The dissertation is devoted to the investigation of biochemical properties of NO biosynthesis in uterine smooth muscle mitochondria, as well as to the study of molecular mechanisms of nitric oxide action as a possible specific endogenous regulator of Ca2+ concentration in myocytes, mitochondrial Ca2+-transport systems functioning, and bioenergetic processes (electron transport chain activity and inner mitochondrial membrane polarization) and to the study of the effect of calix[4]arenes – exogenous non-toxic modulators of mitochondrial functional activity – on NO-synthase capacity, bioenergetics and Ca2+-homeostasis of the organelles.
With the use of NO-sensitive fluorescent probe DAF-FM, laser confocal microscopy and flow cytometry, we demonstrated for the first time the formation of NO in uterine smooth muscle cells. Mitochondria have been shown to be a potent source of NO in uterine smooth muscle cells. The NO synthesis in mitochondria is sensitive to inhibitors of constitutive NO synthases (NG-nitro-L-arginine and 2-aminopyridine) and Ca2+-uniporter. In addition, it is inhibited by calmodulin antagonists (calmidazolium and trifluoperazine), Mg ions, and is largely dependent on the level of inner mitochondrial membrane energization. The values of the apparent Michaelis constant by L-arginine and the activation constant by the Ca2+ for reaction of NO biosynthesis are within the physiological values of their concentrations in the mitochondrial matrix. It has also been shown that NO synthases associated with the plasma membrane and sarcoplasmic reticulum may be nitric oxide sources in my-ocytes.
In uterine smooth muscle mitochondria NO stimulates Ca2+ accumulation through the Ca2+-uniporter activation. The system of Ca2+ release from mitochondria – the H+-Ca2+-exchanger, which is presented by the protein LETM 1, is resistant to nitric oxide influence. NO causes a moderate decrease in the electric potential on the inner membrane of the organelles due to the inhibition of the respiratory chain complexes, namely the inhibition of FADH2 oxidation, and counteracts the swelling of the organelles caused by calcium-rich solution.
It has been shown that nitric oxide inhibits the pathways that increase Ca ions concentration in the myoplasm under the effect of carbachol and oxytocin on myocytes, and also causes an increase in the characteristic size of myocytes. The polarizing effect of nitric oxide is due to the increase in K+-permeability of the plasmalemma and depends on Ca ions, as well as due to the cGMP-dependent stimulation of Na+,K+-ATPase activity. The scheme representing biochemical mechanisms of NO regulatory effects at the level of mitochondria and plasma membrane in uterine myocytes is proposed.
A simulation model in terms of functional hybrid Petri nets was created. The latter reproduces the functional activity of mitochondria, namely the simultaneous oxidation of NADH/FADH2, the formation of reactive oxygen species and changes in their hydrodynamic diameter. The obtained equations formalize and describe the concentration-time dependencies of the course of the listed processes in the medium under the action of sodium azide, which is considered as an indirect donor of NO in the literature, with the possibility of predicting their course intensity. In particular, the response of mitochondria over time to the action of NaN3 at a concentration that has not been used in the experiment was predicted.
The possibility of calix[4]arene C-956 penetration into cell myoplasm and its interaction with mitochondria has been demonstrated. Calix[4]arene C-956 effectively inhibits mitochondrial H+-Ca2+-exchanger, without affecting the energy-dependent Ca2+ accumulation, stimulates mitochondrial NO-synthase, and inhibits the NADH and FADH2 oxidation in the electron transport chain. Compounds C-97, C-99 and C-107 cause depolarization of the uterine myocytes plasma membrane. Upon interaction with mitochondria, they cause Ca2+-uniporter inhibition and simultaneous stimulation of its H+-Ca2+-exchanger (C-97, C-99), and moderate inhibition of mitochondrial NO-synthase activity. In addition, there is a transient hyperpolarization of the mitochondrial membrane, which is associated with an initial increase in NADH oxidation and, possibly, stimulation of the electrogenic H+-Ca2+-exchanger. The ab-sent influence of all studied calix[4]arenes on the formation of reactive oxygen species in mitochondria may indicate that the activated/inhibited in their presence processes do not lead to harmful effects in these organelles (i.e. to the development of mitochondrial dysfunction).
Thus, through NO biosynthesis enhancement/inhibition by altering the activity of Ca2+ transport systems, calix[4]arenes can have a modulatory effect on the respiratory chain complexes functioning and mitochondrial bioenergetics and can be used in biochemical studies as myometrial contraction/relaxation regulators.
