoforms and spatial differences in production of lipid second messengers are likely to be important for agonists that utilise a common signalling system.