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Cardiovascular Research 2004 62(1):194-201; doi:10.1016/j.cardiores.2003.12.028
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Copyright © 2004, European Society of Cardiology

Nitric oxide: inhibitory effects on endothelial cell calcium signaling, prostaglandin I2 production and nitric oxide synthase expression

Kazuhiko Takeuchia, Hiroshi Watanabe*,b, Quang-Kim Trana, Mariko Ozekia, Daigo Sumic, Toshio Hayashic, Akihisa Iguchic, Louis J Ignarrod, Kyoichi Ohashib and Hideharu Hayashia

aDepartment of Internal Medicine III, Hamamatsu University School of Medicine, Handayama, Hamamatsu 431-3192, Japan
bClinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, Handayama, Hamamatsu 431-3192, Japan
cDepartment of Geriatrics, Nagoya University School of Medicine, Nagoya 466-0064, Japan
dDepartment of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA

* Corresponding author. Tel.: +81-53-435-2385; fax: +81-53-435-2384. Email address: hwat{at}hama-med.ac.jp

Received 22 September 2003; revised 13 December 2003; accepted 30 December 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase exerts many harmful effects such as stimulation of inflammation and induction of apoptosis. The effects of excessive NO production on functions of endothelial cells, the major physiological source of NO, are not completely known. The aim of this study was to investigate the role of NO on the regulation of endothelial cell Ca2+ signaling and endothelial cell function. Methods: Primary porcine aortic endothelial cells (PAECs) were used for all these studies. Intracellular Ca2+ concentrations ([Ca2+]i) were measured using fura-2/AM. Production of prostaglandin I2 (PGI2) and cyclic GMP were assessed using enzyme immunoassays, and endothelial NO synthase protein expression was evaluated by Western blotting. Results: Bradykinin (BK, 10 nM) and thapsigargin (TG, 1 µM) provoked large increases in [Ca2+]i. The NO donor NOC12 reduced these responses, respectively, by 21% and 31% at 100 µM, 60% and 55% at 300 µM, and 74% and 78% at 500 µM. These effects were also observed with other NO donors including spermine NONOate and NOC18, and were completely prevented by carboxy-PTIO (200 µM), an NO scavenger. 8-Bromo-cGMP, however, had no effects on BK- and TG-stimulated Ca2+ responses. A 30-fold increase in PGI2 production was observed in cells stimulated with BK. NOC12 again reduced this response by 12%, 54%, 83% and 95% at 10, 100, 300 and 500 µM, respectively. Endothelial NO synthase protein level was reduced by 2%, 15%, 36 and 47% after 2, 6, 12 and 24 h, respectively, of incubation with NOC18, a NO donor with long half-life. Conclusions: NO, when produced in large amounts, can inhibit agonist-induced Ca2+ responses independently of cyclic GMP, reduce the production of endothelium-derived relaxing factors (EDRFs) and interfere with endothelial NO synthase protein expression.

KEYWORDS Calcium (cellular); Endothelial function; Nitric oxide; Prostaglandins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Maintenance of vascular homeostasis is dependent to a great extent on the activities of endothelium-derived relaxing factors (EDRFs), including nitric oxide (NO) and prostaglandin I2 (PGI2), and endothelium-derived hyperpolarizing factor (EDHF). Production of EDRFs requires Ca2+ entry, which represents the major portion of the increase in free intracellular Ca2+ in response to physiological stimuli such as shear stress and receptor agonists. Endothelial NOS (eNOS), the enzyme responsible for the physiological production of NO, also requires Ca2+ entry for sustained activation.

NO produced by eNOS has multiple beneficial effects including modulation of platelet aggregation, inhibition of leukocyte adhesion and control of vascular smooth muscle cell proliferation [1–3]. However, when it is produced or administrated in large quantities, NO could have such harmful effects as causing inflammation and inducing apoptosis [4–6].

The effects of excessive NO on endothelial cell signaling and functions are nevertheless not completely known. Studies examining the effects of NO donors on Ca2+ entry have described controversial findings [7,8], and little is known as to whether NO can affect the production of other EDRFs. In this study, we examined the effects of NO donors on Ca2+ signaling and PGI2 production, and eNOS protein expression in primary cultured endothelial cells. The results indicate that large amounts of NO inhibit agonist-stimulated Ca2+ entry, substantially block the production of PGI2 and interfere with eNOS protein expression.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Cell culture
Porcine aortic endothelial cells (PAECs) were isolated, as previously described [9], by gently scraping the intima of descending aortas from pigs from slaughter house. After centrifugation at 250 x g for 10 min in M199 solution (Boehringer, Mannheim, Germany), the fraction of endothelial cells was purified from this suspension, resuspended in M199 solution with Earle's salts, supplemented with 100 IU/ml penicillin G, 100 µg/ml streptomycin and 20% newborn calf serum (NCS), then seeded onto polybiphenyl dishes fixed on 10 x 10 mm glass cover slips, and cultured at 37 °C under 5% CO2 for 2 days. The medium was renewed everyday.

2.2 Measurement of intracellular Ca2+ concentration
After 2 days of culture, PAECs adhering to glass cover slips were incubated for 45 min in a modified Tyrode's solution (composition in mM: 150.0 NaCl, 2.7 KCI, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2 and 10.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, with pH 7.4 at 25 °C) containing 10% NCS and 2 µM fura-2/AM, a fluorescent Ca2+ indicator. The cells were subsequently washed three times with modified Tyrode's solution to remove the fura-2/AM and the serum from the extracellular fluid, then left to equilibrate in the cell buffer for 20 min before measurements were started. All experiments were performed at 25 °C. The absorption shift of fura-2 that occurs upon binding can be determined by scanning the excitation spectrum between 340 and 380 nm while monitoring the emission at 510 nm. Fluorescent images were analyzed every 30 s from individual cells with an [Ca2+]i analyzer (Argus 50, Hamamatsu Photonics) using an ultrahigh sensitivity television camera (CCD). After background subtraction, the fluorescence ratio (F340/F380) was obtained by dividing, pixel by pixel, the 340 nm image by the 380 nm image. Changes in this ratio were used to express changes in intracellular Ca2+ concentration to eliminate potential artifacts caused by variations in cell thickness, intracellular dye distribution or photobleaching. Bradykinin (BK), thapsigargin (TG), NOC12, NOC18, spermine NONOate, carboxy-PTIO, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 8-bromo-cyclic guanosine-3',5'-monophosphate (cGMP) had no effect on fura-2 fluorescence itself, or on autofluorescence of unloaded cells when examined at concentrations employed in this study.

2.3. Measurement of cGMP concentration
Determinations of cGMP level were performed as previously described [10]. PAECs were plated onto 96-well culture dishes at a density of 104 cells/well and incubated with M199 medium for 2 days at 37 °C under 5% CO2–air and saturated humidity. When PAECs reached confluency, the medium was removed and the cells were washed with cell buffer. PAECs were then incubated for 45 min at room temperature in the cell buffer containing NOC12, an NO donor, and/or ODQ, a guanylyl cyclase inhibitor. The cells were then resolved and cyclic GMP levels were determined using an enzyme-linked immunoassay according to the manufacturer’s instructions (EIA kit, Amersham, USA).

2.4 Measurement of prostaglandin I2
The amount of PGI2 released from PAECs was measured as the concentration of its stable metabolite 6-keto-PGF1{alpha}. PAECs were plated in 35-mm culture dishes and incubated with M199 medium for 2 days at 37 °C under 5% CO2–air and saturated humidity. When PAECs reached confluency, the medium was removed and the cells were washed with cell buffer. PAECs were then incubated for 45 min at room temperature in the cell buffer containing various concentrations of NOC12. Bradykinin (10 nM) was then added, and the incubation was continued for 10 min at room temperature. The concentrations of 6-keto-PGF1{alpha} in the cell medium were then determined using an enzyme-linked immunoassay according to the manufacturer's protocol (EIA, Assay Designs, Amersham).

2.5. Western blotting
After incubation of subconfluent PAECs with NOC18 for 2, 6, 12 and 24 h, the cells were lysed in a lysis buffer containing (in mM) 150.0 NaCl, 1.0 EDTA, 1.0 EGTA, 2.5 sodium pyrophosphate, β-glycerophosphate, 1.0 Na3VO4, 20.0 Tris–HCl, 1.0 PMSF, with 1 µg/ml leupeptin and 1% (vol/vol) Triton X-100. Total cell protein concentration was determined using the Dc protein assay kit (Bio-Rad, CA, USA). Samples containing equal amounts (20 µg) of total cellular protein were loaded and separated on 7.5% SDS-polyacrylamide gel electrophoresis. The gel was then transferred onto a PVDF membrane, which was then incubated at 4 °C for 24 h with a primary anti-eNOS monoclonal antibody (1 µg/ml). After washes in transfer buffer, the membrane was incubated with goat anti-mouse IgG-horseradish peroxidase. The washes were repeated before the membrane was developed by a light-emitting nonradioactive method using ECL reagent (Amersham). The membrane was then subjected to autoluminography for 1–5 min. Band intensities were analyzed densitometrically using the software IMAGE (National Institutes of Health, MD, USA).

2.6. Statistical analysis
Unless otherwise indicated, data are means±S.D. Statistical analysis was performed using Student's t-test for unpaired data or one-way ANOVA followed by post-hoc tests in the Systat MGLH program, where appropriate. Differences with p<0.05 are considered significant.

2.7. Materials
Medium 199 was purchased from Boehringer (Manheim, FRO). Newborn calf serum and penicillin–streptomycin were from GIBCO (New York, USA). Fura-2/AM, NOC12 and NOC18 were purchased from Dojindo (Kumamoto, Japan). Bradykinin, thapsigargin, spermine NONOate and ODQ were from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grades.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Ca2+ signals produced by bradykinin and thapsigargin in PAECs
To trigger increases intracellular Ca2+ concentration in PAECs, we used BK, a receptor agonist, and TG, an irreversible inhibitor of the endoplasmic reticulum Ca2+-ATPase. In the presence of extracellular Ca2+, BK (10 nM) rapidly increased F340/F380 from 0.6±0.1 (basal) to 5.1±0.4 (maximal), which was slowly attenuated (open circles, Fig. 1A). The rapid increase in [Ca2+]i was dependent on both intracellular and extracellular Ca2+, while the sustained increase was dependent mainly on the presence of extracellular Ca2+, because BK induced only a small and transient increase in F340/F380 from 0.5±0.1 (basal) to 1.6±0.5 (maximal) in the absence of extracellular Ca2+.


Figure 1
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  		Fig. 1 Effects of NOC12 on BK- and TG-induced Ca2+ responses in PAECs. Cells were pretreated with 0 µM (open circles), 100 µM (closed circles), 300 µM (closed triangles) or 500 µM NOC12 (closed squares) for 45 min before BK (10 nM, A) or TG (1 µM, B) was added. Values are means±S.D.; n=21 cells from three separate experiments.

 
TG (1 µM) provoked a slightly delayed but long lasting increase in F340/F380 from 0.7±0.1 (basal) to 4.7±0.7 (maximal) (open circles, Fig. 1B) in the presence of extracellular Ca2+. Under Ca2+-free conditions with the cell buffer containing 1 mM EGTA, TG (1 µM) provoked a small transient rise in F340/F380 from 0.5±0.1 (basal) to 1.2±0.2 (maximal) (n=21). Thus, the sustained Ca2+ increases in response to TG depend on Ca2+ entry.

3.2 Effects of different NO donors, NOC12, NOC18 and spermine NONOate, on the BK- and TG-induced Ca2+ responses in PAECs
Pretreatment of PAECs with NOC12 (500 µM) for 45 min almost completely prevented the Ca2+ responses to BK (10 nM) and TG (1 µM). Basal and maximal F340/F380 ratios were, respectively, 0.7±0.1 and 1.3±0.4 for BK (closed squares, Fig. 1A), and 0.8±0.1 and 1.0±0.1 for TG (closed squares, Fig. 1B) in the presence of 1 mM extracellular Ca2+. NOC12 dose-dependently (100–500 µM) inhibited the BK- and TG-stimulated Ca2+ responses, as shown in Fig. 1A and B. The reversibility of the effect of NOC12 was confirmed in the experiments in Fig. 2A, in which BK (10 nM) caused only a very small increase in [Ca2+]i (F340/380 ratio of 1.0±0.4) in the presence of NOC12, but triggered a big increase in [Ca2+]i in the same cells after NOC12 was removed (F340/F380 ratio of 4.2±0.5).


Figure 2
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  		Fig. 2 (A) Reversible inhibition by NOC12 of the BK-induced Ca2+ increases. PAECs were pretreated with 500 µM NOC12 for 45 min. BK (10 nM) was added at 1 min, washed out together with NOC12 at 8 min and re-administered in the absence of NOC12 at 51 min. Values are means±S.D.; n=21 cells from three separate experiments. (B) Effect of carboxy-PTIO on the inhibition by NOC12 of BK-induced Ca2+ responses in PAECs. Cells were incubated with cell buffer only (open circles), 500 µM NOC12 (open triangles), 500 µM NOC12 and 200 µM carboxy-PTIO (closed triangles), or 200 µM carboxy-PTIO (closed circles) for 45 min before the addition of BK (10 nM). Values are means±S.D.; n=21 cells from three separate experiments.

 
To address the specificity issue, NO donors that are structurally different from NOC12, including NOC18 and spermine NONOate, were also tested. Both donors significantly suppressed BK- and TG-induced increases in Ca2+ concentration. Thus, peak F340/F380 ratios in PAECs pretreated with NOC18 (1 mM) and spermine NONOate (500 µM), respectively, were only 2.5±0.5 and 1.4±0.5 in response to BK, and 2.0±0.1 and 1.0±0.1, in response to TG (Table 1). To further confirm the specificity of the effects by NOC12 on the Ca2+ signals, we tested if scavenging NO could prevent the inhibitory effect of NOC12 on the BK-induced Ca2+ signals. As shown in Fig. 2B, carboxy-PTIO (200 µM), an NO scavenger, totally prevented the inhibitory effect of NOC12, with F340/F380 ratio of 4.9±0.4 (closed triangles).


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  		Table 1 Effects of NOC12, NOC18 and spermine NONOate (NONOate) on BK- and TG-induced Ca2+ increases in PAECs

 
3.3 Testing the involvement of cGMP in the inhibition of the Ca2+ signals by NO
To clarify if cGMP is involved in the observed inhibitory effect of NOC12, we tested the effect of the cGMP analog 8-bromo-cGMP on BK- and TG-induced Ca2+ increases in PAECs. As shown in Fig. 3A and B, pretreatment of 2 mM 8-bromo-cGMP had absolutely no effect on the BK- and TG-induced Ca2+ increases; the peak F340/F380 ratios were 5.1±0.4 and 4.3±0.2, respectively, for BK and TG. These data indicate that NOC12 does not inhibit BK- and TG-stimulated Ca2+ responses via the accumulation of cGMP.


Figure 3
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  		Fig. 3 Effects of 8-bromo-cGMP, NOC12 and/or ODQ on BK- and TG-induced Ca2+ increases in PAECs. Cells were incubated with cell buffer only (CTL) and 2 mM 8-bromo-cGMP (cGMP), 300 µM NOC12 (NOC), 20 µM ODQ (ODQ), or 300 µM NOC12 and 20 µM ODQ (NOC+ODQ) for 45 min before the addition of BK (A, 10 nM) and TG (B, 1 µM). Values are means±S.D.; n=21 cells from three separate experiments. *P<0.05 vs. CTL, {dagger}N.S. vs. NOC. (C) Effect of ODQ on NOC12-stimulated cGMP production in PAECs. Cells were incubated with various concentrations of ODQ (0, 10 and 20 µM) for 5 min before 300 µM NOC12 were added. Samples were taken 10 min after the addition of NOC12. Open column, PAECs incubated with cell buffer only. Data are means±S.D. of six independent determinations. *P<0.05 vs. NOC12, {dagger}N.S. vs. cell buffer.

 
To further confirm that the observed inhibitory effect of NOC12 does not involve the cGMP pathway, we tested if the guanylate cyclase inhibitor ODQ could prevent the observed effect of NOC12. The effect of ODQ to inhibit NOC12-stimulated cGMP production was first confirmed. As shown in Fig. 3C, NOC12-induced cGMP accumulation was inhibited by ODQ (375.7±75.9 and 248.1±59.9 pg/106 cells at 0 and 10 µM ODQ, respectively), and was almost completely inhibited at 20 µM ODQ (46.2±8.8 pg/106 cells in control vs. 65.8±24.3 pg/106 cells with 20 µM ODQ, N.S., n=6). However, even at this dose ODQ could not prevent the inhibitory effect of NOC12 on the Ca2+ responses (Fig. 3A and B). These results suggest that the inhibition by NOC12 of the BK- and TG-stimulated Ca2+ responses in PAECs does not involve cGMP accumulation.

3.4 Effect of excessive NO on PGI2 production induced by BK in PAECs
Besides NO, PGI2 plays important role in endothelium-dependent vasorelaxation and its production also depends on increases in cytosolic Ca2+ concentration. Phospholipase A2, the rate-limiting enzyme for the liberation of arachidonic acid from phospholipids to produce PGI2, is strictly Ca2+-dependent [11–14]. We therefore tested the effects of various doses (10–500 µM) of NOC12 on PGI2 production in PAECs. PGI2 production was assessed by measuring its stable metabolite 6-keto-PGF1{alpha}. As shown in Fig. 4, BK (10 nM) greatly increased 6-keto-PGF1{alpha} from 16.0±6.2 ng/106 cells to 481.4±68.0 ng/106 cells. This effect was inhibited by NOC12 in a dose-dependent manner (420.3±27.3, 220.6±55.6, 76.4±8.6 and 21.3±1.8 ng/106 cells at 10, 100, 300 and 500 µM NOC12, respectively). There is a good correlation between the inhibitory effects of NOC12 on BK-stimulated peak [Ca2+]i and PGI2 production (R=0.96, p<0.05).


Figure 4
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  		Fig. 4 Effect of NOC12 on BK-induced PGI2 production in PAECs. Cells were incubated with various concentrations of NOC12 (0, 10, 100, 300 and 500 µM) for 45 min before the addition of BK (10 nM). Open column, PAECs not stimulated by BK. All data shown are means±S.D. of eight independent observations in separate cell culture wells. *P<0.05 vs. cell buffer.

 
3.5. Effect of NOC18 on eNOS protein expression in PAECs
Endothelial NOS is the major physiological source of NO production. We thus examined the effect of excessive NO on eNOS protein levels in PAECs using NOC18, an NO donor with a half-life of 21 h at 37 °C. Subconfluent PAECs were pretreated with NOC18 for various periods of time before the cells were lysed and eNOS protein were blotted. As shown in Fig. 5, NOC18 time-dependently reduced eNOS protein levels by 2%, 15%, 36% and 47% after 2, 6, 12 and 24 h, respectively.


Figure 5
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  		Fig. 5 Time-dependent effect of NOC18, a long half-life NO donor, on eNOS protein expression in PAECs. Cells were incubated with 500 µM NOC18 for 0, 2, 6, 12 and 24 h. Blots are representatives and values are means±S.D. from three separate determinations. *P<0.05 vs. 0 h.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study, we have found that large amounts of NO can substantially inhibit BK- and TG-stimulated Ca2+ responses independently of the cGMP pathway, decrease PGI2 production and suppress eNOS protein expression in vascular endothelial cells.

Changes in Ca2+ concentration, in particular capacitative Ca2+ entry, in response to agonists and shear stress are important for many endothelial cell functions [15]. The literature has been controversial as regards the effects of NO on Ca2+ entry. Several groups have reported that NO donors can inhibit capacitative Ca2+ entry (CCE) through a cGMP-linked pathway [7,16–19]. Dedkova et al. reported that sodium nitroprusside (SNP, 100 µM) and 8-bromo-cGMP (300 µM) inhibited ATP-induced CCE in bovine vascular endothelial cells and concluded that NO could exert an autoregulatory negative feedback on its own Ca2+-dependent synthesis. On the contrary, Gilon et al. [8] reported that neither NO nor cGMP had any effect on CCE in rat pancreas acinar cells. In the present study, we found that excessive NO could inhibit BK- and TG-induced Ca2+ responses independently of cGMP pathway, as evidenced by the observations that NOC12 significantly prevented these responses while 8-bromo-cGMP did not, and that inhibition of guanylyl cyclase with ODQ could not reverse the inhibitory effect of NOC12. While it is possible that differences among cell types could attribute to some extent to the apparently opposing findings in these studies, further studies are needed to address this issue.

Several studies have demonstrated that exogenous NO can inhibit the vascular response to NO. Dinerman et al. [20] demonstrated that endothelium desensitized the relaxation of vascular smooth muscle in response to nitroglycerin and postulated a competitive interaction between endothelial NO and nitroglycerin. Our studies suggest that desensitization might occur at the level of the endothelial cell through attenuation of the Ca2+ response to vasorelaxing agonists.

The mechanism underlying the inhibitory effect of excessive NO on the Ca2+ responses remains unclear. However, it is possible that excessive NO does this by inhibiting cytochrome P450 via NO-heme interactions. Previous studies have suggested that the cytochrome P450 product 5,6-epoxy eicosatrienoic acid (5,6-EET) can regulate Ca2+ entry. Thus, CYP inhibitors were shown to inhibit agonist-induced 5,6-EET production and Ca2+ entry in endothelial cells. Also in these studies, induction of CYP potentiated agonist-induced Ca2+ entry, and nano- to picomolar 5,6-EET induced Ca2+ elevation consistent with Ca2+ entry [21]. The activity of cytochrome P450 isozymes depends on a heme-protein in their structures. NO has been demonstrated to form a tight complex with the heme [22] and inhibit CYP-related reactions [23–25]. Therefore, large quantities of NO may inhibit agonist-induced Ca2+ signals in endothelial cells by suppressing 5,6-EET production. Additionally, excessive NO could inhibit Ca2+ entry via the formation of peroxynitrite. Elliott [26] reported that peroxynitrite decreased BK-induced Ca2+ entry in vascular endothelial cells. Large amounts of NO will diffuse easily through the mitochondrial membrane and reversibly inhibit cytochrome oxidase. This inhibition suppresses the mitochondrial respiratory chain and as a consequence increases mitochondrial O2– radical release, leading to peroxynitrite formation [27,28]. The effects of NO on CYP450 and cytochrome oxidase could converge to bring about its inhibition of the Ca2+ responses in endothelial cells.

Endothelial nitric oxide synthase is activated by increases in cytosolic Ca2+ [29–32] and is dependent on Ca2+ entry for its sustained activation [33]. We now show that NO can inhibit Ca2+ entry independently of cGMP production. However, Parkinson et al. [34] have shown that Ca2+ can directly inhibit NO-stimulated soluble guanylyl cyclase. From these studies, it is possible that cGMP production is balanced in part by Ca2+-dependent eNOS activity and Ca2+-inhibitable soluble guanylyl cyclase activity, with possible superiority of eNOS in this regards [29–32].

The present study also demonstrates that excessive NO can reduce eNOS protein expression. Chen and Mehta [35] previously showed that NO, authentic or derived from nitroglycerin, decreased NOS activity without affecting NOS protein expression in human platelets after incubation for 3 h. These findings suggest that the negative feedback mechanism can be formed in short term. In the present study, a NO donor with a long half-life reduced eNOS protein expression by ~50% after 24-h incubation. This clearly confirms that exogenous NO can interfere with eNOS protein expression and influence intrinsic NO production. The underlying mechanism is still not clear. However, excessive NO has been shown to induce apoptosis [4–6]. It is likely that apoptosis induced by such large amounts of NO over a long period of time could lead to cleavage of eNOS and possibly other proteins in the endothelial cell.

EDRFs other than NO, including PGI2 and EDHF, play important roles in endothelium-dependent vasorelaxation in various physiological and pathological settings [36–38] and the production of these also depends on increases in cytosolic Ca2+ concentration [11–14,38]. The activity of phospholipase A2, and thus PGI2 production, has been shown to strictly require Ca2+ [11–14]. The finding that excessive NO inhibits Ca2+ entry suggests that it could thereby inhibit also the production of PGI2. Indeed, NOC12 dose-dependently inhibited both the BK-stimulated increase in cytosolic Ca2+ concentration and the production of 6-keto-PGF1{alpha}, a stable metabolite of PGI2 in PAECs. The inhibitory effects of NOC12 on Ca2+ entry and PGI2 production appear to be well correlated. We previously demonstrated that Ca2+ entry also plays a predominant role in the effects of EDHF stimulated by agonists. Thus, BK produces very little or no hyperpolarization of porcine coronary artery under Ca2+-free conditions or in the presence of extracellular Ca2+ with Ca2+ entry inhibited by the non-specific Ca2+ channel inhibitor, SKF96365 [38]. This is in line with the observations by Bauersachs et al. [39] that NO attenuates the release of EDHF in cultured human endothelial cells. NO thus probably modifies EDHF production by regulating cytosolic Ca2+ concentration.

In conclusion, we have shown that excessive NO can inhibit Ca2+ entry, PGI2 production and eNOS expression in endothelial cells. The functions of many protein kinases in the cell are dependent on Ca2+ signals and the vasculature has its tone maintained by the production of EDRFs. These effects of excessive NO clearly will disturb many endothelial functions and promote endothelial injury.


   Acknowledgements
 
This study was supported by Grants-in-Aid (14570652) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H. Watanabe.


   Notes
 
Time for primary review 21 days


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

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