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Cardiovascular Research 1998 38(2):405-413; doi:10.1016/S0008-6363(98)00005-4
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes

Tatsuya Shimizua, Koh-ichiro Kinugawaa, Yasuyuki Sugishitaa, Kazuro Sugishitaa, Kazumasa Haradaa, Hiroshi Matsuia, Osami Kohmotob, Takashi Serizawab and Toshiyuki Takahashia,*

aThe Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
bThe Second Department of Internal Medicine, Saitama Medical School, 38 Morohongo, Moroyama Irumagun, Saitama 350-04, Japan

* Corresponding author. Tel. +81 (3) 3815 5411 ext. 3076; Fax +81 (3) 3814 0021; E-mail: takahashit-2im@h.u-tokyo.ac.jp

Received 10 June 1997; accepted 3 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Inducible nitric oxide synthase (iNOS) has been implicated to contribute to myocardial dysfunction in various settings, but considerable species differences have been noted in the levels of iNOS expression and its function in several tissues. The aim of this study was to elucidate evolutional changes in myocardial iNOS expression and function. Methods: An iNOS cDNA clone was isolated by RT-PCR from the 10-day old cultured chick embryonic ventricular myocytes stimulated with 10 µg/ml of lipopolysaccharide. Expression of the iNOS mRNA was analyzed with Northern blot analysis and RNase protection assay. The iNOS activity was estimated from conversion rates of L-arginine to L-citrulline and intracellular cGMP contents were measured with radioimmunoassay. Furthermore, both [Ca2+]i (fluorescent dye indo-1) and cell contraction (video motion detector) were simultaneously recorded. Results: Aside from the primer sequences, the insert (1026 bp) of the cDNA clone showed 66.4% identity at the deduced amino acid level to the human iNOS cDNAs. Northern blot analysis revealed that chicken iNOS mRNA of approximately 4.5 kb was induced by lipopolysaccharide within 6 h in the cultured myocytes. RNase protection assay also showed that lipopolysaccharide provoked 14.6±5.1-fold increases (n=6, p<0.05) in the iNOS mRNA signals within 6 h. The iNOS activity (+300%, P<0.05) as well as the intracellular cGMP contents (+75%, P<0.01) were significantly augmented in the lipopolysaccharide-stimulated cells. Both the cell contraction and [Ca2+]i were significantly reduced after the administration of a large amount (10 mM) of L-arginine in the myocytes pretreated with both lipopolysaccharide and NG-monomethyl-L-arginine (100 µM). Conclusion: As like as the nucleotide and amino acid sequences, the myocardial effects of the iNOS may also be evolutionary conserved.

KEYWORDS Experimental; Heart; Molecular biology; Cell culture/isolation; Calcium (cellular); Myocytes; Nitric oxide; Contractile function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Recently, several clinical studies have implicated that inducible nitric oxide synthase (iNOS or NOS2) may contribute to development of myocardial damage in a variety of settings, including dilated cardiomyopathy [1–3], myocarditis [2], graft rejection [4], septic shock [5]and even failing myocardium in general [6]. On the other hand, expression of iNOS and its functional significance have been studied in various experimental models [7]. Some studies [8–10]have shown that iNOS was highly inducible in rat cardiac myocytes by cytokines and/or lipopolysaccharide (LPS). In our previous study [10], myocardial iNOS exerted negative inotropic, negative chronotropic and [Ca2+]i-lowering effects through a production of a large amount of NO. The results of these in vitro studies may support the hypothesis that iNOS molecule can work as a cardiac depressant in various myocardial disorders. However, the iNOS induction at such high level as shown in rodents has not necessarily been attained in other species, including human subjects [11, 12]. Considerable species differences in the levels of iNOS induction as well as NO production have been reported in several tissues [13], whereas those differences in myocardial iNOS expression and function remain to be elucidated.

On the other hand, our previous study suggested that interleukin-6 induces iNOS activity in the chick embryonic ventricular myocytes and that the iNOS molecule exerts negative myocardial effects [14]. However, quantitative relationships among the levels of iNOS expression, NOS activity and the extent of the myocardial effects have not been well clarified in nonmammalian heart. Although NO is considered as one of the essential mediators in many biological processes, little is known about evolutional changes in structure or function of the iNOS molecules. In this regard, it appeared to be interesting to study iNOS expression and its functional significance in avian cardiac myocytes.

In order to elucidate the myocardial expression and function of avian iNOS, we first isolated an iNOS cDNA clone from the chick embryonic ventricular myocytes stimulated with LPS. Second, we quantified the LPS-induced changes in the levels of the iNOS expression and its activity in the avian cardiac myocytes. Third, we studied effects of the iNOS induction on Ca2+ transient and cell contraction in this well-defined in vitro model of cardiac performance. As the nucleotide and amino acid sequences, the myocardial effects of the iNOS were evolutionally conserved.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Chick embryonic ventricular myocyte culture
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No.85-23, revised 1985). Chick embryonic ventricular myocytes were isolated and cultured as previously described [14]. Briefly, ventricles were isolated from 10-day-old chick embryos, minced, and placed in the Ca2+- and Mg2+-free Hanks' balanced salt solution containing 0.25% trypsin at 37°C for 4 cycles of 8 min each. After a centrifugation, the cells were resuspended in the culture media consisting of 6% fetal calf serum, 40% Medium 199, 0.1% penicillin-streptomycin solution, and 54% balanced salt solution containing (mM) NaCl 116, NaH2PO4 1.0, MgSO4 0.8, NaHCO3 26.2, CaCl2 0.9 and glucose 5. The cell suspensions were then diluted to 1x106 cells/ml and plated in plastic Petri dishes or 24-well dishes. The cells were incubated in 5% CO2 atmosphere for 2 days at 37°C. On day 3, the culture media were exchanged for the serum-free media consisting of 20% Medium 199, 20% F-12 Ham, 0.1% penicillin-streptomycin solution, 54% balanced salt solution, 0.6 mM KCl, and 5 ng/ml insulin-transferrin-selenium mixture.

Fetal calf serum, medium 199, penicillin-streptomycin solution, F-12 Ham were purchased from GIBCO BRL (Gaithersburg, MD). Insulin-transferrin-selenium mixture was obtained from Sigma Chemical.

2.2 RNA preparation
Total cellular RNA was isolated from the cultured cells with the acid guanidium phenol-chloroform method [15]. The concentration of RNA solutions was determined by a spectrophotometer. We verified the quality of the RNA samples by visualization of 27S and 18S rRNA bands after electrophoresis through a 1% agarose/3% formaldehyde gel.

2.3 Reverse-transcription polymerase chain reaction (RT-PCR) and cloning of chick embryo ventricular myocyte iNOS cDNA
RT-PCR was performed with RT-PCR kit (Perkin Elmer). An aliquot (1 µg) of the total RNA sample isolated from the chick embryo ventricular myocytes treated with 10 µg/ml of LPS (Escherichia coli serotype 055:B5; GIFCO Laboratories) for 6 h was reverse-transcribed for 30 min at 42°C with MMLV reverse transcriptase and a downstream primer 5'-GCCGCTGCTGCCAGAAACTTC-3', which corresponds to the nucleotides 1913–1933 of the rat vascular smooth muscle iNOS cDNA [16]. This downstream primer is located in the conserved NADPH ribose-binding site of the rat iNOS. PCR was then performed with the downstream primer and an upstream primer 5'-TCGACCAGAAACTGTCTCACC-3', which corresponds to the nucleotides 2960–2980 of the rat vascular smooth muscle iNOS cDNA [16]. This upstream primer is located in the conserved FMN-binding region of the rat iNOS. After initial denaturalization for 2 min at 95°C, the reverse transcription products were amplified with 40 cycles consisting of denaturalization for 1 min at 95°C, annealing for 2 min at 50°C, and extension for 3 min at 72°C, followed by a final extension for 7 min at 72°C. The PCR products, close to the expected rat iNOS fragment of 1068 bp, were purified and cloned into the pCRTM II vectors with a TA cloning kit (Invitrogen). We first obtained the partial sequences of the inserts using a BcaBest sequencing kit (Takara Shuzo, Japan), and then isolated a clone pCHiNOS, which contained an insert with a similar nucleotide sequence to the rat iNOS. The entire sequence of the pCHiNOS insert was determined with an automated sequencer 373A (Applied Biosystems Perkin Elmer) and a Prism Dye Terminator sequencing kit (Perkin Elmer). Sequence homology search was carried out with a FASTA program at the DNA Information and Stock Center (DISC) in Japan.

2.4 Northern blot analysis
Aliquots (20 µg) of the total RNA samples were size-fractionated by electrophoresis through 1% agarose/3% formaldehyde gels, and blotted on Hybond-NTM nylon membranes (Amersham) in 10xSSC (1xSSC=0.15 M NaCl/0.015 M sodium citrate, pH 7.0). The filters were prehybridized in the buffer solution containing 40% formamide, 5xSSC, 0.5% SDS, 100 µg/ml sonicated salmon sperm DNA, 20 mM NaH2PO4 (pH 7.2), 5xDenhardt's solution overnight at 42°C. The membranes were then hybridized with the labeled probe in the freshly-prepared same solution mix for 18 h at 42°C. The probe used in this study was the pCHiNOS insert liberated by EcoRI digestion and labeled by random priming with a BcaBest DNA labeling kit (Takara Shuzo, Japan) and Formula -dCTP (3000 mCi/mmol, NEN Dupont). Labeling reaction was done for 30 min at 55°C to facilitate the synthesis of longer probes. We used approximately 106 cpm/ml of the probe for hybridization. At the end of hybridization, the filters were washed sequentially with the final stringent wash in 0.5xSSC/0.1% SDS for 15 min at 55°C, and then exposed to X-ray films (New RX, Fuji Photo Film, Japan) with intensifying screens for 2 days at –80°C before development. The radioactivity counts of the iNOS mRNA signals were determined directly with an InstantImagerTM Electronic Autoradiography System (Packard Instrument, Meriden, CT).

2.5 RNase protection assay
We performed RNase protection assay using a RPA II kit (Ambion, Austin, TX). Briefly, aliquots (10 µg) of the total RNA samples were hybridized with the riboprobes in the hybridization buffer (80% deionized formamide, 100 mM sodium citrate, pH 6.4, 300 mM sodium acetate, pH 6.4, 1 mM EDTA) for at least 18 h at 42°C. The riboprobes were synthesized from the following plasmids containing both the specific inserts and promoter (Sp6) with a Maxscript kit (Ambion Inc) and Formula -UTP (800 mCi/mmol, NEN Dupont) for 30–40 min at 37°C. The plasmids used for RNA synthesis in this study were as follows: (1) pCHiNOS2, the pCRTM II vector containing the shorter fragment (340 bp) of the EcoRI-Hind III-digested pCHiNOS insert; (2) pCH{alpha}-actin, the pCRTM II vector containing the chick cardiac {alpha}-actin cDNA (240 bp). The insert of the pCHiNOS2 may produce three nucleotide mismatches (the 1st, 16th, 19th nucleotides from the 5' end) between the riborprobe and mRNA. These mismatches are all located in the sequence of the upstream PCR primer. Because of the mismatch at the 1st nucleotide from the 5' end, full protection from RNase digestion may produce a band of 339 nucleotides. We used the probes of approximately 7x104 cpm for hybridization. At the end of hybridization, the samples were digested with RNase A and T1 mix for 30 min at 37°C. The digested samples were size-fractionated through a denaturing 5% polyacrylamide/8M urea gel at a constant power of 40 W. The gels were dried in a gel drier and exposed to X-ray films with intensifying screens for 10 h to 4 days at –80°C before development. The radioactivity counts of the protected bands were also determined with an InstantImagerTM Electronic Autoradiography System, and the relative abundance of the chick iNOS mRNA fragment to the chick {alpha}-actin mRNA fragment was adopted as a quantitative index of iNOS expression.

2.6 Intracellular NOS activity
We estimated the intracellular NOS activity from the conversion rates of L-arginine to L-citrulline using a NOSdetectTM Assay Kit (Stratagene). The cells cultured in a 24-well plate were washed with PBS, and harvested in 500 µl of PBS containing 1 mM EDTA. Aliquots (100 µl) of these cell suspensions were used for determination of protein content with Lowry's method [17]. The remaining cell suspensions were centrifuged and the pellets were homogenized in 100 µl of the homogenization buffer (25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA). The homogenates were centrifuged for additional 5 min, and the supernatants were used for further analysis. Aliquots (10 µl) of the supernatants were then mixed with 20 µl of the reaction buffer (50 mM Tris-HCl, pH 7.4, 6 mM tetrahydrobiopterin, 2 mM flavin adenine dinucleotides, 2 mM flavin adenine mononucleotide), 4 µl of 10 mM NADPH and 0.5 µl (0.5 mCi) of L-[3H]-arginine. The reaction mixtures were incubated for 60 min at 37°C. After the reaction was stopped, resin suspension (100 µl) was added to each reaction tube. These solution mixtures were then loaded on special spin columns and the radioactivity counts in the elutants were obtained with a scintillation counter (Hewlett-Packard). L-[3H]-arginine can be bound to the resin in the spin column, while L-[3H]-citrulline, which is a product of NOS, can run through the column, because of differences in pKa of these amino acids. The radioactivity count was corrected by the protein content and used as an index of the NOS activity. The NOS activity was determined in the presence or absence of 0.6 mM Ca2+ or 1 mM N{omega}-nitro-L-arginine methyl ester HCl (L-NAME). The Ca2+-independent NOS activity (measured without Ca2+) represents the enzymatic activity of iNOS.

2.7 Intracellular cGMP contents
Intracellular cGMP contents were determined with radioimmunoassay as previously described [14]. Briefly, the cells cultured in 24-well plates were washed with ice-cold PBS, and harvested with 500 µl of 6% trichloroacetic acid (TCA). The cells were then homogenized at 4°C, and stored at –20°C until use. After the homogenates were thawed, aliquots (100 µl) were used for cGMP measurements with a cGMP Assay Kit (Yamasa Shoyu, Japan). Intracellular cGMP contents were also corrected by the protein contents and expressed as pmol/mg protein.

2.8 Simultaneous measurements of [Ca2+]i and cell contraction
Intracellular Ca2+ concentration ([Ca2+]i) was measured with the Ca2+ fluorescent dye indo-1 as previously described [18]. Briefly, coverslips with the cultured chick embryonic ventricular myocytes were incubated for 30 min at 37°C in diluted indo-1 solution (5 µM), and then washed in indo-1-free culture media for 10 min. After the dye was loaded, the coverslip was in a flow-through chamber equipped with a clear glass bottom, and mounted on the stage of an inverted microscope (Nikon Diaphot, Japan). The cells were continuously superfused at 37°C with HEPES-buffered physiological solution containing (mM) NaCl 137, KCl 3.7, MgCl2 0.5, CaCl2 1.8, glucose 5.6, and HEPES 4.0 (pH 7.35). The instrumentation for fluorescence measurement was also described previously [18]. In brief, a high-pressure mercury arc lamp was used as the excitation light source, which provided an intense peak at 360 nm. The cells were illuminated via epifluorescence optics with a Fluor 40x objective lens (Nikon Diaphot, Japan). The fluorescent light was collected by the objective lens and divided with a dichroic mirror system (Rincon Scientific, Japan) to permit simultaneous measurements of both 400- and 500-nm wavelengths by use of two separate photomultiplier tubes. The ratio (R) of emitted fluorescence (400/500) was obtained on-line through an analog-divider circuit. Using the in vitro calibration method, we calculated [Ca2+]i from the R as previously reported [19]. Cell contraction was measured by means of a video motion detector with plastic microspheres (latex beads) attached to the surface of a layer of the cultured ventricular myocytes.

Indo 1-AM was purchased from Dojin Kagaku, Japan. Pluronic F127 was obtained from BASF. The latex beads were from Sigma Chemical.

2.9 Study protocols
The chick embryonic ventricular myocytes were treated with LPS (10 µg/ml) for 6–24 h for cDNA cloning or analyses of RNA expression, in the presence or absence of 100 µM NG-monomethyl-L-arginine (L-NMMA). For cGMP assay, the cells were stimulated with LPS for 24 h in the presence or absence of 100 µM L-NMMA. For the NOS activity assay, the cultured myocytes were treated with LPS for 24 h. For the measurements of [Ca2+]i and cell contraction, the ventricular myocytes were treated with both LPS and L-NMMA for 24 h. This pretreatment created a specific type of ventricular myocytes that expressed iNOS without excessive production of NO or cGMP [10, 14]. To observe the myocardial effects of the iNOS expression, the perfusate containing L-NMMA (100 µM) was rapidly exchanged for that containing a high concentration (10 mM) of L-arginine. This concentration of L-arginine was used, because it can completely reverse the effect of L-NMMA and can allow the ventricular myocytes produce significant levels of NO and cGMP in a brief period [10, 14].

L-NMMA and L-arginine were purchased from Sigma Chemical.

2.10 Statistics
All data are expressed as mean±S.E. Comparison of the mean values was done with the unpaired or paired t-test. Statistical significance was accepted at P<0.05 level.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Chick embryonic ventricular myocytes iNOS cDNA
The pCHiNOS clone contained a 1068 bp-long insert, of which size was exactly the same as that of the rat vascular smooth muscle iNOS. Aside from the primer sequences, the nucleotide sequence of the remaining 1026 bp fragment showed 99.6% identity to that of the recently reported chicken macrophage iNOS cDNA [20]. As in mammalian tissues, the same iNOS molecule may be expressed in various kinds of avian tissues, including cardiac myocytes and macrophages. The chick embryonic ventricular myocyte iNOS cDNA demonstrated 67.4% identity at the nucleotide level and 66.4% identity at the deduced amino acid level to the human iNOS cDNAs [21, 22](Fig. 1). The deduced amino acid sequence of this chick iNOS partial cDNA also showed 61.4%, 61.1%, 46.7%, 51.4% identity to the mouse iNOS [23, 24], rat iNOS [16], human endothelial NOS (NOS3) [25], human neuronal NOS (NOS1) [26], respectively.


Figure 1
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  		Fig. 1 Comparison of the deduced amino acid sequences between the chick embryonic ventricular myocytes iNOS (CHiNOS) cDNA and human iNOS (HUiNOS) cDNA. The CHiNOS cDNA showed 66.4% identity to the human iNOS cDNAs at the amino acid level. The deduced protein sequence of the chick iNOS partial cDNA also demonstrated 61.4%, 61.1%, 46.7%, 51.4% sequence identity to those of the mouse iNOS, rat iNOS, human endothelial NOS (NOS3), human neuronal NOS cDNAs, respectively. The boxes represent putative cofactor binding sites. FMN, flavin mononucleotide binding site; FAD-PPi, flavin adenine-dinucleotide (FAD) pyrophosphate binding site; FAD-ISO, FAD isoalloxazine binding site; NADPH-ribose, nicotinamide adenine dinucleotide phosphate ribose binding site.

 
3.2 Northern blot analysis
Northern blot analysis revealed that LPS (10 µg/ml) induced iNOS mRNA within 6 h in the chick embryo ventricular myocytes (Fig. 2A). The chicken iNOS mRNA species of approximately 4.5 kb was induced, as reported in the LPS-treated avian macrophage cell line [20], although there remains the possibility that the mRNA signal consists of multiple transcripts of similar sizes. The chick iNOS mRNA signal was barely detectable in the control ventricular myocytes or the cells treated only with L-NMMA for 6 h.


Figure 2
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  		Fig. 2 Expression of the chick iNOS mRNA. Northern blot analysis revealed that lipopolysaccharide (LPS) induced the chick iNOS mRNA of approximately 4.5 kb within 6 h (Panel A). This mRNA signal was barely detectable in the control or L-NMMA-treated ventricular myocytes. A picture of ethidium bromide-stained rRNAs is shown as an internal control. RNase protection assay also revealed that LPS provoked significant (14.6±5.1-fold) increases in the levels of the iNOS mRNA at 6 h (Panel B). The protected bands of around 340 and 320 nucleotides (nt) represent the chick iNOS mRNA. Expression of the iNOS mRNA returned nearly to the control level at 24 h after the LPS treatment. Protected bands of 240 nt represent chick cardiac {alpha}-actin mRNA, which are shown as an internal control. For the iNOS mRNA signals, the gel was exposed for 4 days, and for {alpha}-actin signals it was exposed for only 10 h.

 
3.3 RNase protection assay
A representative result of RNase protection assay is shown in Fig. 2B. The protected bands of 339 bp represent the chick iNOS mRNA species. The shorter bands of approximately 320 bp (possibly two bands of 324 and 321 nucleotides) may also represent the chick iNOS mRNA, because of the two nucleotide mismatches (the 16th and 19th nucleotides from the 5' ends) in the riboprobe:mRNA hybrid, as described above. Thus, the total radioactivity counts of these bands were obtained with the InstantImagerTM for the quantification of the iNOS expression. The protected bands of 240 bp representing chick cardiac {alpha}-actin mRNA are shown as an internal control. The chick iNOS mRNA signal was scarcely detected in the control chick embryo ventricular myocytes even by RNase protection assay. LPS provoked 14.6±5.1-fold increases (n=6, p<0.05) in the iNOS messages at 6 h of the stimulation. The levels of the iNOS mRNA returned nearly to the control values at 24 h after the addition of LPS.

3.4 Intracellular NOS activity
The NOS activity, estimated from the conversion rates of L-arginine to L-citrulline, was significantly augmented (from 4.25±0.35 to 16.98±3.72 cpm/min/mg protein, P<0.01, n=5, Fig. 3) by LPS treatment for 24 h. These 4-fold increases in the NOS activity were completely suppressed by L-NAME, but were also detected in the absence of Ca2+, suggesting that the augmented NOS activity was mainly attributable to the iNOS induction.


Figure 3
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  		Fig. 3 Intracellular NOS activity. The NOS activity, estimated from the conversion rates of L-arginine to L-citrulline, was significantly augmented by LPS in the chick embryonic ventricular myocytes. These increase in the NOS activity were completely suppressed by L-NAME. In contrast, they were detected even in the absence of Ca2+ [Ca(–)], suggesting that the augmented NOS activity was mostly attributable to the iNOS induction. *P<0.05, {dagger}P<0.01. The results of 5 separate experiments are shown.

 
3.5 Intracellular cGMP contents
LPS significantly augmented intracellular cGMP contents in the chick embryonic ventricular myocytes from 1.51±0.29 to 2.64±0.28 pmol/mg protein (P<0.05, n=6, Fig. 4) within 24 h. Pretreatment with L-NMMA (100 µM) completely abolished the LPS-induced increases in the cGMP contents.


Figure 4
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  		Fig. 4 Intracellular cGMP contents. LPS significantly augmented intracellular cGMP contents by 75% (P<0.05) in the chick embryonic ventricular myocytes. Treatment with L-NMMA completely abolished the LPS-induced increases in the cGMP contents. *P<0.05. The results of 6 separate experiments are shown.

 
3.6 Changes in [Ca2+]i and cell contraction
In the chick embryonic ventricular myocytes treated with both LPS and L-NMMA for 6 h, iNOS mRNA was induced at the comparable levels as in the cells treated only with LPS for 6 h (Fig. 5A and 5B). The levels of iNOS mRNA in the cells pretreated with both the agents were 96±26% (by Northern blot analysis) and 135±45% (by RNase protection assay) of those in the cells treated only with LPS (n=4). In the cells pretreated with both LPS and L-NMMA for 24 h, exchange of the perfusate containing L-NMMA (100 µM) for that containing a high concentration (10 mM) of L-arginine acutely produced negative inotropic, negative chronotropic, and [Ca2+]i-lowering actions (Fig. 5C), along with increases in cGMP contents by 56% within 5 min (n=2). The amplitude of cell contraction was decreased by 30% (P<0.05, n=3) and the spontaneous beating rate was slowed from 106±23 to 85±19 beats/min (P<0.05, n=3). The peak systolic [Ca2+]i was decreased from 662±32 to 437±45 nM (P<0.01, n=3), and the minimal diastolic [Ca2+]i was also lowered from 224±49 to 147±36 nM (P<0.01, n=3). In the cells pretreated only with L-NMMA did not show any changes in [Ca2+]i, cell contraction or cGMP contents after the L-arginine administration (data not shown).


Figure 5
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  		Fig. 5 Changes in [Ca2+]i and cell contraction in the chick embryonic ventricular myocytes expressing iNOS. The chick embryonic ventricular myocytes pretreated with both LPS and L-NMMA for 24 h were used to study functional significance of the iNOS induction. In these cells, iNOS mRNA was induced at 6 h after the stimulation at comparable levels as in the cells treated only with LPS for 6 h. Representative results of Northern blot analysis (Panel A) and RNase protection assay (Panel B) are shown. Exchange of the perfusate containing L-NMMA for that containing a high concentration (10 mM) of L-arginine produced acute negative inotropic, negative chronotropic, and [Ca2+]i-lowering effects (Panel C). The amplitude of cell contraction was reduced by 30% (P<0.05, n=3) and the spontaneous beating rate was significantly slowed (P<0.05, n=3). Both the peak systolic and minimal diastolic [Ca2+]i were significantly decreased (P<0.01, n=3).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study has demonstrated that iNOS mRNA was induced in response to LPS in the chick embryonic ventricular myocytes. This cardiac iNOS was the same as that induced in the LPS-stimulated avian macrophage cell line [20], and showed moderately conserved nucleotide and deduced amino acid sequences as compared with the mammalian iNOS molecules. LPS induced the chick iNOS mRNA within 6 h in the chick embryonic ventricular myocytes. Increases in the iNOS activity were demonstrated by the arginine-citrulline conversion assay, and intracellular cGMP contents were also augmented in the LPS-stimulated cells. This chick iNOS exerted a significant negative inotropic (30% reduction in the amplitude of cell contraction), negative chronotropic (20% reduction in spontaneous beating rate), and [Ca2+]i-lowering (34% reduction in the peak systolic [Ca2+]i) effects. This is the first report that has shown the myocardial expression and effects of iNOS molecules in nonmammalian species.

We used the chick embryonic ventricular myocytes in this study for the following reasons: First, evolutional changes in structure and function of the iNOS molecule were not well recognized. Considerable species differences have been reported concerning the levels of iNOS expression as well as NO production [11–13]. Furthermore, functional consequences of these species differences are not clear. Quite recently, avian iNOS cDNAs have been partially [27]or completely [20]cloned, sequenced, and their expression has been investigated. However, no study has been performed regarding myocardial expression or function of the chick iNOS. Second, the chick embryonic ventricular myocytes culture is one of the well-defined, in vitro models of cardiac performance and metabolism. The cultured cells isolated from 10-day-old chick embryonic ventricles consist almost exclusively (more than 90%) of cardiac myocytes [14]and contain very few non-muscle cells. Especially, this culture has been assumed to include very few cells of vascular or neuronal origin [28]. Many studies with a variety of disciplines have been successfully performed in this model. Thereby, our data may provide a basis for investigating iNOS expression and roles of NO in the settings suitable for this model, including myocardial growth, metabolic inhibition [19]and hypoxia [28]. Third, transcriptional regulation of the chick iNOS gene may be simpler than that of the mammalian counterparts. A recent study has shown that the 5'-regulatory region of the chick iNOS gene has only one cluster of enhancer binding sequences [20], although the promoter regions of the mouse and rat iNOS genes contain two separate areas of enhancer binding sites [29, 30]. Furthermore, a recent study has shown that enhancer sequences are scattered in as long as 16 kb stretch of the 5'-regulatory region of the human iNOS gene [31]. Thus, the chick iNOS gene may provide us with a simple model for studying the molecular mechanisms of iNOS induction.

Myocardial expression of iNOS and its functional significance have extensively been investigated in rat cardiac myocytes [8–10]. Our previous study has shown that LPS induced very high level of iNOS expression (up to 100-fold) with an increase in iNOS activity and a large amount of NO production in neonatal rat cardiac myocytes [10]. Intracellular cGMP contents were also augmented by 75% in the LPS-stimulated rat myocytes. In the neonatal rat cardiac myocytes pretreated with both LPS (10 µg/ml) and L-NMMA (100 µM), an administration of L-arginine (10 mM) in place of L-NMMA provoked acute decreases in the peak systolic [Ca2+]i by 30% and the relative amplitude of cell contraction by 28%. Although the levels of iNOS expression and iNOS activity appeared to be lower in the chick ventricular myocytes than in the neonatal rat cardiac myocytes, the increase in cGMP contents and the degree of the negative myocardial effects observed in the iNOS-expressing cells were comparable between these two models. Thus, these data suggest that the physiological role of iNOS may be preserved in a wide range of species.

Biological significance of the myocardial iNOS expression remains to be determined. In case of sepsis or specific infections, pathogens can invade the myocardium and proliferate in this hypervascular tissue. Thus, the augmented expression of the myocardial iNOS may be a part of self defense responses [32]. On the other hand, NO may exert a protective effect on the heart by decreasing myocardial oxygen consumption. During septic shock syndrome, peripheral resistant vessels are fully dilated because of the iNOS induction in vascular smooth muscle cells. Consequently, the resistance to left ventricular emptying, that is, the workload on the ventricle, may be markedly decreased in patients with septic shock. By constant, chronic sustained expression of the myocardial iNOS, which has been observed in patients with heart failure, may be disadvantageous, because peripheral vascular resistance is usually increased in this setting. Molecular mechanisms responsible for such sustained iNOS expression should further be examined.

The size and number of iNOS mRNA transcripts may also be different among animal species, tissues and developmental stages. From the results of Northern blot analysis, iNOS transcript is assumed to consist of a single mRNA species in most types of tissues or cells from most animal species [8–10, 12, 16, 21–23]. In contrast, the iNOS mRNA message was reported to be composed of multiple transcripts, which might be created by use of alternative polyadenylation sites, in a murine macrophage cell line [24], whereas another study only showed a single iNOS mRNA species in the same cell line [23]. From the earlier studies [20, 27]and ours, the chick iNOS mRNA appears to consist of a single mRNA species of 4.5 kb or 4.8 kb, although we cannot deny the possibility that multiple iNOS messages of similar sizes are induced in avian tissues. Further studies will also be needed to clarify the post-transcriptional regulation of the chicken iNOS mRNA.

Although the direct functional consequences of the myocardial iNOS expression have not yet been fully established in the clinical settings, our data suggest that myocardial depressant effects of NO produced by iNOS may contribute to development of the failing myocardium in a wide range of evolutional stages.

Time for primary review 28 days.


   Acknowledgements
 
This study was in part supported by Grant-in-Aid of the Ministry of Education and Culture, Japan. The nucleotide sequence reported in this paper has appeared in the DDBJ, EMBL and/GenBank Data Bank with accession number D85422 [GenBank] .


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

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