This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Minamino, T. Articles by Hori, M. Search for Related Content PubMed PubMed Citation Articles by Minamino, T. Articles by Hori, M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE

(Circulation. 1997;96:1586-1592.)
© 1997 American Heart Association, Inc.

Articles

Inhibition of Nitric Oxide Synthesis Increases Adenosine Production via an Extracellular Pathway Through Activation of Protein Kinase C

Tetsuo Minamino, MD, PhD; Masafumi Kitakaze, MD, PhD; Koichi Node, MD; Hiroharu Funaya, MD; ; Masatsugu Hori, MD, PhD

From the First Department of Medicine, Osaka University School of Medicine, Osaka, Japan.

Correspondence to Masafumi Kitakaze, MD, The First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565, Japan. E-mail kitakaze{at}medone.med.osaka-u.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background NO is known to deactivate protein kinase C (PKC). Because we have reported that the activation of PKC activates ecto-5'-nucleotidase, we examined whether the inhibition of NO synthesis increases ecto-5'-nucleotidase activity through the activation of PKC.

Methods and Results The left anterior descending coronary artery (LAD) was cannulated and perfused with blood through a bypass tube from the left carotid artery in 65 open-chest dogs. The intracoronary administration of N^G-nitro-L-arginine methyl ester (L-NAME, 10 µg · kg^-1 · min^-1), an NO synthase inhibitor, for 30 minutes increased (P<.05) adenosine levels in coronary venous blood (123±10 versus 21±3 pmol/mL) and ecto-5'-nucleotidase activity (64±6 versus 41±4 nmol · mg^-1 · min^-1) in the LAD-perfused myocardium. The intracoronary administration of ,ß-methyleneadenosine 5'-diphosphate, an inhibitor of ecto-5'-nucleotidase, or GF109203X or calphostin C, both of which are PKC inhibitors, attenuated the L-NAME–induced increases in adenosine levels and ecto-5'-nucleotidase activity. Treatment of cultured human coronary arterial endothelial cells (HCAECs) with L-NAME for 30 minutes increased ecto-5'-nucleotidase activity, which was inhibited by either GF109203X or calphostin C. NO releasers decreased both ecto-5'-nucleotidase and PKC activities in HCAECs. Treatment of HCAECs with zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase, with or without atrial natriuretic peptide, increased intracellular cGMP concentrations but did not change ecto-5'-nucleotidase activity.

Conclusions These results indicate that the inhibition of NO synthesis increases both adenosine production and ecto-5'-nucleotidase activity through the activation of PKC and that NO modulates ecto-5'-nucleotidase via cGMP-independent mechanisms.

Key Words: adenosine • nitric oxide • proteins • cells

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nitric oxide^{1 2} and adenosine^{3 4} are potent vasodilators involved in modulating coronary circulation. NO is produced by constitutive NOS in endothelial cells.² Adenosine is produced intracellularly by S-adenosyl-L-homocysteine hydrolase and cytosolic 5'-NT and extracellularly by ecto-5'-NT in endothelial cells, smooth muscle cells, and cardiomyocytes.^{4 5} NO and adenosine activate guanylate cyclase and adenylate cyclase, respectively, with the subsequent biosynthesis of cGMP and cAMP.^{2 4 6} These two distinct chemical mediators are known to exhibit similar cardiovascular effects.^{2 4 6} Recently, it has been reported that the inhibition of NO synthesis increases adenosine production under baseline conditions,⁷ during reactive hyperemia,⁸ and during increased myocardial oxygen consumption induced by pacing.⁹ Conversely, both NO and NO donors inhibit ecto-5'-NT activity in cultured cells.¹⁰ These findings suggest that NO may modulate the production of adenosine via an extracellular pathway and that increased adenosine levels may compensate for NO-mediated functions when NO release is diminished. However, little is known about the underlying mechanism of the interaction between NO and adenosine.

We have previously reported that the activation of PKC activates ecto-5'-NT in myocardium^{11 12} and isolated coronary vessels.⁵ Because NO and NO-generating agents deactivate PKC,¹³ we hypothesized that the inhibition of NO synthesis increases ecto-5'-NT activity through the activation of PKC, resulting in increases in adenosine levels. To test this hypothesis, we examined the effects of the inhibition of NO synthesis on adenosine production and ecto-5'-NT activity and the role of PKC in this condition in the in vivo canine heart. We also examined the effects of NO and the increases in intracellular cGMP on ecto-5'-NT activity using cultured HCAECs.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Chemicals
L-NAME, L-NMMA, AMP-CP, Hb, GF109203X, ANP, and SNP were purchased from Sigma Chemical Co. Calphostin C was purchased from Calbiochem. SIN-1 was purchased from Dojinlab. Zaprinast was kindly donated by Ono Pharmaceutical Co, Ltd.

Instrumentation
We anesthetized 65 mongrel dogs of either sex (15 to 21 kg) with sodium pentobarbital (30 mg/kg IV). The anesthetized dogs were prepared as described previously^{11 12} ; the LAD was ligated, cannulated, and perfused with blood from the left carotid artery through an extracorporeal bypass tube. CBF in the perfused area was measured with an electromagnetic flow probe attached to the bypass tube, and CPP was monitored at the tip of the coronary artery cannula. A small coronary vein near the center of the perfused area was cannulated to sample coronary venous blood. A miniature pressure transducer (P-5, Konigsberg Instruments, Inc) was inserted into the LV cavity through the LV apex to determine LV dP/dt. A pair of ultrasonic crystals (5 MHz, 2 mm in diameter; Schuessler) was implanted in the endomyocardial segment of the LV anterior wall in the center of the perfused area to measure segmental length. The lengths of end-diastolic and end-systolic segments were determined, and FS was calculated as described previously.¹⁴ All hemodynamic parameters were recorded on a multichannel recorder (RM-6000, Nihon Kohden).

Human Coronary Arterial Endothelial Cells
HCAECs (Cryo HCAEC; Clonetics Co Ltd, Sanko Jyunyaku Co Ltd) were plated in dishes (10 cm in diameter) and grown at 37°C in a 5% CO₂ humidified incubator in modified MCDB131 medium supplemented with 5% FBS, 10 ng/mL human epidermal growth factor, 1 µg/mL hydrocortisone, 50 µg/mL gentamicin, and 50 µg/mL amphotericin B. We preliminarily confirmed that constitutive NOSs are expressed by immunoblotting analysis and that NO₂^- and NO₃^- measured with the Griess reaction can be released in response to bradykinin (1x10^-4 mol/L) in HCAECs (1.5±0.1 versus 2.1±0.2 µmol · mg protein^-1 · 60 min^-1, n=4). These cells were used at passages 5 through 10.

Experimental Protocols
Protocol 1. Effects of inhibitions of NO synthesis, ecto-5'-NT, or PKC or antagonism of adenosine receptors on coronary hemodynamic and metabolic parameters in vivo. To examine the effects of the inhibition of NO synthesis on coronary hemodynamic and metabolic parameters, we administered L-NAME (10 µg · kg^-1 · min^-1) through the bypass tube for 30 minutes in 5 dogs. CBF, CPP, and the lengths of end-diastolic and end-systolic segments in the LAD-perfused myocardium were monitored throughout the protocol. We measured the time course of changes in lactate concentration, pH, and oxygen content in both coronary arterial and venous blood. To examine the effects of endogenous adenosine on coronary hemodynamic and metabolic parameters, we administered 8-SPT (25 µg · kg^-1 · min^-1), an antagonist of adenosine receptors, into the LAD for 30 minutes in the presence (n=5) and absence (n=5) of L-NAME (10 µg · kg^-1 · min^-1). This dose of 8-SPT completely abolishes the coronary vasodilatory effect of an intracoronary infusion of exogenous adenosine (1 µg · kg^-1 · min^-1). To examine the effects of inhibition of ecto-5'-NT on coronary hemodynamic and metabolic parameters, we administered AMP-CP (40 µg · kg^-1 · min^-1), an inhibitor of ecto-5'-NT, into the LAD for 30 minutes (n=5). The dose of AMP-CP used in the present study inhibits adenosine production via an extracellular pathway.¹⁵ To examine the effects of the inhibition of PKC on coronary hemodynamic and metabolic parameters, we administered GF109203X (300 ng · kg^-1 · min^-1) or calphostin C (400 ng · kg^-1 · min^-1), both of which are extensively used as a specific inhibitor of PKC,^{16 17} through the bypass tube for 30 minutes (n=5, respectively). We measured the time course of changes in coronary hemodynamic and metabolic parameters in all the groups in protocol 1 as in L-NAME.

Protocol 2. Effects of the inhibition of NO synthesis on adenosine production in vivo. To examine whether the inhibition of NO synthesis increases adenosine production in the in vivo hearts, we measured the time course of changes in adenosine levels in both coronary arterial and venous blood with (n=5) and without (n=5) intracoronary administration of L-NAME (10 µg · kg^-1 · min^-1). To examine the mechanistic cellular pathway by which the inhibition of NO synthesis increases adenosine production in the myocardium, we measured the time course of changes in adenosine levels in both coronary arterial and venous blood after intracoronary administrations of AMP-CP (40 µg · kg^-1 · min^-1) and L-NAME (10 µg · kg^-1 · min^-1) in 5 dogs. Furthermore, because both NO and NO-generating agents induce an inactivation of PKC,¹³ we examined whether the inhibition of NO synthesis increased adenosine release through the activation of PKC. We measured the time course of changes in adenosine levels after intracoronary administration of GF109203X (300 ng · kg^-1 · min^-1) or calphostin C (400 ng · kg^-1 · min^-1) through the bypass tube during administration of L-NAME (10 µg · kg^-1 · min^-1) in 5 dogs each.

Protocol 3. Effects of the inhibition of NO synthesis on myocardial 5'-NT and PKC activities. To examine whether the inhibition of NO synthesis modulates ecto-5'-NT and PKC activities, we administered L-NAME (10 µg · kg^-1 · min^-1) through the bypass tube for 30 minutes in 5 dogs. To examine whether the inhibition of NO synthesis increases ecto-5'-NT activity through the activation of PKC, we administered GF109203X (300 ng · kg^-1 · min^-1) or calphostin C (400 ng · kg^-1 · min^-1) through the bypass tube during the administration of L-NAME (10 µg · kg^-1 · min^-1) in 5 dogs each. We sampled the LAD-perfused myocardium and the myocardium perfused through the LCx as a control. The samples were quickly stored in liquid nitrogen for measurement of ecto-5'-NT and PKC activities.

Protocol 4. Effects of NO on 5'-NT and PKC activities in HCAECs. To examine whether the inhibition of NO synthesis modulates ecto-5'-NT and PKC activities in HCAECs, we measured ecto-5'-NT and PKC activities in HCAECs 30 minutes after the addition of L-NAME (1x10^-5 and 1x10^-4 mol/L) or L-NMMA (1x10^-4 mol/L). To examine whether NO itself modulated ecto-5'-NT and PKC activities, we measured ecto-5'-NT and PKC activities in HCAECs 30 minutes after the addition of Hb (1x10^-5 mol/L), which deactivates NO by oxidizing it to NO₂^- and NO₃^-. Furthermore, to examine whether NO releasers decrease 5'-NT and PKC activities, we measured ecto-5'-NT and PKC activities in HCAECs 30 minutes after the addition of either SNP (1x10^-4 mol/L) or SIN-1 (1x10^-3 mol/L). To examine whether the inhibition of NO synthesis increased ecto-5'-NT through the activation of PKC, we measured ecto-5'-NT activity 30 minutes after the addition of GF109203X (1x10^-7 mol/L) or calphostin C (5x10^-7 mol/L) with L-NAME (1x10^-5 mol/L) or L-NMMA (1x10^-4 mol/L).

Protocol 5. Effects of increases in intracellular cGMP concentration on 5'-NT activity. To examine whether NO modulates 5'-NT activity via a cGMP-dependent mechanism, we measured intracellular cGMP concentration and ecto-5'-NT activity in HCAECs 30 minutes after the addition of zaprinast (1x10^-4 mol/L), a selective inhibitor of cGMP-specific phosphodiesterase, with and without SNP (1x10^-5 mol/L) or ANP (1x10^-7 mol/L).

Chemical Analysis
The plasma concentration of lactate was determined enzymatically.¹⁸ The blood oxygen differences of coronary arterial and venous blood were assessed by measurement of differences between coronary arterial and venous oxygen contents. Blood gas analysis was performed with an ABL300 blood gas analyzer (Radiometer A/S). LER and MO₂ (mL · 100 g^-1 · min^-1) were calculated as previously described.¹⁸ The plasma concentration of adenosine was measured as previously described.¹⁴

Measurement of 5'-NT and PKC Activities in Myocardium
The myocardial segments obtained above were separated into membrane and cytosolic fractions as described previously.¹⁹ The activity of 5'-NT was assessed by an enzymatic assay technique²⁰ and is reported in units of mol · mg protein^-1 · min^-1. We defined 5'-NT activity in membrane fractions as ecto-5'-NT activity.

PKC activity was measured by enzyme assay with the RPN 77A kit (Amersham), which provides a simple and reliable method of estimating PKC without extensive purification of the samples.^{11 12} PKC activity in the presence of 12 mmol/L Ca²⁺ and 8 mol% phosphatidylserine was expressed as nmol · mg protein^-1 · min^-1. Protein concentration was measured by the method of Lowry et al²¹ with BSA as the standard.

Measurement of 5'-NT and PKC Activities in HCAECs
To prepare the total cell lysates, freshly isolated HCAECs were suspended in cold lysis buffer (50 mmol/L Tris-HCl, 250 mmol/L sucrose, 5 mmol/L leupeptin, pH 7.4), vortexed, and kept at 4°C. Cell lysates were disrupted by three cycles of rapid freezing and thawing. The methods to prepare membrane and cytosolic fractions from HCAECs and the methods for measurements of PKC and ecto-5'-NT have been described above.

Measurement of cGMP Concentration in HCAECs
After removal of culture medium, HCAECs were washed with HBS supplemented with 15 mmol/L HEPES and 2 mmol/L L-glutamine (HBS-HEPES) and then preincubated for 15 minutes at 37°C in HBS-HEPES. Zaprinast with and without ANP or SNP was then added, and the cells were incubated at 37°C for 5 minutes. The reaction was terminated by aspiration and the addition of 0.1N HCl. The solubilized materials were centrifuged at 2000g for 10 minutes, and the supernatants were lyophilized. The lyophilized materials were resuspended in 5 mmol/L sodium acetate buffer, pH 4.75, and subjected to a radioimmunoassay for cGMP. The radioimmunoassay was performed according to the method described previously.²²

Statistical Analysis
Data are expressed as mean±SEM. The time courses of changes in coronary hemodynamic and metabolic parameters were compared by one-way repeated-measures ANOVA. The time courses of changes in adenosine production were compared by two-way repeated-measures ANOVA. 5'-NT and PKC activities were compared by one-way factorial ANOVA. Bonferroni's test was used to determine significance for group pairs that exhibited statistical significance. A level of P<.05 was accepted as statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Coronary Hemodynamic and Metabolic Functions
Neither L-NAME, AMP-CP, GF109203X, calphostin C, 8-SPT, nor L-NAME+8-SPT altered systemic hemodynamic parameters (Table 1). CPP, CBF, FS, MO₂, LER, and pH of coronary venous blood were similarly unaffected by L-NAME, AMP-CP, GF109203X, calphostin C, 8-SPT, or L-NAME+8-SPT (Table 1).

View this table: [in this window] [in a new window]   Table 1. Coronary Hemodynamic and Metabolic Function

Adenosine Levels
L-NAME increased (P<.05) adenosine levels in coronary venous blood and the difference of adenosine levels between coronary venous and arterial blood. Either AMP-CP, GF109203X, or calphostin C blunted the increases in adenosine levels in coronary venous blood and the difference of adenosine levels between coronary venous and arterial blood induced by L-NAME (Table 2).

View this table: [in this window] [in a new window]   Table 2. Effects of Inhibition of NO Synthesis on Adenosine Levels

Ecto-5'-NT and PKC Activities in Myocardium
L-NAME increased ecto-5'-NT activity in the LAD-perfused myocardium compared with the LCx-perfused myocardium, which was blunted by the concomitant administration of GF109203X or calphostin C (Fig 1). L-NAME increased PKC activity in the membrane but not the cytosolic fraction in the LAD-perfused myocardium compared with the LCx-perfused myocardium (Fig 2).

View larger version (31K): [in this window] [in a new window]   Figure 1. Effects of L-NAME on 5'-NT activity in myocardium with and without GF109203X or calphostin C. Data are mean±SEM. *P<.05 vs control.

View larger version (34K): [in this window] [in a new window]   Figure 2. Effect of L-NAME on PKC activity in membrane (A) and cytosolic (B) fraction in myocardium. Data are mean±SEM. *P<.05 vs control.

Ecto-5'-NT and PKC Activities in HCAECs
Ecto-5'-NT activity was increased (P<.05) by 10^-5 mol/L L-NAME (2.8±0.2 µmol · mg protein^-1 · min^-1, n=6), 10^-4 mol/L L-NAME (2.7±0.2 µmol · mg protein^-1 · min^-1, n=5), L-NMMA (2.6±0.2 µmol · mg protein^-1 · min^-1, n=5), or Hb (2.5±0.3 µmol · mg protein^-1 · min^-1, n=5) compared with control (1.9±0.2 µmol · mg protein^-1 · min^-1, n=6). PKC activity in the membrane fraction in HCAECs was increased (P<.05) by 10^-5 mol/L L-NAME (3.4±0.4 nmol · mg protein^-1 · min^-1, n=6), 10^-4 mol/L L-NAME (3.2±0.4 nmol · mg protein^-1 · min^-1, n=5), L-NMMA (3.0±0.4 nmol · mg protein^-1 · min^-1, n=5), or Hb (2.9±0.4 nmol · mg protein^-1 · min^-1, n=5) compared with control (2.2±0.3 nmol · mg protein^-1 · min^-1, n=5). However, ecto-5'-N activity was decreased (P<.05) by SNP (1.7±0.1 µmol · mg protein^-1 · min^-1, n=5) or SIN-1 (1.5±0.1 µmol · mg protein^-1 · min^-1, n=5). PKC activity in the membrane fraction in HCAECs was decreased (P<.05) by SNP (1.3±0.3 nmol · mg protein^-1 · min^-1, n=5) or SIN-1 (1.2±0.2 nmol · mg protein^-1 · min^-1, n=5). Neither L-NAME (37±4 nmol · mg protein^-1 · min^-1), L-NMMA (34±6 nmol · mg protein^-1 · min^-1), Hb (35±5 nmol · mg protein^-1 · min^-1), SNP (39±4 nmol · mg protein^-1 · min^-1), nor SIN-1 (36±5 nmol · mg protein^-1 · min^-1) affected PKC activity in the cytosolic fraction. GF109203X blunted the increases in ecto-5'-NT activity induced by L-NAME (1.8±0.3 µmol · mg protein^-1 · min^-1, n=5) or L-NMMA (2.1±0.4 µmol · mg protein^-1 · min^-1, n=4). Calphostin C blunted the increases in ecto-5'-NT activity induced by L-NAME (1.9±0.4 µmol · mg protein^-1 · min^-1, n=5) or L-NMMA (2.0±0.3 µmol · mg protein^-1 · min^-1, n=5).

cGMP Concentrations on Ecto-5'-NT
Zaprinast with and without ANP or SNP increased the cGMP concentration in HCAECs (Fig 3A). However, zaprinast alone or zaprinast with ANP did not change ecto-5'-NT activity in HCAECs (Fig 3B).

View larger version (52K): [in this window] [in a new window]   Figure 3. Effects of zaprinast with and without ANP or SNP on intracellular cGMP concentration (A) and ecto-5'-NT activity (B) in HCAECs. Data are mean±SEM. *P<.05 vs control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We demonstrated that the inhibition of NO synthesis increases ecto-5'-NT activity and adenosine production via an extracellular pathway through the activation of PKC in both in vivo canine hearts and in vitro HCAECs. We also demonstrated that NO modulates ecto-5'-NT via a cGMP-independent mechanism in HCAECs.

Role of NO and Adenosine in Coronary Circulation in the In Vivo Hearts
Although NO is known as a potent vasodilator,¹ its role in the coronary circulation is controversial. The intracoronary administration of NO synthesis inhibitors does not change either baseline CBF or CPP in conscious and anesthetized dogs.^{23 24 25} However, Woolfson et al⁷ reported that L-NAME increases CPP in isolated rabbit hearts perfused at a constant flow rate, in which the level of shear stress is greater than that in the in vivo heart. Species difference (dogs versus rabbits) and varied experimental models (in vivo hearts versus isolated perfused hearts) are possible explanations for the differences in the role of NO in the coronary circulation. In the present study, the intracoronary administration of L-NAME did not change either CBF or CPP, which was compatible with the previous studies using in vivo hearts.^{23 24 25} This finding suggests two possible explanations for the role of NO in coronary circulation. One is that NO is not a major regulator of coronary circulation under baseline conditions. However, Jones et al²³ demonstrated that the inhibition of NO synthesis by L-NAME leads to the constriction of small coronary arteries (diameters >100 µm), indicating that the basal flow-dependent activity of NO synthesis reduces the tone of these vessels. This finding suggests the important role of NO in coronary circulation in the in vivo model. Another possible explanation is that the increased adenosine levels compensate for the reduced CBF caused by the inhibition of NO synthesis. However, 8-SPT did not decrease baseline CBF in the presence and absence of L-NAME, suggesting that increased adenosine levels during the inhibition of NO synthesis may not contribute to coronary regulation or that some vasoactive substance(s) other than NO and adenosine may play a compensatory role when both of them are inhibited. Puybasset et al²⁴ recently reported that the cyclooxygenase pathway is also activated when NO synthesis is inhibited, suggesting that eicosanoids, which are also involved in coronary circulation, could play a compensatory role when both NO and adenosine are inhibited. Recently, Matsunaga et al⁹ reported that increases in CBF in response to increased myocardial oxygen consumption during cardiac pacing are decreased by concomitant administration of an NOS inhibitor and an antagonist of adenosine receptors but not by an NOS inhibitor alone in the open-chest dog model. This finding implies that increased adenosine production caused by the inhibition of NO synthesis may compensate for the reduced increases in CBF in response to myocardial oxygen requirement when NO synthesis is diminished. These findings suggest that some vasoactive substance(s) other than NO and adenosine could play a compensatory role only under normal conditions and that the role of increased adenosine during the inhibition of NO synthesis may be augmented under pathophysiological conditions. Further investigations are needed to clarify the interaction of vasoactive substances in the coronary circulation.

Mechanism by Which Adenosine Production Is Increased During the Inhibition of NO Synthesis
Endothelium-derived vasoactive factors may interact with each other and synergistically regulate vascular tone.^{26 27} However, little is known about the potential interactions between NO and adenosine. Importantly, we demonstrated that the inhibition of NO synthesis increases adenosine levels in the in vivo model, which is consistent with previous studies using isolated hearts.^{7 8 25} Adenosine levels are markedly increased in ischemic hearts,^{4 14} suggesting that L-NAME may cause myocardial ischemia. However, we demonstrated that L-NAME did not change hemodynamic and metabolic parameters, including LER, MO₂, and FS, indicating that the inhibition of NO synthesis did not induce myocardial ischemia. Adenosine can be produced intracellularly by cytosolic 5'-NT and S-adenosyl-L-homocysteine hydrolase and extracellularly by ecto-5'-NT.^{3 4} We demonstrated that an inhibitor of ecto-5'-NT, AMP-CP, attenuates the increases in adenosine levels caused by L-NAME. These findings suggest that the inhibition of NO synthesis increases adenosine production via an extracellular pathway without myocardial ischemia. The possible mechanisms by which L-NAME increased adenosine production via an extracellular pathway are by (1) increasing the concentration of adenine nucleotides, the substrates for ecto-5'-NT, and/or (2) by increasing the activity of extracellular ectophosphatases. Bodin et al²⁸ reported that increased blood flow augments ATP release from endothelial cells. In the present study, we have demonstrated that the inhibition of NO synthesis increased ecto-5'-NT activity both in the in vivo heart model and in cultured endothelial cells without changing blood flow. Thus, increased concentrations of adenosine via an extracellular pathway caused by L-NAME may be attributable, at least in part, to the activation of ecto-5'-NT. However, we cannot completely deny the former mechanism.

Activation of Ecto-5'-NT During the Inhibition of NO Synthesis
Ecto-5'-NT is a plasma membrane enzyme attached by a glycosyl phosphatidylinositol anchor.²⁹ We demonstrated that the treatment of L-NAME for 30 minutes increases ecto-5'-NT activity in both the in vivo hearts and in vitro HCAECs. Although the mechanism by which the inhibition of NO synthesis increases ecto-5'-NT activity is not identified at this time, the time course of the activation of ecto-5'-NT suggests that it is not due to the synthesis of new enzyme protein. Because we have shown that okadaic acid inhibits deactivation of ecto-5'-NT caused by ischemia, phosphorylation of ecto-5'-NT itself or phosphorylation of proteins that interact with this enzyme may activate ecto-5'-NT.³⁰ We have also demonstrated in the present study that the inhibition of PKC blunts the L-NAME–induced activation of ecto-5'-NT. We have previously reported that the deactivation of PKC blunts the activation of ecto-5'-NT induced by ischemia or hypoxia in the myocardium^{11 12} and isolated coronary vessels.⁵ These findings suggest that phosphorylation processes mediated by PKC are involved in the activation of ecto-5'-NT.

NO exerts many cardiovascular effects via several signaling pathways: stimulation of guanylate cyclase resulting in an increase of intracellular cGMP concentrations, ADP-ribosylation-like reaction with proteins, and S-nitrosylation of proteins.³¹ Recently, Siegfried et al¹⁰ reported that ecto-5'-NT is inhibited by NO donors via a cGMP-independent mechanism, possibly via S-nitrosylation, in cultured renal epithelial cells. In the present study, we demonstrate that the inhibition of NO synthesis by two different inhibitors or deactivation of NO by Hb increases ecto-5'-NT activity and that two different NO donors decrease the activity of this enzyme, indicating that the inhibition of NO synthesis increases ecto-5'-NT activity and that increases in NO synthesis decrease its activity in HCAECs. It is reported that both NO and NO-generating agents induce an inactivation of PKC, suggesting that PKC is a susceptible target of NO.^{13 32} We demonstrated that the activation of ecto-5'-NT during the inhibition of NO synthesis is blunted by two different PKC inhibitors in both the in vivo hearts and in vitro HCAECs, suggesting that the inhibition of NO synthesis increases ecto-5'-NT activity through the activation of PKC. Furthermore, we demonstrated that increases in intracellular cGMP concentration by zaprinast with and without ANP did not alter ecto-5'-NT activity. These findings suggest that the inhibition of NO synthesis increases ecto-5'-NT activity through the activation of PKC via cGMP-independent mechanisms.

Clinical Implications
Recently, Quyyumi et al³³ demonstrated that increases in CBF during cardiac pacing are not regulated by NO in patients with angiographically normal coronary arteries who have risk factors for coronary artery disease, in whom the release of endothelium-dependent factor/NO is known to be impaired.³⁴ Because adenosine is also a potent vasodilator, increased adenosine levels caused by decreased NO release might compensate for the impairment of NO-mediated function in patients with risk factors for coronary artery disease.

In conclusion, when NO synthesis is suppressed, adenosine production is increased via extracellular pathways through the activation of PKC via cGMP-independent mechanisms. This increased concentration of adenosine may be an important compensatory mechanism under pathophysiological conditions in which NO release and/or NO-mediated cardiovascular functions are diminished.

	Selected Abbreviations and Acronyms

 

AMP-CP = ,ß-methyleneadenosine 5'-diphosphate ANP = atrial natriuretic peptide CBF = coronary blood flow CPP = coronary perfusion pressure FS = fractional shortening Hb = hemoglobin HBS = Hanks' balanced salt solution HCAEC = human coronary arterial endothelial cell LAD = left anterior descending coronary artery LER = lactate extraction ratio L-NAME = NG-nitro-L-arginine methyl ester L-NMMA = NG-monomethyl-L-arginine LV = left ventricular NOS = NO synthase 5'-NT = 5'-nucleotidase PKC = protein kinase C SIN-1 = morpholinosydnonimine SNP = sodium nitroprusside 8-SPT = 8-sulfophenyltheophylline

	Acknowledgments

 
The authors wish to thank Kayoko Yoshida and Yukiyo Nomura for measuring 5'-NT and PKC activities and Makoto Hasegawa for his technical assistance in the dog preparation.

Received January 8, 1997; revision received February 28, 1997; accepted March 14, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Kelm M, Schrader J. Control of coronary vascular tone by nitric oxide. Circ Res. 1990;66:1561-1575.[Abstract/Free Full Text]

2. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43:109-142.[Medline] [Order article via Infotrieve]

3. Berne RM. The role of adenosine in the regulation of coronary blood flow. Circ Res. 1980;47:807-813.[Free Full Text]

4. Kitakaze M, Hori M, Kamada T. Interaction between adenosine and alpha-adrenoceptor activity and its role of ischemia and reperfusion. Cardiovasc Res. 1992;27:18-27.

5. Minamino T, Kitakaze M, Komamura K, Node K, Takeda H, Inoue M, Hori M, Kamada T. Activation of protein kinase C increases adenosine production in the hypoxic canine coronary artery through the extracellular pathway. Arterioscler Thromb Vasc Biol. 1995;15:2298-2304.[Abstract/Free Full Text]

6. Schulz R, Triggle CR. Role of NO in vascular smooth muscle and cardiac muscle function. Trends Pharmacol Sci. 1994;15:255-259.[Medline] [Order article via Infotrieve]

7. Woolfson RG, Patel VC, Neild GH, Yellon DM. Inhibition of nitric oxide synthesis reduces infarct size by an adenosine-dependent mechanism. Circulation. 1995;91:1545-1551.[Abstract/Free Full Text]

8. Kostic MM, Schrader J. Role of nitric oxide in reactive hyperemia of the guinea pig heart. Circ Res. 1992;70:208-212.[Abstract/Free Full Text]

9. Matsunaga T, Okumura K, Tsunoda R, Tayama S, Tabuchi T, Yasue H. Role of adenosine in regulation of coronary flow in dogs with inhibited synthesis of endothelium-derived nitric oxide. Am J Physiol. 1996;270:H427-H434.[Abstract/Free Full Text]

10. Siegfried G, Amiel C, Friedlander G. Inhibition of ecto-5'-nucleotidase by nitric oxide donors: implications in renal epithelial cells. J Biol Chem. 1996;271:4659-4664.[Abstract/Free Full Text]

11. Kitakaze M, Hori M, Morioka T, Minamino T, Takashima S, Okazaki Y, Node K, Komamura K, Iwakura K, Itoh T, Inoue M, Kamada T. ₁-Adrenoceptor activation increases ecto-5'-nucleotidase activity and adenosine release in rat cardiomyocytes by activating protein kinase C. Circulation. 1995;91:2226-2234.[Abstract/Free Full Text]

12. Kitakaze M, Node K, Minamino T, Komamura K, Funaya H, Shinozaki Y, Chujo M, Mori H, Inoue M, Hori M, Kamada T. Role of activation of protein kinase C in the infarct size-limiting effect of ischemic preconditioning through activation of ecto-5'-nucleotidase. Circulation. 1996;93:781-791.[Abstract/Free Full Text]

13. Gopalakrishna R, Chen ZH, Gundimeda U. Nitric oxide and nitric oxide-generating agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding. J Biol Chem. 1993;268:27180-27185.[Abstract/Free Full Text]

14. Minamino T, Kitakaze M, Morioka T, Node K, Shinozaki Y, Chujo M, Mori H, Takeda H, Inoue M, Hori M, Kamada T. Bidirectional effects of aminophylline on myocardial ischemia. Circulation. 1995;92:1254-1260.[Abstract/Free Full Text]

15. Kitakaze M, Hori M, Morioka T, Minamino T, Takashima S, Sato H, Shinozaki Y, Chujo M, Mori H, Inoue M, Kamada T. Infarct size-limiting effect of ischemic preconditioning is blunted by inhibition of 5'-nucleotidase activity and attenuation of adenosine release. Circulation. 1994;89:1237-1246.[Abstract/Free Full Text]

16. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane B, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, Kirilovsky J. The bisindolylmaleimide GF109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991;266:15771-15781.[Abstract/Free Full Text]

17. Kobaysahi E, Nakano H, Morimoto M, Tamaoki T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1989;159:548-553.[Medline] [Order article via Infotrieve]

18. Hohorst HJ. Tissue lactate analysis. In: Bergmeyer H, ed. Methods of Enzymatic Analysis. New York, NY: Academic Press; 1963:266-270.

19. Minamino T, Kitakaze M, Morioka T, Node K, Komamura K, Takeda H, Inoue M, Hori M, Kamada T. Cardioprotection due to preconditioning correlates with increased ecto-5'-nucleotidase activity. Am J Physiol. 1996;270:H238-H244.[Abstract/Free Full Text]

20. Smith K, Varon HH, Race GJ, Paulson DL, Urshel HC, Mallams JT. Serum 5'-nucleotidase in patients with tumor in the liver. Cancer. 1965;19:1281-1285.

21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem. 1951;193:265-275.[Free Full Text]

22. Kato J, Lanier-Smith KL, Currie MG. Cyclic GMP down-regulates atrial natriuretic peptide receptors on cultured vascular endothelial cells. J Biol Chem. 1992;266:14681-14685.[Abstract/Free Full Text]

23. Jones CJH, Kuo L, Davis MJ, Defily DV, Chilian WM. Role of nitric oxide in the coronary microvascular responses to adenosine and increased metabolic demand. Circulation. 1995;91:1807-1813.[Abstract/Free Full Text]

24. Puybasset L, Bea ML, Ghaleh B, Giudicelli JF, Berdeaux A. Coronary and systemic hemodynamic effects of sustained inhibition of nitric oxide synthesis in conscious dogs: evidence for cross talk between nitric oxide and cyclooxygenase in coronary vessels. Circ Res. 1996;79:343-357.[Abstract/Free Full Text]

25. Parent R, Pare R, Lavallee M. Contribution of nitric oxide to dilation of resistance coronary vessels in conscious dogs. Am J Physiol. 1992;262:H10-H16.[Abstract/Free Full Text]

26. Luscher TF, Vanhoutte PM. The endothelium: modulator of cardiovascular function. Boca Raton, Fla: CRC Press; 1990.

27. Pearson PJ, Vanhoutte PM. Vasodilator and vasoconstrictor substances produced by the endothelium. Rev Physiol Biochem Pharmacol. 1993;122:1-67.[Medline] [Order article via Infotrieve]

28. Bodin P, Basiley D, Burnstock G. Increased flow-induced ATP release from isolated vascular endothelial cells but not smooth cells. Br J Pharmacol. 1991;103:1203-1205.[Medline] [Order article via Infotrieve]

29. Low MG, Finean JB. Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C. Biochim Biophys Acta. 1978;508:565-570.[Medline] [Order article via Infotrieve]

30. Kitakaze M, Mori H, Sakamoto H, Inoue M. Disappearance of infarct size-limiting effect is attributable to dephosphorylation process of ecto-5'-nucleotidase. Circulation. 1995;92(suppl I):I-524. Abstract.

31. Stamler JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell. 1994;8:931-936.

32. Murohara T, Parkinson SJ, Waldman SA, Lefer AM. Inhibition of nitric oxide biosynthesis promotes P-selectin expression in platelets: role of protein kinase C. Arterioscler Thromb Vasc Biol. 1995;15:2068-2075.[Abstract/Free Full Text]

33. Quyyumi A, Dakak N, Andrews NP, Gilligan DM, Panza JA, Canon RO. Contribution of nitric oxide to metabolic coronary vasodilation in the human heart. Circulation. 1995;92:320-326.[Abstract/Free Full Text]

34. Forstermann U, Mugge A, Alheid U, Haverich A, Frolich JC. Selective attenuation of endothelium-mediated vasodilation in atherosclerotic human coronary arteries. Circ Res. 1988;62:185-190.[Abstract/Free Full Text]

This article has been cited by other articles:

				J. Satriano, L. Wead, A. Cardus, A. Deng, G. R. Boss, S. C. Thomson, and R. C. Blantz Regulation of ecto-5'-nucleotidase by NaCl and nitric oxide: potential roles in tubuloglomerular feedback and adaptation Am J Physiol Renal Physiol, November 1, 2006; 291(5): F1078 - F1082. [Abstract] [Full Text] [PDF]

				A. Gamboa, R. Abraham, A. Diedrich, C. Shibao, S. Y. Paranjape, G. Farley, and I. Biaggioni Role of Adenosine and Nitric Oxide on the Mechanisms of Action of Dipyridamole Stroke, October 1, 2005; 36(10): 2170 - 2175. [Abstract] [Full Text] [PDF]

				M. Fujita, T. Minamino, H. Asanuma, S. Sanada, A. Hirata, M. Wakeno, M. Myoishi, H. Okuda, A. Ogai, K.-i. Okada, et al. Aldosterone Nongenomically Worsens Ischemia Via Protein Kinase C-Dependent Pathways in Hypoperfused Canine Hearts Hypertension, July 1, 2005; 46(1): 113 - 117. [Abstract] [Full Text] [PDF]

				R. R. Martinez, S. Setty, P. Zong, J. D. Tune, and H. F. Downey Nitric oxide contributes to right coronary vasodilation during systemic hypoxia Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1139 - H1146. [Abstract] [Full Text] [PDF]

				R. Schulz, M. Kelm, and G. Heusch Nitric oxide in myocardial ischemia/reperfusion injury Cardiovasc Res, February 15, 2004; 61(3): 402 - 413. [Abstract] [Full Text] [PDF]

				S. Sanada, K. Node, T. Minamino, S. Takashima, A. Ogai, H. Asanuma, H. Ogita, Y. Liao, M. Asakura, J. Kim, et al. Long-Acting Ca2+ Blockers Prevent Myocardial Remodeling Induced by Chronic NO Inhibition in Rats Hypertension, April 1, 2003; 41(4): 963 - 967. [Abstract] [Full Text] [PDF]

				S. Sanada, K. Node, H. Asanuma, H. Ogita, S. Takashima, T. Minamino, M. Asakura, Y. Liao, A. Ogai, J. Kim, et al. Opening of the adenosine triphosphate-sensitive potassium channel attenuates cardiac remodeling induced by long-term inhibition of nitric oxide synthesis: Role of 70-kDa S6 kinase and extracellular signal-regulated kinase J. Am. Coll. Cardiol., September 4, 2002; 40(5): 991 - 997. [Abstract] [Full Text] [PDF]

				M. Thielmann, H. Dorge, C. Martin, S. Belosjorow, U. Schwanke, A. van de Sand, I. Konietzka, A. Buchert, A. Kruger, R. Schulz, et al. Myocardial Dysfunction With Coronary Microembolization: Signal Transduction Through a Sequence of Nitric Oxide, Tumor Necrosis Factor-{alpha}, and Sphingosine Circ. Res., April 19, 2002; 90(7): 807 - 813. [Abstract] [Full Text] [PDF]

				J. D. Tune, K. N. Richmond, M. W. Gorman, and E. O. Feigl Control of Coronary Blood Flow during Exercise Experimental Biology and Medicine, April 1, 2002; 227(4): 238 - 250. [Abstract] [Full Text] [PDF]

				Y.-F. Chen, P.-L. Li, and A.-P. Zou Oxidative stress enhances the production and actions of adenosine in the kidney Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R1808 - R1816. [Abstract] [Full Text] [PDF]

				S. Sanada, M. Kitakaze, K. Node, S. Takashima, A. Ogai, H. Asanuma, Y. Sakata, M. Asakura, H. Ogita, Y. Liao, et al. Differential Subcellular Actions of ACE Inhibitors and AT1 Receptor Antagonists on Cardiac Remodeling Induced by Chronic Inhibition of NO Synthesis in Rats Hypertension, September 1, 2001; 38(3): 404 - 411. [Abstract] [Full Text] [PDF]

				M. J. Mullen, R. K. Kharbanda, J. Cross, A. E. Donald, M. Taylor, P. Vallance, J. E. Deanfield, and R. J. MacAllister Heterogenous Nature of Flow-Mediated Dilatation in Human Conduit Arteries In Vivo : Relevance to Endothelial Dysfunction in Hypercholesterolemia Circ. Res., February 2, 2001; 88(2): 145 - 151. [Abstract] [Full Text] [PDF]

				J. D. Tune, K. N. Richmond, M. W. Gorman, and E. O. Feigl KATP+ channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H868 - H875. [Abstract] [Full Text] [PDF]

				J. D. Tune, K. N. Richmond, M. W. Gorman, and E. O. Feigl Role of Nitric Oxide and Adenosine in Control of Coronary Blood Flow in Exercising Dogs Circulation, June 27, 2000; 101(25): 2942 - 2948. [Abstract] [Full Text] [PDF]

				M. B. Ganz and A. Seftel Glucose-induced changes in protein kinase C and nitric oxide are prevented by vitamin E Am J Physiol Endocrinol Metab, January 1, 2000; 278(1): E146 - E152. [Abstract] [Full Text] [PDF]

				Y. Hellsten The effect of muscle contraction on the regulation of adenosine formation in rat skeletal muscle cells J. Physiol., August 1, 1999; 518(3): 761 - 768. [Abstract] [Full Text] [PDF]

				K. O. Aley, G. McCarter, and J. D. Levine Nitric Oxide Signaling in Pain and Nociceptor Sensitization in the Rat J. Neurosci., September 1, 1998; 18(17): 7008 - 7014. [Abstract] [Full Text] [PDF]

				M. Takamura, R. Parent, and M. Lavallee Enhanced contribution of NO to exercise-induced coronary responses after alpha -adrenergic receptor blockade Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H508 - H515. [Abstract] [Full Text] [PDF]

				M. Thielmann, H. Dorge, C. Martin, S. Belosjorow, U. Schwanke, A. van de Sand, I. Konietzka, A. Buchert, A. Kruger, R. Schulz, et al. Myocardial Dysfunction With Coronary Microembolization: Signal Transduction Through a Sequence of Nitric Oxide, Tumor Necrosis Factor-{alpha}, and Sphingosine Circ. Res., April 19, 2002; 90(7): 807 - 813. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Minamino, T. Articles by Hori, M. Search for Related Content PubMed PubMed Citation Articles by Minamino, T. Articles by Hori, M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE