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Fig. 11. Detection of NO stores by the BP response to DETC in conscious rats, % of baseline values. Upper panel: (a) 5 h after DNIC with glutathione injection; (b) control. Bottom panel: (1) baseline; (2) BP response to DETC in untreated rats; and (3) BP response to DETC in rats pretreated with DNIC. Significant difference from baseline, p < 0.05 [32].

Fig. 11. Detection of NO stores by the BP response to DETC in conscious rats, % of baseline values. Upper panel: (a) 5 h after DNIC with glutathione injection; (b) control. Bottom panel: (1) baseline; (2) BP response to DETC in untreated rats; and (3) BP response to DETC in rats pretreated with DNIC. Significant difference from baseline, p < 0.05 [32].

Media Luna Gimnasia Metodologia

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Fig. 12. EPR of MNIC-DETC after the depletion of NO stores with DETC [32].

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Fig. 12. EPR of MNIC-DETC after the depletion of NO stores with DETC [32].

detected by EPR signals (Fig. 12). We proposed that the DETC-induced decrease in BP 5 h after the DNIC injection was due to the depletion of NO stores induced by DETC.

We proposed a parameter describing the efficiency of NO storage in vascular wall, the maximum amount of NO stores, which can be accumulated during incubation of an isolated blood vessel with an NO donor, specifically with DNIC. Using this method, we showed that the efficiency of NO store formation in blood vessels from SHR is more than twofold lower than in their genetic control WKY [33].

A tentative relationship between the NO level, NO stores and BP in normotensive and SHRs can be represented as shown in Fig. 13.

Fig. 13. Atentative relationship between the NO level, NO stores and blood pressure (BP) in normotensive and spontaneously hypertensive rats (SHRs) treated with DNIC. SHRSP, stroke-prone spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; HTE, hypotensive effect; NO, nitric oxide.

After administration of the NO donor, a part of the excess NO binds to the NO store whereas the unbound part of NO exerts its biological effect, i.e. induces vasodilation and a decrease in BP. When the efficiency of NO storage is high, the volume of NO stores is large while the hypotensive effect is small. This situation apparently takes place in normotensive rats. On the contrary, when the efficiency of NO storage is low, much NO remains unbound and exerts a more pronounced hypotensive effect. This is the case in SHRs. This may explain why the hypotensive effect of DNIC is much more pronounced in SHRSP than in WKY rats.

The formation of NO stores is apparently an adaptive process. Binding of excess NO to NO stores may protect blood vessels and the body as a whole against NO cytotoxicity. Since SHRs are characterized by NO overproduction in vascular smooth muscles and macrophages resulting in vascular damage, impairment of endothelial nitric oxide synthase (eNOS) activity and endothelial dysfunction [34], the decreased capacity for NO binding observed in SHRSP may facilitate the development of these disorders.

Recently however, a possibility for modulation of NO storage capacity was demonstrated. We showed that chronic treatment of rats with DNIC increased the efficiency of NO storage and vice versa, chronic administration of the NO synthase inhibitor l-NNA reduced it [35]. This mechanism of vascular adaptation to chronic changes in NO level may be potentially used to improve the defense against nitrosative stress of the cardiovascular system in SHRSP.

Therefore, the DNICs can be considered as powerful long-acting hypotensive compounds which can ensure the development of new type of medicines at least with cardiovascular activity.

THE VASODILATORY ACTIVITY OF DNICs

It was reasonable to suggest that high hypotensive activity of the DNICs was caused by their capacity for inducing vasorelaxation. Organ bath experiments performed on pre-contracted denuded isolated ring segments of rat aorta supported this idea completely [20]. Original recordings of ring segment tension for monomeric and dimeric forms of DNIC with cysteine (monomeric DNIC 1:20 and dimeric DNIC 1:2, respectively; see Chapter 2) and comparison with gaseous NO and acetylcholine (Ach) are presented in Fig. 14. The experiments with Ach-induced release of endothelium-derived relaxing factor (EDRF) from endothelial cells [36] were performed on ring segments with preserved endothelium. These preparations were treated with atropine (Atr), 10-5 M to prevent the effect of Ach.

For all vasodilators, superoxide dismutase (SOD) potentiated their vasodilatory action. Similarity between the vasorelaxing activity of DNIC with cysteine and EDRF was observed. This similarity was seen in the slow kinetics of vessel tone restoration after the addition of DNIC or Ach at maximal concentrations (10-5 M) (Fig. 14). For both the agents, 10-5 M hemoglobin (Hb) added to the organ bath, restored the vessel tone, independent of the extent of tension recovery at the time of Hb administration. Finally, pretreatment of aortic ring segments with DETC (10-3 M) led to a decrease in the vasodilatory activity both of DNIC and Ach (EDRF), and prevented the vessel tone recovery (Fig. 15). In contrast, when DETC was applied after the addition of DNIC or Ach (10-5 M), a rapid restoration of vascular tension was observed (Fig. 15).

Fig. 14. Effects of nitric oxide (NO) (A), DNIC with cysteine 1:20 (B) and acetylcholine (Ach) (C) in the absence (-SOD) or in the presence (+SOD) of superoxide dismutase (SOD 30 U/ ml), on norepinephrine (NE, 10-7 M)-induced contractions of isolated ring segments from rat aorta. The dots show the addition of the agents (in -log M) atropine (Atr, 10-5 M) and hemoglobin (Hb, 10-5 M). Dotted line shows passive tension before the addition of NE. Vertical bars, 1 g; horizontal bars, 5 min [20].

Fig. 14. Effects of nitric oxide (NO) (A), DNIC with cysteine 1:20 (B) and acetylcholine (Ach) (C) in the absence (-SOD) or in the presence (+SOD) of superoxide dismutase (SOD 30 U/ ml), on norepinephrine (NE, 10-7 M)-induced contractions of isolated ring segments from rat aorta. The dots show the addition of the agents (in -log M) atropine (Atr, 10-5 M) and hemoglobin (Hb, 10-5 M). Dotted line shows passive tension before the addition of NE. Vertical bars, 1 g; horizontal bars, 5 min [20].

Long-lasting relaxation induced by DNIC or Ach after pretreatment of the blood vessels with DETC was not due to an effect on the contractile ability of preparations, since after 60 min of rest and extensive washing with PBS the vessel segments responded normally to NE and endothelium-dependent and -independent relaxing agents.

We can speculate that the long-lasting vessel relaxation characteristic of the ring segments pretreated with DETC prior to DNIC resulted from the formation of high amounts of RS—NOs during degradation of DNIC induced by DETC as shown in Scheme 1. A similar

Fig. 15. Effect of diethyldithiocarbamate (DETC, 10-3 M) on the relaxation of rat aortic rings with (B) and without (A) endothelium pre-contracted with norepinephrine (NE, 10-7 M). Experiments were carried out in the presence of superoxide dismutase (SOD, 30 U/ml). Relaxation was induced by (A) nitric oxide (NO); acetylcholine (Ach); and (C) dinitrosyl iron complex with cysteine (DNIC) 1:2 and (D) DNIC 1:20. Effects of subsequent addition of DETC and hemoglobin (Hb, 10-5 M) are also shown. In (B), atropine (Atr, 10-5 M) was used to block endothelial muscarinic receptors. The dotted line indicates addition of the agents (in -log M). Vertical bars, 1 g, horizontal bars, 5 min. Numbers indicate the interval between recordings in minutes. The dotted line indicates the passive tension before the addition of norepinephrine (NE). These traces are representative of seven experiments [21].

Fig. 15. Effect of diethyldithiocarbamate (DETC, 10-3 M) on the relaxation of rat aortic rings with (B) and without (A) endothelium pre-contracted with norepinephrine (NE, 10-7 M). Experiments were carried out in the presence of superoxide dismutase (SOD, 30 U/ml). Relaxation was induced by (A) nitric oxide (NO); acetylcholine (Ach); and (C) dinitrosyl iron complex with cysteine (DNIC) 1:2 and (D) DNIC 1:20. Effects of subsequent addition of DETC and hemoglobin (Hb, 10-5 M) are also shown. In (B), atropine (Atr, 10-5 M) was used to block endothelial muscarinic receptors. The dotted line indicates addition of the agents (in -log M). Vertical bars, 1 g, horizontal bars, 5 min. Numbers indicate the interval between recordings in minutes. The dotted line indicates the passive tension before the addition of norepinephrine (NE). These traces are representative of seven experiments [21].

hypothesis was made above to explain the brief drop of BP in anesthetized or conscious rats successively treated with DNIC and DETC (Figs. 3 and 6). In contrast, the relaxation induced by DNIC in isolated vessel segments in the presence of DETC had a long-lasting nature. Possibly, it was due to much more intensive accumulation of RS—NO molecules in the organ bath medium because all added DNIC molecules reacted with DETC. This was not the case when DETC was added 15 min after DNIC administration. This time DNIC decomposition led to a decrease in the amount of RS—NOs, which could have formed during the subsequent degradation of remaining DNICs by DETC. As a result, the vasorelaxation was eliminated.

A striking similarity between the effects of DETC on time course of BP on the addition of DNIC or Ach to the preparations allows to suggest that the compound accumulated in aorta segments by the time of Atr addition could be the DNIC. This is in line with the hypothesis advanced 15 years ago that EDRF was identical to DNIC [37]. Being degraded by DETC this compound produced RS—NOs. When the latter is accumulated in sufficient amounts it exerted a hypotensive effect.

Fully reversible (transient or T-type) or long-lasting (sustained or S-type) vasodilator responses were demonstrated for the pre-contracted, internally perfused rat tail artery after a bolus treatment with two iron-sulfur cluster nitrosyls (heptanitrosyltetra-^3-thioxotetraferrate or tetranitrosyltetra-^3-sulfidotetrahedro-tetrairon) [38]. Both the compounds produced a T-type response only at low doses below a critical threshold concentration, 10 4 or 10-5 M. The S-type response was observed at higher concentrations of both the vasodilators that comprised an initial, rapid drop of vessel tone, followed by incomplete tone restoration, resulting in a plateau of reduced tone persisting for a few hours. SNP or S-nitroso-N-acetylpenicillamine (SNAP) produced T-type responses only. DesoxyHb (NO scavenger) or methylene blue (MB), a guanylate cyclase inhibitor, initiated a prompt and complete recovery of vessel tone of all agonist-induced tone.

The T-type response was attributed to free NO released from added iron-nitrosyl cluster at the time of injection. With respect to S-type response, it was attributed to NO generated by gradual decomposition of a "store" of iron-nitrosyl complexes within the tissue. This idea was supported by the results of histochemical studies on arterial tissue treated with iron-sulfur-nitrosyl clusters. They showed high accumulation of ferrous iron, derived from these clusters. These iron deposits were predominantly located in endothelial cells.

The authors underline the similarity between vasorelaxing responses to the two used iron-nitrosyl complexes and above-considered DNIC with thiol-containing ligands, which can also induce hypotension in animals considered above or inhibit platelet aggregation [15-19,21,22]. The vasodilator and hypotensive actions of the DNICs also exhibit two-phase kinetics resembling the S-type response observed for the two used in [38] iron-nitrosyl complexes. These data also indicate the formation of iron-nitrosyl stores in tissues treated with DNICs. Interestingly, in accordance with the data described in [39] an injection of heptanitrosyltetra-^3-thioxotetraferrate ("Roussin's Black Salt") into the rat led to the appearance of protein-bound DNICs in animal tissues in vivo.

A similar time course type of relaxation was also observed in isolated blood vessels for DNIC with non-thiolic ligands, DNIC with phosphate or bathocuproine disulfonate [40]. It is reasonable to suggest that long-lasting vasorelaxation induced by these DNICs was also due to the formation of protein-bound DNICs in vessel tissues acting as a depot of iron-nitrosyls. This was supported by experiments where DNIC with phosphate was injected into mice: formation of protein-bound DNIC was observed in the animal body [41].

ROLE OF DNIC/NITRIC OXIDE STORES IN PROTECTION AGAINST NITRIC OXIDE OVERPRODUCTION

Formation of NO stores in vascular wall begins after any increase in NO level induced by the stimulation of NO synthesis or administration of NO donors. An efficient method for the stimulation of NO synthesis and correspondingly, formation of NO stores is adaptation to environmental factors such as heat, exercise, mild stress or hypoxia [42]. We tested the hypothesis that DNIC/NO stores not only can provide a hypotensive effect in situations of absolute or relative NO deficiency but also can prevent NO overproduction and related injuries [43,44]. Heat stroke was used as an inductor of NO overproduction, which resulted in acute hypotension and death of some rats [45]. Adaptation to mild restraint stress was used as a means of prior increase in NO level and protection against heat stroke and NO overproduction. It was demonstrated that the protection was associated with the formation of NO stores in the vascular wall. Indeed when small doses of l-NNA were injected prior to each session of adaptation, neither protective effects developed nor NO stores formed. At the same time, injections of the NO donor DNIC with glutathione as ligand induced the formation of NO stores and completely mimicked the protection [46]. The protective effect of NO donor could not be due to the immediate action of NO because the lifetime of intravenous DNIC in the organism was about 3-5 h [29], while the effect of exogenous DNIC on both survival of rats and NO overproduction was observed 24 h after heat stroke [46]. Potential mechanisms of the protective effects of NO stores are the restriction of activity and/or expression of NOS by negative feedback [47] or the removal of excess NO by binding in NO stores. This compensatory mechanism helps in the prevention of toxic effects of increased NO formed in the process of adaptation.

Further evidence for the protective role of NO stores in NO overproduction was obtained on the rat model of Alzheimer's disease [48]. It is known that development of Alzheimer's disease is associated with toxic effects of excess NO overproduced by neurons and glia [49]. Experiments were carried out on anesthetized rats at natural ventilation. Local cerebral blood flow was continuously monitored with a laser Doppler probe. NO stores in cerebral blood vessels were detected by the vasodilatory response to N-acetylcysteine evident as an increase in cerebral blood flow. Since the size of NO stores is statistically significantly correlated with NO production [50], appearance and increase of NO stores may be regarded as an indirect marker of NO overproduction. Furthermore, the overproduction of NO was confirmed by the measurement of nitrite and nitrate in brain tissue.

NO stores were absent in cerebral blood vessels of control rats. In rats pretreated with DNIC, we observed an increase in cerebral blood flow, which indicated the presence of NO stores. In both hypoxia-adapted and Ap-treated rats, N-acetylcysteine also revealed NO stores. In rats injected with Ap after adaptation, the size of NO stores was significantly larger than in non-adapted rats treated with Ap or adapted rats (Fig. 16).

We suggest that adaptation increased the ability of blood vessels for storing NO complexes. Indeed, as shown in the bottom diagram, chronic elevation of NO induced by daily injections of NO donor or by daily sessions of hypoxia, significantly increased the efficiency

Fig. 16. Effect of DNIC, adaptation to hypoxia, Aß(25-35) and Aß(25-35) after pre-adaptation to hypoxia on the formation of NO stores in cerebral blood vessels. Bars reflect increases in cerebral blood flow in response to N-acetylcysteine in percent of baseline value. *Significant difference from control, p < 0.05 [50].

Fig. 16. Effect of DNIC, adaptation to hypoxia, Aß(25-35) and Aß(25-35) after pre-adaptation to hypoxia on the formation of NO stores in cerebral blood vessels. Bars reflect increases in cerebral blood flow in response to N-acetylcysteine in percent of baseline value. *Significant difference from control, p < 0.05 [50].

of binding NO to NO stores. This adaptive mechanism may enhance protection against NO overproduction.

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