Regulation of Ca2+-dependent vasoconstriction in large and small arteries.
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Date
2013
Authors
Chan, Yann Yoong
Editors
Advisors
Wilson, David Peter Murray
Beltrame, John Francis
Rychkov, Grigori
Beltrame, John Francis
Rychkov, Grigori
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Abstract
Vasospasm, of coronary, cerebral or peripheral circulation, is associated with myocardial infarction, stroke and claudication. Many forms of vasospasm are well characterised and can be effectively managed using therapies including nitrates and L-type Ca²⁺ channel blockers. However, some forms of vasoconstriction are less well managed by these therapies. Evidence also indicates that endothelin-1 (ET-1) can potentiate vasoconstriction elicited by other agonists. Consequently, an elevation in local and/or circulating ET-1, for example inflammation associated with acute coronary syndrome, has prompted us to explore the extent to which Ca²⁺ release from the sarcoplasmic reticulum (SR) and Ca²⁺ sensitisation contribute to acute ET-1- mediated vasoconstriction. To examine this hypothesis, we coupled functional measurement of the isometric tension in isolated rat mesenteric arterial segments with biochemical analysis of the downstream target of Rho kinase, myosin phosphatase (MYPT). We identified that SR Ca²⁺ release contributed significantly to ET-1- dependent vasoconstriction. Using a fixed Ca²⁺ concentration coupled with biochemical analysis, we also demonstrated the contribution of RhoA/ Rho kinase-dependent Ca²⁺ sensitisation, consistent with an increased phosphorylation state at Threonine-855 site of MYPT in an ET-1-dependent vasoconstriction in the microvasculature. Extracellular Na⁺ entry and Cl⁻, on the other hand, are not required to mediate ET-1-mediated contractile response. The inflammatory milieu not only contains ET-1, but also platelet-derived thromboxane A₂ (TxA₂). In the context of TxA₂ receptor-mediated vasoconstriction, there remains a gap in our understanding as to how TxA₂ causes membrane depolarisation and subsequent activation of L-type Ca²⁺ channels. Using functional vascular myography, we have identified that extracellular Na⁺ entry through NSCC and Cl⁻ channels are required for the activation of L-type Ca²⁺ channels in TxA₂₋dependent vasoconstriction in the small rat mesenteric arteries; whilst K⁺ fluxes through the BKCa channels activates the L-type Ca²⁺ channels in TxA₂₋dependent vasoconstriction in large rat caudal arterial preparation. In addition, we examined the relative efficacy of short-term, 30-minute high dose simvastatin administration in attenuating vasoconstriction in rat caudal and mesenteric arteries, challenged with the TxA₂ mimetic, U46619. We demonstrated in functional experiments that short-term 30-minute, high dose simvastatin treatment attenuated TxA₂ receptor-mediated vasoconstriction in both rat caudal and mesenteric arteries, with greater efficacy in the small arteries. Specifically, this reduction was endothelium-independent in the mesenteric arterial preparation. However, an increase in the phosphorylation state of eNOS suggested that there may be a tonic nitric oxide release and simvastatin may have the capacity to mediate an increase in the phosphorylation of Serine-1177 eNOS independently. In addition, biochemical western blot analysis revealed that the Threonine-855 phosphorylation state of MYPT was not influenced by 30-minute simvastatin treatment in both arterial preparations. Similarly, Threonine-697 phosphorylation state of MYPT also remained unaffected by either simvastatin treatment or TxA₂ stimulation in rat mesenteric arteries. Perhaps more interestingly, the voltage-dependent L-type, Ca⌄1.2 current was inhibited by simvastatin in a TxA₂ receptor-mediated fashion, consistent with our functional data which showed that simvastatin significantly attenuated the contractile response to high K⁺ depolarisation. Taken together, these data provide both evidence and rationale for the development and short-term use of SR Ca²⁺ release blockade therapy in addition to the implementation of Rho kinase inhibitor for patients with acute vasospastic disorders, who are resistant to conventional nitrate and L-type CCB therapies. Our data also outlined several vasomotor mechanisms including the L-type Ca²⁺ channel blockade and increase in eNOS activity, independent from the statin’s cholesterol lowering property, which may be responsible for the benefit of short-term statin therapy in reducing mortality following cardiac intervention.
School/Discipline
School of Medical Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2013
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Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.