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Departamento de Bioquímica, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, Buenos Aires, Argentina
Address all correspondence and requests for reprints to: Dr. Cora B. Cymeryng, Departamento de Bioquímica, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 5° piso (1121), Buenos Aires, Argentina. E-mail: CCYMERYN{at}fmed.uba.ar
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Abstract |
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Introduction |
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Concerning the mechanism of action of NO, its binding to iron in heme and nonheme iron-containing enzymes has been demonstrated (2, 3). The binding of NO to the heme group of soluble guanylate cyclase, the main target for the physiological effects of NO, alters the enzyme conformation, thus increasing its activity (15, 16). NO also reacts with several other metalloproteins, such as cytochromes P-450 (17) and adrenodoxin, that are essential for steroidogenic reactions (11, 13, 18).
Although many reports demonstrate the inhibitory effect of exogenous NO on the steroidogenic pathway, whether endogenously produced NO is a physiological regulator of corticosterone production remains an open question.
Palacios et al. (19) described NOS activity in the cytosol from adrenal glands and stimulation of a soluble guanylate cyclase activity induced by NO in whole rat adrenal, bovine cortex, and medulla cytosol. NG-Nitro-L-arginine methyl-ester (L-NAME), a NOS inhibitor, was reported to increase serum corticosterone and testosterone levels in rats, suggesting that endogenous NO negatively regulates steroidogenesis (20). Perfusion of L-arginine to rats abolished the angiotensin II-induced, but not the ACTH-induced, increase in aldosterone levels in adrenal zona glomerulosa (21). On the other hand, Cameron and Hinson (22) showed that corticosterone production in isolated perfused adrenals is inhibited by L-NAME. However, they also described an inhibitory effect of 1 mM L-arginine on corticosterone secretion.
The biochemical mechanisms that regulate the intracellular levels of L-arginine, the necessary substrate for NOS reaction, have been poorly investigated. Remarkably little is known about how L-arginine is transported into endocrine cells, whether its availability as a precursor for the synthesis of NO would depend on a specific uptake mechanism, and how the physiological regulation of such process might occur. As L-arginine is an essential amino acid, its intracellular concentration and its availability in all likelihood depend exclusively on its transport. Accordingly, it has been demonstrated that the NOS-mediated formation of NO from L-arginine is dependent upon an adequate and continuing supply of L-arginine in endothelial cells (23) and brain slices (24).
In the present work, the effects of L-arginine in rat adrenal zona fasciculata cells were studied to investigate the role of an endogenous NOS in the modulation of adrenal physiology. Characterization of the transport system for L-arginine in rat adrenal zona fasciculata cells was also performed.
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Materials and Methods |
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-Fluoro-11-ß,17,21-trihydroxy-16--methylpregna-1,4-diene-3,20-dione
(dexamethasone) was purchased from Fluka (Buchs, Switzerland).
L-Arginine, D-arginine,
L-ornithine, L-lysine, BSA,
22(R)-hydroxycholesterol, 8-bromo-cAMP (8Br-cAMP),
pregnenolone, L-NAME, and 3-isobutyl-1-methylxanthine (IBMX) were
purchased from Sigma Chemical Co. (St. Louis, MO).
NG-Nitro-L-arginine (L-NNA) was
obtained from RBI (Natick, MA).
[1,2,6,7-3H]Corticosterone and
L-[2,3-3H]Arginine were purchased from New
England Nuclear Corp. (Boston, MA). Corticosterone antisera were
provided by Dr. A. Bélanger (Laval University, Québec,
Canada). The Nitrate/Nitrite Colorimetric Assay Kit was obtained from
Cayman Chemical Co. (Ann Arbor, MI). Rabbit polyclonal anti-cGMP
antiserum was purchased from Chemicon International, Inc. (Temecula,
CA). All other reagents were commercial products of the highest grade
available.
Animals
Male adult Wistar rats were used in all the experiments. The
animals had full access to water and Purina formula chow (Ralston Purina Co., St. Louis, MO). Dexamethasone (10 µg/ml) was
supplied in the drinking water ad libitum 16 h before
death. This treatment suppresses both circadian rhythm and
stress-induced output of corticosterone. The animals were killed by
decapitation according to protocols for animal use approved by the
institutional animal care and use committee following NIH guidelines.
The adrenals were rapidly excised, kept on ice, and demedullated before
preparation of zona fasciculata cells.
Zona fasciculata cell preparation and treatment
Zona fasciculata cells were isolated using published procedures
(25). The cells were suspended in Krebs-Ringer bicarbonate buffer (pH
7.4) containing 10 mM glucose, 0.5% (wt/vol) bovine
albumin (KRBGA medium) and 0.1 mM IBMX under 95%
O2-5% CO2 and aliquoted in fractions
containing 105 cells/tube.
Preincubation in the presence of L-arginine was carried out for 30 min at 37 C, then ACTH, 8Br-cAMP, 22(R)-hydroxycholesterol, or pregnenolone was added to the cell suspensions, and incubation proceeded at 37 C with shaking (100 cycles/min) for 60 min. In another set of experiments, cell suspensions were preincubated in the presence of D-arginine, L-lysine, or L-ornithine for 15 min, and then L-arginine or KRBGA was added to the cell suspensions. The incubations were stopped by cooling the tubes on ice-water, and the cells were pelleted by centrifugation at 500 x g for 15 min. The supernatants were assayed for corticosterone by RIA after extraction with methylene chloride. Cell viability, as determined by trypan blue exclusion, was greater than 88% and was not significantly affected by any treatment.
cGMP assessment
Zona fasciculata cells were incubated for 90 min in KRBGA medium
(37 C, under O2-CO2) in the presence of 1
mM IBMX and different concentrations of
L-arginine (0–2.5 mM) in the presence or
absence of 100 µM L-NAME or 2.5 mM
D-arginine. After incubation, the cells were centrifuged,
and the pellets were resuspended in ice-cold water with 1
mM IBMX and disrupted by ultrasonic oscillation. The
extracts were heated for 2 min in boiling water. After centrifugation
in an Eppendorf microfuge for 3 min, aliquots from the
supernatants were diluted with sodium acetate (final concentration, 50
mM), pH 6. Samples and standards were acetylated and
assayed by RIA according to the protocol described by Steiner et
al. (26) with slight modifications. At the end of the incubation,
the antigen-antibody complexes were precipitated by the addition of 50
µl 2% BSA and 2 ml cold ethanol (95%) and centrifuged at 2000 rpm
for 20 min. The supernatants were aspirated, and the pellets were
counted in a 1272 Clini-Gamma (LKB, Bromma, Sweden).
Measurement of nitrite levels
Nitrite levels were determined in cell suspension media after
incubation with different L-arginine concentrations in the
presence or absence of 100 µM L-NAME or 2.5
mM D-arginine for 210 min at 37 C. For this
purpose, 80-µl aliquots were mixed with Griess reagent and incubated
at room temperature for 10 min, and absorbance at 570 nm was
determined. Fresh medium served as a blank, and 1- to
5-µM solutions of sodium nitrite were used as
standard.
L-Arginine uptake
L-Arginine uptake was assayed by the method of Rao
and Butterworth (27) with slight modifications. Briefly, adrenal cells
(105 cells/tube) were suspended in 200 µl KRBGA,
preincubated for 15 min at 37 C, and incubated for 2 min in the
presence of L-[3H]arginine (50
µM, 5–7 x 105 dpm).
L-Arginine uptake was stopped by adding 4 ml ice-cold PBS
and rapidly filtering the samples under vacuum through Whatman GF-C
filters (Whatman, Clifton, NJ) soaked in 0.3%
polyethylenimine to reduce nonspecific binding of
L-[3H]arginine. The filters were washed twice
with 4 ml fresh buffer, and radioactivity was counted in a liquid
scintillation spectrometer. Nonspecific uptake of
L-[3H]arginine was assessed in the presence
of 10 mM L-arginine and was subtracted from all
values.
The effects of several amino acids on L-arginine uptake were examined by incubating cells with Krebs solution containing 50 µM L-[3H]arginine in the absence or presence of a L-lysine, L-ornithine, L-valine, and L-leucine (100 µM). To assess the effect of ACTH on L-arginine uptake, the cells were preincubated in the presence of ACTH (500 pg/105 cells) for 30 min at 37 C.
Uptake of 50 µM L-[3H]arginine was also determined in Krebs buffer without NaCl (compensated by isosmolar concentrations of choline chloride). Unlabeled L-arginine (50–500 µM) was used in the uptake kinetics studies. Rates of L-[3H]arginine uptake were determined by double reciprocal plots, the slopes and y-intercepts of linear regression being calculated by the method of least squares.
Statistical analysis
Statistical analysis of results was performed by Student’s
t test or one-way ANOVA followed by Dunnett’s t
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. L-Arginine
significantly decreased steroid production, with threshold
concentrations of 500 and 100 µM for basal and stimulated
corticosterone synthesis, respectively. The inhibition of
steroidogenesis was specific for the L-optical isomer, as
it was not mimicked by D-arginine (Fig. 1). On the other hand, cationic amino
acids such as L-lysine and L-ornithine that
were ineffective per se blocked the effect of
L-arginine on corticosterone biosynthesis (Fig. 1).
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, A and B). A NO synthase inhibitor,
L-NAME (100 µM), although ineffective per se
(not shown), abolished this effect of L-arginine (Fig. 2B). Nitrite levels in the incubation medium were also increased in the
presence of L-arginine (Fig. 3A). Similarly, D-arginine
was ineffective, whereas L-NAME blocked the effect of
L-arginine on nitrite levels (Fig. 3B).
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, L-arginine uptake exhibited monophasic kinetics
(Km = 163 ± 14 µM;
Vmax = 53 ± 6 pmol/min·105 cells). This
mechanism was moderately Na+ dependent, as isoosmotic
substitution with choline chloride decreased it by 35% (10.02 ±
0.3 pmol/min for choline chloride vs. 15.11 ± 0.5
pmol/min for NaCl; P < 0.01). The effect of several
amino acids and NOS inhibitors on L-arginine transport was
investigated. Figure 5 shows that
L-lysine and L-ornithine (basic amino acids)
inhibited L-arginine uptake, whereas L-valine
and L-leucine (neutral amino acids) were ineffective. In
addition, L-NAME, L-NNA, and D-arginine did not impair
L-arginine uptake. ACTH did not modify
L-arginine transport (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO generators have been widely used to study the effect of NO on steroidogenesis. However, conclusive evidence for the participation of an endogenous NOS in the modulation of adrenal physiology had not been previously provided. Several observations support the involvement of NOS in the effects of L-arginine shown herein. 1) Addition of D-arginine, instead of its enantiomer, had no effect on cGMP accumulation, nitrite levels, or steroidogenesis; therefore, the effects of L-arginine are stereospecific. 2) L-Lysine and L-ornithine, two other cationic amino acids, that are not substrates for NO synthesis had no significant effect on steroidogenesis. 3) The increase in cGMP levels indicates activation of a soluble guanylate cyclase, a classical target of NO action; L-NAME, a NOS inhibitor, abolished this effect. 4) L-Arginine increases nitrite levels (a breakdown product of NO); this effect is blocked by L-NAME. Also, in this sense we have previously shown that L-NNA significantly increases corticosterone secretion by zona fasciculata adrenal cells (14).
According to our results, it has been demonstrated that exogenous L-arginine increases NO levels in a variety of cells (23, 24, 28, 29). Furthermore, Hinson et al. (30) demonstrated that addition of L-arginine to the perfusion medium of isolated adrenal glands produces an increase in the medium flow rate that was attributed to an increase in NOS activity.
Purified NOS has been reported to have a low half-saturating arginine concentration (EC50 <20 µM), although high levels of intracellular L-arginine, ranging from 0.1–1 mM, have been measured in many cells (23, 31, 32, 33). It was then expected that endogenous L-arginine would support maximal activation of NOS. However, a number of in vivo and in vitro studies indicate that NO production under physiological conditions can be increased by extracellular arginine despite saturating intracellular arginine concentrations. This has been termed the arginine paradox (34). One possible explanation could be that intracellular arginine is sequestered in one or more pools that are poorly, if at all, accessible to NOS, whereas extracellular arginine transported into the cells is preferentially delivered to NOS (34). Buckley et al. (29) reported that bradykinin-stimulated NO production in bovine aortic endothelial cells was submaximal in the absence of extracellular L-arginine, and they concluded that L-arginine intracellular stores were not fully available to support NO synthesis by the endothelial isoform of NOS or that intracellular L-arginine levels in these cells were lower than previously reported.
To induce the activation of NOS, an obligatory influx of exogenous L-arginine is required. Therefore, we undertook the characterization of the mechanism of its uptake in rat adrenal cells. In our conditions, L-arginine is taken up by a saturable and stereospecific transport system, with a Km on the order of those of high affinity transport systems for other amino acids, i.e. around 100 µM (35, 36). This Km value is also compatible with plasma L-arginine concentrations (20–150 µM). The presence of cationic amino acids (L-lysine and to a lesser extent L-ornithine) decreased L-[3H]arginine uptake, whereas neutral amino acids (L-leucine and L-valine) and NOS inhibitors (L-NNA and L-NAME) were ineffective. Sodium replacement with choline decreased the rate of L-arginine influx by 35%. Classically, the amino acid transport systems have been classified considering two main criteria: 1) substrate specificity, i.e. which amino acids or group of amino acids are transported by the system; and 2) sodium dependence of the transport, generally defined with reference to the rate measured in the presence of choline salts. Four amino acid transport systems have been defined on the basis of substrate specificity and sodium dependence (37, 38). Only one of them (y+) has been shown to be selective for cationic amino acids (albeit it shows weak interaction with neutral amino acids) and is also sodium independent (37, 39). This y+ system was identified, among others, in reticulocytes (40), fibroblasts (41), as well the central nervous system (35). Although the characteristics of L-arginine influx in adrenal cells correlate with those of the y+ amino acid transport system, the presence of multiple carriers with overlapping specificity in these cells, as described for several systems (38), cannot be ruled out. In this sense, the fact that L-arginine influx was reduced by 35% in the absence of sodium suggests that more than one carrier system may coexist in adrenal cells. The results obtained with L-NNA and L-NAME on L-arginine transport also agree with those previously described for the y+ system (35, 42). This transporter encompasses four homologous proteins (CAT-1, CAT-2A, CAT-2B, and CAT-3) that have been characterized in several tissues (38). The identification at the molecular level of the transport protein(s) present in zona fasciculata cells deserves to be examined.
As our results suggest that extracellular L-arginine reduces basal and ACTH-induced corticosterone production via the stimulation of NOS activity, we hypothesized that limiting cellular L-arginine uptake would reduce NO production and consequently restore steroid synthesis. Accordingly, L-lysine and L-ornithine, which were ineffective per se but inhibited L-arginine influx, decreased the effect of L-arginine on corticosterone production. As the Km of NOS is almost 1 order of magnitude less than that for the uptake mechanism, it seems likely that the influx of arginine limits the response elicited by this amino acid. In this sense, our results show that the range of concentrations that significantly affect steroidogenesis correlates with the affinity of the uptake mechanism.
Different threshold concentrations of L-arginine were needed to decrease steroidogenesis in the presence and absence of ACTH. As ACTH had no effect on L-arginine uptake, the higher sensitivity observed in the presence of ACTH could not be attributed to an increase in substrate availability to NOS. The possibility of an increase in NOS activity induced by ACTH in rat adrenal cells is currently under investigation.
In summary, we present evidence for the participation of endogenously generated NO in the modulation of basal and ACTH-induced steroidogenesis. These results support the hypothesis that NO may play a role as a paracrine or autocrine regulator of adrenal steroidogenesis. As ACTH is a pleiotropic regulator in the zona fasciculata of the adrenal gland, the existence of signals able to modulate its function may contribute to prevent an all or none type of response, providing the gland with a higher capacity to respond to a wide range of physiological demands. In this context, we consider that NO should be included in this category of biological modulators of adrenal physiology.
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Acknowledgments |
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Footnotes |
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Received December 17, 1998.
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