Experiments were carried out in 76 male Wistar rats weighing 250 - 300 g. Animals received humane care in compliance with “Guide for the Care and Use of Laboratory animals” (National Institutes of Health publication 86-23, Revised 1996) and was performed with approval of the local Interinsitutional (International Scientific Centre of Introduction of New Biomedical Technology, Department of Pharmacology, Faculty of Medicine, I.Javakhishvili Tbilisi State University and Department of Medical Pharmacology and Pharmacotherapy, Tbilisi State Medical University, Tbilisi) Animal Care and Use Committee. Nitroglycerin-induced tolerance was reproduced by treatment with NTG (10 mg/kg, s.c.) three times a day for 8 days and was confirmed by a reduction in hypotensive responses to intravenous NTG [14] . All animals were randomized into the five groups: Control group―s.c. injection of 0.1 ml normal saline 3 times daily for 8 days; II―were given 0.1 ml 0.9% NaCl in the same frequency and period of administration and then NTG (in form of NITRO 5 mg/ml², Orion Corporation, Finland, 150 mcg/kg) i.v. injection; III―received NTG (10 mg/kg s.c) three times a day for 8 days and then NTG (150 mcg/kg) i.v. injection; IV―received NTG (10 mg/kg s.c.) three times a day for 8 days and NADCIN^Ò, 8.0 mg/kg of body weight resolved in 0.1 ml of water for injection i.v. from the 4^th days of NTG treatment and once daily 20 min after the NTG (150 mcg/kg) i.v. injection; V―were given s.c. injection of 0.1 ml 0.9% NaCl three times a day for 8 days and then received MB −2.5 mg/kg i.v. 20 minute before i.v. injection of NTG (150 mcg/kg). After 8 days the rats were anaesthetized with pentobarbital (60 mg/kg intraperitoneally).

Polyethylene catheter was placed into the right femoral artery and connected to blood pressure transducer for measuring blood pressure (BP) with electromanometer and heart period (HP) with cardiotachometer. Intravenous injections were given via catheter inserted into the rigor ht femoral vein. Baroreflex sensitivity (BRS) was defined by measuring HP in response to rises BP 30 - 50 mm Hg above control after i.v. injection of phenylephrine (3 - 10 mcg/kg). The slope of relationship between BP and HP was used as an index of BRS according to [15] . Splanchnic sympathetic nerve activity (SSNA) was recorded according to [16] . A thin bipolar silver electrode was placed around a branch of the splanchnic nerve and isolated carefully with silicone rubber. Nerve activity was recorded via a cable connected to an adaptor. The nerve signal was amplified and rectified and the mean nerve activity was displayed on a polygraph. BRS was assessed with respect to control of SSNA as percentage of nerve inhibition from control per mmHg of BP rise (% mmHg) and with respect to control of heart rate (HR) as HR decrease per mmHg of BP rise (beats min⁻¹ mmHg⁻¹).

Thoracic aorta was frozen and homogenized in liquid nitrogen, before the experiments homogenate was rapidly placed in modified Krebs’ solution [17] . The powdered tissue was dispersed in 5 vol ice-cold aqueous 30 mM potassium phosphate buffer, pH 7.5, vortexed, sonicated, and centrifuged at 10.000 g for 10 min. Isolation of mitochondria was performed using “Mitochondria isolation kit” (BioChain Institute, Inc.) according to the manufacturer’s instructions. ALDH activity in the supernatant was determined by monitoring NADH formation from NAD⁺. The assay mixture (0.5 mL) contained 100 mM Tris-HCl (pH 8.5), 1 mM NAD⁺, 1 mM 4-methylpyraz- ole, and 100 μg protein. The reaction was started by addition of 1 mM propionaldehyde to the cuvette, and absorbance changes at 340 nm as a result of NADH formation were recorded for 10 min. The mean rate of absorbance change was taken as a measure of ALDH activity (0.0125 A340 was equivalent to 1 nmol/mg/min). The ALDH-2 inhibitor benomyl (10 - 5 mol/L) was used as a negative control. The content of pyridine nucleotide in aortic mitochondries was measured using sspectrofluorimetric methods [13] [18] . The protein concentration was determined with BSA protein assay kit. Superoxide anion generation in isolated rat heart mitochondria was determined immediately following the isolation procedure. Briefly, mitochondria (0.5 mg/ml) were incubated with buffer (6 mM succinate, 70 mM sucrose, 220 mM mannitol, 2 mM, Hepes, 25 mM KH₂PO₄, 2.5 mM MgCl₂, 0.5 mM EDTA, 5 mg/ml catalase, pH 7.4) at 37˚C. At the indicated time points, 40 mM acetylated cytochrome c was added and the change in absorbance at 550 nm was measured for 1 min at 37˚C. Background absorbance for all groups was determined by the addition of SOD (1000 units/ml). Endothelin-1 (ET₁) in aortic homogenate concentration (pg/ml) was measures by R & D Systems for Human endothelin-1 Immunoassay (Great Britain).

All values are expressed as the mean ± standard error deviation of at least 6 experiments. Statistical analysis for comparison between different groups of animals was assessed by two-way unpaired Student t-test. Significance was defined as p < 0.05 or less.

In anaesthetized rats the baseline values of BP and HP did not show any significant differences in all groups of animals (Table 1). The study of BRS revealed great attenuation of its cardiochronotropic vagal component in rats with NTGT (0.62 ± 0.08 ms/Hg mm⁻¹) vs. control animals (0.98 ± 0.1 ms/Hg mm⁻¹, p < 0.05) that correlated with the decreasing the ability to cause bradycardia reflex (1.9 ± 0.09 beats min⁻¹ Hg mm⁻¹⁾ and an inhibition of SSNA (Table 1). The concomitant use of NADCIN^Ò in NTGT rats markedly increased the reduced BRS (+39%, p < 0.001) and its ability with respect to heart rate (HR) control (+65%, p < 0.001) and inhibition of SSNA (+38.2%, p < 0.01) resulting from NTGT animals. Treatment with guanylyl cyclase inhibitor, MB, similarly to NTGT, but in less degree, caused reduction in BRS (0.72 ± 0.4 ms Hg mm⁻¹) and its properties with respect to control SSNA (1.7 ± 0.1 Hg mm⁻¹) vs control group of rats. Acute single dose of NTG (150 mcg/kg i.v) administration was accompanied with significant hypotensive reactions in non-tolerance normal rats and in group treated with NADCIN^Ò. BRS after NTG i.v. injection was more deeply blunted in MB treated group (0.68 ± 0.15 ms Hg mm⁻¹) and especially NTGT rats (0.51 ± 0.09 ms Hg mm⁻¹) vs. in group after single i.v. treatment with NTG. NADCIN^Ò significantly inhibits tolerance-inducing properties of the prolonged nitroglycerin infusion (BP_max, nitroglycerin response in % of normal controls: NTGT 51.2%, NADCIN^Ò-91.6, MB-55.8%). NADCIN^Ò in NTGT rats after NTG i.v. administration increased reduced BRS (+37.8%, p < 0.05), reflex control of SSNA (+29.4%, p < 0.05).

Acute action of NTG did not induce significant changes in redox-potential of mitochondria of thoracic aortic tissue and did not change the rate of superoxide anion generation, with the tendency to decrease of ALDH-2 activity (Table 2). In NTGT rats after the i.v. injection of NTG the changes in BRS and SSNA was accompanied with significantly decrease of redox-potential NAD/NADH by 20% in thoracic aorta tissue (Table 2) without changes in total pyridine nucleotide pool (Table 2) and in the level of vasoconstrictor cytokine (ET-1) production in vessels. The decrease in redox-potential even without changes in the total pool of pyridine nucleotide was accompanied with increased of the rate of superoxide anion generation in 1.5 times vs NTG non tolerated group. Cellular redox status is an important regulator of energy fuel metabolism, and many enzymes are under regul- ation of NAD⁺/NADH and NADP⁺/NADPH ratios not only in normal condition but under various pathological states [19] -[21] . No change in NADP/NADPH redox-potential of mitochondria was observed under acute sin- gle-dose treatment with NTG in normal rats which accompanied with normal rate of superoxide generation and ALDH activity (Table 2). Under continue administration of NTG the reaction of following i/v injection of high

Notes: BP: blood pressure; HP: heart period; HR: heart rate (upon baroreflex activation); MB: methylene blue; n: number of animals in each group; ^*Comparison with control group; ^#With control +NTG 150 mcg group; +: With III(NTGT) group. One symbol: p < 0.05, two: p < 0.01, three: p < 0.001.

dose of NTG disturbed: occurs decreasing in NAD/NADH redox state (without significantly decrease in NADP/ NADPH potential) that leads to a increasing of generation of superoxide anion and to decrease of ALDH2 activity (Table 2) and give grounds to suggest that major source of oxidative stress in normal animals under NTG-tolerance formation is the mitochondrial complex 1 functioning disturbances [11] . Activity of mitoch- ondrial ALDH significantly decrease (by 71%, p < 0.001 in comparison to normal animals and by 67% vs level after single i/v injection of 150 mcg/kg NTG) after the continue administration of NTG in normal animals and fully reverse under treatment with NADCIN^Ò and increased up to level obtained during acute NTG injection under treatment with guanyl cyclase inhibitor, MB. Our data confirmed early hypothesis that mitochondrial complex 1 is one of the targets at which the initial oxidative stress responsible for GTN tolerance takes place. In according with this the treatment with oxidized form NAD-containing drug, NADCIN^®, is a new strategy for prevention and therapy of NTG-tolerance formation, strategy of replacement therapy.