Case 10

The Biological Roles of Nitric Oxide

Focus concept

Nitric oxide, a small, lipophilic and toxic molecule, has the ability to act as a second messenger in blood vessels.

Prerequisites

∙Hemoglobin structure and function concepts.

∙Regulatory schemes and the role of the second messenger.

Background

Nitric oxide (NO), although a small and highly toxic molecule, has recently been found to be important in the regulation of several biological systems. In this case, we focus on the circulatory system where NO is one of the principal regulators of blood pressure. NO is produced from arginine in endothelial cells lining blood vessels, then released from the cells where it then diffuses into the adjacent muscle layer and induces the synthesis of cyclic GMP which promotes relaxation of the blood vessel. (The lipophilic nature of NO obviates the need for a receptor.) NO is short-lived (5-10 seconds) and is rapidly converted by oxygen and water to nitrates and nitrites.

In promoting the relaxation of the blood vessel, NO can clearly facilitate the passage of oxygenated blood through the vessel. Scientists wondered if NO might be involved in oxygen delivery to the cells in a more direct manner–through binding to hemoglobin, for example. This hypothesis was confirmed recently when investigators showed that NO can react with hemoglobin to form S-nitrosohemoglobin. The NO reacts with the sulfhydryl group on Cys 93 on the β chain of hemoglobin to form the S-nitrosohemoglobin. The reaction is readily reversible, with SNO-Hb releasing the NO under certain circumstances.

hemoglobin (Hb) + NO ¾ S-nitrosohemoglobin (SNO-Hb)

In this case, the investigators attempted to determine the reaction conditions favorable for the forward and reverse reactions. Recall that hemoglobin exists in two different conformations: Oxygenated hemoglobin (Oxy-Hb) has the R form and deoxygenated hemoglobin (deoxy-Hb) has the T form.

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Figure 10.1: Modification of cysteine residues in proteins with the alkylating agent N-ethylmaleimide.

Figure 10.2: Rate of reaction of oxy-Hb and deoxyHb with NO to form S-nitrosohemoglobin (based on Jia, et al., 1996).

Questions

1.NO is produced from arginine by the action of the enzyme nitric oxide synthase (NOS). Citrulline is also a product in this reaction. NADPH is required as a cofactor. Write the balanced equation for the reaction.

2.A purified sample of hemoglobin is subjected to a chemical modification experiment in which an alkylating agent is added to the hemoglobin. The alkylating agent N-ethylmaleimide (NEM) reacts with sulfhydryl groups as shown in Figure 10.1. The NEM has the ability to react with the Cysβ93 in oxy-Hb but not deoxy-Hb. What does this tell you about the environment of Cysβ93?

  1. The investigators used the information from Question 2 to determine the rate of formation of S-nitrosohemoglobin. The results are shown in Figure 10.2. How do you interpret these data?
  1. Next, circulatory dynamics were investigated. The scientists collected arterial and venous blood from anesthetized rats. The hemoglobin was then purified and assayed for SNO-Hb content. The results are shown in Table 10.1. How do you interpret these results?

Table 10.1: Endogenous concentrations of S-nitrosohemoglobin in arterial and venous blood. (Based on Jia, et al., 1996.)

Blood / SNO-Hb (nM)
No NOS inhibitor / With NOS inhibitor
Arterial / 311 / 82
Venous / 32 / not measured
  1. Next the investigators injected purified hemoglobin and purified SNO-Hb into the circulation of an experimental animal, then measured the animal’s blood pressure. The results are shown in Table 10.2. How do you interpret these results? (Assume that the injection of the purified proteins did not affect blood volume.)

Table 10.2: Reactivity of Hb and SNO-Hb with endogenous NO. (Based on Jia, et al., 1996.)

Hb / SNO-Hb
Change in arterial pressure / ↑ 20 mm Hg / No change
  1. Oxygen delivery to capillaries is therefore controlled by two factors: The oxygen content of the hemoglobin molecule itself, and the ability of the blood vessel to relax so that the blood can flow through the capillary easily. Through the action of NO, both of these processes are linked. The investigators proposed that the S-nitrosohemoglobin somehow senses the oxygen demand in a particular tissue and acts accordingly.
  1. Describe how oxygen is delivered effectively to cells in hypoxic (oxygen-deprived) tissue. For example, what would be the relative concentrations of SNO-Hb and Hb? What is the effect on the blood vessels?
  2. What would the relative concentrations of SNO-Hb and Hb be in blood circulated through tissue in which oxygen was plentiful? What is the effect on blood vessels in this situation?

References

Blakeslee, S. (July 22, 1997) “What Controls Blood Flow? Blood” The New York Times, p. C1.

Hsia, C. C. W. (1998) New Engl. Jour. Med. 338, pp. 239-247.

Jia, L., Bonaventura, C., Bonaventura, J., and Stamler, J. S. (1996) Nature380, pp. 221-226.

Snyder, S. H., and Bredt, D. S. (1992) Scientific American, 266(5), pp. 68-77.

Stamler, J. S., Jia, L., Eu, J. P., McMahon, T. J., Demchenko, I. T., Bonaventura, J., Gernert, K., and Piantadosi, C. (1997) Science276, pp. 2034-2037.

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