Regulation of Neuronal Nitric Oxide Synthase in Rat Adrenal Medulla
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.
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.
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 0.2× 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.
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 analyses were performed using Student’s t test.
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⇓).
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⇓).
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⇓).
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.
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).
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.
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.
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.
- Revision received June 15, 1994.
- Accepted November 11, 1994.
Knowles RG, Moncada S. Nitric oxide synthetase in mammals. Biochem J. 1994;298:249-258.
Dinerman JL, Lowenstein CJ, Snyder SH. Molecular mechanisms of nitric oxide regulation: potential relevance to cardiovascular disease. Circ Res. 1993;73:217-222.
Kilbourn RG, Gross SS, Jubran A, Adams J, Griffith OW, Levi R, Lodato RF. NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implication for the involvement of nitric oxide. Proc Natl Acad Sci U S A. 1990;87:3629-3632.
Rees DD, Palmer RMJ, Moncada S. Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci U S A. 1989;86:3375-3378.
Togashi H, Sakuma I, Yoshioka M, Kobayashi T, Yasuda T, Kitabatake A, Saito H, Gross SS, Levi R. A central nervous system action of nitric oxide in blood pressure regulation. J Pharmacol Exp Ther. 1992;262:343-347.
Dipaola ED, Vidal MN, Nistico G. L-glutamate evokes the release of an endothelium-derived relaxing factor-like substance from rat nucleus tractus solitarius. J Cardiovasc Pharmacol. 1991; 17:5269-5272.
Iwai N, Inagami T. Isolation of preferentially expressed genes in the kidneys of hypertensive rats. Hypertension. 1991;17:161-169.
Schmidt HHHW, Pollock JS, Nakane M, Gorsky LD, Forstermann U, Murad F. Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase. Proc Natl Acad Sci U S A. 1991; 88:365-369.
Pettinger WA, Campbell WB, Keeton K. Adrenergic component of renin release induced by vasodilating antihypertensive drugs in the rat. Circ Res. 1973;33:82-86.
Linet O, van Zweiten PA, Hertting G. Effect of hydrazinophthalazines on catecholamines in rats. Eur J Pharmacol. 1968; 6:121-124.
Wong PC, Price WA, Chiu AW, Duncia JV, Carini DJ, Wexler RR, Johnson AL, Timmermans PBMWM. Nonpeptide angiotensin II receptor antagonists, IX: antihypertensive activity in rats of Dup 753, an orally active antihypertensive agent. J Pharmacol Exp Ther. 1990;252:726-732.
Thatikunta P, Chakder S, Rattan S. Nitric oxide synthase inhibitor inhibits catecholamine release caused by hypogastric sympathetic nerve stimulation. J Pharmacol Exp Ther. 1993;267:1363-1368.
Mueller RA, Thoenen H, Axelrod J. Increase in tyrosine hydroxylase activity after reserpine administration. J Pharmacol Exp Ther. 1969;169:74-79.
Joh TH, Geghman C, Reis D. Immunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine. Proc Natl Acad Sci U S A. 1973;70:2767-2771.
Donohue SJ, Stitzel RE, Head RJ. Time course of changes in the norepinephrine content of tissues from spontaneously hypertensive and Wistar-Kyoto rats. J Pharmacol Exp Ther. 1988;245:24-31.