The regulation of mitochondrial NO synthase activity under nitroglycerine application in rat heart and liver mitochondria

The mechanisms of lipid oxidation under the application of nitroglycerine (NG) were studied in isolated rat heart and liver mitochondria. Dose-dependent formation of diene conjugates (DC), leukotriene C 4 (LTC 4 ) and thromboxane B 2 (TxB 2 ) was shown. To disclose the mechanisms regulating lipid peroxidation in mitochondria, we studied the effect of NG application on the formation of prooxidants (H 2 O 2 and free Fe 2+ ), as well as xanthine oxidase and mtNOS activity as main sources of ROS and RNS. Based on the correlation dependences, we have found that DC, LTC 4 , and TxB 2 formation was strongly dependent on hydroperoxide production and free divalent iron release in mitochondria. Also, DC formation exhibited the dependence on Ca 2+ uptake in mitochondria. No dependence of lipid oxidation on xanthine oxidase activity was found. In heart, but not liver mitochondria, DC, LTC 4 , and TxB 2 exhibited strong dependence on mtNOS activity, but were independent of nitrosothiols formation. This indicated that lipid oxidation was independent of direct protein nitrosylation caused by NG application. No dependence of lipid oxidation on mtNOS activity in liver was found, which agreed with much higher mtNOS activity in heart mitochondria, and suppression of mtNOS activity in liver mitochondria at high doses of NG. So, we came to the conclusion that under NG application ROS overproduction and free Fe 2+ release promoted both enzymatic and non-enzymatic lipid oxidation in heart and liver mitochondria. Also, we hypothesized that RNS overproduction due to the elevated mtNOS activity in the heart could largely contribute to lipid peroxidation and promote much faster increase in the formation of lipid oxidation products in heart as compared to liver mitochondria, especially at high doses of NG. Obtained correlation dependences allowed us disclose free iron, hydroperoxide, and mtNOS activity as principal factors affecting lipid peroxidation in mitochondria under NG application.

Section: Resultsmentioning

confidence: 67%

Section: Introductionmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Untitled

2022

“…Наявність у мітохондріях конститутивної ізоформи NO-синтази (mtNOS) була доведена імуногістохімічними методами для окремих тканин: печінки, серця [1,2], мозку [3], нирок [4], скелетного м'яза (діафрагми) [5] та тимуса [6]. mtNOS зумовлює можливість ендогенного біосинтезу оксиду азоту енергізованими мітохондріями в кисневмісному середовищі.…”

Section: вступunclassified

Possible Importance of Adenylate Cyclase Signaling Pathway in the Synthesis of Nitric Oxide by Myometrium Mitochondria

Danylovych¹,

Danylovych²,

Костерін³

2022

NO synthase activity (mtNOS) in uterine smooth muscle mitochondria under the action of the cAMP/protein kinase A signaling system modulators was studied. The experiments were performed on isolated mitochondria from rat myometrium using the NO-sensitive fluorescent probe DAF-FM-DA. NO synthesis in mitochondria was increased by adenylate cyclase activators NaHCO3 (30 mM) and forskolin (10 μM), as well as phosphodiesterase inhibitor caffeine (1 mM). The addition of ATP (0.5-5 mM) caused a slight increase in nitric oxide synthesis. The effect of ATP was enhanced in the presence of NaHCO3 and caffeine. The intensity of NO formation in mitochondria decreased by approximately 50 % in the case of inhibition of adenylate cyclase activity by the compound KH7 (25 μM). In the presence of the protein kinase A inhibitor PKI (10 nM) NO synthesis in mitochondria was also significantly reduced. When the constitutive NO-synthase inhibitor L-NAME (100 μM) was introduced into the incubation medium, the stimulating effect of the studied compounds on NO synthesis in mitochondria was not observed. These data suggests a possible dependence of mtNOS function on the activity of the cAMP/protein kinase A signaling system in smooth muscle mitochondria.

Ecdysterone treatment restores constitutive NO synthesis and alleviates oxidative damage in heart tissue and mitochondria of streptozotocin-induced diabetic rats

Акопова¹,

Korkach²,

Nosar³

et al. 2023

Self Cite

Constitutive NO synthases (cNOS) are the primary targets of diabetes mellitus and the impairment of cNOS functioning in cardiovascular system is one of the hallmarks of this disease. The aim of this work was to study the effect of a plant sterol ecdysterone (20-β-hydroxyecdysterone) on the NO synthases functioning and RNS metabolism in heart mitochondria and the heart tissue in the rat model of streptozotocin-induced type I diabetes. Diabetes development resulted in cNOS dysfunction both in heart mitochondria and heart tissue. cNOS activity was dramatically suppressed, but 3-fold and 6-fold rise of iNOS activity was observed in mitochondria and heart tissue respectively. Also, in mitochondria there was ~2.5 time’s increase in urea content and the activity of arginase 2 (ARG2), which could compete with NOS for the common substrate L-arginine. Total RNS production was dramatically elevated in mitochondria of diabetic animals, which well agreed with iNOS activation. Unlike this, in heart tissue dramatic increase of iNOS activity increased the content of nitrosothiols (RSNO), while total RNS production remained close to control. Both in the heart tissue and mitochondria, there was dramatic augmentation of superoxide production that correlated with sharp elevation of iNOS activity and steep rise of diene conjugates (DC) content, which indicated strong lipid oxidation. Ecdysterone treatment resulted in the reduction of iNOS activity and twofold elevation of mtNOS activity as compared to control. However, in the whole heart tissue eNOS was restored only by half of control level, which indicated specific action of ecdysterone on mtNOS isoform. RNS production returned to control in mitochondria, and was by half reduced in the heart tissue, which indicated the abolition of nitrosative stress. Correlation dependence between iNOS activity and superoxide production was found in mitochondria, which could indicate iNOS uncoupling. The restoration of cNOS activity and the reduction of iNOS activity to control level after ecdysterone treatment well correlated with the reduction of superoxide production and indicated possible ‘iNOS re-coupling‘, which resulted in the reduction of DC formation to control level. So, STZ-induced type I diabetes dramatically up-regulated iNOS activity and suppressed cNOS activity. Ecdysterone treatment reduced iNOS activity and restored constitutive NO biosynthesis to control level, which abolished oxidative and nitrosative stress in cardiac mitochondria and heart tissue of STZ-induced diabetic animals. Possible pathways involved in ecdysterone action on constitutive NO biosynthesis were discussed.