An investigation of NAADP-dependent Ca²⁺ signalling mechanisms in arterial smooth muscle
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Previous investigations on pulmonary artery smooth muscle cells have shown that nicotinic acid adenine dinucleotide diphosphate (NAADP) evokes highly localised intracellular Ca²⁺ bursts by mobilising thapsigargin-insensitive Ca²⁺ stores. Such localised Ca²⁺ signals may initiate global Ca²⁺ waves and contraction of the myocytes through the recruitment of ryanodine receptors (RyR) located on the sarcoplasmic reticulum (SR) via Ca²⁺-induced Ca²⁺-release (CICR). In this thesis I have shown that NAADP evokes localised Ca²⁺ signals through the mobilisation of a bafilomycin A1-sensitive, lysosome-related Ca²⁺ store. Lysosomal Ca²⁺ stores facilitate this process by colocalising with a subpopulation of RyRs on the surface of the SR to comprise a highly specialised trigger zone for Ca²⁺ signalling by NAADP. I have also shown that the proposed trigger zone for NAADP-dependent Ca²⁺ signalling may be formed between lysosomes and clusters of RyR subtype 3 (RyR3) located in close proximity to one another in the perinuclear region of cells. Localised Ca²⁺ bursts generated by NAADP-dependent Ca²⁺ release from acidic Ca²⁺ stores and subsequent CICR via RyR3 on the SR may then amplify Ca²⁺ bursts into a propagating Ca²⁺ signal by recruiting clusters of RyR subtype 2 (RyR2) located in the perinuclear and extra-perinuclear regions of the cell. The presence of this trigger zone may explain, in part, why Ca²⁺ bursts by NAADP induce, in an all-or-none manner, global Ca²⁺ signals by CICR via RyRs on the SR. Consistent with a role for NAADP and lysosomes as a discrete and agonist-specific Ca²⁺ signalling pathway utilised by vasoconstrictors, I have shown that endothelin-1 (ET-1), but not phenylephrine or prostaglandin-F2α, mobilises Ca²⁺ stores by NAADP, and that ET-1 initiates Ca²⁺ signalling by NAADP in a receptor subtype-specific manner through the activation of ETB receptors. These findings further advance our understanding of how that spatial organisation of discrete, organellar Ca²⁺ stores underpin the generation of differential Ca²⁺ signalling patterns by different Ca²⁺-mobilising messengers.
Thesis, PhD Doctor of Philosophy
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