AJP - Heart Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 289: H916-H923, 2005. First published April 8, 2005; doi:10.1152/ajpheart.01014.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/2/H916    most recent
01014.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Orie, N. N.
Right arrow Articles by Moore, K. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Orie, N. N.
Right arrow Articles by Moore, K. P.

S-nitroso-albumin carries a thiol-labile pool of nitric oxide, which causes venodilation in the rat

Nelson N. Orie,1 Patrick Vallance,2 Dean P. Jones,3 and Kevin P. Moore1

1Centres for Hepatology, and 2Clinical Pharmacology, Department of Medicine, Royal Free and University College Medical School, University College, London, United Kingdom; and 3Department of Biochemistry, School of Medicine, Emory University, Atlanta, Georgia

Submitted 5 October 2004 ; accepted in final form 4 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is now established that S-nitroso-albumin (SNO-albumin) circulates at low nanomolar concentrations under physiological conditions, but concentrations may increase to micromolar levels during disease states (e.g., cirrhosis or endotoxemia). This study tested the hypothesis that high concentrations of SNO-albumin observed in some diseases modulate vascular function and that it acts as a stable reservoir of nitric oxide (NO), releasing this molecule when the concentrations of low-molecular-weight thiols are increased. SNO-albumin was infused into rats to increase the plasma concentration from <50 nmol/l to ~4 µmol/l. This caused a 29 ± 6% drop in blood pressure, 20 ± 4% decrease in aortic blood flow, and a 25 ± 14% reduction of renal blood flow within 10 min. These observations were in striking contrast to those of an infused arterial vasodilator (hydralazine), which increased aortic blood flow, and suggested that SNO-albumin acts primarily as a venodilator in vivo. This was confirmed by the observations that glyceryl trinitrate (a venodilator) led to similar hemodynamic changes and that the hemodynamic effects of SNO-albumin are reversed by infusion of colloid. Infusion of N-acetylcysteine into animals with artificially elevated plasma SNO-albumin concentrations led to the rapid decomposition of SNO-albumin in vivo and reproduced the hemodynamic effects of SNO-albumin infusion. These data demonstrate that SNO-albumin acts primarily as a venodilator in vivo and represents a stable reservoir of NO that can release NO when the concentrations of low-molecular-weight thiols are elevated.

S-nitrosothiols; cardiac physiology; N-acetylcysteine

THE MAJOR CIRCULATING PLASMA S-nitrosothiol is S-nitroso-albumin (SNO-albumin) (30), which has been proposed to act as a reservoir of nitric oxide (NO) within the circulation, transporting and releasing NO into vascular beds to cause vasodilation (9, 26). Although it was initially suggested that the concentrations of S-nitrosothiols in plasma are as high as 7 µmol/l (30), the most recent reports agree that the concentrations are in the nanomolar range in health (19, 23, 33, 37) but may increase to low micromolar concentrations in diseases such as endotoxemia (16, 22). For example, Jourd’heuil et al. (16) observed that plasma concentrations increased from ~120 to >400 nmol/l at 5 h after injection of lipopolysaccharide (LPS), and our group (22) observed that plasma concentrations of S-nitrosothiols increased to >1.2 µmol/l after injection of LPS into cirrhotic rats at 2 h. Because both sepsis and cirrhosis are associated with major changes in vascular function, including increased cardiac output and systemic vasodilatation (21, 32, 35), this suggested to us, and to others (33), that these hemodynamic changes may be secondary to increased circulating levels of S-nitrosothiols.

However, one of the striking findings of the study on the effects of LPS in cirrhosis was that these rats did not develop hypotension, despite very high plasma concentrations of S-nitrosothiols (22). This was surprising because both Scharfstein et al. (26) as well as Keaney et al. (9) reported that the bolus injection of SNO-albumin at 300 nmol/kg into rabbits or dogs caused a ~35% decrease in blood pressure, and our group (22) had also observed that the bolus injection of SNO-albumin caused a transient decrease in mean arterial pressure (MAP) in normal rats. This observation led us to investigate the hemodynamic responses of normal rats to infusion of SNO-albumin to achieve steady-state concentrations similar to those observed in disease states. This approach was more "physiological" because it enabled plasma concentrations to increase gradually over 1–2 h, thus mimicking what actually happens in vivo.

Circulating SNO-albumin can undergo transnitrosation reactions with low molecular weight thiols in plasma to form less stable intermediates such as S-nitrosoglutathione or S-nitrosocysteine, which in turn can undergo reductive or transition metal-dependent decomposition to release NO (26, 27, 34, 36). This may be important therapeutically, because cysteine (Cyst) may be infused as part of an intravenous infusion of amino acids and N-acetylcysteine (NAC) is frequently infused into patients with severe liver disease or multiorgan failure in Europe. Thus it has been shown that infusion of NAC in acute liver failure or cirrhosis causes transient vasodilatation and an increase in plasma cGMP (6, 13). One possible explanation for these data is that infusion of NAC may have caused the transient release of NO and vasodilatation in patients with liver disease from circulating S-nitrosothiols.

The aim of the present study was to test the hypothesis that the concentrations of SNO-albumin that are found in disease states modulate vascular function and second to determine whether SNO-albumin acts as a stable reservoir of NO that releases NO when the concentrations of low-molecular-weight thiols are increased.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Synthesis of SNO-albumin. SNO-albumin was prepared as previously described (20). In brief, human albumin (20 mg/ml) was initially treated briefly with 2 mmol/l dithiothreitol in PBS to reduce the Cys-34 thiol group. It was then dialyzed for 48 h against 5 x 3 liters of PBS containing diethylenetriaminepentaacetic acid (100 µmol/l) and diluted to a final concentration of albumin at 0.15 mmol/l. S-nitrosocysteine (~100 mmol/l) was prepared fresh by reacting L-cysteine hydrochloride (100 mmol/l) with sodium nitrite (100 mmol/l) at pH 2. S-nitrosocysteine was added to reduced albumin in a ratio of 9 to 1 and stirred at room temperature for 30 min in the dark to form SNO-albumin. The yield of SNO-albumin is usually >80% (with respect to reactive thiols). Any unreacted thiol groups were then alkylated with N-ethylmaleimide (NEM; 1 mmol/l) at room temperature, followed by dialysis at 4°C in the dark against 4 x 3 liters of PBS containing 100 µmol/l diethylenetriaminepentaacetic acid for 48 h. SNO-albumin was stored at –80°C, and its concentration (~120–140 µmol/l) was determined immediately before use by the Saville reaction (25).

Animal studies. All experiments were performed in male Sprague-Dawley rats (Harlan) weighing 280–350 g; animal experiments were approved by the Home Office. Rats were housed in the central animal facility of the university with food and water available ad libitum. They were anesthetized with isoflurane (1.5%, Baxter) vaporized with air (Vatex 3 calibrated vaporizer, Cheshire, UK). Delivery of the anesthetic was maintained via a tracheal cannula while the animal respired spontaneously. Body temperature was maintained at ~37°C with the use of a heated pad. The right jugular and right femoral veins were cannulated for drug infusion and bolus injection, respectively. The jugular vein cannula was connected to a motor-driven syringe pump (model SE200B, Vial Medicals) for infusion of fluids or drugs. The left carotid artery was cannulated, and blood pressure was monitored using a Sensonor pressure transducer and the PowerLab data acquisition system (AD Instruments). A midline laparotomy was carried out, and 1) the urinary bladder was cannulated to prevent urinary retention, and 2) transonic flow probes (Transonics System) were placed on the lower abdominal aorta (below the left renal artery) and on the left renal artery. Arterial blood flow rates were measured by a flow meter (model T206, Transonics System). Three needle electrodes were implanted subcutaneously at the two upper limbs and one at the lower limb for ECG monitoring and measurement of heart rate.

Vascular resistance was calculated as follows:

where RBF is renal blood flow and ABF is aortic blood flow.

Hemodynamic studies. After surgery, normal saline was infused continuously at 20 ml·kg–1·h–1 until drug infusions were commenced. Baseline measurements were made after stabilization of basal blood flows and pressure for at least 15 min.

There were four groups of normal rats studied. These were 1) rats infused with albumin in saline (control-albumin, 90 µmol/l; this was identical to the albumin base concentration in the SNO-albumin infusion; n = 4), 2) rats infused with SNO-albumin (1.8 µmol·kg–1·h–1; n = 5), 3) rats infused with glyceryl trinitrate (GTN; 6 mg·kg–1·h–1; n = 4), and 4) rats infused with hydralazine (6 mg·kg–1·h–1; n = 6). All infusions were carried out in normal saline at a volume rate of 20 ml·kg–1·h–1. Further studies were carried out as described for the measurements of plasma thiols or to assess the effects of volume challenge.

Sixty minutes after the onset of SNO-albumin or drug infusion, NAC (0.8 mmol/kg in saline, injection volume = 1.25 ml/kg) or an equal volume of saline was injected as a bolus via the femoral vein, and the hemodynamic effects were monitored for a further 30 min. Infusion of SNO-albumin or other drugs was continued during this time. At the end of each experiment, blood was collected via the carotid artery into a tube containing EDTA (5 mmol/l final concentration) and NEM (5 mmol/l final concentration). The blood was centrifuged at 8,500 g in an Eppendorf Microfuge for 1 min to separate the plasma. NEM was added to the plasma to give a final concentration of 5 mmol/l and thus stabilize and prevent the decomposition of S-nitrosothiols. Samples were stored at –20°C until assay. Thus these data give the plasma S-nitrosothiol concentration at the end of drug infusion and assess the effects of a bolus injection of NAC on circulating RSNO concentrations.

Effect of SNO-albumin on plasma thiol concentrations. To measure the effect of infusion of SNO-albumin on plasma concentrations of thiols, a further series of experiments (n = 4 each) were carried out in which no hemodynamic measurements were made. This was to avoid the effects of blood sampling and volume replacement on systemic hemodynamics in the preceding studies. Fifteen minutes after the start of saline infusion (20 ml·kg–1·h–1), a single blood sample (1 ml) was taken from the carotid artery for measurement of baseline concentrations of plasma thiols, followed by bolus injection of 1 ml of saline to replace volume. Fifteen and sixty minutes after the onset of SNO-albumin or control-albumin infusion, a blood sample (1 ml) was collected as above. Sixty-five minutes after the start of SNO-albumin infusion, a bolus of NAC (0.8 mmol/kg) was injected, and a further 1 ml of blood was collected 10 min later. Each blood sample was quickly transferred to two tubes containing a preservative solution (vol/vol) previously described by Jones et al. (14) and gently inverted for mixing. The tubes were then spun at 8,500 g in a microcentrifuge for 1 min to remove the cells. Aliquots (200 µl) of supernatant were transferred to another set of two tubes containing perchloric acid and inverted to mix. These were then frozen in dry ice and stored at –80°C. All samples were analyzed within 1 mo of collection.

Measurement of plasma S-nitrosothiol concentrations. Plasma S-nitrosothiols were measured using a chemiluminescence-based assay previously described (19). In brief, samples were stabilized with NEM (5 mmol/l), and any nitrite contaminant was removed by adding 2.5% sulfanilamide in 1 M hydrochloric acid to the sample in a ratio of 1:5. Each sample was then injected into a purge vessel containing refluxing glacial acetic acid, potassium iodide, and copper sulfate. This method eliminates interference from circulating nitrite or nitrate, and the NO signal is quantified by its gas-phase chemiluminescence reaction with ozone in an NO analyzer.

Measurement of plasma thiol concentrations. Thiol concentrations were measured by HPLC according to the method described by Jones et al. (14, 15). In brief, after centrifugation of the samples to pellet protein, aliquots (300 µl) were transferred to fresh tubes and derivatized with iodoacetic acid followed by dansyl chloride. Thereafter, aliquots (20 µl) were analyzed on a 3-aminopropyl column (5 µm; 4.6 mm x 25 cm; Custom LC, Houston, TX) with a gradient formed from 80% methanol-water and 4 M sodium acetate, pH 4.6, containing 64% methanol. Fluorescence was monitored with bandpass filters, at 305- to 395-nm excitation and 510- to 650-nm emission (Gilson Medical Electronics, Middleton, WI), with two detectors in series with different sensitivity settings to facilitate simultaneous measurement of cysteine disulfide (Cyss) and glutathione disulfide (GSSG). Quantitation was obtained by integration relative to the internal standard, with validation relative to external standard.

Statistics. Data were acquired with Chart version 4.1.2 software from AD Instruments. MAP was calculated according to this formula: 1/3 pulse pressure (systolic – diastolic pressures) + diastolic pressure. Data are presented as means ± SE. MAP, ABF, RBF, and heart rate values presented were taken every 5 min for the duration of each experiment. Statistical analysis was by Student’s t-test (unpaired) and ANOVA as appropriate. P values <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Infusion of SNO-albumin lowers blood pressure. At baseline, the heart rate was 372 ± 4 beats/min and MAP was 135 ± 2 mmHg, with an ABF of 35.4 ± 0.9 ml/min and a RBF of 6.3 ± 0.4 ml/min. Within 10–15 min of commencement of infusion of SNO-albumin, there was a transient 29 ± 6% decrease in MAP (Fig. 1), and this was associated with a 4 ± 2% decrease in heart rate and 20 ± 4% decrease in ABF (Fig. 1). Aortic vascular resistance (AVR) was unchanged. Infusion of SNO-albumin also caused a 25 ± 14% decrease of RBF, which was associated with a moderate increase in renal vascular resistance (RVR) (see Fig. 2). These hemodynamic changes returned toward basal levels over 25–35 min.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 1. Summary of the changes in mean arterial blood pressure (MAP), heart rate (HR), aortic blood flow (ABF), and renal blood flow (RBF) during the infusion of control-albumin (n = 4; open squares) or S-nitroso (SNO)-albumin (n = 5; closed squares) in rats. Infusion of SNO-albumin or control-albumin was commenced at 15 min (1st arrows). A bolus of N-acetylcysteine (NAC) was injected 65 min from the onset of agent infusion (2nd arrows). All values were recorded every 5 min and represented as percentage of baseline values.

 


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Summary of the changes in aortic and renal vascular resistance (AVR and RVR, respectively) during the infusion of SNO-albumin (top; n = 6) or hydralazine (bottom; n = 4) in rats. Infusions were commenced at 15 min of stable baseline recording (1st arrows). A bolus of NAC was injected at the times indicated (2nd arrows).

 
Comparison of SNO-albumin with other vasodilators. The initial hemodynamic changes caused by infusion of SNO-albumin were surprising because they are not characteristic of an arterial vasodilator. Normally, a drug that causes arterial vasodilatation leads to an increase in cardiac output and a decrease in systemic vascular resistance, whereas the decrease in ABF after SNO-albumin infusion suggested that cardiac output had decreased. Therefore, we first compared and contrasted the responses observed with the arterial vasodilator hydralazine. Infusion of hydralazine caused an immediate fall in blood pressure that was slow to recover (Fig. 3), with a compensatory 7 ± 3% increase of ABF and a 54 ± 10% decrease of RBF, which remained depressed throughout the infusion period (Fig. 3). Infusion of hydralazine led to a 32 ± 6% decrease in AVR and a 75 ± 26% increase in RVR. These data are shown in Fig. 2, bottom, to enable direct comparison with the effects of SNO-albumin infusion. These results are exactly as one would predict for an arterial vasodilator and suggested that SNO-albumin was behaving differently.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 3. Summary of the changes in MAP, HR, ABF, and RBF during the infusion of glyceryl trinitrate (GTN; top; n = 5) or hydralazine (bottom; n = 6) in rats. Infusion of both agents was commenced at 15 min (1st arrows). A bolus of NAC was injected 65 min from the onset of agent infusion (2nd arrows). All values were recorded every 5 min and represented as percentage of baseline values.

 
We next considered the possibility that SNO-albumin was behaving as a venodilator and therefore compared its effects to infusion of GTN. Infusion of GTN evoked similar hemodynamic changes to those observed with SNO-albumin (Fig. 3). However, there were some differences, in particular the decrease in MAP did not recover fully during GTN infusion and the decrease in MAP was greater with GTN, presumably because the dose was not equivalent. One of the characteristics of a venodilator is that their hemodynamic effects can be reversed by volume expansion. Therefore, in a separate series of studies, the effect of colloid infusion on systemic hemodynamics was determined. SNO-albumin or GTN were infused as previously, but after ~15 min, when their hemodynamic changes were maximal, a bolus of 6 ml/kg of a synthetic hydroxyethyl starch (EloHaes, Fresenius Kabi) was injected via the femoral vein. This volume challenge caused an immediate reversal of the hemodynamic changes, with concomitant and immediate increases in MAP, ABF, and RBF in animals infused with SNO-albumin as well as those infused with GTN. A typical tracing showing the response to this test is shown in Fig. 4. To seek further evidence of venodilation, the central venous pressure (CVP) was monitored in five rats during infusion of SNO-albumin. SNO-albumin infusion caused a small but significant decrease in CVP consistent with venodilation (Fig. 5).



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 4. Typical tracing showing the effect of a volume challenge produced by a bolus injection of EloHaes (6 ml/kg) on the changes in MAP, ABF, and RBF produced by SNO-albumin infusion.

 


View larger version (9K):
[in this window]
[in a new window]
  		Fig. 5. Changes in central venous pressure (CVP) after the infusion of SNO-albumin (SNALB) in normal rats. There was a 10–30% decrease in CVP in all animals studied. Right: typical tracings of the changes in MAP and CVP.

 
The hemodynamic effects of SNO-albumin are enhanced by NAC. Circulating SNO-albumin can undergo rapid transnitrosation reactions with low-molecular-weight thiols to form a less stable species. However, this is limited by the concentration of low-molecular-weight thiols (typically <20 µmol/l). To test the hypothesis that infusion of a low-molecular-weight thiol would cause NO liberation and further hemodynamic changes and catalyze the decomposition of SNO-albumin, a bolus injection of NAC or saline was administered after 60 min of infusion of SNO-albumin. Injection of NAC caused a 24 ± 7% decrease in arterial pressure and a 2 ± 4% and 24 ± 10% decrease of ABF and RBF, respectively, within 5 min of its administration. Both AVR and RVR were relatively unaffected (see Fig. 2). Infusion of NAC into rats infused with control albumin, hydralazine, or GTN was associated with variable but minor hemodynamic changes. Thus infusion of NAC caused a modest increase of ABF in animals infused with hydralazine or GTN and a modest reduction of RBF during GTN or hydralazine infusion (Fig. 3).

Effects of NAC on plasma S-nitrosothiol concentrations. To determine whether infusion of NAC caused decomposition of circulating plasma S-nitrosothiols, the plasma concentrations of S-nitrosothiols (predominantly SNO-albumin) were measured in samples collected in the absence or after a bolus injection of NAC. The plasma concentrations of S-nitrosothiols were 48 ± 10 nmol/l for animals infused with control albumin and 4.1 ± 0.7 µmol/l for those infused with SNO-albumin without NAC (Fig. 6). For animals that received a bolus injection of NAC during SNO-albumin infusion, the plasma concentration of S-nitrosothiols decreased from 4.1 to 1.0 ± 0.2 µmol/l (P < 0.001, Fig. 6).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 6. Concentration of circulating plasma S-nitrosothiols during an infusion of control-albumin or SNO-albumin (1.8 µmol·kg–1·h–1) and after a bolus injection of NAC (n = 5).

 
Plasma thiol concentrations. A key observation during these studies was the rapid decrease of blood pressure and subsequent recovery during continued SNO-albumin infusion. This was not observed with GTN (Fig. 3). One possible explanation for the rapid hemodynamic recovery observed is that SNO-albumin leads to the consumption and oxidation of the low-molecular-weight thiols, and this can be reversed by NAC, leading to further hemodynamic change. To test this hypothesis, the plasma concentrations of the major low-molecular-weight thiol pool were measured during the infusion of SNO-albumin or control-albumin alone in five animals. Low-molecular-weight thiols measured included Cys and glutathione (GSH) and their disulfides, (Cyss and GSSG, respectively) before and during SNO-albumin infusion. The results are shown in Table 1. The concentrations of Cys, GSH, Cyss, and GSSG remained relatively unchanged during SNO-albumin infusion. Similarly, there were no changes in the redox potentials of the two thiol-disulfide pools (GSSG/GSH and Cyss/Cys) during SNO-albumin infusion. However, after NAC injection Cys, Cyss and redox potential of Cyss/Cys increased significantly (P < 0.05). These data do not support the concept that SNO-albumin infusion leads to consumption of low-molecular-weight thiols in vivo.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of SNO-albumin infusion on plasma concentrations of low-molecular-weight thiols

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
SNO-albumin is the most abundant circulating S-nitrosothiol in plasma, but its role in the control of vascular function remains unknown. Several studies have shown that the bolus injection of S-nitrosothiols, including SNO-albumin, causes an immediate reduction of blood pressure (8, 9, 22, 24, 26), and the general assumption has been that this is secondary to systemic vasodilation. This assumption is based partly on the finding that various S-nitrosothiols have been shown to cause relaxation of preconstricted vascular rings (9, 11, 12). Other studies have shown that infusion of SNO-albumin into dogs whose reflex responses had been prevented by autonomic blockade resulted in a modest dose-dependent increase in coronary blood flow, and systemic infusion of SNO-albumin led to a sustained decrease of blood pressure, together with a modest decrease in left ventricular end diastolic pressure and pulmonary capillary wedge pressure (9).

Although it is clear that injection of a bolus of SNO-albumin causes an acute decrease in blood pressure, no study has evaluated its effects as a "vasodilator." In preliminary studies, we found that infusion of SNO-albumin to achieve a plasma concentration of ~1 µmol/l over 60–90 min had no effect on blood pressure or ABF in normal rats (data not shown). These data are consistent with our previous study (22) in which we showed that injection of endotoxin into cirrhotic rats led to a marked increase of plasma S-nitrosothiols from 50 nmol/l to ~1 µmol/l, and yet these animals did not develop hypotension. Therefore, in the present study, we infused SNO-albumin to achieve a higher plasma concentration (~4 µmol/l), which was sufficient to cause a fall in blood pressure and alter aortic and RBF. Contrary to the general perception that SNO-albumin is a systemic arterial vasodilator, the present study shows that infusion of a high dose of SNO-albumin predominantly causes venodilation and a secondary decrease in cardiac output and blood pressure. Three observations support our conclusion that venodilation is the most dominant vascular response. First, the vascular effects of SNO-albumin were similar to those of GTN, a known venodilator (11). Second, blood pressure and ABF after SNO-albumin were normalized by infusion of colloid to increase plasma volume. Third, infusion of SNO-albumin led to a modest decrease of CVP. The mechanisms that lead to an agent such as SNO-albumin or GTN to act predominantly as a venodilator are not clear. One major difference between arteries and veins is the intensity or level of exposure to endogenous NO. Thus veins are exposed to relatively little NO under normal conditions, and this may increase the sensitivity of guanylate cyclase to NO-mediated vasodilation. Thus Kojda et al. (10) have shown that exposure of coronary arteries to micromolar concentrations of NO inhibits vascular bioactivation of GTN and leads to desensitization of soluble guanylate cyclase. Most studies that have tried to explain the venoselectivity of nitrates have suggested that there may be increased venous activity of the enzymes necessary for the bioactivation of nitrates. GTN undergoes biotransformation in vascular tissue to yield 1,2-glyceryl dinitrate (1,2-GDN), 1,3-GDN, inorganic nitrite, and NO or an S-nitrosothiol (2, 7). Early studies suggested that deoxyhemoglobin may be involved in the bioactivation of GTN because GTN breaks down in the presence of deoxyhemoglobin to yield nitrite (1, 18). Other studies have suggested that bioactivation of GTN may involve a glutathione S-transferase, which also forms inorganic nitrite from GTN (17, 28), and recent studies from the laboratory of Stamler and colleagues (2, 31) identified a nitrate reductase (mitochondrial aldehyde dehydrogenase) that specifically catalyzes the formation of 1,2-GDN and nitrite from GTN.

Although much of the focus of this work has centered on the formation of 1,2-GDN and subsequent venodilation, this type of pathway is unlikely to be invoked as the mechanism of action for SNO-albumin. However, one emerging theme in all of these pathways is the formation of inorganic nitrite, which we now know can be converted to NO through a deoxyhemoglobin-dependent pathway (3). Although SNO-albumin can release NO when exposed to low-molecular-weight thiols, much of the NO appears as inorganic nitrite, at least in vitro (data not shown); the formation of nitrite during SNO-albumin infusion was not measured. Thus nitrite, released through thiol-dependent metabolism of SNO-albumin (or by the bioactivation of GTN), may lead to venodilation through a deoxyhemoglobin-dependent pathway under the relatively hypoxic conditions of the venous circulation and explain the predominant venoselectivity of SNO-albumin observed in this study.

A second observation of this study is that the reduction of blood pressure produced by SNO-albumin was transient even though the plasma concentrations remained high. We have previously shown that SNO-albumin is relatively stable, with a half-life of ~30 min in vivo, and that plasma S-nitrosothiols undergo rapid decomposition in the presence of low-molecular-weight thiols (19, 20). Studies by Scharfstein et al. (26) and Tsikas et al. (34) have previously demonstrated that Cys or NAC can undergo transnitrosation reactions with SNO-albumin in vivo, and we observed that infusion of NAC was accompanied by a marked decrease of plasma SNO-albumin concentrations (Fig. 6), consistent with the concept that NAC reacts with SNO-albumin to form S-nitroso-N-acetylcysteine, which then releases NO acutely (Fig. 7). The fact that bolus injection of SNO-albumin, which leads to a transient but very high plasma concentration of SNO-albumin, causes transient hypotension in vivo suggests that SNO-albumin may undergo transnitrosation to form a more labile but finite pool of S-nitrosothiols, which then releases NO. To investigate whether a sustained increase in plasma S-nitrosothiol concentration could lead to consumption of circulating redox-active thiols, which normally catalyze the decomposition or transfer of NO into cells, we measured the concentration of redox active thiols during infusion of SNO-albumin and observed no change. There are several potential mechanisms to explain this. For example, the measurement of plasma thiols over the time of infusion may not be sufficiently accurate to detect the changes in plasma thiol concentration, particularly if the reductive pathways that lead to the maintenance of extracellular thions keep pace with the changes induced by SNO-albumin infusion. Second, there are data to suggest that circulating S-nitrosothiols may release NO at the cell interface through cell surface thiols. Thus Zai et al. (38) have shown that a protein disulfide isomerase catalyzes the transnitrosation of a cell surface thiol and regulates intracellular transfer of NO. Whether this pathway is saturable is unknown, but, if so, it would explain why the effects of SNO-albumin are limited or transient. This system may also confer venoselectivity, because there may be differential expression on venous endothelium compared with arterial endothelium.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 7. Schematic representation of transnitrosation of low-molecular-weight thiol (LMW-SH) by SNO-albumin (alb-SNO) and the subsequent release of NO, leading to venodilation. VSM, vascular smooth muscle cell.

 
The present study evaluated the effect of "high" plasma concentrations of SNO-albumin on systemic hemodynamics. There has been much debate about the concentration of circulating S-nitrosothiols ever since the first publication reported concentrations of SNO-albumin at ~7 µmol/l (30). Most investigators in the field, who use assays that have been well validated, have observed concentrations that are 100- to 500-fold lower than those first reported (19, 23, 33, 37). The validity and specificity of the method based on photolysis have also recently been questioned in a detailed study (4). Despite this, publications are still emerging that challenge the findings of numerous investigators, without an equally rigorous validation of the assays under contention (5, 29). The observation that high plasma concentrations of SNO-albumin are needed to cause venodilation suggests that endogenous circulating SNO-albumin has little impact on vascular function in vivo. However, whether the human circulation has similar sensitivity to SNO-albumin is unknown at present. The present findings demonstrate why high circulating plasma concentrations of SNO-albumin are not associated with catastrophic cardiovascular collapse in certain diseases and may explain why infusion of NAC causes a decrease in blood pressure in patients with acute or chronic liver failure but not in healthy individuals (6, 13).

We conclude that SNO-albumin acts primarily as a venodilator in vivo, leading to a reduction of ABF and RBF. These effects can be enhanced in vivo by infusion of a low-molecular-weight thiol such as NAC.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Medical Research Council, UK, and National Institutes of Health Division of Research Resources Grant M01 RR-00039.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. P. Moore, Centre for Hepatology, Dept. of Medicine, Royal Free and Univ. College Medical School, Univ. College London, London NW3 2PF, UK (e-mail: k.moore{at}medsch.ucl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bennett BM, Kobus SM, Brien JF, Nakatsu K, and Marks GS. Requirement for reduced, unliganded hemoprotein for the hemoglobin- and myoglobin-mediated biotransformation of glyceryl trinitrate. J Pharmacol Exp Ther 237: 629–635, 1986.[Abstract/Free Full Text]
  2. Chen Z, Zhang J, and Stamler JS. Identification of the enzymatic mechanism of nitroglycerin bio-activation. Proc Natl Acad Sci USA 99: 8306–8311, 2002.[Abstract/Free Full Text]
  3. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO III, and Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasolidates the human circulation. Nat Med 9: 1498–1505, 2003.[CrossRef][ISI][Medline]
  4. Dejam A, Kleinbongard P, Rassaf T, Hamada S, Gharini P, Rodriguez J, Feelisch M, and Kelm M. Thiols enhance NO formation from nitrate photolysis. Free Radic Biol Med 35: 1551–1559, 2003.[CrossRef][ISI][Medline]
  5. Foster MW, Pawloski JR, Singel DJ, and Stamler JS. Role of circulating S-nitrosothiols in control of blood pressure. Hypertension 45: 15–17, 2005.[Free Full Text]
  6. Harrison PM, Wendon JA, Gimson AE, Alexander GJ, and Williams R. Improvement by acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure. N Engl J Med 324: 1852–1857, 1991.[Abstract]
  7. Hill KE, Hunt RW, Jones R, Hoover RL, and Burk RF. Metabolism of nitroglycerin by smooth muscle cells. Involvement of glutathione and glutathione S-transferase. Biochem Pharmacol 43: 561–566, 1992.[CrossRef][ISI][Medline]
  8. Kashiba M, Kasahara E, Chien KC, and Inoue M. Fates and vascular action of S-nitrosoglutathione and related compounds in the circulation. Arch Biochem Biophys 363: 213–218, 1999.[CrossRef][ISI][Medline]
  9. Keaney JF Jr, Simon DI, Stamler JS, Jaraki O, Scharfstein J, Vita JA, and Loscalzo J. NO forms an adduct with serum albumin that has endothelium-derived relaxing factor-like properties. J Clin Invest 91: 1582–1589, 1993.[ISI][Medline]
  10. Kojda G, Patzner M, Hacker A, and Noack E. Nitric oxide inhibits vascular bio-activation of glyceryl trinitrate: a novel mechanism to explain preferential venodilatation of organic nitrates. Mol Pharmacol 53: 547–554, 1998.[Abstract/Free Full Text]
  11. Kowaluk EA, Poliszczuk R, and Fung HL. Tolerance to relaxation in rat aorta: comparison of an S-nitrosothiol with nitroglycerin. Eur J Pharmacol 144: 379–383, 1987.[CrossRef][ISI][Medline]
  12. Kukreja RC, Wei EP, Kontos HA, and Bates JN. Nitric oxide and S-nitroso-L-cysteine as endothelium-derived relaxing factors from acetylcholine in cerebral vessels in cats. Stroke 24: 2010–2014, 1993.[Abstract/Free Full Text]
  13. Jones AL, Bangash IH, Bouchier IA, and Hayes PC. Portal and systemic haemodynamic action of N-acetylcysteine in patients with stable cirrhosis. Gut 35: 1290–1293, 1994.[Abstract/Free Full Text]
  14. Jones DP, Carlson JL, Mody VC, Cai J, Lynn MJ, and Sternberg P. Redox state of glutathione in human plasma. Free Radic Biol Med 28: 625–635, 2000.[CrossRef][ISI][Medline]
  15. Jones DP, Carlson JL, Samiec PS, Sternberg P Jr, Mody VC Jr, Reed RL, and Brown LA. Glutathione measurement in human plasma. Evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin Chim Acta 275: 175–184, 1998.[CrossRef][ISI][Medline]
  16. Jourd’heuil D, Gray L, and Grisham MB. S-nitrosothiol formation in blood of lipopolysaccharide-treated rats. Biochem Biophys Res Commun 273: 22–26, 2000.[CrossRef][ISI][Medline]
  17. Lau DT, Chan EK, and Benet LZ. Glutathione S-transferase-mediated metabolism of glyceryl trinitrate in subcellular fractions of bovine coronary arteries. Pharm Res 9: 1460–1464, 1992.[CrossRef][ISI][Medline]
  18. Marks GS, McLaughlin BE, MacMillan HF, Nakatsu K, and Brien JF. Differential biotransformation of glyceril trinitrate by red blood cell-supernatant fraction and pulmonary vein homogenate. Can J Physiol Pharmacol 67: 417–422, 1989.[ISI][Medline]
  19. Marley R, Feelisch M, Holt S, and Moore K. A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols. Free Radic Res 32: 1–9, 2000.[ISI][Medline]
  20. Marley R, Patel RP, Orie N, Ceaser E, Darley-Usmar V, and Moore K. Formation of nanomolar concentrations of S-nitroso-albumin in human plasma by nitric oxide. Free Radic Biol Med 31: 688–696, 2001.[CrossRef][ISI][Medline]
  21. Martin PY, Gines P, and Schrier RW. Nitric oxide as a mediator of hemodynamic abnormalities and sodium and water retention in cirrhosis. N Engl J Med 339: 533–534, 1998.[Free Full Text]
  22. Ottesen LH, Harry D, Frost M, Davies S, Khan K, Halliwell B, and Moore K. Increased formation of S-nitrothiols and nitrotyrosine in cirrhotic rats during endotoxemia. Free Radic Biol Med 31: 790–798, 2001.[CrossRef][ISI][Medline]
  23. Rassaf T, Bryan NS, Kelm M, and Feelisch M. Concomitant presence of N-nitroso and S-nitroso proteins in human plasma. Free Radic Biol Med 33: 1590–1596, 2002.[CrossRef][ISI][Medline]
  24. Ricardo KF, Shishido SM, de Oliveira MG, and Krieger MH. Characterization of the hypotensive effect of S-nitroso-N-acetylcysteine in normotensive and hypertensive conscious rats. Nitric Oxide 7: 57–66, 2002.[CrossRef][ISI][Medline]
  25. Saville B. A scheme for the colorimetric determination of microgram amounts of thiols. Analyst 83: 670–672, 1958.[CrossRef]
  26. Scharfstein JS, Keaney JF Jr, Slivka A, Welch GN, Vita JA, Stamler JS, and Loscalzo J. In vivo transfer of nitric oxide between a plasma protein-bound reservoir and low molecular weight thiols. J Clin Invest 94: 1432–1439, 1994.[ISI][Medline]
  27. Singh RJ, Hogg N, Joseph J, and Kalyanaraman B. Mechanism of nitric oxide release from S-nitrosothiols. J Biol Chem 271: 18596–18603, 1996.[Abstract/Free Full Text]
  28. Singhal SS, Piper JT, Srivastava SK, Chaubey M, Bandorowicz-Pikula J, Awasthi S, and Awasthi YC. Rabbit aorta glutathione S-transferases and their role in bioactivation of trinitroglycerin. Toxicol Appl Pharmacol 140: 378–386, 1996.[CrossRef][ISI][Medline]
  29. Stamler JS. S-nitrosothiols in the blood: roles, amounts, and methods of analysis. Circ Res 94: 414–417, 2004.[Free Full Text]
  30. Stamler JS, Jaraki O, Osborne J, Simon DI, Keaney J, Vita J, Singel D, Valeri CR, and Loscalzo J. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci USA 89: 7674–7677, 1992.[Abstract/Free Full Text]
  31. Sydow K, Daiber A, Oelze M, Chen Z, August M, Wendt M, Ullrich V, Mülsch A, Schulz E, Keaney JF Jr, Stamler JS, and Münzel T. Central role of mitochondrial aldehyde dehydrogenase and reactive oxygen species in nitroglycerin tolerance and cross-tolerance. J Clin Invest 113: 482–489, 2004.[CrossRef][ISI][Medline]
  32. Thijs LG, Schneider AJ, and Groeneveld AB. The haemodynamics of septic shock. Intensive Care Med 16, Suppl 3: S182–S186, 1990.[CrossRef]
  33. Tsikas D, Sandmann J, and Frolich JC. Measurement of S-nitrosoalbumin by gas chromatography-mass spectrometry. III. Quantitative determination in human plasma after specific conversion of the S-nitroso group to nitrite by cysteine and Cu2+ via intermediate formation of S-nitrosocysteine and nitric oxide. J Chromatogr B Analyt Technol Biomed Life Sci 772: 335–346, 2002.[CrossRef][ISI][Medline]
  34. Tsikas D, Sandmann J, Rossa S, Gutzki FM, and Frolich JC. Investigations of S-transnitrosylation reactions between low- and high-molecular-weight S-nitroso compounds and their thiols by high-performance liquid chromatography and gas chromatography-mass spectrometry. Anal Biochem 270: 231–241, 1999.[CrossRef][ISI][Medline]
  35. Vallance P and Moncada S. Hyperdynamic circulation in cirrhosis: a role for nitric oxide? Lancet 337: 776–778, 1991.[CrossRef][ISI][Medline]
  36. Wolzt M, MacAllister RJ, Davis D, Feelisch M, Moncada S, Vallance P, and Hobbs AJ. Biochemical characterization of S-nitrosohemoglobin. Mechanisms underlying synthesis, no release, and biological activity. J Biol Chem 274: 28983–28990, 1999.[Abstract/Free Full Text]
  37. Yang BK, Vivas EX, Reiter CD, and Gladwin MT. Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls, and nitrite in biological samples. Free Radic Res 37: 1–10, 2003.[ISI][Medline]
  38. Zai A, Rudd MA, Scribner AW, and Loscalzo J. Cell-surface protein disulfide isomerase catalyzes transnitros and regulates intracellular transfer of nitric oxide. J Clin Invest 103: 393–399, 1999.[ISI][Medline]



This article has been cited by other articles:


Home page
 J. Gerontol. A Biol. Sci. Med. Sci.Home page
D. Giustarini, I. Dalle-Donne, S. Lorenzini, A. Milzani, and R. Rossi
Age-related influence on thiol, disulfide, and protein-mixed disulfide levels in human plasma.
J. Gerontol. A Biol. Sci. Med. Sci., October 1, 2006; 61(10): 1030 - 1038.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/2/H916    most recent
01014.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Orie, N. N.
Right arrow Articles by Moore, K. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Orie, N. N.
Right arrow Articles by Moore, K. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.