Secondary Logo

Journal Logo

S-nitroso-N-acetylpenicillamine (SNAP) During Hemorrhagic Shock Improves Mortality as a Result of Recovery From Vascular Hyporeactivity

Sato, Shigehito MD*; Suzuki, Akira MD*; Nakajima, Yoshiki MD*; Iwamoto, Tatsuaki MD*; Bito, Hiromichi MD*; Miyabe, Masayuki MD

doi: 10.1213/00000539-200002000-00023
CRITICAL CARE AND TRAUMA
Free
SDC

Nitric oxide donors are protective against hemorrhagic shock (HS). However, no detailed investigation has been performed. We investigated this mechanism using S-nitroso-N-acetylpenicillamine (SNAP). HS (mean arterial pressure: 40 mm Hg) was induced in 20 dogs. Sixty min after HS, the animals were treated with saline (Cont-Gr:n = 7) or SNAP; 5 μg · kg−1 · 10 min−1 fol- lowed by 5 μg · kg−1 · h−1 (SNAP-Gr:n = 7). After another 60 min, the shed blood was reinfused. Reactivities to noradrenalin (NA), changes in hemodynamics, the plasma catecholamines, and nitric oxide derivatives were determined. In Cont-Gr, 3 dogs died at 90, 98, and 102 min after HS. In Cont-Gr, % changes of systolic arterial blood pressure to 1 and 2.5 μg/kg of NA after the recovery from HS decreased from 23.7% ± 4.1% (before HS) to 6.5% ± 0.6% and from 50.1% ± 7.7% (before HS) to 14.5% ± 2.6%, respectively (P < 0.01). In SNAP-Gr, reactivity to NA was maintained. At 120 min after HS, mean arterial pressure and cardiac output in SNAP-Gr increased but not in Cont-Gr. Plasma catecholamine levels in SNAP-Gr were suppressed compared with those of Cont-Gr. In conclusion, a small dose of SNAP during HS decreased the mortality of the dogs. This might have been caused in part by residual vascular hyporeactivity.

Implications The administration of a small dose of S-nitroso-N-acetylpenicillamine (a nitric oxide donor), a dose which did not exert a significant vasodilator effect, was administered during hemorrhagic shock in dogs. S-nitroso-N-acetylpenicillamine improved the vascular hyporeactivity to noradrenaline and decreased the mortality rate.

*Department of Anesthesiology and Intensive Care, Hamamatsu University School of Medicine, Hamamatsu; and †Department of Anesthesiology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan

October 26, 1999.

Supported by a Grant-in-Aid (B) 10470312 from the Ministry of Education, Science and Culture, Japan.

Address correspondence and reprint requests to Shigehito Sato, MD, Department of Anesthesiology and Intensive Care, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan. Address e-mail to ssato@hama-med.ac.jp.

It has been shown that nitric oxide (NO) plays an important role in various types of shock (1). NO from inducible NO synthase (i-NOS) has been reported to increase in several models of endotoxin-induced septic shock and the overproduction of NO to contribute to the pathophysiology of septic shock (2,3). NO from the constitutive, endothelial NO synthase (ec-NOS) is also important in the pathophysiology of hemorrhagic shock (HS) (4–8), although this role is not fully understood (4,6,7,9).

However, the experimental administration of NO precursors, such as L-arginine, or NO donors are beneficial in improving the survival rate of animals ex- hibiting HS (10–13). In particular, a small dose of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) has been shown to improve the survival rate of rats in HS models (10,13). However, the mechanism of these protective effects of SNAP during HS have not been evaluated. We investigated the effect of SNAP administration during HS on the vascular reactivity to noradrenaline (NA) before and after HS, as well as changes in the hemodynamic state, and in the levels of plasma catecholamine (CA) and NO derivatives.

Back to Top | Article Outline

Methods

The study protocol was approved by the University of Tsukuba.

Twenty mongrel dogs (9.5–12.5 kg) were anesthetized with pentobarbital sodium 20 mg/kg IV, and intubated tracheally. IV administration, through the antecubital vein, of lactated Ringer’s solution 20 mL · kg−1 · h−1, pentobarbital sodium 3 mg · kg−1 · h, and pancuronium bromide 0.1 mg · kg−1 · h was then started and continued throughout the study. Mechanical ventilation (fraction of inspired oxygen: 0.25) was performed by a respirator, and PaCO2 was maintained between 35 and 40 mm Hg. Catheters for arterial blood pressure measurement and blood withdrawal were placed in the femoral artery and vein, respectively. The right external jugular vein was also exposed, and a flow-directed pulmonary artery catheter was inserted into the pulmonary artery. All catheters for pressure monitoring were attached to pressure transducers and connected to an eight-channel recorder. ETCO2 and inspiratory oxygen concentration were continuously monitored.

Electrocardiogram (lead II), heart rate, cardiac output (CO), mean arterial blood pressure (MAP), central venous pressure (CVP), mean pulmonary artery pressure, and pulmonary capillary wedge pressure were measured continuously and/or intermittently. Before and during the examination, the presence of hypoxia, hypercapnia, and metabolic acidosis was evaluated by sampling arterial blood for blood gas analysis.

The protocol is shown in Figure 1. After the surgical procedures, the dogs were allowed to rest for 1 h before the start of the protocol, after which the first NA test was performed. NA, 1μg/kg, was administered, followed by 2.5 μg/kg of NA after 5 min. Reactivity was expressed as the ratio of change in systolic arterial blood pressure (SBP). The ratio was expressed as:

Figure 1

Figure 1

MATH

Twenty minutes after the first NA test, following administration of 1,000 U of heparin, HS was induced by withdrawing blood from the femoral vein catheter (12-gauge, multi-orifice catheter) placed in the inferior vena cava. The blood was withdrawn and pooled spontaneously into a disinfected plastic bag (700 mL). The plastic bag was placed approximately 50–70 cm lower than the operating table until the MAP reached 40 mm Hg. The start of HS was defined as the period at which the MAP decreased to 40 mm Hg. Thereafter, the plastic bag was elevated to almost the same level as the animal’s heart, and an attempt was made to maintain the MAP at 35–40 mm Hg by adjusting the level of the plastic bag. HS of 35–40 mm was maintained for 60 min. The blood-withdrawing line was closed by using a three-way stopcock to stop the blood flow. The bag with the withdrawn blood was placed on the operating table and maintained at 37°C. Immediately after the closing of the three way stopcock, the animals were administered saline or SNAP, and observed for an additional 60 min.

Concerning the dose of SNAP, Symington et al. (10) reported that, in rats, 10 μg/kg per bolus and the same dose per hour as a maintenance was effective in improving the survival rate after HS. In a preliminary study, we administered the same dose to dogs. However, the administration of 10 μg/kg of SNAP during HS decreased the MAP more than 10 mm Hg, and the animals died because of progressive hypotension. We found that a dose of SNAP consisting of a loading dose of 5 μg/kg over 10 min, followed by a maintenance dose of 5 μg · kg−1 · h−1, did not decrease the MAP more than 3 mm Hg. Therefore, we decided that this dose of SNAP would be appropriate for the purpose of the study. We used SNAP 2 mg which was dissolved into 20 mL of saline (SNAP concentration: 100 μg/mL).

The animals were divided randomly into three groups. In the SNAP-treated group (SNAP-Gr:n = 7), HS was induced for 60 min, after which SNAP was administered. HS was not induced in the sham operation group (Sham-Gr :n = 6), but SNAP was administered. In the control group (Cont-Gr :n = 7), after 60 min of HS, the animals were administered saline in a volume equivalent to that of SNAP in the other two groups. After another 60 min of observing HS, the withdrawn blood was reinfused at a rate which prevented any CVP increase of more than 3 mm Hg over a short period. After the blood reinfusion, metabolic acidosis was treated with sodium bicarbonate for another 30 min to achieve an arterial pH (pHa) > 7.25, and then the second NA test (1 and 2.5 μg/kg of NA) was performed.

The plasma levels of CA and NO derivatives were measured from arterial blood. Blood sampling for these measurements was performed before the first NA test (baseline), 60 and 120 min after the induction of HS (HS-60, HS-120), and just after the end of blood reinfusion and 30 min after the recovery from HS (Rec-0, Rec-30). All blood samples taken in the course of the experiment were immediately centrifuged, and the plasma was pipetted into plastic storage vials and stored at −42°C. The assay was performed on the day after each experiment by using high-performance liquid chromatography for plasma CA levels and the method used by Griece et al. (14) for levels of NO derivatives.

All data were presented as mean ± SEM. The data were compared by using analysis of variance with Fisher’s protected least significance difference. P < 0.05 was the criterion for statistical significance.

Back to Top | Article Outline

Results

In both Cont-Gr and SNAP-Gr, HS was induced in 281 ± 43 s, and withdrawn blood was reinfused in 623 ± 71 s (mean ± SEM). Withdrawn blood was 560 ± 54 mL and 546 ± 44 mL in Cont-Gr and SNAP-Gr, respectively (P > 0.05). In two of seven animals in the SNAP-Gr, the initial dose of SNAP had to be discontinued and changed to a maintenance dose at 7 and 9 min after the start of SNAP administration to avoid a decrease in MAP of more than 3 mm Hg. However, in the other five dogs in the SNAP-Gr, the decrease of MAP did not exceed 3 mm Hg during the administration of the loading dose. In the Sham-Gr, the decrease in MAP caused by SNAP administration was so small that it was not necessary to stop the administration. In the Cont-Gr, three of seven dogs died at 90, 98, and 102 min after the induction of HS. In contrast, all animals in the SNAP-Gr and Sham-Gr survived. The mortality in the Cont-Gr (43%) was significantly greater than that in the SNAP-Gr (0%;P < 0.05).

Table 1 shows the hemodynamic changes that occurred during the study. The induction of HS occurred smoothly, and at the start of HS, ETCO2 in both Cont-Gr and SNAP-Gr decreased significantly compared with that of the Sham-Gr and then recovered at HS-60. SNAP administration did not decrease MAP, systemic vascular resistance, or pulmonary vascular resistance in any of the three groups. MAP and CO at HS-120 in SNAP-Gr increased significantly compared with the value at the start of HS (HS-0). However, in Cont-Gr, MAP and CO did not increase as observed in SNAP-Gr. CVP at HS-120 in Cont-Gr increased significantly compared with the value at HS-0, but not in the other groups. In both Cont-Gr and SNAP-Gr, the MAP of animals that survived for 120 min of HS recovered rapidly with reinfusion of withdrawn blood.

Table 1

Table 1

Although the pHa in Cont-Gr and SNAP-Gr at Rec-30 was less than that of Sham-Gr (P < 0.01), the pHa was corrected to more than 7.25, as expected. There was no significant difference in pHa at Rec-30 between Cont-Gr and SNAP-Gr (Table 2). The base excess at HS-60 in Cont-Gr (−12.8 ± 1.4) decreased to −17.0 ± 2.7 at HS-120 (P = 0.07). However, the change in base excess in SNAP-Gr during this period was small. Sodium bicarbonate used to correct the metabolic acidosis after blood reinfusion was 46.1 ± 2.5 mEq in Cont-Gr and 34.9 ± 3 mEq in SNAP-Gr (P < 0.01).

Table 2

Table 2

In Cont-Gr, the ratio of changes in SBP (%) in response to 1 and 2.5 μg/kg of NA after HS was decreased from 23.7% ± 4.1% to 6.5% ± 0.6% and from 50.1% ± 7.7% to 14.5% ± 2.6%, respectively (P < 0.01) (Figure 2). However, in SNAP-Gr, reactivity to NA was maintained even after HS. Moreover, reactivity in the second NA test in Sham-Gr increased significantly compared with those before SNAP administration.

Figure 2

Figure 2

Although plasma adrenalin, NA, and dopamine levels increased after the induction of HS, those CA levels in SNAP-Gr significantly decreased compared with those of Cont-Gr at HS-120, Rec-0, and Rec-30 (Table 3). NO derivatives, nitrite (NO2) and nitrate (NO3), did not show any significant changes throughout the study (Table 4).

Table 3

Table 3

Table 4

Table 4

Back to Top | Article Outline

Discussion

In the present study of HS for 120 min, the administration of 5 μg/kg for 10 min plus 5 μg · kg−1 · h−1 of SNAP at 60 min after the induction of HS significantly reduced the mortality in animals and improved the reactivity to NA after HS in SNAP-Gr. The hemodynamic state in CO and MAP during HS improved in SNAP-Gr. Doses of sodium bicarbonate required to correct metabolic acidosis after HS in SNAP-Gr were less than those needed for Cont-Gr. These results suggest that a small dose of SNAP, which in this study did not exert a significant vasodilator effect, may have a beneficial effect during HS in dogs. Our findings partially support the results from a previous study on rats (10), as well as the report of a similar protective effect by another organic NO donor, C87-3754, administered to cats exhibiting superior mesenteric occlusion shock (15). In the present study, there might have been increased production of NO from endothelial cells at the beginning of HS (9). This NO production affected protectively to maintain microcirculation because increasing NO production improves survival in experimental HS in rats (16). However, later in HS, NO production might have been exhausted because of prolonged ischemia to endothelial cells. Lefer et al. (17) reported that the interaction of polymorphonuclear neutrophils (PMNs) with vascular endothelial cells plays an important role in ischemia-reperfusion tissue injury. We therefore investigated how a small dose of external NO administration might have protected the endothelial function and attenuated tissue injury. First, external NO administration may result in more even microcirculation blood flow caused by constricted capillaries, without affecting larger vessels responsible for vascular resistance. Second, NO may decrease PMNs adhesion and capillary plugging.

In a previous report of HS using SNAP, Symington et al. (10) stressed the possibility of dual endothelial protective mechanisms. First, by quenching the superoxide radicals produced in the ischemic vascular endothelium, SNAP may directly maintain NO production and preserve control of splanchnic vascular conductance. Second, inhibition of platelet aggregation, which is an important endogenous inhibitor of PMN adherence to endothelial cells, may play an important role. We investigated whether the small dose of NO administered externally might have recovered the suppressed endothelial cell function and increased the vascular reactivity to NA, thereby leading to the observed improvement of CO and MAP at HS-120. That a reduced amount of sodium bicarbonate was required in SNAP-Gr to restore the pHa to more than 7.25 after HS also suggests an improvement in tissue perfusion. Moreover, the significant decrease of plasma CA at HS-120, Rec-0, and Rec-30 in SNAP-Gr also suggests the possibility of improved vascular reactivity by SNAP administration. In SNAP-Gr, the plasma CA levels were suppressed significantly compared with those of Cont-Gr. The suppression of the increase in CA levels might have been caused by the increase of MAP and CO, after administration of SNAP.

The best known property of SNAP is its vasodilator activity (18,19). SNAP is different from many other NO donors with a half-life of 4.6 h in vitro (20). SNAP liberates NO spontaneously without any requirement for enzyme degradation. Moreover, recent studies have shown that SNAP produces significantly less tolerance than nitroglycerin and retains its relaxation potency and ability to produce cyclic-guanosine monophosphate in nitroglycerin-tolerant vessels (21,22). Because we administered a small dose of SNAP which did not decrease MAP, SVR, or PVR, we can speculate that SNAP did not act as a vasodilator in the present study. The development of vascular hyporeactivity might have been protected by the improvement of microcirculation caused by SNAP administration. It has been reported that SNAP produces anti-PMN effects at concentrations below that which dilated cat coronary arteries (23). The cellular protective effect of NO is thought to be a result of its ability to prevent PMN adherence and preserve vascular endothelial cell function. Thus NO prevents the development of ischemia-reperfusion-induced tissue injury (12,13). Kubes et al. (18,19) reported that NO, which has been well recognized as an inhibitor of platelet aggregation, is an important endogenous inhibitor of PMN adherence to endothelial cells. Therefore, we can speculate that SNAP administration might have inhibited PMN and platelet adherence to vascular endothelial cells and prevented PMN-mediated damage to the vascular endothelium. Moreover, a small dose of external NO may increase the reactivity to NA even in normal vasculature as observed in Sham-Gr.

Thiemermann et al. (24) investigated the role of NO in the development of vascular hyporeactivity to NA during HS in rats. According to their report, HS for 120 min caused a time-dependent reduction of the presser responses to NA in anesthetized rats. They suggested that this hyporeactivity is mediated by an enhanced release of NO from the ec-NOS, because it was reversed by L-NAME, an inhibitor of both ec-NOS and i-NOS, but it was not prevented by dexamethasone, an inhibitor of i-NOS induction. Thus, the external administration of SNAP in the present study might have inhibited the enhancement release of NO from the ec-NOS, and improved the vascular reactivity to NA.

However, a small dose of a NO donor can inhibit the activation of i-NOS (25). Kelly et al. (26) determined the role of NO in decompensated and irreversible HS using rats subjected to HS for three or five hours. Despite the hemodynamic decompensation at three hours of HS, no increase was observed in either i-NOS, messenger ribonucleic acid, or NO derivatives. They concluded that hemodynamic decompensation in HS for three hours is not mediated by i-NOS induction. In the present study, because the NO metabolite did not increase, i-NOS was not induced in our model of HS. SNAP probably did not inhibit the induction of i-NOS and improved the vascular hyporeactivity to NA.

Because most previous investigations have described the effectiveness of NOS inhibitor (NOSI) during and/or after HS, administration of NOSI would seem to be a useful tool in HS therapy (4,7,27). For example, Zingarelli et al. (4) reported that NG-nitro-L-arginine methyl ester, a NOSI, improves the outcome of HS in rats. In contrast, NOSI in HS provides no hemodynamic benefit (5) and has even been shown to result in cytotoxicity in the liver (6). Thus, it has been suggested that the administration of NOSI might help alleviate the HS, although detailed investigation of the effect of NOSI on HS is difficult. Both the NO donor and NOSI may have benefits in treating HS, but the complete deficit of NO production, as well as the excessive release of NO, may induce vascular hyporeactivity and decompensation in HS.

In conclusion, the administration of a small dose of SNAP, a dose which did not exert a significant vasodilator effect, during HS improved the vascular hyporeactivity to NA and decreased the mortality rate of dogs.

Back to Top | Article Outline

References

1. Quinn AC, Petrol AJ, Vallance P. Nitric oxide: an endogenous gas. Br J Anaesth 1995; 74:443–51.
2. Ayuse T, Brienza N, Revelly JP, et al. Role of nitric oxide in porcine liver circulation under normal and endotoxemic conditions. J Appl Physiol 1995; 78:1319–29.
3. Landin L, Lorente JA, Renes E, et al. Inhibition of nitric oxide synthesis improves the vasoconstrictive effect of noradrenaline in sepsis. Chest 1994; 106:250–6.
4. Zingarelli B, Squadrito F, Altavilla D, et al. Evidence for a role of nitric oxide in hypovolemic hemorrhagic shock. J Cardiovasc Pharmacol 1992; 19:982–6.
5. Brown IP, Williams RL, McKirman MD, et al. Nitric oxide synthesis inhibition does not improve the hemodynamic response to hemorrhagic shock in dehydrated conscious swine. Shock 1995; 3:292–8.
6. Harbrecht BG, Wu B, Watkins SC, et al. Inhibition of nitric oxide synthase during hemorrhagic shock increase hepatic injury. Shock 1995; 4:332–7.
7. Klabunde RE, Slayton KJ, Ritger RC. NG-methyl-L-arginine restores arterial pressure in hemorrhaged rats. Circ Shock 1993; 40:47–52.
8. Yao YM, Bahrami S, Leichtfried G, et al. Significance of NO in hemorrhage-induced hemodynamic alterations, organ injury, and mortality in rats. Am J Physiol 1996; 270:H1616–23
9. Sato S, Miyabe M, Saito S, Yamaguchi H. Nitric oxide in the brain during short period of hemorrhagic shock in the rabbit. Shock 1997; 8:136–40.
10. Symington PA, Ma XL, Lefer AM. Protective actions of S-nitroso-N-acetyllpenicillamine (SNAP) in a rat model of hemorrhagic shock. Meth Find Exp Clin Phramacol 1992; 14:789–97.
11. Christofer TA, Ma H-L, Lefer AM. Beneficial actions of S-Nitroso-N-acetylpenicillamine, a nitric oxide donor, in murine traumatic shock. Shock 1994; 1:19–24.
12. Kurose I, Wolf R, Grisham MB, Granger DN. Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide. Circ Res 1994; 74:376–82.
13. Carey C, Siegfried MR, Ma-XL, Weyrich AS. Antishock and endothelial protective actions of a NO donor in mesenteric ischemia and reperfusion. Circ Shock 1992; 38:209–16.
14. Green LC, Wagner DA, Glogowski J, et al. Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Anal Biochem 1982; 126:131–8.
15. Aoki N, Johnson G III, Lefer AM. Beneficial effects of two forms of NO administration in feline splanchnic artery occlusion shock. Am J Physiol 1990; 258:G275–81
16. Daughters K, Waxman K, Nguyen H. Increasing nitric oxide improves survival in experimental hemorrhagic shock. Resuscitation 1996; 31:141–4.
17. Lefer AM, Tsao PS, Lefer DJ, Ma XL. Role of endothelial dysfunction in the pathogenesis of reperfusion injury myocardial ischemia. FASEB J 1991; 5:2029–34.
18. Kubes P, Granger DN. Nitric oxide modulates microvascular permeability. Am J Physiol 1992; 262:H611–5.
19. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991; 88:4651–5.
20. Ignarro LJ, Lippton H, Edwards JC, et al. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for involvement of S-nitrosothiols as active intermediates. J Pharmacol Exp Ther 1981; 218:739–49.
21. Shaffer JE, Han BJ, Chern WH, Lee FW. Lack of tolerance to a 24 hour infusion of S-nitroso-N-acetylpenicillamine (SNAP) in conscious rabbits. J Pharmacol Exp Ther 1992; 260:286–93.
22. Bauer JA, Fung HL. Differential hemodynamic effects and tolerance properties of nitroglycerine and an S-nitrosothiol in experimental heart failure. J Pharmacol Exp Ther 1991; 256:249–56.
23. Ma XL, Tsao PS, Viehman GE, Lefer AM. Neutrophil mediated vasoconstriction and endothelial dysfunction in low flow perfusion reperfused cat coronary artery. Circ Res 1991; 69:95–106.
24. Thiemermenn C, Szabo C, Mitchel JA, Vane JR. Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide. Proc Acad Sci USA 1993; 90:267–71.
25. Colasanti M, Persichini T, Menegazzi Met, al. Induction of nitric oxide synthase mRNA expression: suppression by exogenous nitric oxide. J Biol Chem 1995; 270:26731–3.
26. Kelly E, Shah NS, Morgan NN, et al. Physiological and molecular characterization of the role of nitric oxide in hemorrhagic shock: evidence that type II nitric oxide synthase does not regulate vascular decompensation. Shock 1997; 7:157–63.
27. Vromen A, Szabo C, Southan GJ, Salzman AL. Effects of S-isopropyl isothiourea, a potent inhibitor of nitric oxide synthase, in severe hemorrhagic shock. J Appl Physiol 1996; 81:707–15.
© 2000 International Anesthesia Research Society