The Effects of Oxygen on the Nitric Oxide Pathway During Hypovolemic Shock

by Gregory M. Helbig

Introduction:Hypovolemic shock is a life threatening condition in which the blood pressure drops to a level that makes oxygen-to-tissue perfusion inefficient. With a blood loss of 30% of the total blood volume, the shock-state can be induced (Bitterman et. al., 1996). This shock state is indicated when the body is unable to efficiently perfuse oxygen to the tissues. If 50% of the blood volume is lost, death is a possible outcome (O’Keefe et. al., 1998). Studies using the male Sprague-Dawley rat (as a hemorrhage model) have shown that administration of 100% O₂ at 1 atm can increase hindquarter vascular resistance without a significant effect in the splanchnic (visceral organs), renal and other vital organ perfusion (Bitterman et. al., 1996 & Atkins et. al., 1998). Similar studies conducted at the Walter Reed Army Institute of Research demonstrated that oxygen inhalation after hemorrhage dramatically increases mean arterial blood pressure (MABP) in rats. They demonstrated that breathing oxygen increases the MABP from 70 + 0 to 114 + 3 mmHg (Atkins et. al., 1998). These results indicate that oxygen is a potent vasoconstrictor in non-vital vascular beds during hypovolemic shock.

It has been suggested that inactivation of nitric oxide (NO) by reactive oxygen species may explain the effects of oxygen on hindquarter vascular tone during shock (Bitterman et. al., 1996). Nitric oxide acts by relaxing smooth muscle thus causing vasodilatation, this results in a drop in blood pressure, which is commonly seen with the administration of medicinal nitroglycerine (Karp, 1996). Oxygen, at a 100% blood concentration, may inhibit the effects of NO, thus causing vasoconstriction and an increase in perfusion of blood to the vital organs. With the known physiological effects of oxygen during hemorrhagic events and the biochemical effects of NO on smooth muscle, I intend to examine the relationship between O₂, NO and vasoconstriction during hypovolemic shock.

Dual enzymatic activity during vasodilatation: There are currently two enzymes that are identified in the NO pathway that causes vasodilatation by smooth muscle relaxation. This discussion is limited to the presence of the NO pathway in vascular smooth muscle, due to the interest in hypovolemic shock. The two enzymes are nitric oxide synthase (sNO) and soluble guanylyl cyclase (sGC) (Knowles & Moncada, 1992). sNO is commonly found in the endothelium of arteries and veins. sGC is typically found in the underlying smooth muscle of the luminal endothelium. The NO produced by sNO acts as an intercellular messenger to stimulate guanylate cyclase (Zubay, 1998). Guanylate cyclase then produces cGMP to cause smooth muscle relaxation.

Nitric Oxide synthase is stimulated by an increase in the intracellular Ca⁺² concentration or by the presence of calcium ionophore A23187 (Knowles & Moncada, 1992). In the case of Ca⁺², a cytokine (such as acetylcholine (ACh) or bradykinin) binds to a plasma membrane receptor on the endothelial cell causing an influx of Ca⁺² from the extracellular space. An intracellular change of Ca⁺² from 100 to 500 nM changes the rate of NO synthesis from <5% to >95% of maximum (Knowles & Moncada, 1992). The influx of Ca⁺² stimulates sNO to convert L-Arg to citrulline and NO (figure 1, Knowles & Moncada, 1992).

Figure 1 depicts how nitric oxide synthase produces NO. BH₄ (present in endothelial cells at a concentration of 600 nM) and Ca²⁺/CaM are cofactors for nitric oxide synthase that can disassociate from the enzyme. The three known prosthetic groups of sNO are flavin mononucleotide (FMN), non-hemoglobin iron (Fe), and flavin adenine dinucleotide (FAD). These prosthetic groups are thought to donate electrons to help the formation of reaction intermediates (Knowles & Moncada, 1992). The sNO activity is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. N^G-mono-methyl-L-arginine (L-NMMA) is known to inhibit sNO and decrease NO production and it has been used to demonstrate the importance of NO in vascular tone. Experiments have shown that L-NMMA causes an increase in coronary perfusion pressure. The increase in blood pressure is likely accomplished by inhibition of the release of NO into the coronary effluent (Moncada, et. al, 1991). With the decrease in production of NO there is a responding increase in blood pressure. The smooth muscle is not being relaxed.

Figure 2. Conversion of L-Arginine to Citrulline and NO.

N^G-hydroxy arginine is an intermediate in the reaction of L-Arg to Cit and NO. Knowing that O₂ and L-Arg are reactants and that Cit and NO are the products, the reaction depicted in figure 2 is possible for the production of NO by nitric oxide synthase.

After the production of NO occurs in the endothelial cells, it must then diffuse into the underlying smooth muscle cells. NO has a half-life of 5 to 10 seconds (Zubay, 1998). When the NO enters into the smooth muscle cell it interacts with soluble guanylyl cyclase (sGC). When sGC is stimulated, it converts guanylyl triphosphate (GTP) to cyclic guanylyl monophosphate (cGMP). Much like nitric oxide synthase, sGC has a prosthetic heme group. The stimulation of sGC occurs as a consequence of the reaction of NO with the heme prosthetic group of sGC to form nitrosoheme. This reaction results in a 50-fold greater rate of cGMP synthesis than when catalyzed by unnitrosylated sGC. The resulting rise in cGMP is responsible for smooth muscle relaxation. (Knowles & Moncada, 1992)

Figure 3 depicts the nitric oxide synthase and soluble guanylyl cyclase functioning together to cause smooth muscle relaxation. This is a simplistic picture of what goes on in the blood vessel of a mammal when acetylcholine is the cytokine and causes the initial influx of Ca⁺² and the sequential signaling to lead to smooth muscle relaxation (vasodilatation).

The reactivity between oxygen and nitric oxide: Knowles and Moncada, 1992 presented four predominant reactions that can occur between oxygen species and nitric oxide (NO). These reactions ultimately limit the muscle relaxing effects of NO. (1) Nitric oxide is quickly converted to nitrate due to a high concentration of oxyhaemoglobin (O₂Hb) (>mM) in the red blood cells in the arterial blood vessels.

NO + O₂Hb à  NO₃^-. + Hb⁺

(2) In the venous blood vessels deoxyhemoglobin reacts with NO and NO is removed rapidly from the smooth muscle.

NO + Hb à  NOHb

(3) Within the vessel wall, the reaction between NO and superoxide is very important for removing NO from smooth muscle.

NO + O₂^-.   à   ONOO^-.   +    NO₃^-.

Experiments have shown that vascular cells and tissues that reduce endogenous O₂^-. availability (such as superoxide dismutase) are more prone to vascular relaxation, whereas O₂^-. generating compounds are inhibitory (such as xanthine oxidase) (Granger, et. al., 1986). (4) Finally, when cells are in the absence of hemoglobin or O₂^-. and are in oxygenated media at pH 7.4 the predominant reaction of NO is as follows:

8NO + 4O₂ +   4N₂O₄        à    6NO₂^-. + 2NO₃^-.

These reactions explain the effects of oxygen therapy during hypovolemic shock. If oxygen is able to decrease NO concentrations and ultimately inhibit the vasodilatation effects of NO then vasoconstriction will occur. Inhibitory effects have been observed with the L-NMMA inhibition of nitric oxide synthase. When NO levels decrease due to the inhibitory effects of L-NMMA, vasoconstriction occurs and there is an increase in blood pressure. The same assumption can be made for the inhibitory reaction between oxygen species and NO. If the NO concentration is limited, much like when L-NMMA limits cellular NO concentration, then it would be expected that oxygen species have a vasoconstriction effect. Increased mean arterial blood pressure after oxygen inhalation has been documented in mammalian physiology studies (Bitterman, et. al, 1996).

Relationship between oxygen therapy, NO, and vasoconstriction: When mammals are in the hypovolemic shock state the blood O₂ saturation is not as high as when the mammal is not in hypovolemic shock (Bitterman, et. al., 1991). Blood pressure is significantly low during hypovolemic shock, as stated in the introduction. When 100% oxygen is administered to a hemorrhaged mammal (male Sprague-Dawley rat) via inhalation, the mean arterial blood pressure increases dramatically (Adir, et. al., 1995). From the previous analysis of the various reactivities of oxygen species with NO and the documented experimental results of mammalian hemorrhage models, there is a clear correlation between the administration of oxygen and the blocking of the NO pathway that produces vasoconstriction.

Another relationship that correlates with sNO inhibition and vasoconstriction is when blood is at 100% O₂ saturation. It has been found that oxygen free radicals have a greater tendency to form (Jamieson, et. al., 1986) when blood O₂ saturation is at 100%. In the reactions between NO and oxygen previously presented, the oxygen free radicals were the most likely to react the fastest with NO and inhibit the smooth muscle relaxation effects of NO.

A possible experiment that would show that sNO regulation is required for the increase in blood pressure is one that would require the inhibition of the nitric oxide synthase pathway with L-NMMA. After inhibition of the sNO, the mammal would then be hemorrhaged while breathing room air and induced into hypovolemic shock. If the mean arterial blood pressure remained constant (remained in the shock state) after the administration of 100% oxygen it would then be conclusive that the inhibition of nitric oxide synthase is responsible for vasoconstriction and the increase in blood pressure.

Conclusion: Nitric oxide and its effects in biochemistry and physiology are a relatively new phenomenon. It is important to make correlations between observed physiological effects of NO in specific settings (such as hypovolemic shock) and be able to relate these observations to the biochemical effects of NO and oxygen. There is a strong relationship between the inhibitory effects of oxygen on nitric oxide and vasoconstriction during oxygen administration in hypovolemic shock. Current research with sNO inhibitors (N^G-monomethyl-L-arginine) is being used to treat circulatory shock in critically ill patients. These inhibitors have demonstrated enhanced vasoconstriction (Griffiths, 1993). If oxygen can decrease cellular NO concentration then oxygen has the potential to be a nitric oxide pathway inhibitor. The only problem with inhibiting sNO on a long-term basis is that NO is an important signaler for macrophages to kill invading microorganisms (Zubay, 1998).

In conclusion, oxygen is a potent drug that has many beneficial effects. It appears that inhibiting NO via oxygen during hypovolemic shock is beneficial. Studies have shown that the long-term outcome of hemorrhaged rats is better when oxygen is administered than when it is not administered (Adir, et. al., 1995). By making the correlation that O₂ interacts with NO to inhibit the nitric oxide pathway and cause vasoconstriction, the relationship between vasoconstriction and oxygen therapy during hypovolemic shock is understood.

Adir, Y., Bitterman, N., Katz, E., Melamed, Y., Bitterman, H. 1995. Salutary consequences of oxygen therapy on the long-term outcome of hemorrhagic shock in awake, unrestrained rats. Undersea & Hyperbaric Medicine 22:23-30

Atkins, J., Lee, W., Scott, Z., Johnson, K., and Pearce, F. 1998. Oxygen inhalation after hemorrhage increases mean arterial blood pressure (MABP) and carotid flow. The FASEB Jouranl 11(3), A286

Bitterman, H., Brod, V., Weisz, G., Kushnir, D., and Bitterman, N. 1996. Effects of oxygen on regional hemodynamics in hemorrhagic shock. American Journal of Physiology 271: H203-H211

Bitterman, H., Reissman, P., Bitterman, N., Melamed, Y., Cohen, L. 1991. Oxygen therapy in hemorrhagic shock. Circulatory Shock 33:183-191

Granger, Neil D., Hollwarth, Michael E., and Parks, Dale E. 1986. Ischemia-reperfusion injury: role of oxygen-derived free radicals. Acta Physiologica Scandinavia Supplemental 548:47-63

Griffiths, Mark 1993. NO business. The Lancet 341: 364(2)

Jamieson, Dana, Chance, Britton, Cadenas, Enrique, Boveris, Alberto 1986. The relation of free radical production to hyperoxia. Annual Review of Physiology 48:703-719

Karp, G. (1996) Cell and Molecular Biology: Concepts and Experiments. New York, NY: John Wiley & Sons, Inc. (679-680)

Knowles, Richard G. and Moncada, Salvador 1992. Nitric oxide as a signal in blood vessels. Trends in Biochemical Science 17: 399-402

Moncada, S., Palmer, R. M. J., and Higgs, E. A. 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews 43: 109-142

O’Keefe, M. F., Limmer, D., Grant, H. D., Murray, R. H., and Bergeron, J. D. 1998. Emergency care: eigth edition. Upper Saddle River, NJ: Prentice-Hall, Inc. (486-487)

Zubay, G.L. (1998) Biochemistry, Fourth Edition. Wm. C. Brown Publishers. (917-919)