(Hypertension. 1995;25:431-436.)
© 1995 American Heart Association, Inc.
Articles |
Regulation of Neuronal Nitric Oxide Synthase in Rat Adrenal Medulla
From The First Department of Internal Medicine (N.I., M.K.) and Institute for Molecular Neurobiology (K.H., I.T.), Shiga University of Medical Sciences, Ohtsu, Shiga, and the Department of Neurobiology, Kyoto Pharmaceutical University (Y.K.), Yamashina, Kyoto, Japan.
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Abstract |
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Abstract Neuronal nitric oxide synthase (nNOS) has been suggested to be involved in cardiovascular homeostasis. We studied the regulation of nNOS expression, determining nNOS mRNA expression levels in various tissues in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). We also investigated the effects of antihypertensive treatment with the angiotensin II antagonist hydralazine or reserpine on nNOS mRNA expression. The expression levels of nNOS mRNA and nNOS protein were determined by Northern and Western blot analysis, respectively. NADPH-diaphorase histochemistry was used to identify cells in the adrenal medulla that expressed nNOS. No significant differences in expression levels in SHR and WKY were observed in the cerebellum and brain stem. nNOS mRNA expression levels in the decapsular portion of the adrenal gland were developmentally modulated and in a 24-week-old WKY were 2.5 times higher than in an age-matched SHR. This reduced expression of nNOS mRNA in the decapsular portion of the adrenal gland of SHR seemed to be a result of hypertension in the SHR, because administration of either an angiotensin II antagonist (TCV-116) or hydralazine upregulated nNOS mRNA expression in both SHR and WKY. Marked augmentation of nNOS mRNA expression in the decapsular portion of the adrenal gland by reserpine treatment suggested an intimate relation between nNOS in the decapsular portion of the adrenal gland and the sympathoadrenal system. Reserpine treatment also increased the expression of nNOS protein; however, reserpine treatment did not affect the distribution pattern of nNOS-positive cells (NADPH-diaphorase–positive cells) in the adrenal medulla. The present results suggest that nNOS gene expression in the decapsular portion of the adrenal gland is not constitutive and that an intimate relation may exist between nNOS in the decapsular portion of the adrenal gland and the sympathoadrenal system.
Key Words: nitric oxide • rats, inbred SHR • adrenal medulla • reserpine • angiotensin II • hydralazine
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Introduction |
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Nitric oxide (NO) has recently been recognized as a regulatory molecule for endothelial cells in blood vessels, white blood cells, and neurons in the brain and the peripheral nervous system. Three distinct forms of NO synthase (NOS) have been identified.1 2 NOS induced in macrophages and vascular smooth muscle cells by various cytokines or lipopolysaccharides has been referred to as inducible NOS, whereas that in neuronal cells (nNOS) and endothelial cells (eNOS) appears to be expressed constitutively.
Since NO seems to participate in the physiology or pathophysiology of nearly every organ system, there is great interest in how NO biosynthesis can be regulated. The induction of inducible NOS by lipopolysaccharides or various cytokines may cause hypotension in septic shock.3 4 NO formed by eNOS has been considered to be a physiological regulator of basal blood vessel tone,5 6 and the regulation of eNOS activity or eNOS biosynthesis is now under intense investigation.
nNOS has also been shown to play an important role in cardiovascular homeostasis. For example, NO has been reported to decrease central sympathetic outflow7 and mediate an L-glutamate–elicited decrease in blood pressure and heart rate through baroreceptor-like reflexes in the nucleus tractus solitarius.8
Although there are a growing number of histochemical studies on nNOS, few reports exist on the regulation of the activity or biosynthesis of nNOS. In the present study, we investigated the expression and regulation of nNOS. Since our interests lie in the field of blood pressure regulation, nNOS expression levels in various tissues were compared between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Pharmacological interventions revealed an intimate relation between nNOS and the sympathetic nervous system.
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Methods |
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Animals
SHR and WKY were obtained from Charles River Laboratories (Atsugi, Kanagawa, Japan). Reserpine was administered at 5 mg/kg IP. The angiotensin II (Ang II) antagonist TCV-116 (Takeda Chemical Industries)9 was suspended in arabic gum solution (5%) and administered orally once a day (10 mg/kg). Hydralazine was dissolved in water and administered orally once a day (10 mg/kg). Blood pressure was measured by the tail-cuff method at the time of death, roughly 12 hours after the last drug administration.
RNA Isolation and Analyses
RNA was isolated as previously reported.10 Northern
blot analysis was performed as previously reported.11
Briefly, RNAs were denatured with glyoxal, electrophoresed on a 0.7%
agarose gel with 10 mmol/L phosphate buffer (pH 7.0), and blotted on a
nylon membrane (GeneScreen Plus, DuPont). The blotted membranes were
hybridized with a 32P-labeled nNOS cDNA fragment or GAPDH
cDNA fragment in a solution containing 10% polyethylene glycol, 7%
sodium dodecyl sulfate (SDS), and 50 mmol/L phosphate buffer (pH 7.0)
at 65°C for 12 hours. The hybridized membranes were washed three
times in 0.2x SSC (30 mmol/L NaCl, 3 mmol/L sodium citrate) containing
0.1% SDS at 65°C for 30 minutes each. The rat nNOS cDNA fragment
(2461-3062)12 used as a probe was synthesized by the
polymerase chain reaction using the following two primers:
5'-GAATACCAGCCTGATCCATGGAACACC-3' and 5'-CTCCAGGAGGGTGTCCACCGCATGCC-3'.
The validity of the synthesized cDNA fragment was confirmed by partial
sequencing. The expression levels of mRNA were determined with a
densitometer.
Western Blot Analysis
The expression level of nNOS protein was determined by
Western blot analysis. The decapsular portion of the adrenal gland
(D-AD) was homogenized in 10 vol of a solution containing 10 mmol/L
Tris-HCl (pH 7.4), 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl
fluoride (PMSF), 2.2 µmol/L leupeptin, 15 µmol/L pepstatin, 0.3
µmol/L aprotinin, and 5 mmol/L 2-mercaptoethanol and was centrifuged
for 1 hour at 10 000g. Twenty micrograms of protein in the
supernatant was subjected to SDS–polyacrylamide gel electrophoresis
(PAGE). The resolved proteins were transferred to a piece of
polyvinylideine difluoride membrane (Immobilon P, Millipore) using a
semidry blotting apparatus. Anti-rabbit IgG antibody conjugated with
alkaline phosphatase was obtained from GIBCO-BRL. The nNOS antibody
(rabbit) was purchased from Transduction Laboratories and diluted 1:250
for the Western blot analysis as recommended by the supplier.
Immunoreactive bands were visualized with 5-bromo-4-chloro-3-indolyl
phosphate and nitro blue tetrazolium. Protein concentration was
assessed with BCA protein assay reagent (Pierce Chemical Co).
Purification of Rat Cerebellar nNOS
nNOS was purified from rat cerebella by the method of Schmidt et
al.13 Briefly, rat cerebella (5 g) were thawed and
homogenized in 4 vol of ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4)
containing 0.5 mmol/L EGTA, 0.5 mmol/L EDTA, 0.1 mmol/L PMSF, and 0.3
µmol/L aprotinin (buffer A). The homogenate was centrifuged at
18 000g for 30 minutes, and the supernatant was applied to
a column packed with 2',5'-ADP–agarose gel (1 mL) equilibrated with
buffer A. The column was sequentially washed with 10 mL of buffer A, 3
mL of buffer A containing 0.5 mol/L NaCl, and then again with 10 mL of
buffer A. Subsequently, the nNOS-rich fraction was eluted with 2 mL of
buffer A containing 5 mmol/L NADPH. The fraction thus obtained was
incubated with 2 mmol/L CaCl2 and applied to a column
packed with calmodulin-agarose gel (1 mL) equilibrated with buffer A
containing 2 mmol/L CaCl2. The column was washed with 10 mL
of buffer A containing 2 mmol/L CaCl2. nNOS was eluted with
2 mL of buffer A containing 5 mmol/L EGTA. The purified nNOS consisted
of a single 160-kD polypeptide by SDS-PAGE that could form
L-[14C]citrullin from
L-[14C]arginine in the presence of 1 mmol/L
Ca2+, 10 µg/mL calmodulin, 1 mmol/L NADPH,
and 100 µmol/L tetrahydrobiopterin.
NADPH-Diaphorase Histochemistry
Rats were deeply anesthetized with sodium pentobarbital (70
mg/kg), perfused through the ascending aorta with 100 mL of 10 mmol/L
phosphate-buffered saline (pH 7.4), and then perfused with 300 mL of
ice-cold 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4).
The adrenal glands were excised and postfixed for 24 hours with 4%
paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). After
cryoprotection for 48 hours with 0.1 mol/L phosphate buffer (pH 7.4)
containing 15% sucrose and 0.1% sodium azide, 20-µm-thick sections
were cut with a cryostat (Yamato).
NADPH-diaphorase activity was rendered visible by incubating the sections for 60 minutes in phosphate buffer (pH 8.0) containing 0.01 mmol/L NADPH (Kohjin Co), 0.02 mmol/L nitro blue tetrazolium (Sigma Chemical Co), and 0.3% Triton X-100 at 37°C.14 The reaction was terminated by washing the sections in 0.1 mol/L phosphate-buffered saline. After several washes with distilled water, the sections were air dried and cover slipped with Entellan (Merck). No positive staining was observed when NADPH was omitted from the reaction mixture.
Statistical Analysis
Statistical analyses were performed using Student's
t test.
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Results |
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Expression Levels of nNOS mRNA in SHR and WKY
Expression levels of nNOS mRNA were determined in the cerebellum, brain stem (pons plus medulla oblongata), and D-AD in 4-, 16-, and 24-week-old SHR and WKY. No significant differences in nNOS mRNA expression in the cerebellum and brain stem were observed between SHR and age-matched WKY (Fig 1). The expression level of nNOS mRNA in the D-AD was developmentally regulated, being higher in younger rats in both strains (Fig 2). In addition, the expression level in the D-AD of 24-week-old WKY (n=4) was 2.5-fold higher (P<.01) than that in age-matched SHR (n=4, Fig 2).
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Effects of Antihypertensive Treatment on nNOS Expression
To determine whether the reduced expression of nNOS mRNA in the
adrenal gland of 24-week-old SHR was due to hypertension, the effects
of treatment with antihypertensive drugs on nNOS mRNA expression were
investigated. Administration (7 days) of TCV-116 to 24-week-old SHR
(n=4) and WKY (n=4) increased nNOS mRNA expression levels about twofold
in both strains (P<.01, SHR and WKY). However, SHR
continued to show relatively reduced expression (Fig 3).
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The above observation might indicate that Ang II directly modulates nNOS mRNA expression. However, blood pressure reduction might also be involved in the modulation of nNOS mRNA expression. To evaluate these two possibilities, we investigated the effects of hydralazine treatment on nNOS mRNA expression. Administration (7 days) of hydralazine to 24-week-old SHR (n=4) and WKY (n=4) increased nNOS mRNA expression levels by about fivefold and threefold, respectively (Fig 4). The blood pressure levels of hydralazine-treated SHR and WKY were not significantly different from those of TCV-116–treated SHR and WKY, respectively (Table).
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Effects of Reserpine Treatment
The above observations suggest that a reduction in blood pressure
itself might trigger the augmentation of nNOS mRNA expression. The
sympathetic nervous system is usually activated along with a decrease
in blood pressure. Moreover, several histochemical analyses have
suggested that nNOS-positive ganglion cells and fibers are intimately
related to chromaffin cells in the adrenal medulla, and nNOS in the
adrenal medulla has been suggested to be intimately related to the
sympathetic nervous system. Therefore, the effects of reserpine
treatment on nNOS mRNA expression in the D-AD were investigated (Fig 5). A single administration of reserpine to 24-week-old
SHR (n=5) and WKY (n=5) markedly increased nNOS mRNA expression in both
strains (P<.01) at 24 hours after treatment. The effects of
reserpine on nNOS mRNA expression were both dose dependent and time
dependent (Fig 6). A single dose of reserpine of as low
as 1 mg/kg increased nNOS mRNA expression in 24-week-old WKY.
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To investigate whether the increase in nNOS mRNA expression could lead to an increase in nNOS protein expression, we analyzed nNOS protein expression by Western blot analysis. The expression level of nNOS protein was also increased at 24 hours after reserpine administration (Fig 7, top). The intensity of the immunoreactive bands in reserpine-treated rats (n=3) was approximately four times higher than that in vehicle-treated rats (n=3, P<.001). The validity of the antibody for nNOS was confirmed by using purified nNOS; the antibody recognized the purified nNOS protein (Fig 7, bottom).
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Histochemical Analysis
Since reserpine significantly induced nNOS mRNA and nNOS protein,
the distribution patterns of NADPH-diaphorase–positive ganglion cells
in the adrenal medulla were assessed in reserpine-treated and untreated
WKY (Fig 8). No significant difference in the
distribution patterns was observed between reserpine-treated and
untreated rats.
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Discussion |
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The major finding of the present study was that nNOS expression in the D-AD was modulated by pharmacological antihypertensive treatment. Our results indicate that nNOS expression was not necessarily constitutive and modulation of nNOS expression might also be a mechanism for regulation of NO biosynthesis by nNOS.
Antihypertensive Treatment and nNOS Expression
The short-term (7 days) administration of either an Ang II
antagonist or hydralazine increased nNOS mRNA expression. With the dose
used in the present study, no significant difference in
antihypertensive effect was observed between the two drugs. However,
hydralazine had a stronger potency in increasing nNOS mRNA expression
than TCV-116. Although a reduction in blood pressure itself might be a
factor in increasing nNOS mRNA expression, it is not the sole factor
because the potency of hydralazine in increasing nNOS expression was
stronger than that of TCV-116 despite their similar antihypertensive
effects. Hydralazine and Ang II antagonists have different effects on
neurohumoral factors. Blood pressure reduction with hydral- azine is
usually accompanied by activation of the renin-angiotensin
system,15 whereas that with Ang II antagonists is not. It
is unlikely that Ang II makes a large contribution to the regulation of
nNOS expression because hydralazine and TCV-116 have opposite effects
on the renin-angiotensin system. However, activation of the sympathetic
nervous system may be involved in the augmentation of nNOS mRNA
expression, since blood pressure reduction by hydralazine is known to
activate the sympathetic nervous system16 and that by Ang
II antagonists is not.17
Effects of Reserpine Treatment
The results from hydralazine and TCV-116 treatment strongly
suggest that there may be an intimate relation between the sympathetic
nervous system and nNOS expression in the D-AD. This hypothesis seems
to be supported by several previous histological and biochemical
studies. For example, immunoreactive nNOS in the adrenal medulla has
been reported to exist in ganglion cells and in bundles and single
fibers preferentially located around norepinephrine-storing
cells.18 NO has been reported to potentiate catecholamine
secretion from the adrenal gland.19 In addition, NO and
its second messenger cyclic GMP have been reported to potentiate
nicotine-induced catecholamine secretion from adrenal chromaffin
cells.20 Recently, NO has been reported to play a
facilitative role in the release of norepinephrine from the sympathetic
nervous system in smooth muscle in the gut.21
Reserpine is a classic pharmacological agent widely used for its action on storage vesicles for monoamines, which leads to a depletion of dopamine, norepinephrine, epinephrine, and 5-hydroxytryptamine. Reserpine administration is known to augment the expression of catecholamine-synthesizing enzymes.22 23 The present study showed that nNOS mRNA and protein expression levels in the D-AD were markedly increased by reserpine treatment, which suggests that nNOS in the adrenal medulla may either facilitate or stimulate catecholamine secretion from the adrenal medulla. This hypothesis is consistent with the previous studies cited above.18 19 20
Reserpine administration is known to deplete catecholamines in catecholaminergic neurons and the adrenal medulla. The profound increase in nNOS mRNA expression in the D-AD after reserpine treatment indicated that catecholamines themselves did not directly increase nNOS expression. Thus, the augmented expression of nNOS mRNA in hydralazine-treated rats is not a result of activation of the sympathetic nervous system.
nNOS Protein Expression
Reserpine treatment induced nNOS protein, consistent with the
induction of nNOS mRNA by this treatment. The histochemical analyses
using NADPH-di- aphorase staining confirmed that the nNOS protein
induced in the adrenal medulla by reserpine treatment was expressed in
cells that originally expressed nNOS and not in other cells.
Comparison of SHR with WKY
Since no significant differences in nNOS expression levels
were observed between 4- and 16-week-old SHR and WKY, it is unlikely
that nNOS has a primary role in the pathogenesis of hypertension in
SHR. The reduced nNOS expression in the D-AD of 24-week-old SHR may
compensate for high blood pressure or increased norepinephrine content
in the adrenal medulla of SHR.24 25
The present study has demonstrated that nNOS expression is not necessarily constitutive and rather is dynamically regulated in the D-AD. The present study has also suggested an intimate relation between the sympathoadrenal system and nNOS in the adrenal medulla. The precise mechanisms of the regulation of nNOS expression in the adrenal medulla remain to be determined.
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Footnotes |
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Reprint requests to Naoharu Iwai, MD, First Department of Internal Medicine, Shiga University of Medical Sciences, Tsukinowa Seta, Ohtsu-city 520-21, Shiga, Japan.
Received May 3, 1994; first decision June 15, 1994; accepted November 11, 1994.
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