Increased Ca2+-dependent vascular tone is a hallmark feature of hypertension, which relies on enhanced Ca2+ influx through L-type Ca2+ (Cav1.2) channels in vascular smooth muscle cells (VSMCs). The upregulation of the main pore-forming α 1C subunit of the Cav1.2 channel augments voltage-dependent Ca2+ influx into VSMCs to promote Ca2+-dependent tone. This abnormality of Ca2+ signaling is evident in many animal models of hypertension and appears to be a component of essential hypertension in humans. However, the mechanism by which high blood pressure drives Ca v1.2 channel expression in small arteries and arterioles has been difficult to identify and may involve changes either in the functional role of accessory Cavβ subunits of the Cav1.2 channel and/or in post-translational modifications of the Cav1.2 α1C pore protein. Studies in heterologous expression systems and neuronal tissues have documented a chaperone role of β subunits in the trafficking of the α 1C subunit to the plasma membrane (PM), an interaction that results in the formation of functional Cav1.2 channels. However, two studies in expression systems also have emphasized the importance of posttranslational events including ubiquitination and degradation as regulators of the half-life and abundance of Cav1.2 channels. Based on these findings, we hypothesized that the half-life of vascular Cav1.2 channels at the PM is regulated by Cavβ subunits and the UPS (ubiquitin proteasome system), but a defect in these processes during hypertension results in a longer half-life and increased expression of Cav1.2 channels in VSMCs. In our first study, we showed that of the four known Cavβ subunits, the Cavβ3 subunit is principally expressed in the vasculature and it is required for upregulation of Cav1.2 channel α1C proteins in an angiotensin II (Ang II)-dependent mouse model of hypertension. Accordingly, Ang II -infused Cavβ3-/- mice failed to upregulate vascular Ca v1.2 channels in response to Ang II infusion and also exhibited lower levels of Ang II-dependent hypertension compared to Ang II-infused wild-type (WT) mice (149±4 mm Hg in Cavβ3-/- vs. 180±5 mm Hg in WT). In our subsequent studies, we explored the role of ubiquitination and degradation in regulating the half-life of Ca v1.2 channels at the PM of VSMCs. We found a stark contrast between the half-life of the Cav1.2 channel α1C protein in cultured A7r5 VSMCs compared to a newly devised in vivo model in which we evaluated Cav1.2 channel half-life in rat mesenteric arteries of exposed intestinal loops. The half-life of the Cav1.2 channel α1C protein was between 12h and 24h in cultured A7r5 aortic cells, but only ∼3h in arteries in vivo, suggesting that physiological conditions support a highly dynamic turnover of vascular Cav1.2 channels. However, in both the in vitro and in vivo conditions, we obtained biochemical evidence that the Cav1.2 channel α1C protein was subjected to ubiquitination and proteasomal degradation, suggesting a similar disposal pathway of α1C pore proteins, although the extent of ubiquitination was different between cultured A7r5 cells and mesenteric arteries in vivo. In our last set of studies, we evaluated whether short-term exposure (3h) of mesenteric arteries in vivo to three stimuli associated with hypertension results in Cav1.2 channel upregulation, possibly by inhibiting ubiquitination and proteosomal degradation as an unrecognized mechanism. We observed that 3h exposure to VSMC depolarization or ?1-adrenergic receptor activation resulted in the upregulation of Cav1.2 channel α 1C pore proteins in rat mesenteric arteries in vivo. However, elevated blood pressure established for 3h by Ang II infusion failed to upregulate Cav1.2 channels α1C proteins in similar preparations, suggesting that pressure per se may not be as effective in promoting Cav1.2 expression compared to other endogenous stimuli associated with the development of hypertension. In summary, we propose a new role for Cavβ3 subunits in the development of hypertension and also an undescribed mechanism of Cav 1.2 channel disposal by the ubiquitin-proteasome machinery in the A7r5 VSMC line and in the VSMCs of rat mesenteric arteries. Funded by NIH R01 HL064806 (NJR) and AHA 14PRE18580039 (AKS).
|Advisor:||Rusch, Nancy J.|
|Commitee:||Baldini, Giulia, Marsh, James D., Palade, Philip, Rhee, Sung W., Sturek, Michael|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 77/11(E), Dissertation Abstracts International|
|Keywords:||L-type calicum channels, Vascular smooth muscle|
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