ARTERIAL STORE-OPERATED Ca2+ ENTRY
Chronic hypertension is typically associated with increased peripheral vascular resistance. This is due, in part, to enhanced arterial smooth muscle contractility, which is regulated by intracellular Ca2+. Recent findings suggest that Ca2+ influx through transient receptor potential gene (TRPC)-encoded store-operated channels (SOCs) in the plasma membrane (PM) may play a role in regulating myogenic tone and vascular contractility. The project focuses on properties and PM distribution of TRPC-encoded SOCs in freshly isolated rat and mouse arterial smooth muscle cells (ASMCs) and its role in dysregulation of Ca2+ homeostasis observed during chronic in vivo ouabain treatment (manifested as ouabain-induced hypertension, an animal model of human essential hypertension).
This project tests the hypothesis that the altered Ca2+ homeostasis in ASMCs from ouabain hypertensive (OH) rats is due, in part, to upregulated expression of TRPC-encoded SOCs. Changes in TRPC expression under conditions of α2 Na+ pump inhibition may result from elevated sarcoplasmic reticulum (SR) Ca2+([Ca2+]SR).
Project 2 Has Four Specific Aims
- For Aim 1, we are determining which TRPC proteins (TRPC1-7) are involved in Ca2+ entry following unloading of distinct SR stores [(IP3)/cyclopiazonic acid (CPA)- and caffeine/ryanodine (CAF/RY)-sensitive)] in freshly isolated ASMCs. Early results demonstrate that the amplitude of store-operated Ca2+ entry (SOCE) is related to expression of IP3 receptors (IP3Rs), ryanodine receptors (RYRs) and TRPC proteins (Berra-Romani et al., 2006).
- For Aim 2, we are testing the hypothesis that TRPC-encoded SOCs are localized to PM microdomains adjacent to underlying junctional SR and are functionally linked to distinct SR Ca2+ stores. Immunofluorescent experiments revealed that TRPC1, TRPC3, TRPC4 and TRPC6 labels in cultured ASMCs are distributed in a distinct reticular pattern that parallels the organization of the underlying ER-Tracker-stained SR (Fig. 1). Similar TRPC localization was observed earlier in primary cultured astrocytes (Golovina, 2005). This specific localization of TRPC proteins in the PM is consistent with the proposed role of these proteins as components of a store-operated Ca2+ influx channel.
- For Aim 3, we are addressing the question: Which Ca2+ transport and storage mechanisms are altered in ASMCs from OH rats? Early results suggest that ouabain-induced dysregulation of Ca2+ homeostasis is associated with increased Ca2+ influx via Na+/Ca2+ exchanger due to Na+ pump inhibition by ouabain. Moreover, elevated [Ca2+]SR may upregulate expression of TRPC channels, which further increases Ca2+ influx in ASMCs from OH rats (Berra-Romani et al., 2005).
- For Aim 4, we are testing the hypothesis that nanomolar ouabain affect expression of TRPC-encoded SOCs in ASMCs in vitro. Mice with genetically altered Na+ pumps and Na/Ca exchanger are being used. The results of this project will likely lead to new insights into the role of localized Ca2+ transport mechanisms in the regulation of vascular tone and may suggest innovative approaches for the development of novel therapeutics for vascular dysfunction.
- Berra-Romani, R., J. Hamlyn, M.P. Blaustein, and V.A. Golovina. Abnormal calcium homeostasis in arterial myocytes from ouabain hypertensive rats. 15th European Meeting on Hypertension, Milan, 2005. J. Hypertension, 23(suppl 2):S353, 2005.
- Golovina, V.A. Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum. J. Physiology (London) 564:737-749, 2005. http://jp.physoc.org/cgi/reprint/564/3/737
- Berra-Romani, R., A. Mazzocco-Spezzia, and V.A. Golovina. Independent regulation of store-operated Ca2+entry activated by the depletion of IP3- and ryanodine-sensitive stores in arterial myocytes. Biophysical Soc. 50th Annual Meeting, Salt Lake City, p. 415a, January 2006.
- Golovina, V.A. and M.P. Blaustein. Preparation of primary cultured mesenteric artery smooth muscle cells for fluorescent imaging and physiological studies. Nat. Protoc. 1:2681-2687, 2006.
- Berra-Romani, R., A. Mazzocco-Spezzia, M.V. Pulina, and V.A. Golovina. Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture. Am. J. Physiol. Cell Physiol. 295:C779-90, 2008.http://ajpcell.physiology.org/cgi/reprint/295/3/C779
- Pulina, M.V., A. Zulian, R. Berra-Romani, O. Beskina, A. Mazzocco-Spezzia, S.G. Baryshnikov, I. Papparella, J.M. Hamlyn, M.P. Blaustein, and V.A. Golovina. Up-regulation of Na+ and Ca2+ transporters in arterial smooth muscle from ouabain hypertensive rats. Am. J. Physiol. Heart Circ..Physiol., 2009. [Epub ahead of print].http://ajpheart.physiology.org/cgi/reprint/298/1/H263
- Baryshnikov, S.G., M.V. Pulina, A. Zulian, C.I. Linde, and V.A. Golovina..Orai1, a critical component of store-operated Ca2+ entry, is functionally associated with Na+/Ca2+ exchanger and plasma membrane Ca2+ pump in proliferating human arterial myocytes. Am. J. Physiol. Cell Physiol.297:C1103-1112, 2009. http://ajpcell.physiology.org/cgi/reprint/297/5/C1103