Products of oxidative and non-oxidative metabolism of L-arginine as potential regulators of Ca2+ transport in mitochondria of uterine smooth muscle.

Mitochondria play a crucial role in maintaining Ca2+ homeostasis in cells. Due to the critical regulatory role of the products of oxidative and non-oxidative metabolism of L-arginine, it is essential to clarify their effect on Ca2+ transport in smooth muscle mitochondria. Experiments were performed on the uterine myocytes of rats and isolated mitochondria. The possibility of NO synthesis by mitochondria was demonstrated by confocal microscopy and spectrofluorimetry methods using the NO-sensitive fluorescent probe DAF-FM and Mitotracker Orange CM-H2TMRos. It was shown that 50 µM L-arginine stimulates the energy-dependent accumulation of Ca2+ in mitochondria using the fluorescent probe Fluo-4 AM. A similar effect occurred when using nitric oxide donors 100 µM SNP, SNAP, and sodium nitrite (SN) directly. The stimulating effect was eliminated in the presence of the NO scavenger C-PTIO. Nitric oxide reduces the electrical potential in mitochondria without causing them to swell. The stimulatory effect of spermine on the accumulation of Ca2+ by mitochondria is attributed to the enhancement of NO synthesis, which was demonstrated with the use of C-PTIO, NO-synthase inhibitors (100 µM NA and L-NAME), as well as by direct monitoring of NO synthesis fluorescent probe DAF-FM. A conclusion was drawn about the potential regulatory effect of the product of the oxidative metabolism of L-arginine - NO on the transport of Ca2+ in the mitochondria of the myometrium, as well as the corresponding effect of the product of non-oxidative metabolism -spermine by increasing the synthesis of NO in these subcellular structures.

Biochemical properties of H+-Ca2+-exchanger in the myometrium mitochondria.

Some biochemical properties of the H+-Ca2+-exchanger in uterine smooth muscle mitochondria have been described. The experiments were performed on a suspension of isolated mitochondria from the myometrium of rats. Methods of confocal microscopy, spectrofluorimetry and photon correlation spectroscopy were used. Fluo-4 probe was used to record changes in ionized Ca2+ in the matrix and cytosol; pH changes in the matrix were evaluated with BCECF. It was experimentally proved that in the myometrium instead of Na+-Ca2+-exchanger the H+-Ca2+-exchanger functions. It was activated at a physiological pH value, was carried out in stoichiometry 1: 1 and was electrogenic. The transport system was modulated by magnesium ions and the diuretic amiloride, but was not sensitive to changes in the concentration of extra-mitochondrial potassium ions. H+-Ca2+-exchanger was suppressed by antibodies against the LETM1 protein. Calmodulin may act as a regulator of H+-Ca2+-exchanger by inhibiting it.

NO-synthase activity in mitochondria of uterus smooth muscle: identification and biochemical properties.

Information about the catalytic and kinetic properties of mitochondria NO-synthase from uterus smooth muscle is missing currently. According to the data on MitoTracker Orange CM-H2TMRos and 4-аmino-5-methylamino-2',7'-difluorescein, diaminofluorescein-FM (DAF-FM) dye co-localization in uterine smooth muscle cells, presented in this paper, NO can be synthesized in their mitochondria. High activity of NO synthase requires the presence of substrates of respiration, L-arginine, Ca2+ and NADPH. It is established that the dependence of NO production on the concentration of L-arginine has a bell-shaped character with a maximum of 75 µM, and the apparent affinity constant for L-arginine is 28.9 ± 9.1 µM. The dependence of NO production on Ca2+ concentration has a maximum at 100-250 µM; the activation constant for Ca2+ is 44.4 ± 14.5 µM. The inhibitor of Ca2+ transport in mitochondria ruthenium red (RuR), as well as the inhibitor of NO-synthase NG-nitro-L-arginine (NA), reduces NO production. The biosynthesis of NO by mitochondria depends on its energized level: it is stimulated by the addition of respiration substrates, suppressed with specific inhibitors of the electron transport chain (rotenone and antimycin A) and carbonyl-cyanide 3-chlorophenylhydrazone (CCCP) protonophore.