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Cardiovascular Research 1999 44(1):207-214; doi:10.1016/S0008-6363(99)00193-5
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Adrenomedullin augments nitric oxide and tetrahydrobioptein synthesis in cytokine-stimulated vascular smooth muscle cells

Yoshiyuki Hattoria,*, Nobuo Nakanishib, Steven S Grossc and Kikuo Kasaia

aDepartment of Endocrinology, Dokkyo University School of Medicine, Tochigi, Mibu 321-02, Japan
bDepartment of Biochemistry, Meikai University School of Dentistry, Sakado, Japan
cDepartment of Pharmacology, Cornell University Medical College, New York, NY, USA

* Corresponding author. Tel.: +81-282-87-2150; fax: +81-282-86-4632

Received 8 March 1999; accepted 26 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Immunostimulants increase nitric oxide (NO) and tetrahydrobiopterin (BH4) synthesis in vascular smooth muscle cells (VSMC) by coinducing expression of an isoform of NO synthase (iNOS) and GTP cyclohydrolase I (GTPCH). GTPCH is the first and rate-limiting enzyme in the synthesis of BH4, a cofactor of NO synthases. Given that adrenomedullin (AM) increases NO production, this effect of AM may involve modulation of BH4 synthesis in cytokine-stimulated VSMC. Methods: We investigated the effects of AM on the synthesis of NO and BH4, the expression of iNOS and GTPCH mRNA, and the promoter activity of iNOS and GTPCH genes in rat VSMC stimulated with interleukin-1 (IL-1). Results: IL-1 increased both NO and BH4 synthesis as well as the abundance of iNOS and GTPCH mRNA. AM significantly increased both NO and BH4 synthesis caused by IL-1 stimulation. AM also augmented the IL-1-induced increase in the abundance of iNOS and GTPCH mRNA. IL-1 activated the iNOS promoter activity as well as the GTPCH promoter activity in VSMC. AM alone had no effect on the activity of either the iNOS or the GTPCH promoter, nor did it potentiate the activation by IL-1 of either of these promoters. Conclusion: These results suggest that AM increases IL-1-induced NO and BH4 synthesis by enhancing the expression of iNOS and GTPCH genes at the post-transcriptional level. Thus, the potentiating effect of AM on NO synthesis appears to be associated with an increased expression of both genes necessary for cellular NO synthesis in VSMC.

KEYWORDS Cytokines; Gene expression; Nitric oxide; Septic shock; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Bacterial lipopolysaccharide (LPS) and other immunostimulants induce an isoform of NO synthase (iNOS) in vascular smooth muscle cells (VSMC) [1,2]. The induction of iNOS and the overproduction of nitric oxide (NO) in VSM have been implicated in the genesis of septic and cytokine-induced circulatory shock [3,4]. Whereas the induction of iNOS is necessary for immunostimulant-mediated NO overproduction, de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor of all isoforms of NO synthase, is also elicited in VSM by immunostimulants and is essential for the iNOS activity [2]. We recently showed that immunostimulants coinduce the expression of iNOS and GTP cyclohydrolase I (GTPCH) in VSM; GTPCH is the enzyme that is normally rate-limiting for the synthesis of BH4 [5].

Adrenomedullin (AM), a novel vasodilatory peptide originally isolated from a human pheochromocytoma, has subsequently been shown to be expressed in a variety of tissues and to circulate in plasma [6–8]. It also has been shown that AM is produced and secreted from the vasculature, and AM production in cultured VSMC is stimulated by bacterial lipopolysaccharide (LPS) and other proinflammatory cytokines such as TNF{alpha} and interleukin-1 (IL-1) [9,10]. Intravenous administration of LPS induces a marked increase in plasma AM concentrations and in tissue AM mRNA levels in rats [11,12]. It has recently been shown that circulating AM in patients with septic shock is markedly increased [13,14]. Therefore, AM may be part of the mechanism causing the cardiovascular instability associated with sepsis.

Both NO and AM are potently stimulated by LPS and other cytokines in VSMC in vitro and in vivo in animals and humans with septic shock. Thus, it is possible that the action and/or production of both vasoactive substances may be modulated by each other. AM has been shown to enhance the formation of NO in VSMC and cardiac myocytes [12,15,16]. However, the molecular mechanism of this effect is poorly understood. In this study we investigated the mechanism of AM potentiation of NO synthesis in cytokine-stimulated VSMC. We show that this effect is caused by enhanced induction of iNOS and is accompanied by an increase in GTPCH, resulting in augmented production of BH4.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
VSMC were isolated by elastase and collagenase digestion of thoracic aortae from male Wistar rats as previously described [17]. Cells from the 10th to 15th passage were seeded onto 96-well plates for nitrite assays or onto 10-cm dishes for biopterin measurements and RNA preparation.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985).

2.2 Nitrite measurement
Nitrite production, an indicator of NO synthesis, was measured in the supernatant of VSMC, as previously described [2]. Nitrite was measured by adding 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid) to 100-µl samples of cell culture medium. Absorbance at 550 nm was determined with a microplate reader. Nitrite concentrations were calculated by comparison with the absorbance of standard solutions of sodium nitrite prepared in cell culture medium.

2.3 Biopterin assay
Total cellular biopterin (biopterin plus BH2 and BH4) was measured after acidic oxidation of the reduced forms of biopterin with iodine, as previously described [18]. Briefly, cells were treated with 0.2 M perchloric acid (PCA) and oxidized by exposure to 0.2 M PCA containing 0.2% I2 and 0.4% KI for 1 h at room temperature in the dark. Ascorbate (2%) was added to remove residual free I2 and the mixture was centrifuged for 10 min at 10 000xg. The amount of biopterin in the supernatant was quantitated by C18 reversed-phase high-performance liquid chromatography with fluorescence detection using authentic biopterin as a standard. Protein concentrations were measured by the Lowry method with bovine serum albumin as a standard.

2.4 Competitive PCR for quantitation of GTPCH and iNOS mRNA
Competitive PCR for quantitation of GTPCH and iNOS mRNA was performed as we recently reported [19]. Synthetic competitors for GTPCH and iNOS were prepared using a Competitive DNA construction kit (Takara, Tokyo, Japan). In brief, DNA fragments which contained the same primer binding sites as the natural segments of GTPCH (372 bp) and iNOS (807 bp) [5] but with shorter sizes were amplified by PCR using {lambda}DNA as a template. The resulting PCR products, sizes of 342 and 542 bp, were used as competitors for GTPCH and iNOS, respectively, after spin column purification. Using a mixture of the cDNA from the reverse transcription reaction and the competitors prepared for GTPCH and iNOS, we determined the ratio of signal intensities of the detected bands after PCR for 30 cycles. The concentrations of iNOS and GTPCH in a cDNA solution from VSMC were determined by extrapolating the data to the point where the amplification of the cDNA and the synthetic fragment were identical.

2.5 iNOS and GTPCH promoter analysis
To study iNOS and GTPCH promoter function, rat VSMC were stably transfected with a construct containing a 1.7-kb fragment of the mouse iNOS promoter or a 3-kb fragment of the rat GTPCH promoter each of which was cloned upstream of a reporter gene that encodes the secreted form of human placental alkaline phosphatase (SEAP) [21]. SEAP activity, which is released into the cell culture medium was measured by a sensitive chemiluminescent assay (Phospha-Light, TROPIX, Bedford, MA, USA).

2.6 Statistical analysis
Data are presented as mean±S.D. Multiple comparisons were evaluated by ANOVA followed by Fisher’s protected least significant difference test. Student’s unpaired t-test was used for comparisons between two experiments. A value of P<0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
First, we investigated the effects of IL-1 on nitrite production by VSMC. Addition of IL-1 to the cultures for 24 h increased nitrite production by VSMC. AM alone had no effect. However, the IL-1-stimulated nitrite production was significantly increased by simultaneous treatment of the cells with AM. After a 24-h incubation, the level of IL-1-stimulated nitrite accumulation in the presence of AM was about twice that in the absence of AM. As shown in Fig. 1 (inset), AM dose dependently enhanced the synthesis of nitrite induced by IL-1.


Figure 1
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  		Fig. 1 Effect of AM on nitrite synthesis by cultured rat VSMC. After treatment with IL-1 (10 ng/ml) or AM (1 µM) or both for 24 h, nitrite accumulation in the medium was measured. Inset: Cells were incubated for 24 h with the indicated concentrations of AM in the presence of IL-1 (10 ng/ml), after which nitrite accumulation in the culture medium was assayed. Data are means±S.D. of triplicate observations (n=3). * P<0.01 compared with cells exposed to IL-1 in the absence of AM.

 
Next, we investigated IL-1-stimulated BH4 synthesis in VSMC. As reported previously, de novo BH4 synthesis is elicited in VSMC by immunostimulants and is essential for iNOS activity [2]. After stimulation of the cells with IL-1 for 24 h, intracellular BH4 concentrations significantly increased from very low basal levels. Treatment with AM alone did not result in increased BH4 content in the cells. However, the IL-1-elicited increase in BH4 content of the cells was augmented by simultaneous treatment of the cells with AM. After a 24-h incubation, the intracellular level of IL-1-stimulated BH4 in the presence of AM was about twice that in the absence of AM. As shown in Fig. 2 (inset), when AM was added at increasing intervals after the stimulation of VSMC with IL-1, the enhancement in BH4 levels decreased as the interval lengthened. When AM was added 8 h after stimulation with IL-1, the intracellular BH4 content was the same as in cells from the activated controls (no AM).


Figure 2
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  		Fig. 2 Effect of AM on BH4 synthesis by cultured rat VSMC. After treatment with IL-1 (10 ng/ml) or AM (1 µM) or both for 24 h, biopterin content of the cells was measured. Inset: Effect of adding AM after stimulation of VSMC with IL-1. AM (0.5 µM) was added at the indicated times after stimulation of VSMC with IL-1 (10 ng/ml). Cellular BH4 content was measured 24 h after administration of IL-1. Data are means±S.D. of triplicate observations (n=3). * P<0.01 compared with cells exposed to IL-1 in the absence of AM. For inset, # P<0.05, * P<0.01 compared with that of time=0 h.

 
We examined the effect of AM on iNOS and GTPCH mRNA levels in VSMC stimulated with IL-1. Unstimulated cells or those treated with AM alone express neither iNOS mRNA nor GTPCH mRNA (data not shown). Incubation with IL-1 for 24 h resulted in induction of the expression of iNOS mRNA as well as GTPCH mRNA. Simultaneous treatment of the cells with AM augmented the expression of both iNOS and GTPCH mRNAs by 2.7- and 3.0-fold, respectively (Fig. 3). This stimulatory effect of AM on IL-1-induced iNOS mRNA was clearly inhibited by Rp-8-Br-cAMPS (1 µM, a competitive inhibitor of protein kinase A, Biolog Life Science Institute, Bremen, Germany) as shown in Fig. 4 (inset), while IL-1-induced iNOS mRNA levels was not affected by Rp-8-Br-cAMP (1 µM) (data not shown). Augmentation by AM of IL-1-induced GTPCH mRNA was also reduced by Rp-8-Br-cAMP in a dose-dependent manner (Fig. 4).


Figure 3
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  		Fig. 3 Effect of AM on iNOS and GTPCH mRNA levels in cultured rat VSMC. The iNOS and GTPCH mRNA levels were evaluated by competitive PCR analysis. Total RNA was prepared from cells treated with IL-1 (10 ng/ml) alone or plus AM (1 µM) for 24 h. Depicted are relative differences in the concentrations in the cDNAs between the group in the absence (white bar) or presence (black bar) of AM, expressed as a percentage of the results from the cells treated with IL-1 alone (the means±S.D. of three determinations). Inset shows a representative quatitative assessment. T, target; C, competitor; log(T/C)=0 is the point where the amplification of the cDNA and the synthetic fragment were identical. IL-1 alone, open circles;, IL-1 plus AM, closed circles. * P<0.01.

 

Figure 4
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  		Fig. 4 Effect of Rp-8-Br-cAMPS on iNOS and GTPCH mRNA levels in cultured rat VSMC. The iNOS and GTPCH mRNA levels were evaluated by competitive PCR analysis. Total RNA was prepared from cells treated with IL-1 (10 ng/ml) plus AM (1 µM) with the indicated concentrations of Rp-8-Br-cAMPS (0.1–10 µM) for 24 h. GTPCH mRNA levels analysed were expressed as a percentage of the results from the cells treated with IL-1 alone. Inset: Effect of Rp-8-Br-cAMPS (1 µM) on iNOS mRNA in VSMC stimulated with IL-1 (10 ng/ml) plus AM (1 µM). Data are means of two experiments that yielded similar results.

 
As previously reported, IL-1 activates iNOS as well as GTPCH promoter activity in VSMC. As shown in Fig. 5, IL-1 increased iNOS and GTPCH promoter activity by 5- and 2-fold relative to unstimulated levels, respectively. AM alone had no effect on the activity of either promoter, nor did it potentiate the activation by IL-1 of either promoter.


Figure 5
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  		Fig. 5 Effect of AM on iNOS (A) and GTPCH (B) promoter/SEAP reporter expression in stably transfected rat VSMC. After treatment with IL-1 (10 ng/ml), AM (1 µM) or both for 24 h, SEAP activity in the cell culture medium was measured. Data are means±S.D. of triplicate observations (n=3).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the present study, we demonstrate that AM increases NO synthesis as well as BH4 synthesis in IL-1-stimulated VSMC and that it enhances iNOS mRNA levels as well as GTPCH mRNA levels in these cells.

While increased NO synthesis by AM in IL-1-stimulated VSMC appears to be mediated by enhanced induction of iNOS, potentiation of the formation of BH4 in VSMC is likely to be mediated by increased GTPCH activity [2]. This potentiating effect on BH4 synthesis by AM was most marked when the peptide was added to the cells simultaneously with IL-1, and decreased as the interval between addition of IL-1 and that of AM increased. Thus, AM seems to enhance the induced formation of BH4 in VSMC by promoting the expression of GTPCH rather than by increasing its catalytic activity. This is also the case in the effect of AM on immunostimulant-induced NO synthesis, suggesting that AM augments the induction of iNOS [12].

Using promoter function studies of the iNOS and GTPCH genes, we found that AM alone had no effect on the activity of these promoters, nor did it potentiate the activation by IL-1 of either promoter. Therefore, AM is likely to potentiate the gene expression of iNOS and GTPCH in IL-1-stimulated VSMC at the post transcriptional level.

Rat VSMC possess specific AM receptors that are functionally coupled to adenylate cyclase, and the exposure of cultured VSMC to AM thus results in marked intracellular accumulation of cAMP [21,22]. AM appears to augment NO synthesis in VSMC under cytokine-stimulated conditions, at least partially through a cAMP-dependent pathway [12,16]. Interestingly, cAMP enhances iNOS mRNA stability after cytokine-stimulation in cardiac myocytes [23]. This is likely to be the mechanism by which AM affects iNOS mRNA expression in IL-1 treated VSMC, and is consistent with the results of our iNOS promoter analysis. Sequencing of the GTPCH promoter revealed a consensus sequence for binding of CREB, a transcriptional activator that mediates the effects of cAMP [20]. A cAMP analog, dibutyryl-cAMP, has been shown to cause a modest but concentration-dependent increase in GTPCH promoter activity in VSMC [20]. In addition, cAMP has been shown to induce GTPCH gene and protein expression as well as enzyme activity in rat renal mesangial cells [24]. In the present study, we did not observe an enhancing effect of AM on IL-1-stimulated GTPCH promoter activity, but the effect of AM was inhibited by Rp-8-Br-cAMPS, suggesting that the potentiated GTPCH expression, resulting in increased biosynthesis of BH4, caused by AM, may be mediated by the post transcriptional effect at least partially though a cAMP-dependent pathway.

Our results show that AM has the potential to enhance cytokine-induced NO synthesis in VSMC. AM may directly increase the induction of iNOS elicited by cytokine stimulation, or, AM may upregulate cytokine-induced NO synthesis by stimulating BH4 synthesis.

BH4 synthesis is required for the induction of iNOS activity in VSMC [2]. The amount of NO generated appears to be limited by the amount of BH4 present in cells [2,25]. The main role of the increased pteridine synthesis observed during cytokine stimulation is to provide a cofactor for the BH4-dependent generation of NO by cytokine-induced iNOS. Moreover, the biosynthesis of BH4 may contribute to cytokine induction of iNOS expression by stabilization of iNOS mRNA [26,27].

In conclusion, AM increases the generation of NO and BH4 as well as the abundance of both iNOS mRNA and GTPCH mRNA in cytokine-stimulated VSMC. AM may contribute to vascular pathophysiology during septic shock by exerting a direct vasorelaxant effect as well as by regulating NO and BH4 synthesis in VSMC.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported in part by a grant from Japan Private School Promotion Foundation. S.G. is supported by NIH grant HL46403 and HL50656.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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