85 ± 5

on hypotensive activity of these DNICs in comparison to the well-known hypotensive and vasodilatory agents, sodium nitroprusside (SNP) and nitroglycerol (NG).

All DNICs were proved to be less efficient than SNP or NG at the initial stage characterized by a rapid drop and the subsequent recovery of blood pressure (BP). The main feature of the DNICs was the prolonged character of their hypotensive action. After a bolus injection of these compounds, the animals exhibited a reduced stationary BP level retained up to awakening. For repeated injections, the plateau level was observed to decrease, each time with the proceeding rapid reversible drop of BP (Fig. 1). The injection of SNP resulted only in a short-time (2-4 min) decrease in BP, not affected by the proceeding administration of the same agent. Unlike this, in the case of NG the preliminary injection affected the subsequent reaction (the effect of tolerance to NG) as illustrated in Fig. 1.

The injection of any DNIC type into animals led to the appearance of protein-bound DNICs in blood, liver and other tissues characterized with the EPR signal at gav = 2.03 (gx = 2.04, g|l= 2.014). Such type of EPR signal, recorded in liver is shown in Fig. 2, curve a.

As was demonstrated later in similar experiments, the shape of the signal did not change while the recording temperature was increased from 77 K to the ambient one [22,23]. Namely, this fact pointed unequivocally to the protein-bound nature of DNIC: The mobility of protein molecules at room temperature was not sufficiently high to ensure the averaging of anisotropy g-factor and hyperfine structure (HFS) tensors. In contrast, the low-molecular DNICs are rapidly tumbling and show a motionally narrowed isotropic line with a half-width of 0.7 mT and resolved 13- or 5-component HFS [24].

The formation of protein-bound DNICs was due to the rapid transfer of Fe(NO)2 groups from low-molecular DNICs to paired RS-groups of protein. Thereby, something resembling a pool of nitric oxide (NO) appeared, which was represented by protein-bound DNICs characterized by much higher stability than that of low-molecular counterparts. As protein-bound DNICs decompose, the released nitrosyl-iron groups act as a direct source of NO ensuring the retention of decreased BP in animals for 30 min and above after a bolus injection of low-molecular DNICs [15-17].

The transition of nitrosyl-iron groups from low-molecular DNICs to proteins is confirmed by the following experimental results. The animals received injections of DNIC with thiosulfate, where 56Fe was substituted by the 57Fe isotope with nuclear spin I = 1/2. Preparations of blood, liver and other tissues showed the DNICs with a broadened EPR signal.

Fig. 2. The EPR spectra of DNIC with 56Fe (curve a) or Fe (curve b) formed in the rat liver after injections of DNIC with thiosulfate and mononitrosyl iron complex (MNIC) with diethyldithiocarbamate (DETC) appeared in the liver after injections of DETC into the same animals [(c) and (d), respectively]. Recordings were made at 77 K [15].

The broadening was especially pronounced at gx component due to the non-resolved HFS from 57Fe (Fig. 2, curve b).

The hypothesis that the nitrosyl-iron group in DNIC was responsible for the hypotensive effect might be verified by adding sodium diethyldithiocarbamate (DETC) (formula: (C2Hs)^=N—C-) capable of strong selective binding of Fe—NO groups that results in the formation of mononitrosyl iron complexes (MNIC) with DETC, formula: {(C2H5)2=N—CS-}2—Fe+—NO+. These complexes are characterized by the EPR signal with gx = 2.045, g||= 2.02 and a well-resolved triplet HFS at gx [25] (Fig. 2, curves c,d). Earlier [26] it was demonstrated that DETC readily captures iron and NO radical from DNIC to form corresponding MNIC-DETC. A similar result was obtained on rats injected with DNIC and DETC [15,27]. For example, i.a. injection into the anesthetized rats of DETC 10 min after administration of the DNIC changed the shape of the EPR spectrum of the blood: instead of the EPR signal of the DNIC the MNIC-DETC was observed localized mainly (up to 90%) in the red blood cells [27]. Within 30-40 min the MNIC-DETC content in the blood fell sharply and in place of these complexes DNIC appeared, also localized in the red blood cells. The shape of the EPR signal was similar to that characteristic of the EPR signal of DNIC bound with Hb [28] (see also Chapter 2). The intensity of the signal was far lower compared with that of the initial signal of DNIC in blood before the addition of DETC. In the liver of these animals by this time, judging from the EPR spectrum of this organ, MNIC-DETC persisted [15,27].

No hypotensive effect was observed in the rats injected with "ready-to-use" MNIC-DETC in the physiological salt solution (the dose injected was the same as in the case of DNIC) [15]. Apparently, this was due to the strong binding of Fe—NO group to DETC, preventing the liberation of this group from MNIC in the organism. For this reason, it should be anticipated that the introduction of DETC to animals injected with DNIC would normalize the BP level. Indeed, experiments have confirmed this hypothesis. Irrespective of the time of DETC addition, whether prior to the introduction of DNIC or after establishing a stationary BP level induced by the injection of DNIC, the BP level either recovered or tended to the normalization (Fig. 3, curves b,c) [15,27].

Fig. 3. Blood pressure changes (ABP, mmHg) in rats induced by DNIC with thiosulfate (400 nM) injected at time points 1 or 2 without DETC (curves a,b) or after DETC (5 mg) i.p. injections (40 mg) (curve b). DETC was injected i.v. at time point 3 on curve c [15].

Interestingly, the recovery of BP induced by DETC after DNIC injection was preceded as a rule by a brief drop of BP [15,17] (Fig. 3, curve c). This brief drop of BP can be attributed to different levels of NO in MNIC and DNIC (low-molecular or protein-bound DNICs). When a DETC molecule interacts with DNIC, the former takes a Fe ion and one NO molecule from DNIC to form MNIC-DETC (Scheme 2).

(RS-)2 Fe+(NO+)2}+ + 2{(C2H5)2=N-CS2-}->{(C2H5)2=N-CS2-}2-Fe+-NO+ + RS-—NO+ + RS-

Scheme 2.

The second NO+ ligand (nitrosonium ion) becomes free and nitrosates low-molecular or protein-bound thiols forming respective S-nitrosothiols (RS—NOs). Due to the high vasodilatory activity [12], RS—NOs induce brief hypotension. The source of thiols recombining with nitrosonium ions remains obscure. With regard to the hypotensive effect, obviously only low-molecular RS—NOs could be responsible for it due to their free motion in the intracellular space. Rapid degradation of low-molecular RS—NO under the action of iron or copper ions in animal organisms could sharply shorten the duration of hypotensive effect by these compounds.

Interestingly, administration of DETC prior to SNP (MNIC with cyanide) to the animals completely eliminated the hypotensive effect of SNP, but without the initial brief drop of BP [15,27]. No hypotensive activity is seen in rats even after an injection of 1 ^M SNP [15] (Fig. 4, curve b). For comparison, note that in the absence of DETC an injection of only 200 nM SNP was lethal due to the drop of BP below the permissible level. Apparently, the total elimination of hypotensive activity by DETC was due to the release of Fe+—NO+ group from SNP. This group was completely transferred to DETC without the evolution of free nitrosonium ions, in contrast to the events that could occur during the interaction of DETC with Fe+ (NO+)2 groups in DNICs.

Interestingly, Fe2+ ions themselves exhibited a noticeable hypotensive activity (Fig. 4, curve a). However, these ions differed in behavior from DNIC in that their action has no prolonged character and can be completely suppressed by DETC (Fig. 4, curve a).

Fig. 4. Elimination of the hypotensive effects of FeSO4 (curve a) or sodium nitroprusside (SNP) (curve b) by an injection of DETC (40 mg, i.p.) at time point 2 or before the nitroprusside administration. Doses of FeSO4 or nitroprusside were equal to 100 nM (1), 200 nM (3), 200 nM (4) or 2000 nM (5) [15].

Fig. 4. Elimination of the hypotensive effects of FeSO4 (curve a) or sodium nitroprusside (SNP) (curve b) by an injection of DETC (40 mg, i.p.) at time point 2 or before the nitroprusside administration. Doses of FeSO4 or nitroprusside were equal to 100 nM (1), 200 nM (3), 200 nM (4) or 2000 nM (5) [15].

The idea that protein-bound DNICs as NO donors can determine long-lasting hypotension was supported by experiments with injection of "ready-to-use" protein-bound DNICs to anesthetized rats or dogs [16]. These complexes were synthesized by the administration of low-molecular DNIC with thiosulfate or phosphate to bovine serum or plasma from dogs or rats (0.4 ^M DNIC/ml of serum or blood plasma). This amount of proteins in serum or plasma preparations was sufficiently high to ensure acceptance of all Fe+(NO+ )2 groups from low-molecular DNICs by respective protein ligands. Prolonged hypotension induced by both types of preparations was virtually similar to that induced by the addition of low-molecular DNICs (Fig. 5). The difference was a slower time course of BP response to the injection of preparations with protein-bound DNIC. Interestingly, a bolus injection of noradrenaline to dogs or rats with reduced BP resulted in a brief recovery of BP (within 3-4 min) followed by a fall of BP to the level induced by the preceding DNIC injection (Fig. 5). Administration to animals of serum or plasma not containing protein-bound DNICs virtually left the BP level unchanged.

Hypothetically, the hypotensive activity of protein-bound DNICs could be ensured by back transfer of Fe+(NO+)2 groups from protein to low-molecular ligands with the formation of more mobile low-molecular DNICs. Specifically, the latter donated NO molecules to special enzymes such as guanylate cyclase providing a relaxation response of vascular smooth muscle to NO.

Hypotensive changes in BP evoked by low-molecular DNICs were also observed in conscious normotensive (Wistar) and spontaneously hypertensive (SHRs) rats weighing

Fig. 5. Top panel: Blood pressure changes (ABP, mmHg) in anesthetized dogs induced by an i.v. injection of blood plasma from an animal pretreated with either DNICs (10 ^M/350 ml, curve 1) or DNIC with thiosulfate in phosphate-buffered saline (PBS) (10 ^M/animal, curves 2,3). Subsequently epinephrine or DNIC was added at time points a and b, respectively. Bottom panel: Blood pressure changes (ABP, mmHg; baseline BP = 130 ± 10 mmHg) in anesthetized Wistar rats (n = 5) induced by an i.a. injection of either rat blood plasma containing DNICs (1.5 ^M/kg in 1.5 ml, curve 4) or DNIC with thiosulfate in PBS (1.5 ^M/kg in 1.5 ml, curve 5) [16].

o 20 40 min

200-250 g [17]. A bolus i.v. injection of all used DNICs with glutathione, dithiothreitol or thioglycolate ligands exerted a hypotensive effect in conscious stroke-prone SHR (SHRSP) rats with initial BP value of 180 ± 10 mmHg. At DNIC doses of 1, 3 or 10 ^M/kg, the decrease in BP reached 10-20, 20-30 or 70-80 mmHg. Further increment of the DNIC dose up to 20 ^M/kg did not virtually intensify the effect. The dynamics of BP decrease is shown in Fig. 6, top panel. In contrast to hypotension induced by DNICs in anesthetized animals, a relatively rapid recovery of BP (within 1-1.5 h) was observed in conscious SHR rats. Normotensive Wistar rats with baseline BP of 110-120 mmHg proved more resistant to the effect of DNICs. When 5 ^M/kg DNIC-thioglycolate was administered, the maximal BP decrease did not exceed 30-40 mmHg with hypotension lasting for 1-1.5 h. A bolus injection of SNP or NG into conscious animals produced only a brief hypotensive effect (1-2 min).

Prior DETC injection into SHR (150 ^M/kg) sharply attenuated the hypotensive effect of DNIC with thioglycolate administered at 10 ^M/kg (Fig. 6, bottom panel). Similar effect was observed when DETC was injected 5 min after DNICs. However, DETC injected 20 min after DNICs did not significantly affect intensity or duration of the hypotension induced by DNICs with the exception of a brief BP drop (Fig. 6, bottom panel). Similar effect of DETC mentioned above was characteristic of anesthetized animals (Fig. 3). Obviously, in both cases the reason was formation of short-living RS—NO due to DNIC degradation under the action of DETC as shown in Scheme 1.

The EPR assay demonstrated the formation of protein-bound DNICs in blood and other tissues from conscious animals injected with DNICs (Fig. 7) [17].

The complexes rapidly (within 1 h) disappeared from blood but remained in other tissues (liver, kidney and others) for 2-3 h despite the full recovery of BP. If DETC was administered to animals before DNIC, the latter was transformed into MNIC-DETC in all organs except for blood where DNIC disappeared from plasma but remained unchanged in red cells (Fig. 8). When DETC was injected into animals 20 min after DNIC the major part of DNIC remained unchanged in liver or kidney (Fig. 8).

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