No abstract

An effect of appearance of new band in the excitation spectra of 3-hydroxy-4 -(dimethylamino)flavone (FME probe) in presence of adenosine triphosphate (ATP) is described. Considerable shift of new band up to the red and increase of fluorescence intensity points to the formation of FME-ATP associate, in which FME molecule undergoes to a strong electrostatic stabilization by tetra-charged ATP anion. It is shown the FME anion formation is possible under influence of ATP in the studied conditions. The dynamics of the observed effect is studied in mitochondria. The registered phenomenon allows the quantitative evaluation of ATP concentration in the range of 10 −3 -10 −5 M. In contrast to ATP, other nucleoside phosphates do not give a new band in the excitation spectra of FME probe. This implies the possibility of the in vivo determination of the ATP concentration.

Reactive oxygen species (ROS) overproduction may severely affect cell functions and even provoke cell death. Diazoxide, an opener of ATP-sensitive potassium channels, induces pharmacological preconditioning in different types of cells. However, the target of action of diazoxide is not clear enough because this substance can activate ATP-sensitive potassium channels localized in mitochondria (mitoK ATP ) as well as in plasma membranes (K ATP channels). It has not yet been established if diazoxide activates mitoK ATP in uterus cells. Our objective was to examine the influence of diazoxide on ROS production and on the viability of rat uterus cells under oxidative stress conditions. Using an ROS-sensitive fluorescent probe, we found that diazoxide enhances ROS production under normal conditions, but attenuates this process under conditions of ROS overproduction. Cells pretreated with diazoxide demonstrated lower levels of ROS production and of cell death under oxidative stress, in comparison with conditions where diazoxide was not present. The effects of diazoxide were eliminated by 5-hydroxydecanoate (5-HD), a blocker of mitoK ATP . The principal conclusion of the present study is that decreased ROS production and increased cell survival in the presence of diazoxide under oxidative stress conditions are mediated by the activation of mitoK ATP in rat uterus cells.

We demonstrated using PBFI k +-sensitive fluorescent probe an enhancement of both components of k +-cycle-the aTP-sensitive k +-uptake and quinine-sensitive K + /H +-exchange-under the Ca 2+ induced opening of mitochondrial permeability transition pore (MPTP) in rat myometrium mitochondria. Addition of CaCl 2 (100 μM) to K +-free medium results in the enhancement of reactive oxygen species (ROS) production, which was eliminated by cyclosporine A. Addition of CaCl 2 to k +-rich medium did not increase the rate of ROS production, but blocking of mitoK + aTP-channels with glybenclamide (10 mcM) increased production of ROS. We conclude that k +-cycle exerts a protective influence in mitochondria from rat myometrium by regulation of matrix volume and rate of ROS production under the condition of Ca 2+-induced MPTP.

The objective of this study was to detect ATP-sensitive K uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate) we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of K-channel (ATP-sensitive K-channel) - ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate). Our data demonstrates that K uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species) and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of K-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of K-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane.

No abstract

No abstract

No abstract