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Am J Physiol Heart Circ Physiol 290: H1826-H1829, 2006. First published November 18, 2005; doi:10.1152/ajpheart.00938.2005
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Inhaled nitric oxide does not reduce systemic vascular resistance in mice

Ryuji Hataishi,1 Warren M. Zapol,1 Kenneth D. Bloch,1,2 and Fumito Ichinose1,2

1Department of Anesthesia and Critical Care and 2Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 1 September 2005 ; accepted in final form 11 November 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inhaled nitric oxide (NO) is a highly selective pulmonary vasodilator. It was recently reported that inhaled NO causes peripheral vasodilatation after treatment with a NO synthase (NOS) inhibitor. These findings suggested the possibility that inhibition of endogenous NOS uncovered the systemic vasodilating effect of NO or NO adducts absorbed via the lungs during NO inhalation. To learn whether inhaled NO reduces systemic vascular resistance in the absence of endothelial NOS, we studied the systemic vascular effects of NO breathing in wild-type mice treated without and with the NOS inhibitor N{omega}-nitro-L-arginine methyl ester and in NOS3-deficient (NOS3–/–) mice. During general anesthesia, the cardiac output, left ventricular function, and systemic vascular resistance were not altered by NO breathing at 80 parts/million in both genotypes. Breathing NO in air did not alter blood pressure and heart rate, as measured by tail-cuff and telemetric methods, in either awake wild-type mice (whether or not they were treated with N{omega}-nitro-L-arginine methyl ester), or in awake NOS3–/– mice. Our findings suggest that absorption of NO or adducts during NO breathing is insufficient to cause systemic vasodilation in mice, even when endogenous endothelial NO production is congenitally absent.

peripheral vasodilation; endothelial nitric oxide synthase; telemetry; tail cuff

NITRIC OXIDE (NO) is produced from L-arginine by NO synthases (NOS1, NOS2, and NOS3). NO derived from NOS3 in vascular endothelial cells relaxes subjacent smooth muscle cells, thereby modulating pulmonary and systemic vascular tone. Administration of NOS inhibitors or a congenital deficiency of NOS3 has been shown to cause pulmonary and systemic hypertension (8, 14).

Due to the rapid reaction of NO with hemoglobin in the pulmonary capillaries, the vascular effects of breathing NO gas appear to be restricted to the pulmonary circulation, thereby making it a uniquely selective pulmonary vasodilator. Accordingly, inhalation of NO gas is now an established treatment for persistent pulmonary hypertension of the newborn, and NO inhalation therapy is widely used for the treatment of respiratory failure and pulmonary hypertension (9). While inhaled NO concentrations <20 parts/million (ppm) are often used clinically, higher concentrations of NO (up to 80 ppm) have been administered to patients with a variety of severe disorders, including pulmonary hypertension and right ventricular myocardial infarction. Concentrations of NO higher than 100 ppm are not used clinically due to the increased risk of nitrogen dioxide (NO2) formation and methemoglobinemia.

This conventional view of the intrapulmonary selectivity of inhaled NO has been challenged. It was recently reported that inhaled NO causes peripheral vasodilatation in the presence of a NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), in human volunteers (4).

While these findings suggest the possibility that inhibition of endogenous NOS, especially endothelial NOS (NOS3), uncovers the systemic vasodilator effect of NO absorbed from the lungs during NO inhalation, NOS inhibitors may have nonspecific effects other than inhibition of NOS (2, 3).

Therefore, the objective of the present investigation was to examine whether or not inhaled NO affects peripheral vascular tone in the absence of endogenous NOS activity. To test this hypothesis, we studied mice genetically deficient for NOS3 (NOS3–/–). The mouse is an advantageous animal model in which to test whether or not NO breathing alters systemic vascular resistance because of its very short circulation time (~6 s). Specifically, we studied the effects of inhaled NO on blood pressure (BP) and systemic vascular resistance in NOS3–/– mice and in mice chronically treated with N{omega}-nitro-L-arginine methyl ester (L-NAME). We report that breathing 80-ppm NO does not affect systemic hemodynamic parameters in NOS3-deficient mice.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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After approval by the Massachusetts General Hospital Subcommittee on Research Animal Care, we studied 2- to 3-mo-old male C57BL/6J wild-type (WT) mice and male NOS3–/– mice, backcrossed 10 generations onto a C57BL/6J background.

Invasive measurements of cardiac output and systemic vascular resistance. Seven WT and five NOS3–/– mice were anesthetized with an intraperitoneal injection of urethane, etomidate, and morphine and placed supine on a heated operating table to maintain core body temperature between 37 and 38°C. The trachea was orally intubated with a 20-gauge Angiocath (Beckton-Dickinson, Sandy, UT), and volume-controlled ventilation was initiated at a respiratory rate of 100 breaths/min with an inspired O2 fraction (FIO2) of 0.8 (80% oxygen and 20% nitrogen). A 1.4-French conductance catheter (SPR-839, Millar Instruments, Houston, TX) was inserted to measure left ventricular (LV) function via the right carotid artery. A custom-made catheter (PE10, Becton Dickinson) was inserted via the left carotid artery to monitor mean arterial pressure (MAP). Albumin (12.5% in normal saline) was infused via the left jugular vein to maintain intravascular volume.

Following 10 min of stabilization after surgical preparation, baseline hemodynamic parameters were obtained for 10 min while mice were ventilated at 0.8 FIO2. Then, NO gas was added to the inspiratory gas to deliver 80-ppm NO at 0.8 FIO2 for 10 min. Finally, NO was removed from the inspiratory gas mixture, and mice were again ventilated at 0.8 FIO2 for 10 min. Pressure and flow signals were continuously displayed and digitally recorded at 1,000 Hz (Powerlab, ADInstruments). Concentrations of NO and NO2 in inspiratory gas mixture were continuously monitored with electrochemical analyzer (INOvent, INOTherapeutics LLC, Clinton, NJ). NO2 concentration was <3 ppm at all times in all experiments. Cardiac output (CO), LV end-systolic pressure (LVESP), maximum first derivative of the developed LV pressure (dP/dtmax), and minimum first derivative of the developed LV pressure (dP/dtmin) were calculated with analysis software (PVAN, Millar Instruments, Houston TX). Total systemic vascular resistance (TSVR) was calculated as MAP divided by CO.

In five additional WT mice, effects of administration of sodium nitroprusside (SNP) were examined. Following the measurements of baseline hemodynamic parameters, SNP (25 µg/kg in 1 µl saline/g body wt) was administered as a bolus, and hemodynamic parameters were continuously recorded while mice were ventilated at 0.8 FIO2.

Measurements of tail-cuff BP in awake mice. Systolic BP was measured with a tail cuff (Kent Scientific, Torrington, CT) in five WT and five NOS3–/– mice before and while breathing NO at 80 ppm in air via a nose cone for 5 and 10 min.

Telemetric measurements of BP in freely moving mice. Under general anesthesia, telemetric BP transmitters (TPA-C20, DSI, St. Paul, MN) were implanted in six WT and six NOS3–/– mice with the tip of the transmitter catheter positioned in the aortic arch via the left carotid artery. Mice were allowed to recover in individual cages for 10 days to ensure the return of a normal diurnal sleep/awake rhythm. On the 11th day, mouse cages were placed in a Plexiglas chamber in which mice breathed air or 80-ppm NO in air, while BP, heart rate (HR), and activity level were continuously recorded. The chamber was placed in a quiet room where mice were left undisturbed during the recording sessions. Based on pilot experiments, recording was performed in the afternoon when activity of mice is relatively low and BP and HR are stable (data not shown). After a few hours of air breathing, mice breathed NO at 80 ppm in air for 1 h followed by several hours of air breathing. At 1-min intervals, the system samples the mean, systolic, and diastolic BP and HR at 500 Hz for 10 s and records average values. The WT group underwent recording sessions before and after treatment with L-NAME for 6 days. L-NAME was mixed in drinking water at a dose that has been reported to increase BP in mice (0.5 mg/ml of drinking water) (12).

Statistical analysis. All values are reported as means ± SE. Between-genotype differences in hemodynamic parameters were analyzed by repeated-measures ANOVA with post hoc Scheffé's test. The difference in hemodynamic parameters between pretreatment and during NO inhalation was examined by a paired t-test. P values < 0.05 were considered significant.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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In vivo hemodynamic measurements under general anesthesia. At baseline during ventilation at 0.8 FIO2, MAP, LVESP, and TSVR were higher in NOS3–/– than in WT mice (P < 0.05 for all), whereas HR, CO, dP/dtmax, and dP/dtmin did not differ between the two genotypes. NO inhalation for 10 min did not change any of the parameters examined from their respective baseline values. Repeated-measures ANOVA revealed that MAP and LVESP were higher in NOS3–/– than in WT mice (P < 0.05 and P < 0.00001, respectively; see Table 1) throughout the experiments. TSPVR tended to be higher in NOS3–/– than in WT mice (P = 0.11).


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  		Table 1. Hemodyanamic parameters during NO inhalation

 
In contrast, bolus administration of SNP decreased MAP, LVESP, TSVR, and dP/dtmax and increased CO and dP/dtmin (P < 0.05 for all; Table 2) in WT mice, demonstrating that our experimental model is sensitive enough to detect changes in pressure and flow in response to a vasodilator.


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  		Table 2. Hemodynamic parameters after SNP bolus

 
Tail-cuff BP. Before NO inhalation, systolic BP was greater in NOS3–/– mice than in WT mice (157 ± 7 vs. 117 ± 1 mmHg, P = 0.006, n = 5). Breathing NO at 80 ppm for 5 and 10 min did not affect systolic BP in either genotype (Fig. 1).


Figure 1
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  		Fig. 1. Systolic blood pressure (BP) measured by the tail-cuff method in wild-type (WT) mice (n = 5, bullet) and in endothelial nitric oxide (NO) synthase-deficient (NOS3–/–) mice (n = 5, {circ}) before and during NO breathing at 80 parts/million (ppm). Values are means ± SE. *P < 0.05 vs. WT mice by ANOVA.

 
Telemetric BP measurements. During air breathing, MAP in NOS3–/– mice (130 ± 3 mmHg) and L-NAME-treated WT mice (124 ± 3 mmHg) was greater than that in WT mice (109 ± 2 mmHg, P < 0.05 for both). HR did not differ between groups while they breathed air without added NO (NOS3–/–: 591 ± 3, L-NAME-treated WT: 605 ± 3, and WT: 646 ± 5 beats/min). Breathing NO at 80 ppm for 1 h did not affect BP or HR in either genotype (Fig. 2). Breathing 80-ppm NO for longer periods (2–3 h) also did not affect BP or HR in conscious mice of either genotype (data not shown).


Figure 2
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  		Fig. 2. A: systolic ({circ}) and diastolic (bullet) BP as measured by a telemetric method in untreated WT mice (n = 6), NOS3–/– mice (n = 6), and in N{omega}-nitro-L-arginine methyl ester (L-NAME)-treated WT mice (n = 6). Each data point represents averaged pressure from 6 mice in each group in a 10-min period. Summary of mean BP (B) and heart rate (HR; C) changes in WT mice (bullet, n = 6), NOS3–/– mice ({circ}, n = 6), and L-NAME-treated WT mice ({blacktriangleup}, n = 6) is shown. Each data point represents averaged data from 6 mice in each group in a 60-min period. The hatched area shows the duration of NO inhalation at 80 ppm in air. Values are means ± SE. *P < 0.05 vs. WT mice by ANOVA.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we found that inhalation of 80-ppm NO for 10 or 60 min did not affect mean BP and HR in awake and anesthetized WT mice, NOS3-deficient mice, and in WT mice treated for 6 days with L-NAME. Breathing 80-ppm NO did not change CO and TSVR in anesthetized WT and NOS3–/– mice. These observations suggest that congenital endothelial NO deficiency per se was not sufficient to reveal any acute vasodilator properties of inhaled NO in mice.

Although the vasodilator effects of breathing NO are believed to be largely limited to the lung, several lines of evidence suggest that NO inhalation can have systemic effects. In 1992, Stamler and colleagues (13) proposed that NO undergoes S-nitrosylation with protein-bound thiol groups, forming stable S-nitrosothiols, including S-nitrosohemoglobin and S-nitrosoalbumin, that could conceivably deliver NO molecules to the periphery. Subsequently, Hogman and colleagues (7) reported that breathing high concentrations of NO prolonged the bleeding time in rabbits. This study led us to investigate whether or not breathing NO has anti-thrombotic effects in vivo. Our laboratory reported that breathing 80-ppm NO decreased neointima formation in rat carotid arteries after balloon injury (10) and decreased thrombosis in a canine coronary artery model of thrombosis and thrombolysis (1). In both models, breathing NO did not cause systemic hypotension or prolong the bleeding time. In 1998, Fox-Robichaud et al. (5) showed that inhalation of 80-ppm NO was able to prevent reduction in intestinal blood flow and leukocyte rolling and adhesion in ischemia-reperfusion (I/R) injury in cat mesentery. This work was extended by Cannon and colleagues (4), who reported that, after blockade of NO production and forearm exercise, NO breathing increased forearm blood flow. Furthermore, we have recently observed that breathing NO decreased murine myocardial infarction size after transient left anterior descending coronary artery occlusion associated with preservation of LV function (6). Taken together, the bulk of evidence supports the view that certain biological activities of inhaled NO can extend to the peripheral circulation, especially under conditions of NO depletion such as I/R injury and NOS inhibition.

While our current results do not suggest that enough NO or NO adducts reach the periphery to cause vasodilation, these findings are not surprising in light of recent reports by Ng and colleagues (11), who found that mesenteric I/R, but not NOS inhibition by L-NAME, was associated with sufficient production and consumption of S-nitrosoalbumin during NO inhalation to increase intestinal blood flow. Similarly, Cannon and colleagues (4) reported that breathing 80-ppm NO failed to prevent the L-NMMA-induced increase of forearm vascular resistance in the absence of forearm exercise. It has been suggested that O2– may be required to produce sufficient S-nitrosothiols in the lung to influence NO levels in the periphery. If that were the case, NO breathing in the absence of any significant hemodynamic stress, as in the present study, would not be expected to cause significant systemic vasodilation.

It is also of note that previous studies investigated whether or not NO breathing modified the acute vasoconstrictor effects of NOS inhibitors compared with breathing air (4, 11). In contrast, the present study was designed to investigate the effects of NO inhalation in mice with life-long or chronic NOS deficiency. It is possible that upregulation of unknown compensatory mechanisms induced by chronic NOS deficiency may have blunted the peripheral vasodilation caused by NO inhalation.

In summary, the present study revealed that breathing 80-ppm NO for 10 min or 1 h does not cause systemic vasodilation in mice, even in the congenital absence of endogenous NOS3 activity in mice. While these observations do not refute the possibility that biological effects of inhaled NO may extend to peripheral vascular beds that have an impaired capacity of NO production, our data support the hypothesis that inhaled NO does not modify vascular resistance in the intact peripheral circulation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by US Public Health Service Grants HL-42397 (W. M. Zapol), HL-70896 (K. D. Bloch), and HL-71987 (F. Ichinose) and by a sponsored research agreement from INOTherapeutics LLC.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Ichinose, Dept. of Anesthesia and Critical Care, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114 (e-mail: ichinose{at}etherdome.mgh.harvard.edu)

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  1. Adrie C, Bloch KD, Moreno PR, Hurford WE, Guerrero JL, Holt R, Zapol WM, Gold HK, and Semigran MJ. Inhaled nitric oxide increases coronary artery patency after thrombolysis. Circulation 94: 1919–1926, 1996.[Abstract/Free Full Text]
  2. Archer SL and Hampl V. NG-monomethyl-L-arginine causes nitric oxide synthesis in isolated arterial rings: trouble in paradise. Biochem Biophys Res Commun 188: 590–596, 1992.[CrossRef][ISI][Medline]
  3. Buxton IL, Cheek DJ, Eckman D, Westfall DP, Sanders KM, and Keef KD. NG-nitro L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists. Circ Res 72: 387–395, 1993.[Abstract/Free Full Text]
  4. Cannon RO III, Schechter AN, Panza JA, Ognibene FP, Pease-Fye ME, Waclawiw MA, Shelhamer JH, and Gladwin MT. Effects of inhaled nitric oxide on regional blood flow are consistent with intravascular nitric oxide delivery. J Clin Invest 108: 279–287, 2001.[CrossRef][ISI][Medline]
  5. Fox-Robichaud A, Payne D, Hasan SU, Ostrovsky L, Fairhead T, Reinhardt P, and Kubes P. Inhaled NO as a viable antiadhesive therapy for ischemia/reperfusion injury of distal microvascular beds. J Clin Invest 101: 2497–2505, 1998.[ISI][Medline]
  6. Hataishi R, Morgan JG, Rodorigues ACT, Ichinose F, Zapol WM, Picard MH, Bloch KD, and Scherrer-Crosbie M. Inhaled nitric oxide limits murine ischemia-reperfusion injury (Abstract). Circulation 234: 2004.
  7. Hogman M, Frostell C, Arnberg H, Sandhagen B, and Hedenstierna G. Prolonged bleeding time during nitric oxide inhalation in the rabbit. Acta Physiol Scand 151: 125–129, 1994.[ISI][Medline]
  8. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, and Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 377: 239–242, 1995.[CrossRef][Medline]
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  10. Lee JS, Adrie C, Jacob HJ, Roberts JD Jr, Zapol WM, and Bloch KD. Chronic inhalation of nitric oxide inhibits neointimal formation after balloon-induced arterial injury. Circ Res 78: 337–342, 1996.[Abstract/Free Full Text]
  11. Ng ES, Jourd'heuil D, McCord JM, Hernandez D, Yasui M, Knight D, and Kubes P. Enhanced S-nitroso-albumin formation from inhaled NO during ischemia/reperfusion. Circ Res 94: 559–565, 2004.[Abstract/Free Full Text]
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  14. Steudel W, Ichinose F, Huang PL, Hurford WE, Jones RC, Bevan JA, Fishman MC, and Zapol WM. Pulmonary vasoconstriction and hypertension in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circ Res 81: 34–41, 1997.[Abstract/Free Full Text]



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This Article
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