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 Snitrosohemoglobin. 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.
1
2
O
NH
CH
O
+
C
NH
CH
C
O
CH2
CH2
O
SH
S
CH3
N-ethylmaleimide
O
O
Figure 10.1: Modification of cysteine residues in proteins with the alkylating agent Nethylmaleimide.
CH3
Moles S-NO/tetramer
2
1.5
1
0.5
0
0
10
20
30
40
50
60
Time, minutes
Figure 10.2: Rate of reaction
of oxy-Hb
and deoxyHb with NO to
oxy-Hb
deoxy-Hb
form S-nitrosohemoglobin (based on Jia, et al., 1996).
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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 Cys β93
in oxy-Hb but not deoxy-Hb. What does this tell you about the environment of Cys β93?
3. The investigators used the information from Question 2 to determine the rate of formation of Snitrosohemoglobin. The results are shown in Figure 10.2. How do you interpret these data?
4. 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
5. 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
Change in arterial pressure
↑ 20 mm Hg
4
SNO-Hb
No change
6. 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.
a.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?
b.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) Nature 380, 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) Science 276, pp. 2034-2037.
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