Mechanisms of asynchronous Ca²⁺ oscillations and their role in (mal)function of vascular smooth muscle

• Main Author: Syyong, Harley Thomas Tan
• Language: English
• Published: University of British Columbia 2010
• Online Access: http://hdl.handle.net/2429/24253

Contraction of vascular smooth muscle is regulated by fluctuations in the cytosolic concentration of Ca²⁺. The spatio-temporal regulation of Ca²⁺ relies on the subcellular architecture of the smooth muscle cell and the juxtaposition of the opposing plasmalemma, sarcoplasmic reticulum, and mitochondria. This thesis addresses two related aspects of Ca²⁺ signaling in vascular smooth muscle: 1) Reversal of the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) during agonist-mediated stimulation in cultured rat aorta smooth muscle cells, and 2) the primary function of agonist-stimulated asynchronous Ca²⁺ waves and the signaling pathway(s) underlying them in the intact tissue. Evidence for functional coupling of reverse-mode NCX with canonical transient receptor potential channels (TRPC), specifically TRPC6, was provided in rat aortic smooth muscle cells by demonstrating that NCX reversal was increased following stimulation with ATP and 1-Oleoyl-2-acetyl-sn-glycerol, a diacylglycerol analog. However, this was attenuated by blockade of non-selective cation channels with SKF-96365 and by activation of protein kinase C. These data are consistent with the known properties of TRPC6 and further support that functional coupling of TRPC6 and NCX occurs via a receptor-operated cascade. A combination of wire myography and confocal microscopy determined that uridine 5’-triphosphate (UTP)-induced tonic contractions in rat basilar artery were associated with sustained repetitive oscillations in cytosolic Ca²⁺ which propagated along the length of the smooth muscle cells as Ca²⁺ waves. Pharmacological characterization of the mechanism of Ca²⁺ waves revealed that they are a result of repetitive cycles of sarcoplasmic reticulum (SR) Ca²⁺ release via inositol 1,4,5-trisphosphate-sensitive channels followed by the refilling of the SR. Plasmalemmal Ca²⁺ entry via the reverse-mode NCX coupled with the receptor-operated and L-type Ca²⁺ channels is involved in replenishing the SR and supporting the ongoing Ca²⁺ waves. Finally, phenylephrine-stimulated vascular smooth muscle contraction in mesenteric arteries of a mouse model of Marfan syndrome was significantly inhibited and associated with reduced frequency of Ca²⁺ waves. In addition, endothelium-dependent and endothelium-independent vasodilation was impaired, and vessel stiffness was increased. Together, these vasomotor abnormalities in the resistance vessel may have a negative and detrimental impact on the overall cardiovascular function in Marfan syndrome.