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Invited Symposium: Signal Transduction in Endothelium: Mechano-Sensing, Ion Channels and Intracellular Calcium






Abstract

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Materials & Methods

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The Involvement Of Localized Ca2+ Gradients In Subcellular Ca2+ Distribution, Activity Of Capacitative Ca2+ Entry And Nitric Oxide Synthase In Endothelial Cells

Graier, WF (Department of Medical Biochemistry, University of Graz, Graz, Austria)
Paltauf-Doburzynska, J. (Department of Medical Biochemistry, University of Graz, Graz, Austria)
Paltauf, G. (Department of Experimental Physics, University of Graz, Graz, Austria)
Posch, K. (Department of Medical Biochemistry, University of Graz, Graz, Austria)
Kostner, GM (Department of Medical Biochemistry, University of Graz, Graz, Austria)

Contact Person: Wolfgang F Graier (wolfgang.graier@kfunigraz.ac.at)


Abstract

Recently evidence for an insulated and localized Ca2+ signaling in the subplasmalemmal space of endothelial cells was provided (Graier et al., J. Physiol. Lond. 506: 109-125, 1998). This study was designed to investigate the contribution of the two major phenomena of the subplasmalemmal Ca2+ control, ryanodine-sensitive Ca2+ release (RsCR) and Na+/Ca2+ exchange (Na/CaX) in bradykinin (Bk)-induced Ca2+ release, capacitative Ca2+ entry (CCE) and nitric oxide synthase (eNOS) in freshly isolated bovine coronary artery endothelial cells. 1. Using deconvolution microscopy fura-2 was found throughout the whole cytosol, while FFP-18 was exclusively in the cell membrane. Thus, perinuclear ([Ca2+]peri) and subplasmalemmal ([Ca2+]sub) Ca2+ concentration were monitored using fura-2 and FFP-18. 2. Inhibition of Na/CaX by lowering extracellular Na+ concentration augmented the Bk-induced [Ca2+]peri signal in Ca2+-free solution. This effect was abolished when RsCR was prevented with 25 Ámol l-1 ryanodine, while inhibition of RsCR had no effect on Bk-induced increase in [Ca2+]peri without inhibition of Na/CaX. 3. Initiating RsCR by 200 nmol l-1 ryanodine increased [Ca2+]sub, while [Ca2+]peri remained constant. However, when Na/CaX was prevented, ryanodine was also able to elevate [Ca2+]peri. 4.Blockage of RsCR diminished Ca2+ extrusion to stimulation with Bk in normal Na+ containing solution. 5. Inhibition of RsCR blunted Bk-activated CCE, while inhibition of Na/CaX during stimulation enhanced CCE. 6. Although direct activation of RsCR failed to activate eNOS, inhibition of RsCR diminished the effect of ATP and Bk on eNOS, while the effect of thapsigargin remained unchanged. These data suggest that during stimulation subplasmalemmal RsCR occurs, which contributes to the activities of CCE and eNOS. Thus, the function of the subplasmalemmal Ca2+ control unit (SCCU; Graier et al., J. Physiol. Lond. 506: 109-125, 1998.) must be extended as a regulator for CCE and eNOS.

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Presentation Number SAgraier0377
Keywords: localized Ca2+, nitric oxide, ryanodine, Na+/Ca2+ exchange, endothelial cells


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Graier, WF; Paltauf-Doburzynska, J.; Paltauf, G.; Posch, K.; Kostner, GM; (1998). The Involvement Of Localized Ca2+ Gradients In Subcellular Ca2+ Distribution, Activity Of Capacitative Ca2+ Entry And Nitric Oxide Synthase In Endothelial Cells. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/nilius/graier0377/index.html
© 1998 Author(s) Hold Copyright