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Regulation of intracellular calcium concentration in vascular endothelial cells and valvular myofibroblasts

University of British Columbia

Background Some aspects of Ca2 + signaling in vascular endothelial cells (ECs) and cardiac valvular myofibroblasts (VMFs) were examined in this study. Over the last decade, the relationship between endothelial dysfunction, smooth muscle proliferation, and development of vascular diseases has been relatively well elucidated. Although the specific mechanisms remain unclear, it is generally agreed that alterations in endothelial Ca regulation play an important role in vascular pathophysiology. However, information regarding the underlying causes of valvular heart diseases is very limited, despite the large number of clinical cases reported. In the cardiac valve, ECs serve as a physical barrier between the VMFs underneath and the circulating blood. In addition, ECs secrete vasoactive substances in response to the enormous hemodynamic forces generated by the cardiac cycle. The endothelial secretions may have a general effect on the whole body or they may act locally on the cardiac valve. The VMFs, being the most predominant cells in the cardiac valve, perform a variety of functions to maintain normal valvular physiology. These functions, such as contraction, proliferation, and wound repair, are all directly or indirectly mediated by intracellular Ca2 + concentrations ([Ca2+]i). Thus, knowing how [Ca2+]i is regulated by vasoactive agents in VMFs becomes a critical step towards understanding valvular biology in both health and diseases. Methods Both VMFs and ECs were loaded with Ca2+-sensitive fluorescent indicators fiira-2 and/or fluo-4. The cells were visualized with a fluorimeter-coupled microscope system. Measurements of [Ca2+]i upon different pharmacological stimulations were done at room temperature (23 °C). Raw [Ca2+]i data were analyzed with respect to i) their amplitudes and curve areas (as for the VMF experiments) and ii) their decline rates (as for the EC experiments). Also in the VMF experiments, the Mn2+-quenching method was used to study divalent cation entry. Quantitative data were subjected to the appropriate statistical tests including Student's /-tests and one-way ANOVA. Results and Conclusions Part I: Ca2 + Mobilization bv Pharmacological Agents in Valvular Myofibroblasts The fundamentals of Ca2 + signaling properties in cultured human VMFs were studied. In VMFs, two agonist-induced and one store-operated Ca2 + signaling pathways were identified, and observations of spontaneous Ca2 + releases were made. The main findings are listed below. Histamine, upon binding to Hi receptors, elicited [Ca2+]i increase solely from ER Ca2 + release in normal VMFs. In rheumatic VMFs, however, histamine also stimulated Ca2 + influx. The purinergic agonists ATP and UTP activated P2Y2 receptors and induced both ER Ca2 + release and Ca2 + influx. The ER Ca2 + emptying stimulated by histamine and ATP/UTP was limited to an IP3-sensitive Ca2 + store. A second ER compartment, the IP3-insensitive Ca2 + store, released Ca2 + in response to SERCA blockade, which depletes the ER Ca2 + content and stimulates storeoperated Ca2 + entry. There was also evidence for the presence of RyRs in VMFs, as shown by the effect of RyR modulation on spontaneous Ca2 + releases. Data in support of the above findings are summarized in brief below. The selective Hi antagonist pyrilamine, but not the selective H2 antagonist cimetidine, abolished the histamineinduced [Ca2+]j response, which showed no difference in the presence or absence of extracellular Ca2 + or following treatment of SKF-96365, a blocker of Ca2 + entry. In a Ca2+-free environment (or in the presence of SKF-96365), the duration of ATP/UTP-induced [Ca2+]i response was shorter than in normal Ca2+-containing solution. This suggested a Ca2 + influx component regulated by purinergic receptor stimulation, as confirmed by accelerated Mn/ + entry as well. The purinergic receptor subtype was characterized based on the following results: i) [Ca2+]; response elicited by the selective P2Y2 agonist UTP; and ii) the lack of [Ca2+]i response in VMFs challenged with the selective P2Y1 agonists ADP(3S and 2-Me-S-ATP. The presence of the IPsinsensitive ER Ca2 + store was revealed by additional Ca2 + release stimulated by CPA, following repeated applications of histamine and ATP. Store-operated Ca2 + entry was activated by application of CPA which increased the rate of Mn2 + entry. Unlike in normal VMFs, the curve areas of histamine-elicited [Ca2+]i responses in rheumatic VMFs showed a significant increase between Ca2+-containing and Ca2+-free environments, indicating that in the diseased cells histamine also induced Ca2 + entry. Finally, in a small number of VMFs spontaneous Ca2 + releases in localized areas were observed. Blockade of the RyR elongated the latency period between each Ca2 + releasing event, demonstrating the presence of functional RyRs in VMFs. Part II: Mechanisms of Ca2 + Removal in Vascular Endothelial Cells How endothelial Ca2 + homeostasis is maintained is still an unresolved issue, an important one though since cardiovascular diseases often prevail in the absence of controlled [Ca2+]i regulation. In contrast to most published studies that investigated Ca2 + entry pathways in ECs, the Ca2 + removal mechanisms, which have equally vital roles in Ca2 + homeostasis, were examined in this study. In an effort to study cells that resemble physiological conditions as much as possible, a preparation of freshly isolated ECs was used. It was found that two pathways of extruding cytosolic Ca2 + were present after agonist stimulation in ECs. The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), ryanodine receptor (RyR), and Na+/Ca2 + exchanger (NCX) were components of the first pathway. The SERCA actively transported cytosolic Ca2 + into the endoplasmic reticulum (ER). The RyR is a Ca2 + release channel on the ER membrane. Ca2 + exited the ER via the RyR and diffused across a subplasmalemmal restricted space to the plasmalemmal NCX, where Ca2 + was extruded to the extracellular space. In parallel to the SERCA-RyR-NCX pathway, Ca2 + extrusion also occured via the plasma membrane Ca2 +- ATPase (PMCA), constituting the second Ca2 + removal pathway in ECs. Data supporting the above findings are summarized as follows. Inhibitors of the SERCA, RyR and NCX, which respectively were cyclopiazonic acid (CPA), ryanodine (at the high concentration of 100 pM) and Na+-free solution (ONaPSS), each slowed Ca2 + removal rate to the same extent. Combinations of these treatments, e.g. CPA+ONa and ryanodine+ONa, did not reduce Ca2 + removal rate any further, indicating that the SERCA, RyR, and NCX are arranged in series. When the RyR was activated with caffeine or the low concentration of ryanodine (1 pM), Ca2 + extrusion was facilitated. On the contrary, stimulation of RyR (with 1 pM ryanodine) and inhibition of SERCA (with CPA) together slowed down Ca2 + extrusion, showing the sequential delivery of Ca2 + from SERCA to RyR. When the SERCA-RyR-NCX pathway was blocked, Ca2 + extrusion could be further inhibited by blocking the PMCA with carboxyeosin, suggesting the contribution of the plasmalemmal Ca2 + pump in removing cytosolic Ca2 + .

Thesis/Dissertation

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2009-11-27

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http://hdl.handle.net/2429/15831

Pharmacology

University of British Columbia

UBCV

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