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Mitochondria and microdomains in vascular smooth muscle cell Ca²⁺ signalling Poburko, Damon Todd


Contraction of vascular smooth muscle (VSM) is regulated by fluctuations in the intracellular concentration of free ionic calcium ([Ca²⁺][sub i]). The spatio-temporal regulation of [Ca²⁺][sub i] relies on the sub-cellular architecture of the smooth muscle cell and the juxtaposition of opposing plasmalemma (PM), sarcoplasmic reticulum (SR) and mitochondria. This thesis addresses two related aspects of Ca²⁺-signaling in VSM: 1) basal Ca²⁺-entry across the PM and 2) mitochondrial Ca²⁺-uptake during agonist mediated stimulation in cultured rat aorta smooth muscle cells. Basal Ca²⁺-entry into resting cells, measured with radio-labeled ⁴⁵Ca²⁺, was blocked (~80%) by organic inhibitors of L-type voltage-gated Ca²⁺-channels (nifedipine), store-/receptoroperated Ca²⁺-channels (SKF-96365) and inositol-1,4,5-trisphosphate receptors (IP₃R) (2-APB). At increasing concentrations, gadolium (Gd³⁺) biphasically inhibited Ca²⁺-uptake. The maximal effect of the first phase (100μM Gd³⁺) was equally as effective as combined treatment with 2-APB and SKF-96365. At 0.2-10mM, Gd³⁺ inhibited Ca²⁺ influx to a greater extent than the organic inhibitors. We concluded that basal Ca²⁺ entry primarily occurred via basal activity of excitable channels, and PCR analysis suggests this influx to involve L-type voltage-gated Ca²⁺-channels, and TRPC1, TRPC4 and TRPC6. Next, we used Ca²⁺-sensitive proteins (aequorins and pericams) and dyes (fura-2) to measure parallel [Ca²⁺] changes in the mitochondrial matrix, subplasmalemmal cytosol and bulk cytosol. Replacing extracellular Na⁺ with n-methyl-d-glucamine (NMDG) caused Ca²⁺-entry by reversal of the Na⁺/Ca²⁺-exchanger (NCX), which was selectively blocked by KB-R7943. NCX-reversal increased mitochondrial and subplasmalemmal but not bulk cytosolic [Ca²⁺] revealing a local interaction of the SR, NCX and mitochondria. Furthermore, NCX-reversal and mitochondrial Ca²⁺-uptake appear to occur during the [Ca²⁺][sub i] plateau phase of the response to purinergic stimulation (ATP). However, this mitochondrial Ca²⁺-uptake does not increase [Ca²⁺][sub MT] because of a compensatory stimulation of mitochondrial Ca²⁺-extrusion (blocked by CGP-37157). Finally, we dissected the [Ca²⁺][sub MT] response to SR Ca²⁺-release in response to agonist-mediated stimulation. ATP and [ARG⁸]-vasopressin transiently increased [Ca²⁺][sub MT] by activating both IP₃R and ryanodine receptors (RyR) (selectively inhibited by 2-APB and procaine). Image analysis of fluorescently labeled mitochondria, IP₃R and RyR corroborated functional evidence that IP₃R and RyR release Ca²⁺ from separate sub-compartments of the SR and that physiological [Ca²⁺][sub MT] elevations rely on IP₃R-RyR cross-talk.

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