Modulation of mitochondrial Ca²⁺ by nitric oxide in cultured bovine vascular endothelial cells

Abstract

-In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca 2ϩ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100 -500 M) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca 2ϩ uptake and Ca 2ϩ extrusion rates, whereas low concentrations of Sper/NO (Ͻ100 M) had no effect on mitochondrial Ca 2ϩ levels ([Ca 2ϩ ]mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca 2ϩ uptake, whereas inhibition of mtNOS with 10 M L-N 5 -(1-iminoethyl)ornithine resulted in a significant increase of [Ca 2ϩ ]mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca 2ϩ -induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100 -500 M) induced PTP opening, whereas lower concentrations (Ͻ100 M) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca 2ϩ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca 2ϩ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca 2ϩ overload under pathological conditions where cellular [NO] can reach very high levels. nitric oxide synthase; permeability transition pore; endothelium IN THE VASCULAR ENDOTHELIUM, nitric oxide (NO) plays an important regulatory role. NO, which is synthesized in a Ca 2ϩ -dependent manner by the endothelial NO synthase (eNOS), acts on vascular smooth muscle cells in the vessel wall as an endothelium-derived relaxing factor (40). We have demonstrated previously (19) that in vascular endothelial cells NO synthesis is not only Ca 2ϩ dependent but also under an autoregulatory control that involves NO-dependent regulation of cytoplasmic Ca 2ϩ . Considering the known inhibitory effects of NO on cell respiration (6,7,18) and the recent discovery of a mitochondrial NOS (mtNOS) in endothelial cells (20), the question arises whether a similar autoregulatory function of NO in endothelial cells also affects mitochondrial Ca 2ϩ uptake.Although mtNOS has been discovered in a variety of cell types (3,4,20,21,25,26,35,36,38,49,52), the functional implications of NO produced locally by mitochondria for mitochondrial energy metabolism and Ca 2ϩ homeostasis are much less explored and not well understood. NO is known to be a mediator of Ca 2ϩ homeostasis in a highly complex and cell-specific manner (17)...

Reduction of Mitochondria–Endoplasmic Reticulum Interactions by Acetylcholine Protects Human Umbilical Vein Endothelial Cells From Hypoxia/Reoxygenation Injury

“…Previous studies have demonstrated that in vascular endothelial cells, NO donor enhances ER Ca 2+ uptake and inhibits mitochondrial Ca 2+ overload. 38,39 Importantly, acetylcholine prevented the increased interaction within the VDAC1/Grp75/IP3R1 complex and mitofusin 2 expression, which explains, at least in part, its protective effect against endothelial injury induced by H/R. It is noteworthy that the decrease in mitochondrial Ca 2+ content paralleled a similar decrease in mitochondrial ROS levels by acetylcholine, suggestive of a possible correlation between mitochondrial Ca 2+ levels and ROS generation.…”

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

“…Previous methods for evaluating stimulation-or stress-induced alterations in pulmonary endothelial cell ⌬ m using rhodamines or ratiometric probes have, in general, provided only indexes of relative changes, estimated from changes in fluorescence intensity (19,24,28,43,69). A common pitfall in interpreting such data is that the logarithmic form of the Nernst equation specifies that changes in fluorescence intensity (and, for ratiometric dyes, ratios derived from such changes) are not linearly proportional to changes in ⌬ m (45).…”

“…mathematical modeling; rhodamine 123; tetramethylrhodamine ethyl ester; multidrug transporter P-glycoprotein; hyperoxia PULMONARY ENDOTHELIAL mitochondrial and plasma membrane potentials (⌬ m and ⌬ p , respectively) are implicated in bioenergetic, metabolic, and signaling processes contributing to normal lung function and in injury (16,24,33,54,67,69). In most eukaryotic cells, ⌬ m is the major component of the mitochondrial electrochemical transmembrane potential and, as such, is involved in pulmonary endothelial mitochondrial ATP generation, regulation of calcium homeostasis, apoptosis, nitric oxide signaling, and other functions (19,58,60,61). Dissipation of ⌬ m is considered a hallmark of mitochondrial dysfunction in diverse cell types, including pulmonary endothelial cells exposed to oxidative stresses and bleomycin (24,33,43,54,69).…”

Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes

“…Subsequent surface membrane permeabilization with the nonionic detergent digitonin removed cytoplasmic fluo-3. We developed this method and used it successfully to estimate [Ca 2ϩ ] m of endothelial cells (12,48). ] em from 0.1 to 10 M resulted in the increase of the mitochondria-trapped fluo-3 fluorescence (Fig.…”