Synthesis of L-arginine and nitric oxide
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 1, January, 2019
179
presence of citrulline and ammonium chloride as the
nitrogen source.
blood cells and MPH, which we found previously, is probably
also associated with occurrence of this cycle.29 Ascorbic
acid can participate in maintenance of iNOS cofactors (in
particular, NADPH and ВН4) in the reduced state.
An increase in the NO production in the cells under
the action of citrulline was also observed previously,30 but
the authors considered this cycle to be secondary and
insignificant for NO synthesis. The assumption that the
citrulline-involving cycle does not play a significant role
in NO synthesis was mainly supported by detection of
a certain (small) amount of citrulline in the cells during
NO synthesis using arginine. Meanwhile, our results
strongly suggest that citrulline is not completely consumed
and is accumulated in the cells due to the deficiency of
substrates (substrate depletion) of argininosuccinate and
arginine synthesis enzymes.
The nitric oxide level synthesized in MPH was also
determined by the Griess assay. Macrophages were incu-
bated in the presence of ammonium chloride, arginine,
and citrulline. Figure 7 shows the results of measurement
of nitrite content in the MPH supernatant. Nitrite is
a stable product of NO oxidation reflecting the amount of
NO synthesized by macrophages. Thus, the use of citrul-
line leads to nearly the same amount of MPH-based nitric
oxide as the use of arginine. This implies that a special
(separate) cycle of arginine and nitric oxide synthesis
functions in MPH. This cycle is triggered upon iNOS
induction and can be activated by enzyme substrates syn-
thesizing arginine, i.e., NO synthesis from L-arginine
involving NO-synthase is probably not a linear process,
but represents a closed cycle in which citrulline formed in
the NO synthesis (and considered earlier to be a side
product) is used again for arginine and then NO synthesis.
The amount of resulting NO is still limited by the avail-
ability of substrates for arginine synthesis enzymes (other
than citrulline) and by accessibility of co-enzymes of
iNOS, which is a thoroughly regulated enzyme, like other
NO synthases, and requires six cofactors for normal func-
tioning: flavine mononucleotide (FMN), flavinadenine
dinucleotide (FAD), NADН, BH4, NADPH, and active
site-bound calmodulin. Furthermore, effective function-
ing of iNOS requires oxygen. At a reduced oxygen content,
enzyme operation efficiency decreases. The ability of
ascorbic acid to promote the formation of NO in white
Thus, the results indicate that NO synthesis from
L-arginine involving inducible NO synthase in peritoneal
MPH and liver cells is not a linear process, as was believed
previously, but is a closed cycle in which L-citrulline
formed in the NO synthesis and considered to be a side
product is utilized again in the arginine and then nitric
oxide synthesis. This biochemical cycle seems to function
in all cells capable of iNOS expression. Functioning of
a separate (small) cycle of arginine and nitric oxide syn-
thesis in the peritoneal MPH and liver cells makes these
cells able to produce NO for long periods of time in con-
siderable amounts. The existence of arginine and NO
synthesis cycles in the cells with inducible NO synthase
may account for the difference between the amounts of
NO formed by constitutive and inducible NO synthases.
A
References
0.6
1. A. A. Ustyugov, G. M. Aliev, Russ. Chem. Bull., 2016, 65, 1151.
2. D. J. Stuehr, M. A. Marletta, PNAS USA, 1985, 82, 77383.
3. D. A. Geller, P. D. Freeswick, D. Nguyen, A. K. Nussler,
M. Disilvio, R. A. Shapiro, S. C. Wang, R. L. Simmons,
T. R. Billiar, Arch. Surg., 1994, 129, 165.
4. A. K. Nussler, M. Disilvio, T. R. Billiar, R. A. Hoffman,
D. A. Geller, R. Selby, J. Madariage, R. L. Simmons, J. Exp.
Med., 1992., 176, 261.
5. Y. Vodovotz, N. S. Kwon, M. Pospischil, J. Manning, J. Paik,
C. Nathan, J. Immunol., 1994, 152, 4110.
6. I. Vouldoukis, V. Riveros-Moreno, B. Dugas, F. Ouaaz,
P. Becherel, P. Debre, S. Moncada, M. D. Mossalayai, PNAS
USA, 1995, 92, 7804.
0.5
0.4
0.3
0.2
0.1
7. D. L. Granger, J. B. Hibbs, J. R. Perfect, D. T. Durack,
J. Clin. Invest., 1988, 81, 1129.
8. I. Vouldoukis, D. Mazier, P. Debre, M. D. Mossalayai, Res.
Immunol., 1995, 146, 689.
9. T. R. Billiar, R. D. Curran, D. J. Stuehr, M. A. West, B. G.
Bentz, R. L. Simmons, J. Exp. Med., 1989, 169, 1467.
10. R. D. Curran, T. R. Billiar, D. J. Stuehr, K. Hoffman, R. L.
Simmons, J. Exp. Med., 1989, 170, 1769.
1
2
3
Fig. 7. Formation of nitric oxide by peritoneal MPH activated
by interferon-γ during incubation at room temperature for 40 h:
(1) control (interferon-γ); (2) with arginine; (3) with citrulline
and ammonium chloride. The ordinate axis shows absorbance
at 540 nm.
11. J. B. Hibbs, Z. Vavrin, R. R. Taintor, J. Immunol., 1987,
138, 550.