Departamento de Fisiología, Instituto de
Biotecnología (J.L., Ma.M., G.E., H.K., Mi.M., D.A.-C.) and
Departamento de Química Orgánica y Farmacéutica,
Facultad de Farmacia (E.C., A.E., M.A.G.) Universidad de Granada,
Granada, Spain
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Introduction |
Activation
of the N-methyl-D-aspartate (NMDA)
receptor by glutamate causes a Ca2+ influx into
cells, resulting in the formation of nitric oxide (NO) from
L-arginine (Bredt et al., 1992
). The enzyme
involved in this pathway is a calmodulin
(CaM)/Ca2+/O2
and NADPH-dependent cytosolic nitric-oxide synthase (nNOS), which requires tetrahydrobiopterin (H4-biopterin) for
the expression of its activity (Knowles et al., 1989). The NO may act
intracellularly or it may diffuse extracellularly to adjacent neurons,
acting as a retrograde messenger (Garthwaite and Boulton, 1993).
NO modulates several processes in the central nervous system (CNS) such
as pain perception, long term potentiation and memory, and cerebral
blood flow (Garthwaite and Boulton, 1993). In addition, NO has been
specifically implicated in the glutamate-dependent excitotoxicity and
neuronal death (Dawson et al., 1991). Excessive activation of NMDA has
been associated with a wide range of neurological disorders and
neurodegenerative diseases, including hypoxic-ischemic brain injury,
trauma, epilepsy, Parkinson's disease, Huntington's disease and AIDS
dementia (Herrling, 1994). Thus, selective inhibition of nNOS may
provide a novel therapeutic approach to these pathological situations.
Melatonin (aMT; N-acetyl-5-methoxytryptamine) exerts
neuroprotective properties reflecting both antioxidant (Reiter et al., 1995, 1997, 1998) and inhibitory effects on the CNS
(Acuña-Castroviejo et al., 1986, 1995; Gomar et al., 1994).
Electrophysiological experiments have shown that in rats, the
iontophoretic ejection of aMT attenuates the excitatory response of
striatal neurons to sensorimotor cortex stimulation (Castillo-Romero et
al., 1995; Escames et al., 1996). This excitatory response is mainly
mediated by glutamate acting on the NMDA receptors (Alexander et al.,
1986; Escames et al., 1996). It was suggested that some effects of aMT may be derived from some endogenous brain metabolites of the hormone such as N-acetyl-5-methoxy-kynurenamine (aMK; Hirata et al.,
1974; Acuña-Castroviejo et al., 1994). Therefore, we showed that
some synthetic kynurenines affect the excitatory response of striatal neurons in a structure-related manner. Further experiments proved that
the NMDA-subtype of glutamate receptor is involved in these effects of
aMT and related kynurenines (León et al., 1998a,b).
aMT binds CaM with high affinity (Benítez-King et al., 1993)
and the binding is saturable, reversible, and
Ca2+-dependent. It was suggested that the
inhibitory effect of aMT on nNOS activity in some brain areas (Bettahi
et al., 1996; Pozo et al., 1997) might be produced by removing free
cytosolic CaM through a CaM-aMT interaction (Pozo et al., 1997). Thus,
we consider it worthwhile to investigate the effect and the mechanism
of action of aMT and four synthetic kynurenines on nNOS activity in
homogenates of rat striatum and in a commercially available purified
nNOS from rat brain. Besides, experiments with urea-polyacrylamide gel
electrophoresis (PAGE) were carried out to further assess the possible
interaction of these compounds with CaM.
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Experimental Procedures |
Materials.
L-Arginine,
L-citrulline, HEPES, DL-dithiothreitol (DTT),
leupeptin, aprotinin, pepstatin, phenylmethylsulfonyl fluoride, hypoxantine-9-
-D-ribofuranosid (inosine), EGTA, BSA,
Dowex-50W (50 × 8-200) resin, FAD, NADPH,
5,6,7,8-tetrahydro-L-biopterin dihydrocloride
(H4-biopterin), bovine brain CaM (>98%
SDS-PAGE), trifluoperazine, calmidazolium, 5-hydroxytryptamine
(serotonin), and aMT were obtained from Sigma-Aldrich (Madrid,
Spain). L-[3H]Arginine (58 Ci/mmol) was obtained from Amersham (Amersham Pharmacia Biotech GmbH,
Barcelona, Spain). Tris·HCl and calcium chloride were obtained
from Merck. Purified rat brain nNOS (>98% SDS-PAGE) was obtained from
Alexis Biochemicals (San Diego, CA). Six kynurenine derivatives were
used: 2-acetamide-4-(3-methoxyphenyl)-4-oxobutiric acid (aM);
2-acetamide-4-(2-amine-5-methoxyphenyl)-4-oxobutiric acid (aAM);
2-acetamide-4-(2-dimethylamine-5-methoxyphenyl)-4-oxobutiric acid
(aDAM); 2-acetamide-4-(2-benzylamine-5-methoxyphenyl)-4-oxobutiric acid
(aBAM); 2-butyramide-4-(3-methoxyphenyl)-4-oxobutiric acid (bM), and
2-butyramide-4-(2-amine-5-methoxyphenyl)-4-oxobutiric acid (bAM). They
were synthesized in the Department of Pharmacological Chemistry,
University of Granada; their preparation and purification will be
described elsewhere.
nNOS Activity Determination in Rat Striatum.
Male Wistar
rats (200-250 g) housed under a 12-h:12-h light/dark cycle and given
free access to food and water were used. Animals were sacrificed by
cervical dislocation and striata were quickly collected and immediately
used to measure nNOS activity. Upon removal, tissues were cooled in an
ice-cold buffer (25 mM Tris, 0.5 mM DTT, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride, pH 7.6). Two striata were placed in 1.25 ml of the same
buffer and sonicated (10 s × 6). The crude homogenate was
centrifuged 5 min at 1000g, and an aliquot of the
supernatant was frozen at 
20°C for total protein determination with
the Folin phenol reagent.
NOS activity was measured by the method of Bredt and Snyder (1989),
monitoring the conversion of
L-[3H]arginine to
L-[3H]citrulline. The final
incubation volume was 100 µl and consisted of 10 µl of crude
homogenate added to buffer to give a final concentration of 25 mM Tris,
1 mM DTT, 30 µM H4-biopterin, 10 µM FAD, 0.5 mM inosine, 1 mg/ml BSA, 1 mM CaCl2, 10 µM
L-arginine, 50 nM
L-[3H]citrulline, at pH 7.6. When
required, increasing concentrations of arginine (up to 10 µM) were
also added to the incubation medium. The reaction was started by the
addition of 10 µl of NADPH (0.75 mM final) and continued for 30 min
at 37°C. Control incubations were done by omission of NADPH. The
reaction was stopped by the addition of 400 µl of cold 0.1 M HEPES,
10 mM EGTA, 0.175 mg/ml L-citrulline, pH 5.5. The reaction
mixture was decanted into a 2-ml column packed with Dowex-50W ion
change resin (Na+ form) and eluted with 1.2 ml
water. L-[3H]Citrulline was
quantified by liquid scintillation spectroscopy. The retention of
L-[3H]arginine was greater than
98%. Enzymatic activity was determined subtracting the control value,
usually less than 1% of the radioactivity added. The activity of nNOS
was expressed as picomoles of
L-[3H]citrulline produced per
milligram of protein per minute.
Purified Rat Brain nNOS Activity Determination.
The activity
of nNOS present in the commercial source was also measured by the
method of Bredt and Snyder (1989). After resuspension of nNOS stock
solution in 50 mM HEPES buffer, pH 7.4, aliquots (0.0125U, 0.35 µg of
protein) were incubated by 15 min at 37°C in the presence of 15.5 µM CaCl2, 30 µM
H4-biopterin, 10 µM FAD, 1 mg/ml BSA, 0.5 mM
inosine, 10 µM L-arginine, 10 ng/ml CaM, 0.75 mM NADPH,
and 50 nM L-[3H]arginine, in a
total volume of 100 µl. When required, increasing concentrations of
L-arginine (0-10 µM), FAD (0.1-10 µM),
H4-biopterin (0.3-30 µM), CaM (0-10 ng/ml),
and CaCl2 (0-0.350 mM) were also added to the
incubation medium. The reaction was started by the addition of NADPH.
The other steps in the procedure were the same as described for nNOS
activity determination in rat striatum.
Electrophoresis Studies.
Urea-PAGE was done as described
previously (Erickson-Vhtanen and De Grado, 1987). To study CaM
electrophoretic migration, slab gels (0.75-mm thickness) of 15%
polyacrylamide, 4 M urea, 375 mM Tris, pH 8.8, and 1 mM
CaCl2 or 2 mM EGTA, in the absence or presence of
1 mM of each compound studied (i.e., aMT, serotonin, or
kynurenine derivatives) were run at constant voltage of 100 V. Electrode buffer contained 25 mM Tris, 192 mM glycine, pH 8.3, and 0.1 mM CaCl2 or 2 mM EGTA, in the absence or presence
of either 1 mM aMT, 1 mM serotonin, or 1 mM each kynurenine. Samples
containing 10 µg of CaM in 100 mM Tris, 4 M urea, pH 7.2, and 0.1 mM
CaCl2 in the absence or presence of either 1 mM
aMT, 1 mM serotonin, or 1 mM each kynurenine, were incubated at room
temperature for 60 min, in a total volume of 50 µl. One half-volume
of 50% glycerol with tracer bromphenol blue was added and samples were
applied to wells. The gels were fixed in 43% (v/v) methanol/1.6 M
acetic acid and stained with 0.1% (w/v) Coomassie brilliant blue
R-250. When required, gels were scanned using the QuantiScan System
software (Biosoft, Cambridge, UK).
Statistical Analysis.
Data are expressed as the mean ± S.E.M. Statistics included two-way analysis of variance and a posthoc
test to assess any significant difference between CaM concentrations.
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Results |
Fig. 1 shows the structure of the
six synthetic kynurenines tested. It can be seen that the structure of
aAM is the same as that of aM except for the presence of the
NH2 group in position R1.
Similarly, the structure of the bAM is the same as that of bM except
for the amino group in position R1. The
structures of aDAM and aBAM are the same as those of aAM, except that
the NH2 group is blocked by a dimethyl or a
benzyl group, respectively.
In previous experiments, we found that aMT and kynurenines aAM and bAM
inhibit nNOS activity in a dose-dependent manner (León et al.,
1998a,b). The calculated IC50 values were aMT,
>1 mM; aAM, 40.9 µM; and bAM, >1 mM. The other kynurenines (i.e.,
aM, bM, aDAM, and aBAM) did not affect nNOS activity at concentrations of up to 1 mM. To investigate the mechanism by which aMT and
kynurenines aAM and bAM inhibit nNOS activity, rat striatal homogenates
were incubated with each drug (1 mM) in the presence of increasing concentrations of L-arginine (0-10 µM). Striatal nNOS
activity was saturable and proportional to substrate concentration
(Fig. 2A). The activity of the enzyme,
however, was significantly decreased in the presence of 1 mM aMT, aAM,
or bAM, as assessed by the Lineweaver-Burk double-reciprocal analysis
of the data (Fig. 2B). Although Km values
of control, aMT, aAM, and bAM (Table 1)
were similar, the Vmax values for these
compounds were lower than the control. The results suggest that aMT and
kynurenines aAM and bAM behave as noncompetitive inhibitors of nNOS
activity.
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Fig. 2.
Experiments with homogenates from rat striatum
showing the kinetics of enzyme-substrate reaction in the presence of
the indicated drugs. A, effect of aMT ( ), aAM ( ), and bAM ( )
on rat striatal nNOS activity. Homogenates from rat striatum were
incubated for 30 min at 37°C with increasing concentrations of
L-arginine and in the absence ( ) or in the presence of 1 mM drug. Each point is the mean ± S.E.M. of three experiments
done in triplicate. B, double reciprocal plot of the data showing that
the drugs tested modify Vmax and not
Km values of the enzyme-substrate
reaction.
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TABLE 1
Km and Vmax values for nNOS
activity measured in rat striatal homogenates incubated in the presence
of a 1 mM concentration of each indicated drug
Kinetic parameters were calculated from Lineweaver-Burk analysis of the
data showed in Fig. 2. Each value represents the mean ± S.E.M. of
three experiments with four striata, each one performed in triplicate.
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We next tested for the possible existence of any interaction between
aMT and the kynurenines aAM and bAM with nNOS cofactors. We used a
commercially available purified rat nNOS. In preliminary experiments we
found that concentrations of Ca2+ between 0 and
17.5 µM in the incubation medium did not affect [3H]citrulline production by purified nNOS.
Concentrations of Ca2+ above 17.5 µM produced a
dose-related inhibition of [3H]citrulline
formation with this commercial source of purified nNOS (Fig.
3A). However, inclusion of 1 mM EGTA
abolished nNOS activity, highlighting the Ca2+
dependence of this NOS isoform (Fig. 3B). The CaM dependence of nNOS
activity in our conditions was assessed with the CaM antagonists trifluoperazine and calmidazolium. In the presence of 1 mM
trifluoperazine or 100 nM calmidazolium, the activity of nNOS was
significantly reduced at CaM concentrations of 0.1 and 10 µg/ml,
whereas 1 mM aMT inhibited nNOS at 0.1 but not at 10 µg/ml of CaM
(Fig. 4). Figure 4 shows that 100 nM
calmidazolium significantly reduced nNOS activity at both
concentrations of CaM. In light of these preliminary findings, the
effect of aMT and kynurenines aAM and bAM on nNOS activity was
investigated in an incubation medium containing 0.0125 U of purified
nNOS, 17.5 µM CaCl2, 10 µg/ml CaM, 10 µM
FAD, and 30 M H4-biopterin.
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Fig. 3.
Experiments with purified rat brain nNOS carried out
in presence of 10 µg/ml CaM, 10 µM FAD and 30 µM
H4-bioperin. A, effect of different concentrations of
Ca2+ on nNOS activity. **P < .01 and
***P < .001 versus Ca2+ = 0. B,
effect of 1 mM EGTA, a specific Ca2+ chelator, in the
incubation medium. ***P < .001 versus nNOS
activity in absence of EGTA. Each point is the mean ± S.E.M. of
three experiments done in triplicate.
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Fig. 4.
Effect of aMT (1 mM), trifluoperazine (TFP, 1 mM),
and calmidazolium (CDZ 100 nM) on the activity of purified rat brain
nNOS incubated in presence of 0.1 or 10 µg/ml CaM, 10 µM FAD, and
30 µM H4-bioperin. Results show percentage of inhibition
of control nNOS activity and are the mean ± S.E.M. of three
experiments done in triplicate. **P < .01 and
***P < .001 versus control.
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In absence of added CaM, aMT inhibited purified nNOS activity in a
dose-dependent manner, the effect being significant at 1 nM
(P < .05, Fig. 5A). The
incorporation of increasing amounts of CaM into the incubation medium
resulted in a progressive loss of aMT efficiency to inhibit nNOS (Table
2). At 10 µg/ml of CaM, aMT was unable
to inhibit the enzyme activity. Fixing the CaM concentration at 0.1 µg/ml, different concentrations of FAD (0.1-10 µM) (Fig. 5B) or
H4-biopterin (0.3-30 µM) (Fig. 5C) into the
incubation medium did not modify the enzyme inhibition by aMT.
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Fig. 5.
Effect of aMT (109 to 103
M) on nNOS activity in the presence of increasing concentrations of
nNOS cofactors. A, CaM ( , 0 µg/ml; , 0.1 µg/ml; , 1 µg/ml; , 10 µg/ml); FAD, 10 µM; and H4-biopterin,
30 µM. B, FAD (, 0.1 µM; , 1 µM; , 10 µM); CaM, 0.1 µg/ml; and H4-biopterin, 30 µM. C, H4-biopterin (,
0.3 µM; , 3 µM; , 30 µM); CaM, 0.1 µg/ml; and FAD, 10 µM. Results show the percentage of inhibition of control nNOS
activity and are the mean ± S.E.M. of three experiments done in
triplicate. Comparisons between aMT doses: *P < .05 versus  ; **P < .01 versus ;
***P < .001 versus . Comparisons between CaM
doses: #P < .05 versus CaM 10 µg/ml;
##P < .01 versus CaM 0.1, 1 and 10 µg/ml; ###P < .001 versus CaM 0.1, 1 and 10 µg/ml.
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TABLE 2
IC50 values for nNOS activity inhibition calculated from
experiments with purified rat enzyme incubated with aMT, aAM, or bAM at
different concentrations of calmodulin
Experiments were performed in the presence of 10 µM FAD, 30 µM
H4-biopterin, and 17.5 µM Ca2+.
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Kynurenines aAM and bAM inhibited nNOS activity in a dose-dependent
manner when CaM was absent from the incubation medium, and the effect
was significant at 1 nM each compound (P < .05, Figs.
6A and 7A,
respectively). Increasing the concentration of CaM resulted in a loss
of inhibitory potency of aAM and bAM (Table 2) on purified nNOS
activity. The behavior of these kynurenines was similar to that of aMT.
However, in the presence of 10 µg/ml CaM, aAM (10 µM) inhibited
nNOS activity (P < .05), an effect absent for bAM and
aMT at this concentration of CaM. After fixing the concentration of CaM
at 0.1 µg/ml, neither changes in FAD (0.1 - 10 µM) nor in
H4-biopterin (0.3 - 30 µM) in the incubation medium affected the inhibitory effect of either aAM and bAM on purified
nNOS activity (Fig. 6, B and C, and Fig. 7, B and C, respectively).
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Fig. 6.
Effect of aAM (109 to 103
M) on nNOS activity in the presence of increasing concentrations of
nNOS cofactors. A, CaM (, 0 µg/ml; , 0.1 µg/ml; , 1 µg/ml; , 10 µg/ml); FAD, 10 µM; and H4-biopterin,
30 µM. B, FAD (, 0.1 µM; , 1 µM; , 10 µM); CaM, 0.1 µg/ml; and H4-biopterin, 30 µM. C,
H4-biopterin (, 0.3 µM; , 3 µM; , 30 µM);
CaM, 0.1 µg/ml; and FAD, 10 µM. Results show the percentage of
inhibition of control nNOS activity and are the mean ± S.E.M. of
three experiments done in triplicate. Comparisons between aMT doses:
*P < .05 versus ; **P < .01 versus ; ***P < .001 versus 10 µg/ml.
Comparisons between CaM doses: #P < .05 versus CaM 1 µg/ml; ##P < .01 versus CaM 0 µg/ml; ###P < .001 versus CaM 0 µg/ml.
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Fig. 7.
Effect of bAM (109 to 103
M) on nNOS activity in the presence of increasing concentrations of
nNOS cofactors. A, CaM (, 0 µg/ml; , 0.1 µg/ml; , 1 µg/ml; , 10 µg/ml); FAD, 10 µM; and H4-biopterin,
30 µM. B, FAD (, 0.1 µM; , 1 µM; , 10 µM); CaM, 0.1 µg/ml; and H4-biopterin, 30 µM. C,
H4-biopterin (, 0.3 µM; , 3 µM; , 30 µM);
CaM, 0.1 µg/ml; and FAD, 10 µM. Results show the percentage of
inhibition of control nNOS activity and are the mean ± S.E.M. of
three experiments done in triplicate. Comparisons between aMT doses:
*P < .05 versus ; ***P < .001 versus 10 µg/ml. Comparisons between CaM doses:
#P < .05 versus CaM 0.1 and 1 µg/ml;
##P < .01 versus CaM 0.1 and 1 µg/ml; ###P < .001 versus CaM 0 µg/ml.
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To further study any possible interaction of aMT, aAM, and bAM with
CaM, urea-PAGE of CaM was made. The electrophoretic migration mobility
of CaM was studied in the absence or presence of both 2 mM EGTA and 1 mM aMT (Fig. 8A), 1 mM aAM (Fig. 8B), and
1 mM bAM (Fig. 8C). The presence of aMT, aAM, or bAM produced a similar CaM mobility into the gel that was faster than the CaM mobility obtained in control gels in which these compounds were absent. In the
presence of EGTA, the migration of CaM was slower than the observed in
the absence of EGTA. In these conditions (i.e., in the presence of
EGTA), aMT, aAM, and bAM were unable to modify the migration pattern of
CaM.
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Fig. 8.
Effects of aMT (A) and kynurenines aAM (B) or bAM (C)
on the electrophoretic mobility of CaM. Urea-PAGE gels were made in the
presence of 1 mM Ca2+ (control) or 2 mM EGTA and in the
absence or presence of 1 mM each compound. See Results for
explanation.
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To assess whether the interaction of aMT and the kynurenines aAM and
bAM with the calcium-CaM (CaCaM) complex is related to their inhibition
of nNOS activity, a series of urea-PAGE of CaM were made in the
presence of the kynurenines aM and bM. Besides, to test the hypothesis
that the differences in the molecular structure (i.e., differences in
hydrophobicity) may account for the interaction between these compounds
and CaM, a urea-PAGE of CaM with serotonin was also made. The results
suggest that none of these compounds affected the migration pattern of
CaM either in the presence or absence of EGTA (Fig.
9A, B, and C, respectively). Moreover,
blocking the NH2 group of kynurenine aAM with a
dimethyl (aDAM) or a benzyl (aBAM) group made the resultant compounds
unable to change the migration pattern of CaM (Fig.
10A and B, respectively).
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Fig. 9.
Effects of serotonin (A) and the kynurenine aM (B) or
bM (C) on the electrophoretic mobility of CaM. Urea-PAGE gels were made
in the presence of 1 mM Ca2+ (control) or 2 mM EGTA and in
the absence or presence of 1 mM each compound. See Results
for explanation.
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Fig. 10.
Effects of the kynurenine aDAM (A) or aBAM (B) on
the electrophoretic mobility of CaM. Urea-PAGE gels were made in the
presence of 1 mM Ca2+ (control) or 2 mM EGTA and in the
absence or presence of 1 mM each compound. See Results for
explanation.
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A dose-dependent study of the interaction of aMT, aAM, and bAM with
CaCaM was made. Fig. 11 shows the
urea-PAGE gels of these experiments, proving that the kynurenines aAM
and bAM bind CaCaM with more affinity than aMT. In fact, at
concentrations of 250 µM, the kynurenines aAM and bAM but not aMT
were able to affect the migration behavior of CaCaM.
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Fig. 11.
Dose-response effects of aMT and the kynurenine aAM
or bAM on electrophoretic mobility of CaM. Urea-PAGE gels were made in
the presence of 1 mM Ca2+ (lanes 1) and in the absence or
presence of aMT, aAM or bAM at the following doses: 1 mM (lanes 2); 500 µM (lanes 3); 250 µM (lanes 4); 100 µM (lanes 5).
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Discussion |
The major findings of this study are the demonstration that aMT
and some synthetic kynurenines, structurally similar to the natural
brain aMT metabolite (Hirata et al., 1974), inhibit nNOS activity
through a mechanism involving a complex formation with CaCaM. The
results also show interesting structure-related effects of these
kynurenines in their ability to bind CaCaM. Although kynurenines
carrying an NH2 group on their molecule (aAM and
bAM) bind CaCaM inhibiting nNOS activity, kynurenines lacking the amino group (aM and bM) are unable to bind CaCaM, losing their inhibitory effect on nNOS. That the NH2 group is decisive
for the inhibitory effect on nNOS activity was also demonstrated by
blocking it with either a dimethyl or a benzyl group (aDAM and aBAM,
respectively); in this case nNOS was not inhibited further. Our results
also show that aMT inhibits nNOS activity in a dose-dependent manner. The hormone significantly reduced nNOS activity at 1 nM, corresponding to the concentration of aMT during its nocturnal peak (Reiter, 1991;
Bettahi et al., 1996). Interestingly, aAM and bAM showed a 20%
reduction in nNOS activity at concentration of 10 pM, reflecting 100 times more activity than aMT itself to inhibit the enzyme (León
et al., 1998a).
Kinetic studies showed that the inhibitory effect of aMT and
NH2-kynurenines on nNOS activity measured in rat
striatal homogenates could not be prevented by increasing
L-arginine concentration, the natural substrate of the
enzyme. Similar results were found using a purified rat brain nNOS as a
source of the enzyme. The data suggest that these compounds behave as
noncompetitive inhibitors of the nNOS. It was surprising that purified
nNOS converted L-[3H]arginine to
L-[3H]citrulline in the absence of
Ca2+ or the presence of CaM. These results
confirm data elsewhere reported and presumably suggest a significant
carry-over of these compounds during enzyme purification (Handy and
Moore, 1997). The inclusion of Ca2+ up to 17.5 µM did not affect
L-[3H]citrulline production; above
this concentration, a Ca2+-dependent inhibition
of nNOS activity was found. The precise mechanism of this inhibition is
poorly understood (Mittal and Jadhav, 1994). Calcium ions and CaM are
required for the catalytic conversion of L-arginine to
nitric oxide and L-citrulline (Knowles et al., 1989; Mittal
and Jadhav, 1994). High Ca2+ concentration may
account for Ca2+ itself or some
Ca2+-derivative intermediates interacting with a
presumed regulatory (noncatalytic) site of nNOS. In turn, the
Ca2+-dependent interaction leads to a diminished
generation of oxidizing equivalents at the catalytic site of the
enzyme, decreasing nNOS activity. However, the concentration of
Ca2+ within the cell in vivo is considerably
lesser than that required to inhibit nNOS in the in vitro experiments
here described. Thus, it is unlikely that
Ca2+-induced nNOS inhibition was of physiological
significance (Handy and Moore, 1997). Anyway, the activity of nNOS was
completely blocked in presence of EGTA in the incubation medium,
further supporting a constitutive, Ca2+-dependent
nNOS isoenzyme.
The inhibitory role of aMT and NH2-kynurenines on
nNOS activity was independent of FAD or
H4-biopterin concentration in the incubation
medium. Increasing concentrations of CaM, however, resulted in a loss
of the inhibitory ability of these compounds. That the decrease in nNOS
activity was caused by CaCaM inhibition was shown by incubation of nNOS
with the CaM antagonists trifluoperazine and calmidazolium. Thus, other
experimental approaches were made to further investigate a possible
interaction between aMT and NH2-kynurenines with
CaM. Urea-PAGE experiments proved an increase in the mobility of CaM in
the presence of either aMT, aAM, or bAM. CaM migration pattern in the
presence of these drugs was similar for all of them. In the absence of
Ca2+, aMT and the kynurenines aAM and bAM were
unable to modify the mobility of CaM alone. With the use of NMR,
circular dichroism, and fluorescence spectroscopy, it was recently
shown that aMT binds CaCaM but neither the CaCaM-NOS complex nor NOS
alone (Ouyang and Vogel, 1998). These data together with our results
suggest an interaction between aMT and
NH2-kynurenines and the CaCaM complex that may
account for the changes in the peptide electrophoretic migration. The
results also suggest that Ca2+ ions are necessary
for the interaction between aMT and
NH2-kynurenines with CaM. Dose-dependent
experiments with urea-PAGE demonstrate that the kynurenines aAM and bAM
bind CaCaM with higher affinity than aMT, thus supporting the results
of kinetic experiments.
In response to high intracellular Ca2+ levels,
CaM binds Ca2+ ions and undergoes a major
conformational change after the exposure of two hydrophobic regions on
the protein surface (Ouyang and Vogel, 1998). Studies on peptide-CaM
interactions show that CaM-binding peptides are normally positively
charged, hydrophobic 
-helices (O'Neil and DeGrado, 1990). Small,
hydrophobic molecules can also bind to CaM and this may account for the
interaction between CaCaM and aMT, aAM, and bAM (Ouyang and Vogel,
1998). Besides hydrophobic interactions, the electrostatic interaction
between basic residues on the molecule and acidic residues of CaM also
contribute to the binding (Crivici and Ikura, 1995). This may be the
case for some tryptophan metabolites such as 5-hydroxytryptophan, in
which the extra negative charge from its carboxyl group is likely to be
repelled by the negative charges from the acidic residues on CaM
surrounding the hydrophobic clefts (Ouyang and Vogel, 1998). For
serotonin, its hydrophilicity (Benítez-King et al., 1993, 1996)
may account for its very weak bind to the C-terminal domain of CaM
(Ouyang and Vogel, 1998). Thus, the relative hydrophobic interactions
and electrostatic repulsion might be the main reason that serotonin and
kynurenines aM and bM did not bind apparently to CaM in our experiments.
Upon NMDA activation and Ca2+ influx into the
cell, Ca2+ binds to CaM, producing the CaCaM
complex responsible for the activation of nNOS (Moncada et al., 1991;
Garthwaite and Boulton, 1993). We showed that microiontophoresis of aMT
inhibits the excitatory response of the striatal neurons to the
sensory-motor cortex stimulation acting via the NMDA receptor in a
dose-dependent manner (Castillo-Romero et al., 1995; Escames et al.,
1996). The kynurenines aAM and bAM were more potent than aMT in
inhibiting this striatal excitatory response; aM displayed potency
similar to that of aMT, and bM seemed to act as a functional aMT
antagonist (León et al., 1998a,b). Comparison of the
electrophysiological data with the results reported here suggest that
the break of the pyrrolic ring of the indoleamine during kynurenine
synthesis leads to the loss of aMT's ability to bind to CaM. In turn,
the addition of an NH2 group in position R1 of the kynurenine molecule recovers this
ability. Consequently, the synthetic kynurenines here described keep
some of the properties of aMT in a structure-related fashion;
NH2-kynurenines keep the ability of aMT to
inhibit both NMDA-dependent excitatory response and nNOS activity,
whereas kynurenines lacking the NH2 group or with
this group blocked retain the ability to affect NMDA response but do
not further affect nNOS activity.
A relationship between aMT, CaM, and cytoskeleton has been demonstrated
(Benítez-King et al., 1993; Benítez-King et al., 1996).
Glutamate receptors are associated with cytoskeletal proteins, and CaM
may inactivate NMDA receptor interacting with the NR1 subunit, an
effect involving -actinin, another cytoskeletal protein (Wyszynski
et al., 1997). However, CaM reduces NMDA channel activity but CaM
inhibitors were unable to counteract the inactivation of CaM-dependent
NMDA activity in whole-cell recordings (Krupp et al., 1999). These data
suggest that NMDA inactivation in the intact cell was not a simple
phenomenon and the participation of aMT and aMT metabolites in this
process should be taken into account. In fact, recent data based on a
carefully conducted kinetic study suggest that CaM may act as a
membrane/cytosolic aMT receptor (Romero et al., 1998).
One last consideration that should be addressed is the relation between
the effects of aMT and kynurenines here described in vitro and their
physiological relevance in vivo. Melatonin, aAM, and bAM significantly
inhibit recombinant nNOS activity at 1 nM. These effects shown with
recombinant nNOS confirm similar results in homogenates of rat
striatum, although in this case, aAM and bAM were effective at
concentrations of 10 pM (León et al., 1998a,b). Because 1 nM may
be considered a physiological concentration of aMT during the night
(Reiter, 1991), the inhibition of nNOS activity by the nocturnal peak
of the indoleamine might be a mechanism involved in the depressive
effect of aMT on brain excitability. Perhaps this mechanism explains,
at least in part, the sedative and hypnotic effects of the indoleamine
at night. Anyway, aMT levels into the cell are not clearly stated and
aMT may concentrate in such cellular structures as membranes and
mitochondria, where it may reach concentrations of 200 nM
(Martín et al., 2000). These data lead one to re-examine the
"physiological" levels of the hormone. Furthermore, aMT is
metabolized to aMK by 2,3-dioxygenase, an enzyme of the cytosolic side
of the cellular membrane. It was hypothesized that aMK could have some
of the physiological functions assigned to aMT (Kennaway and Hugel,
1991; Acuña-Castroviejo et al., 1994). In experiments in our
laboratory, we have found that aMK displays an
IC50 value of 40 nM for nNOS inhibition in rat
striatal homogenates [i.e., more than 25,000 times the
IC50 for aMT (D. Acuña- Castroviejo, unpublished observations)]. The action's
mechanism of aMK is the same as aMT (i.e., the aMT metabolite forms a
complex with CaM that hampers the activation of nNOS). Consequently,
the actions of aMT in vivo may depend not only on its
"physiological" concentrations, but also on the aMK produced endogenously. Because the kynurenines assayed in our study are synthetic, we cannot compare their effects at the doses used with similar compounds produced endogenously. The interest of these synthetic kynurenines was the search for neuroprotective compounds with
potential clinical use.
The data discussed highlight the importance of brain tryptophan
metabolism in modulating CNS activity. The methoxyindole pathway of
tryptophan produces aMT, with inhibitory effects on brain homeostasis, acting on NMDA via CaCaM interaction. The kynurenine pathway produces a
series of compounds with both excitatory and inhibitory effects on
glutamate receptors (Fukui et al., 1991). In addition, some kynurenines
are bioactive metabolites of aMT, and may mediate some actions of aMT
(Kennaway and Hugel, 1991; Acuña-Castroviejo et al., 1994;
Acuña-Castroviejo et al., 1995). Thus, an imbalance of this
tryptophan metabolic pathways may produce widespread changes in CNS
excitability (Muñoz-Hoyos et al., 1997). The results also account
for the potential use of aMT and kynurenines as neuroprotective drugs
in human clinical studies (Molina-Carballo et al., 1997; Crespo et al.,
1999) and suggest a pathway for the design and synthesis of other new ones.
We thank Drs. David Pozo and Francisco Martínez for help
in the preparation of urea-PAGE experiments.
This work was partially supported by grants from the Ministerio
de Educación y Cultura (SAF98-0156), and from the Junta de Andalucía (CTS-101). J.L. and Ma.M. are a fellows from
the Programa de Formación de Personal Investigador, Ministerio de
Educación y Cultura, Spain; H.K. is a fellow from the Ministerio
de Asuntos Exteriores, Spain; Mi.M. is a fellow from the
Universidad de Granada.
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Abstract
Introduction
