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Fig. 6. Top panel: Blood pressure changes in conscious SHR rats (n = 6, baseline BP = 180 ± 10 mmHg) induced by an i.v. injection of DNIC with glutathione (10 ^M/kg). (1) and (2) are changes in ABP (mmHg) and ABP/BP (%), respectively. Bottom panel: Blood pressure changes in conscious SHR rats induced by an i.v. injection of DNIC with thioglycolate (10 ^M/kg) followed by the addition of DETC (30 mg/kg) (1 min after DNIC injection). Arrows indicate time points of subsequent additions of DETC [17].

Fig. 7. EPR spectra of blood (curve a), liver (curve b) and kidney (curve c) from Wistar rats injected i.v. with DNIC with glutathione; (curves d,e) are the EPR spectra of DNIC with glutathione or thiosulfate, respectively. Recordings were made at 77 K (left side) and ambient temperature (right side) [17].

Fig. 8. EPR spectra of blood (curves a,d), liver (curves b,e) or kidney (curves c,f) from Wistar rats injected with DNIC with thiosulfate (5 ^M/kg) 1 min after or 30 min before the addition of DETC (30 mg/kg) (curves a-c or d-f, respectively). Recordings were made at 77 K [17].

Thus, the attenuation of DNIC hypotension induced by the prior administration of DETC correlated with the disappearance of protein-bound DNICs as possible NO donors producing hypotension. This was not the case in experiments where DETC was added 20 min and above after a DNIC injection or DNICs were administered without DETC. In this case, BP recovered to the baseline level despite persisting protein-bound DNIC in animal tissues. The baroreflex mechanism maintaining BP could be responsible for the BP restoration. It was sufficiently strong to get over the hypotensive action of NO released from protein-bound DNICs.

The hypotensive activity of DNIC with cysteine in normotensive Wistar-Kyoto (WKY) rats was compared to DNIC distribution in organs and tissues in order to propose an optimum DNIC dosage, which would, on the one hand, provide an efficient tissue level of DNIC and, on the other hand, would not exert adverse effects on animals [29]. Blood pressure (BP) of rats injected intravenously with 0.5, 2, 4 and 6 ^M/kg DNIC was monitored continuously for 3 h following the injection and measured again in 24 h. Fig. 9 shows the time course of BP after DNIC injection into conscious rats. Injection of 0.5 ^M/kg DNIC did not significantly influence BP. After injection of 2 ^M/kg DNIC, BP gradually decreased but remained above 90-95 mmHg in all animals. After injection of 4 ^M/kg DNIC, BP significantly decreased as soon as in 10 min and reached a nadir of 80-85 mmHg in 2-2.5 h. The dose of 6 ^M/kg DNIC almost immediately induced a dramatic drop of BP to 75-80 mmHg. The BP variation at the latter dose was high; in some animals BP fell to 55-60 mmHg. The decreased BP persisted for the entire period of monitoring (3 h) and was accompanied with considerable disorders of heart rhythm. Twenty-four hours after injection, BP returned to the baseline level irrespective of the DNIC dose.

Fig. 9. Time course of BP after DNIC injection. (1) control; (2) 0.5 M-M/kg; (3) 2 M-M/kg; (4) 4 M-M/kg; and (5) 6 M-M/kg. *Significant difference from control, p < 0.05 [29].

Data on distribution of DNIC in tissues show that the EPR signal from DNIC was undetectable 1.5 h after the injection of 0.5 ^M/kg DNIC in all analyzed organs (liver, kidneys, heart, spleen, small intestine, blood, lungs and brain). After the injection of 2 ^M/kg DNIC, the EPR signal was consistently recorded in blood, kidneys and brain. After the injection of 4 and 6 ^M/kg DNIC, a typical EPR signal was recorded in all the organs and tissues studied at 1.5 h and disappeared at 24 h.

Therefore, intravenous DNIC distributed to organs and tissues in a dose-dependent manner and exerted a dose- and time-related hypotensive effect, which corresponded to the content of DNIC in tissues. The dose of 4 ^M/kg DNIC was efficient and did not exert adverse effects. The use of DNIC as a NO donor in diseases and conditions associated with absolute or relative NO deficiency seemed promising.

The effect of DNIC on development of hypertension was studied in SHRSP rats aged 5-6 weeks, which corresponded to the early hypertension stage. DNIC (3 ^M/kg, i.p.) was injected into SHRSP and their normotensive control WKY rats every fourth day for 40 days [30]. During the development of hypertension, BP of untreated SHRSP increased from 136 ± 3 mmHg at the early hypertension stage to 216 ± 7 mmHg at the established hypertension stage (p < 0.001) (Fig. 10). In SHRSP treated with DNIC, BP increased only to 168 ± 17 mmHg (p < 0.01). Interestingly, the course of DNIC did not result in any significant changes in BP of WKY rats (124 ± 3.2 mmHg vs. 113 ± 2.5 mmHg before and after the DNIC course, respectively).

Therefore, DNIC exerted a selective anti-hypertensive effect in SHRs and left the BP unchanged in normotensive rats. This phenomenon may be attributed to different capacity for binding of free NO released from DNIC to NO stores in blood vessels of SHRSP and WKY rats.

NO stores primarily in the form of DNICs, which have been formed in vivo in vascular walls after the injection of DNIC or stimulation of endogenous NO synthesis, can be detected on isolated blood vessels [31]. The method used is based on the interaction of DETC of

Baseline

Untreated control

DNIC treatment

Fig. 10. Effect of DNIC with glutathione on development of hypertension in SHRSP rats. Empty bars, control; dashed bars, SHRSP. *Significant difference from baseline value, p < 0.05 [30]. Reprinted with permission from "Role of nitric oxide in adaptation to hypoxia and adaptive response" (Physiol. Res. 2002; 49: 89-97).

Baseline

Untreated control

DNIC treatment

Fig. 10. Effect of DNIC with glutathione on development of hypertension in SHRSP rats. Empty bars, control; dashed bars, SHRSP. *Significant difference from baseline value, p < 0.05 [30]. Reprinted with permission from "Role of nitric oxide in adaptation to hypoxia and adaptive response" (Physiol. Res. 2002; 49: 89-97).

N-acetylcysteine with NO stores, which results in the release of free NO and vasorelaxation. This method was successfully modified for detecting NO stores in conscious rats pretreated with DNIC [32]. NO stores were detected 5 h after the DNIC injection (4 ^M/kg, i.p.) with prior (1 h before experiment) inhibition of NO synthase with NW-nitro-L-arginine (l-NNA) to exclude contribution of de novo synthesized NO to the vasodilatory response. The DNIC injection induced a moderate hypotensive response which lasted for 1.5 h. Infusion of DETC (100 ^M/kg, i.v.) resulted in a transient 13.0 ± 3.4% decrease in BP, which returned to the baseline level in approximately 40 min (Fig. 11). The hypotensive response was accompanied by the formation of MNIC-DETC in all analyzed organs, heart, liver, kidneys and brain as

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