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Nitric oxide plays a minimal role in hypoxic pulmonary vasoconstriction in isolated rat lungs
Accepted Manuscript Title: Nitric oxide plays a minimal role in hypoxic pulmonary vasoconstriction in isolated rat lungs Author: T.S. Hakim A. Pedoto D. Mangar E.M. Camporesi PII: DOI: Reference:
S1569-9048(13)00234-6 http://dx.doi.org/doi:10.1016/j.resp.2013.06.028 RESPNB 2137
To appear in:
Respiratory Physiology & Neurobiology
Received date: Revised date: Accepted date:
28-3-2013 20-5-2013 28-6-2013
Please cite this article as: Hakim, T.S., Pedoto, A., Mangar, D., Camporesi, E.M., Nitric oxide plays a minimal role in hypoxic pulmonary vasoconstriction in isolated rat lungs, Respiratory Physiology & Neurobiology (2013), http://dx.doi.org/10.1016/j.resp.2013.06.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights Hypoxic pulmonary vasoconstriction was independent of the existing NO production or the change in NO production during hypoxia. Potentiation of hypoxic pulmonary vasoconstriction by nitric oxide synthase inhibitors may be due to effects other than NO inhibition. L-arginine supplement may increase NO production only when iNOS is upregulated and especially when blood L-arginine is depeleted. Isolated lung preparation is ideal to examine the direct effect of various factors on NO production from lung cells in their normal environment. The rat is ideal for increasing NO production by iNOS upregulation using endotoxin.
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Nitric oxide plays a minimal role in hypoxic pulmonary vasoconstriction in isolated rat lungs
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T. S. Hakim, A. Pedoto, D. Mangar, and E. M. Camporesi
Department of Surgery and Anesthesiology, University of South Florida, Tampa, FL, Sloan
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Kettering Cancer Center, New York, NY, and Sleep Apnea Treatment Center, Phoenix AZ
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Short title: nitric oxide production in the isolated lung.
Correspondence: T.S. Hakim Ph.D. Sleep Apnea Treatment Center 10443 N Cave Creek Rd Suite 110 Phoenix, AZ 85020
Tel (602) 944-0847 Fax (602) 944-1014 Email: [email protected]
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Abstract: The goal of this study was to elucidate the importance of nitric oxide production during hypoxic pulmonary vasoconstriction (HPV). One group of Sprague Dawley rats received an ip
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injection of saline (controls), while a second group received an ip injection of E Coli
lipopolysacharides (LPS-treated) to render them septic. Three hours later, the animals were
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anesthetized and prepared for the isolated lung experiment. The lungs were ventilated and
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perfused with diluted autologous blood (Hct 23%) at constant flow rate while monitoring pulmonary arterial (Pa). Nitric oxide production from the lungs was monitored by measuring its
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concentration in the mixed exhaled gas (NOe) offline. NOe in the isolated lungs was 2 ppb in controls and 90 ppb in the LPS treated lungs. Hypoxia caused the pulmonary arterial pressure to
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rise from 10 to 17 mmHg in control lungs, and from 10 to 27 mmHg in the LPS treated lungs. NO production was then manipulated to determine if it affects HPV. NOe was increased by adding
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L-arginine to the blood, and was blocked by adding nitro-L-arginine (LNA). L-arginine had
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minimal effect on NOe in control lungs, but increased NOe in LPS treated lungs, and yet HPV was
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similar in the 2 groups. Despite inhibition of NO synthesis with nito-L-arginine (LNA), HPV was potentiated equally in control lungs and in LPS treated lungs (Pa rose by 23 mmHg). Thus potentiation or reduction in NO production did not affect the difference in HPV between control and LPS treated lungs. With the exception of the observation that HPV was potentiated after treatment with LNA, no other data supported the notion that NO production plays an important role in HPV. The results suggest that NO does not plays a primary role in HPV. Keywords: pulmonary vascular resistance, lung, hypoxia, hypoxic vasoconstriction, nitric oxide, temperature, L-arginine, isolated lung.
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1. Introduction Hypoxic pulmonary vasoconstriction (HPV) is a local response that helps divert blood flow from poorly ventilated areas to better ventilated regions, thus optimizing matching of
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perfusion to ventilation. The mechanisms of HPV have been a subject of research for many
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years and yet uncertainties remain. The state of our understanding of the mechanisms of HPV
has recently been reviewed by Sylvester et al (2012). Nitric oxide (NO) affects vascular tone and
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vascular responses in a variety of conditions (Ignarro 1989, Moncada et al 1991). Nitric oxide production is one factor that has been thought to be involved during HPV (Archer et al 1989,
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Persson et al 1990, Robertson et al 1990, Ferrario et al 1996). NO synthesis is oxygen
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dependent (Moncada et al 1991) leading some investigators to embrace the idea that hypoxia reduces NO synthesis leading to increased pulmonary vascular resistance. The conclusions from
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such studies were usually based on indirect evidence that HPV becomes potentiated after
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inhibition of NO synthesis with L-arginine analogs (Archer et al 1989, Persson et al 1990, Robertson et al 1990). Studies that have used direct measurement of nitric oxide in the exhaled
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gas showed that hypoxia was associated with a small reduction in NO release, and the reduction in NO was used as evidence to support the role NO in HPV (Cremona et al 1995, Carlin et al 1997, Grimminger et al 1995, Persson et al 1990). It is important to note that the change in NO concentration during hypoxia in these studies was usually small and cannot adequately explain the magnitude of the hypoxic response. For example in normal rat (Stewart et al 1995), rabbit lung (Carlin et al 1997, Grimminger et al 1995) and pig lungs (Cremona et al 1995), in which HPV is relatively brisk, NO concentration in the exhaled gas is very small and changes only by a few ppb ( < 5 ppb) during hypoxia, which is barely adequate to have any influence on vascular tone or resistance. Thus even with direct measurement of NO production there is some doubt about the conclusions of many studies regarding the role of NO during HPV. Reversal of pulmonary
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hypoxic vasoconstriction in dogs and minipigs by inhalation of nitric oxide requires much higher concentrations of NO in the inhaled gas (up to 100 ppm) than are released endogenously (Maggiorini et al 1998). Therefore, there is some confusion regarding the relationship of HPV
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and the NO levels that need further explanation.
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The isolated lung preparation is a closed system and involves only cells within the lungs.
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Any stimulus used in such a preparation, would reflect the direct effect on lung cells. We utilized this isolated lung preparation to examine the relationship of NO production and HPV. This
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preparation allows us to measure NOe immediately while monitoring small changes in vascular resistance. The goal of this study was to elucidate the relationship between NO production and
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HPV in conditions where NO production is normal or elevated.
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2. Material and Methods This study was approval by our institutional review board. Adult male Sprague Dawley rats (Taconic, Germantown NY) weighing 350 to 450 g were utilized for this study. The animals
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were injected ip either with saline (Controls, n=12), or with 20 mg/kg E Coli lipopolysacharides
(LPS treated, n=17) and left in their cages for 3 hours. LPS (Sigma Chemical L4130, Lot 76H4071)
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was dissolved in saline such that both groups received equal volume of fluid (0.5 ml). Three
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hours later, the animals were anesthetized with an ip injection of sodium pentobarbital (50 mg/kg). A midline incision on the ventral surface of the neck was made and the trachea was
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cannulated . Animals were mechanically ventilated with a gas mixture from a bag containing 5% CO2, 35% O2 and nitrogen using a rodent ventilator (Harvard Apparatus Model 683 South Natick
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Mass) with a tidal volume of 3 ml and a respiratory rate of 45 breaths /min. Airway pressure (Paw) was monitored by a Gould pressure transducer (Gould IN, Cleveland OH) and recorded on
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a chart paper recorder (Grass Model 7D polygraph, Quincy, Mass).
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Exhaled gas from the expiratory port of the ventilator was collected as described below for measurement of average nitric oxide concentration in the exhaled gas of the anesthetized intact animal. This was done in all animals prior to preparing the isolated lung. NO concentration was measured offline as described below. The carotid artery and jugular vein were exposed and cannulated. Heparin was injected (1000 units/kg), and the animal was exsanguinated slowly through the carotid artery cannula. During bleeding a total of 30 ml of lactated ringer was injected gradually into the animal to expand its blood volume. A total of 40 ml of diluted blood (Hct 23±1%) was collected from the animal for use in the isolated lung preparation which was described in more details previously (Ferrario et al 1996, Hakim et al 1997). The blood was added to the reservoir in the perfusion system which was kept at 37º C and was used to prime
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the perfusion system. The chest was opened and two cannulas, connected to the perfusion system, were placed in the pulmonary artery and the left atrium and secured in place. Blood flow through the lungs was reestablished quickly (< 3 min) using a roller pump. A pressure of 1.5
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cm H2O was applied at end expiration. The blood flow was increased gradually (up to 40±3
ml/min). The final flow was selected to give a near normal pulmonary artery pressure of 10
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mmHg and was kept constant for the duration of the experiment. During the initial stabilization
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period, and when necessary, blood pH was adjusted to 7.35 to 7.45 by adding bicarbonate to the reservoir. Pulmonary arterial pressure (Pa) and venous pressure (Pv) were measured from
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side ports in the cannulas using pressure transducers and were recorded continuously . Pv was kept at 1 mmHg by adjusting the level of the reservoir. Because perfusion rate and Pv were kept
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constant, a change in Pa represented a change in pulmonary vascular resistance. Adequate time was allowed for the isolated lung preparation to stabilize (10 to 15 min).
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Baseline measurements of blood gases and pH, Paw, and exhaled NO (NOe) were obtained.
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Exhaled gas from the isolated lungs was collected for > 3 minutes using a 1 L polyvinyl bag
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connected to the expiratory port of the ventilator. After collecting > 0.5 L of exhaled gas, the bag was removed and the concentration of NO in the exhaled gas was measured immediately offline using chemiluminescence NO analyzer (Siever 270B). The NO analyzer was calibrated daily using nitrogen for zero and a mixture of 248 ppb NO in nitrogen as described in more details elsewhere (Carlin et al 1997, Pedoto et al 1998). Lung ventilation was then switched to a low oxygen bag containing 3%O2, and 5% CO2 in nitrogen to test the effect of hypoxia on pulmonary vascular response and on NO concentration in the exhaled gas. After Pa rose to a new steady level (within 5 to 10 min), measurements were repeated and exhaled gas was collected as above. Ventilation was then switched back to the normoxic gas. From here onward, the control lungs or the LPS treated lungs were assigned to receive either L-arginine or nitro-L-arginine
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(LNA), thus generating 4 groups. Group 1 consisted of controls (n=6) and Group 2 consisted of LPS treated lungs(n=6). Groups 1 and 2 were monitored before and after adding L-arginine. Group 3 was a second control group (n=6) but instead of L-arginine, the lungs received LNA after
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the initial baseline measurements, while Group 4 consisted of LPS treated lungs (n=6) and
received LNA after baseline measurements. L-arginine (10 mg) was added to the blood in the
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perfusion system to increase NO production. This dose of L-arginine consistently increased NO
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production and prevented any spontaneous decline in NOe with time. LNA (2.5 mg added to perfusion system) was used to block NO production. After 10 minutes, a new set of baseline
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measurements were made including NOe, and hypoxic ventilation. During normoxic gas ventilation in the isolated lungs, PO2 was 252±3 mmHg, PCO2 was 39±1 mmHg, and pH was
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7.36±0.01, while during hypoxic gas ventilation PO2 was 34±3 mmHg, PCO2 was 34±3 mmHg,
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2.1 Statistical Analysis:
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and pH was 7.38±0.02.
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Statistical analysis of the results was done using repeated measures analysis of variance (ANOVA) and a paired Student’s t test. All results are given as mean plus or minus SEM. A p value of < 0.05 was considered as being significant. The Tukey post-hoc test was performed for multiple comparisons.
3. Results
In the intact anesthetized animals prior to isolating the lungs, NOe in the control animals was 2.2±0.4 ppb, and in the LPS treated animals, NOe was 131±11 ppb. After preparation of the isolated lungs, NOe in the control lungs was 1.8±0.3 ppb, while in LPS treated lungs, NOe was 99±17 ppb (lower than in intact animals), and usually continued to decline gradually with time
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as illustrated in 5 individual experiments in Fig 1. However, this decline in NOe could be prevented when L-arginine is added to the blood. Fig 2 illustrates the effects of adding L-arginine on NOe. In control lungs, addition of L-arginine affected neither NOe nor the perfusion pressure,
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while in LPS lungs, addition of L-arginine, increased NOe markerdly to 143±27 ppb (p<0.05) with minimal decrease in pressure. These results show that increasing L-arginine concentration
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resulted in a significant increase NO production only in LPS treated lungs. Note that, L-arginine
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concentration in the blood (not measured) was likely to have been half the normal plasma concentration because the blood was diluted with Ringer’s lactate.
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The effects of hypoxia are shown in figs 3, 4 and 5. The 3 figures compare responses to
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hypoxia under conditions where NOe was increased or decreased. The scales in these 3 figures were kept similar to allow comparison among the 3 figures. The baseline values and the hypoxic
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responses in the two control groups preceding addition of L-arginine and LNA, were not
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significantly different, therefore, the values from 12 lungs were combined and are shown in Fig 3. Hypoxia, did not change NOe in control lungs, but decreased it slightly in the LPS treated
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lungs from 111±19 to 90±15 ppb (p<0.05). Hypoxia caused the pulmonary arterial pressure to rise from 9.7±0.5 to 17.4±2.7 mmHg in control lungs, and from 10.5±0.5 to 27.1±2.4 mmHg in the LPS treated lungs. Thus HPV was potentiated in LPS treated lungs, despite the elevated level of NO production. Fig 4 shows the responses to hypoxia after NOe was increased by adding Larginine to the blood. Hypoxia had a minimal effect in controls, but decreased NOe in the LPS treated lungs from 143±27 to 115±22 ppb (p<0.05), but the increase in pressure in the LPS treated lungs was about similar to that in controls. After NOe was reduced with LNA (to 32±7 ppb), hypoxia had no effect on NOe in either the control group or the LPS treated group, however, HPV was potentiated equally in control lungs and in LPS treated lungs (Pa rose by approximately 23 mmHg in both groups), despite the marked difference in NOe levels.
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Therefore, based on the data in these 3 figures, we suggest that potentiation or reduction in NO synthesis, did not affect the difference in HPV between control and LPS treated lungs. These results suggest that hypoxia may affect NO production slightly, but NO levels play a minimal role
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in HPV.
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4. Discussion The main findings from the present study is that HPV was unrelated to the existing level
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of NOe or the change in NOe during hypoxia suggesting that NO production is not an important factor in HPV. Despite the marked difference in NOe in control lungs and LPS treated lungs, LNA
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enhanced HPV equally in these 2 groups, further corroborating the minimal role of NO in HPV. It is possible that LNA may potentiate HPV by mechanisms other than inhibition of NO synthesis.
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4.1. Isolated lung preparation: Rat lungs were ideal for this study because they exhibit a brisk pulmonary hypoxic vasoconstriction (HPV) response and NO production can be reliably
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upregulated with endotoxin. Because this is an isolated lung preparation, any change in NOe
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observed would have to be attributed to a direct effect on cells within the lungs that are capable of producing NO or capable of expressing iNOS. In the lung many cells are capable of doing
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doing so including bronchial epithelia , endothelial cells, pulmonary artery smooth muscle cells ,
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macrophages , or neutrophils (Scumpia et al 20022002, Szabo and Salzman 1997). In isolated lung preparation, vascular response to hypoxia (HPV) can also be measure simultaneously while
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measuring NO production, allowing more precise conclusions about the relationship of NO production to vascular responses. The results lead us to conclude that although NO production maybe diminished slightly by hypoxia, the change in NO levels that are sometimes discernible during hypoxia are too small to have a significant influence on vascular tone or on HPV. We are able to make such conclusions with confidence because of the isolated lung preparation where changes in vascular resistance and NOe can be measured with accuracy, and can be reproduced in the same preparation. We have successfully examined the effects of L-arginine supplement, and the effect of hypoxia on NO production in the lung. The limitation of the isolated lung preparation is that we cannot say which cells are involved but it is likely to involve all cells capable of producing NO and expressing iNOS. The cells are in their natural environment and
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usual milieu and their response would reflect best the response in intact animal. Most NO produced in the lungs diffuses easily into the alveoli and exit in the exhaled gas. A small amount of NO diffuses into the blood and is scavenged by hemoglobin but it is possible to perfuse
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isolated lungs with red blood cells free solutions (Grimminger et al 1995). In the isolated lung preparation, the vascular responses can be identified in terms of arteries or veins using the
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arterial, venous, or double occlusion technique (Hakim et al 1997). The isolated lung provided
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an excellent model to explore the role of NO because the changes in NOe can be measured immediately and repeatedly under different conditions, in control lungs with cNOS or LPS
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treated lungs with iNOS upregulated. This isolated lung model is relatively easy to prepare and
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would allow more firm conclusions about the role of NO.
4.2 Effect of L-arginine: The isolated lung was ideal to test the effect of L-arginine on NO
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production from lung cells by measuring NO production immediately. Clearly any effect would
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have to be due to effect on many cells in the lung. To begin with, we found that NO production
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in the isolated lungs that were perfused with diluted blood (i.e with low L-arginine concentration) could exhibit a lower NO production than in intact animal. When L arginine was added to the blood, NO production in the LPS treated lungs increased subtantially. This was not surprising because the blood was diluted and hence L-arginine concentration was low. In contrast to the LPS treated lungs, NO production in control lungs is very low, and was not affected by addition of L-arginine to the blood. In the intact rat, Wu et al (1999) showed that dietary arginine deficiency, limited NO production by cNOS and iNOS. Our results are in partial agreement and show that in the LPS treated lungs (iNOS present), L-arginine supply may be a limiting factor, but L-arginine does not seem to affect NO production in control lungs (cNOS present). Our conclusions are supported by studies in isolated vessels (Schini and Vanhoutte
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1991, Julou-Schaeffer et al 1990). This information may be relevant to issues related to arginine supplementation in healthy subjects (Alvares et al 2012), in patients (Jabecka et al 2012, Antosova and Starpkova 2013) or in sepsis (Luiking and Deitz 2007, Kalil and Danner 2006). The
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results from this study suggest that arginine supplements would have little effect on NO production in healthy subjects and patients with normal plasma arginine levels. Clearly,
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supplementation with L-arginine can elevate plasma arginine levels, but may not necessarily
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increase NO production. In intact rats, we have found that injection of supplemental L-arginine has minimal effect on NOe in normal or septic animals that have normal l-arginine (unpublished
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observation).
As pointed out in Fig 1, NOe concentration in LPS lungs declined slowly with time during
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the course of the experiment. This is important to remember when performing studies on isolated lungs with iNOS upregulated and NO production is elevated. In contrast, we found that
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NOe concentration in control rat lung remained low and stable. Furthermore, we previously
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found that in isolated rabbit lungs that inherently have an elevated level of NOe due to cNOS
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remained fairly stable (Carlin et al 1997). One explanation for the decline in NOe in LPS treated isolated lungs is the high NO production rate in the presence of a limited supply of L-arginine in the blood.
4.3 Effect of hypoxia: HPV in LPS treated lungs was potentiated or remained as brisk as in controls despite the presence of markedly elevated NO levels. That in itself suggests that HPV is independent of NO levels or the change in NO production during hypoxia. Furthermore, LPS treated lungs showed that indeed, hypoxia caused a small decrease in NO production but such a change in NOe does not justify the magnitude of the HPV. Previous investigators examined the role of NO during HPV by comparing HPV before and after blockade with L-arginine analog and
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reported potentiation in HPV after blocking NO synthesis. This indirect finding lead them to conclude that HPV maybe mediated by NO production (Archer et al 1989, Robertson et al 1990, Persson et al 1990). Most studies agree that NOS inhibition augments HPV including this
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present study (Archer et al 1989, Persson et al 1990, Robertson et al 1990, Rodman et al 1990) but it is not clear if this augmentation in HPV is due to the small reduction in NO synthesis. Fig 5
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shows that HPV after LNA, was indeed potentiated but was similar in the control group as in the
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LPS treated group. In most animal species, basal NO production due to cNOS is very low (Gustafson et al 1991), and blocking its synthesis is not likely to have much influence on
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pulmonary vascular resistance that can be attributed to the small reduction in NO synthesis. Although NOS blockers affect vascular resistance slightly, they have a more impressive effect on
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HPV, but the potentiation in HPV after NOS inhibitors, may be due to a nonspecific effect not related to NO production. Some studies reported attenuation in HPV following treatments with
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endotoxins, and attributed it to upregulation of NO synthesis (Weir et al 1976), while others
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reported potentiation in HPV (Castaneda et al 2001). We find that HPV could become enhanced
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in LPS treated lungs in the presence of high NO. Several investigators, demonstrated a small decrease in NOe during hypoxia, but the decrease was usually very small (less than 5 or 10 ppb), it could hardly be responsible for changes in vascular resistance or explain the magnitude of the HPV. There is little doubt that NOe decreases during hypoxia, especially in LPS treated lungs where it becomes more discernible than in control lungs . The cause of such a decrease is thought to be due to less oxygen being available for NO synthesis process. The study by Hemmingsson and Linnarsson (2009) suggest that hypoxia may affect NOe because of changes in diffusion that leads to less NO emerging in the exhaled gas. Such a finding supports our conclusions and suggests that the HPV and reduction in NOe do not necessarily have a cause and effect relationship. NO inhalation therapy requires NO levels much higher (up 100 ppm) to
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lower pulmonary vascular resistance (Lundin et al 1999, Marriagoni et al 1998). Our results are more consistent with HPV being primarily independent of what happens to NO levels. Thus it appears HPV becomes potentiated whether NO becomes elevated or diminished. One may
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think that the issue is confounded by the fact that LPS treated lungs are sick lungs and may involve complicated events. This may not be so, because healthy rabbit lungs that have
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elevated levels of NOe (much higher than in rat lungs), also exhibit potentiation in HPV after
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inhibition of NO production (Carlin et al 1997) as it did in the LPS treated rats lungs. This leads us to the inescapable conclusion that NO production does not play a significant role in HPV,
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most likely because the changes in NO production are too small to have any influence on pulmonary vascular resistance. The most that can be said is that hypoxia decreases NO
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production, and HPV may be slightly modulated by the presence of NO, but NO does not play a significant role during HPV.
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In conclusion, we find that hypoxic pulmonary vasoconstriction is independent of
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existing NO production or the changes in NO production during hypoxia . Hypoxia may lower NO
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production but such an effect may be unrelated to the intensity of the pressor response. Investigators who report enhancement in hypoxic pulmonary vasoconstrions following NOS inhibition, should explore explanations other than NO production. Clearly, the results form rats lungs should be extrapolated with caution to other animal species.
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Persson MG, Gustafsson LE, Wiklund NP, Moncada S, Hedqvist P. 1990. Endogenous nitric oxide as a possible modulator of pulmonary circulation and hypoxic pressor response in vivo. Acta Physiol Scand 140, 449-457.
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Figure Legends Fig 1: Decline of exhaled nitric oxide in 5 individual isolated lungs from LPS treated rats. The
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solid line indicates the average exhaled nitric oxide in intact animal before preparing the isolated lung.
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Fig 2: Effect of adding L-arginine on exhaled nitric oxide and perfusion pressure in control lungs
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(n=6) and LPS treated lungs (n=6). Lightly shaded bars are values before adding L-arginine and densely shaded bars are values after adding L-arginine. Asterisks indicate significant difference
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(p< 0.05)
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Fig 3: Effect of hypoxia on exhaled nitric oxide and on perfusion pressure in control lungs (n=12) and LPS treated lungs (n=12) during baseline at start of the experiment before L-arginine or LNA
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was added to the perfusion system. Clear bars are values during normoxia, and shaded bars are
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values during hypoxia. Asterisks indicate significant difference (p< 0.05).
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Fig 4: Effect of hypoxia on exhaled nitric oxide and perfusion pressure in control lungs (n=6) and LPS treated lungs (n=6) after adding L-arginine to enhance NO production. Clear bars are values during normoxia, and shaded bars are values during hypoxia. Asterisks indicate significant difference (p< 0.05).
Fig 5: Effect of hypoxia on exhaled nitric oxide and perfusion pressure in control lungs (n=6) and LPS treated lungs (n=6) after adding nitro-L-arginine to block NO production. Clear bars are values during normoxia, and shaded bars are values during hypoxia. Asterisks indicate significant difference (p< 0.05).
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S1569-9048(13)00234-6 http://dx.doi.org/doi:10.1016/j.resp.2013.06.028 RESPNB 2137
To appear in:
Respiratory Physiology & Neurobiology
Received date: Revised date: Accepted date:
28-3-2013 20-5-2013 28-6-2013
Please cite this article as: Hakim, T.S., Pedoto, A., Mangar, D., Camporesi, E.M., Nitric oxide plays a minimal role in hypoxic pulmonary vasoconstriction in isolated rat lungs, Respiratory Physiology & Neurobiology (2013), http://dx.doi.org/10.1016/j.resp.2013.06.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights Hypoxic pulmonary vasoconstriction was independent of the existing NO production or the change in NO production during hypoxia. Potentiation of hypoxic pulmonary vasoconstriction by nitric oxide synthase inhibitors may be due to effects other than NO inhibition. L-arginine supplement may increase NO production only when iNOS is upregulated and especially when blood L-arginine is depeleted. Isolated lung preparation is ideal to examine the direct effect of various factors on NO production from lung cells in their normal environment. The rat is ideal for increasing NO production by iNOS upregulation using endotoxin.
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Nitric oxide plays a minimal role in hypoxic pulmonary vasoconstriction in isolated rat lungs
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T. S. Hakim, A. Pedoto, D. Mangar, and E. M. Camporesi
Department of Surgery and Anesthesiology, University of South Florida, Tampa, FL, Sloan
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Kettering Cancer Center, New York, NY, and Sleep Apnea Treatment Center, Phoenix AZ
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Short title: nitric oxide production in the isolated lung.
Correspondence: T.S. Hakim Ph.D. Sleep Apnea Treatment Center 10443 N Cave Creek Rd Suite 110 Phoenix, AZ 85020
Tel (602) 944-0847 Fax (602) 944-1014 Email: [email protected]
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Abstract: The goal of this study was to elucidate the importance of nitric oxide production during hypoxic pulmonary vasoconstriction (HPV). One group of Sprague Dawley rats received an ip
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injection of saline (controls), while a second group received an ip injection of E Coli
lipopolysacharides (LPS-treated) to render them septic. Three hours later, the animals were
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anesthetized and prepared for the isolated lung experiment. The lungs were ventilated and
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perfused with diluted autologous blood (Hct 23%) at constant flow rate while monitoring pulmonary arterial (Pa). Nitric oxide production from the lungs was monitored by measuring its
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concentration in the mixed exhaled gas (NOe) offline. NOe in the isolated lungs was 2 ppb in controls and 90 ppb in the LPS treated lungs. Hypoxia caused the pulmonary arterial pressure to
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rise from 10 to 17 mmHg in control lungs, and from 10 to 27 mmHg in the LPS treated lungs. NO production was then manipulated to determine if it affects HPV. NOe was increased by adding
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L-arginine to the blood, and was blocked by adding nitro-L-arginine (LNA). L-arginine had
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minimal effect on NOe in control lungs, but increased NOe in LPS treated lungs, and yet HPV was
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similar in the 2 groups. Despite inhibition of NO synthesis with nito-L-arginine (LNA), HPV was potentiated equally in control lungs and in LPS treated lungs (Pa rose by 23 mmHg). Thus potentiation or reduction in NO production did not affect the difference in HPV between control and LPS treated lungs. With the exception of the observation that HPV was potentiated after treatment with LNA, no other data supported the notion that NO production plays an important role in HPV. The results suggest that NO does not plays a primary role in HPV. Keywords: pulmonary vascular resistance, lung, hypoxia, hypoxic vasoconstriction, nitric oxide, temperature, L-arginine, isolated lung.
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1. Introduction Hypoxic pulmonary vasoconstriction (HPV) is a local response that helps divert blood flow from poorly ventilated areas to better ventilated regions, thus optimizing matching of
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perfusion to ventilation. The mechanisms of HPV have been a subject of research for many
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years and yet uncertainties remain. The state of our understanding of the mechanisms of HPV
has recently been reviewed by Sylvester et al (2012). Nitric oxide (NO) affects vascular tone and
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vascular responses in a variety of conditions (Ignarro 1989, Moncada et al 1991). Nitric oxide production is one factor that has been thought to be involved during HPV (Archer et al 1989,
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Persson et al 1990, Robertson et al 1990, Ferrario et al 1996). NO synthesis is oxygen
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dependent (Moncada et al 1991) leading some investigators to embrace the idea that hypoxia reduces NO synthesis leading to increased pulmonary vascular resistance. The conclusions from
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such studies were usually based on indirect evidence that HPV becomes potentiated after
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inhibition of NO synthesis with L-arginine analogs (Archer et al 1989, Persson et al 1990, Robertson et al 1990). Studies that have used direct measurement of nitric oxide in the exhaled
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gas showed that hypoxia was associated with a small reduction in NO release, and the reduction in NO was used as evidence to support the role NO in HPV (Cremona et al 1995, Carlin et al 1997, Grimminger et al 1995, Persson et al 1990). It is important to note that the change in NO concentration during hypoxia in these studies was usually small and cannot adequately explain the magnitude of the hypoxic response. For example in normal rat (Stewart et al 1995), rabbit lung (Carlin et al 1997, Grimminger et al 1995) and pig lungs (Cremona et al 1995), in which HPV is relatively brisk, NO concentration in the exhaled gas is very small and changes only by a few ppb ( < 5 ppb) during hypoxia, which is barely adequate to have any influence on vascular tone or resistance. Thus even with direct measurement of NO production there is some doubt about the conclusions of many studies regarding the role of NO during HPV. Reversal of pulmonary
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hypoxic vasoconstriction in dogs and minipigs by inhalation of nitric oxide requires much higher concentrations of NO in the inhaled gas (up to 100 ppm) than are released endogenously (Maggiorini et al 1998). Therefore, there is some confusion regarding the relationship of HPV
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and the NO levels that need further explanation.
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The isolated lung preparation is a closed system and involves only cells within the lungs.
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Any stimulus used in such a preparation, would reflect the direct effect on lung cells. We utilized this isolated lung preparation to examine the relationship of NO production and HPV. This
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preparation allows us to measure NOe immediately while monitoring small changes in vascular resistance. The goal of this study was to elucidate the relationship between NO production and
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HPV in conditions where NO production is normal or elevated.
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2. Material and Methods This study was approval by our institutional review board. Adult male Sprague Dawley rats (Taconic, Germantown NY) weighing 350 to 450 g were utilized for this study. The animals
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were injected ip either with saline (Controls, n=12), or with 20 mg/kg E Coli lipopolysacharides
(LPS treated, n=17) and left in their cages for 3 hours. LPS (Sigma Chemical L4130, Lot 76H4071)
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was dissolved in saline such that both groups received equal volume of fluid (0.5 ml). Three
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hours later, the animals were anesthetized with an ip injection of sodium pentobarbital (50 mg/kg). A midline incision on the ventral surface of the neck was made and the trachea was
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cannulated . Animals were mechanically ventilated with a gas mixture from a bag containing 5% CO2, 35% O2 and nitrogen using a rodent ventilator (Harvard Apparatus Model 683 South Natick
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Mass) with a tidal volume of 3 ml and a respiratory rate of 45 breaths /min. Airway pressure (Paw) was monitored by a Gould pressure transducer (Gould IN, Cleveland OH) and recorded on
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a chart paper recorder (Grass Model 7D polygraph, Quincy, Mass).
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Exhaled gas from the expiratory port of the ventilator was collected as described below for measurement of average nitric oxide concentration in the exhaled gas of the anesthetized intact animal. This was done in all animals prior to preparing the isolated lung. NO concentration was measured offline as described below. The carotid artery and jugular vein were exposed and cannulated. Heparin was injected (1000 units/kg), and the animal was exsanguinated slowly through the carotid artery cannula. During bleeding a total of 30 ml of lactated ringer was injected gradually into the animal to expand its blood volume. A total of 40 ml of diluted blood (Hct 23±1%) was collected from the animal for use in the isolated lung preparation which was described in more details previously (Ferrario et al 1996, Hakim et al 1997). The blood was added to the reservoir in the perfusion system which was kept at 37º C and was used to prime
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the perfusion system. The chest was opened and two cannulas, connected to the perfusion system, were placed in the pulmonary artery and the left atrium and secured in place. Blood flow through the lungs was reestablished quickly (< 3 min) using a roller pump. A pressure of 1.5
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cm H2O was applied at end expiration. The blood flow was increased gradually (up to 40±3
ml/min). The final flow was selected to give a near normal pulmonary artery pressure of 10
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mmHg and was kept constant for the duration of the experiment. During the initial stabilization
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period, and when necessary, blood pH was adjusted to 7.35 to 7.45 by adding bicarbonate to the reservoir. Pulmonary arterial pressure (Pa) and venous pressure (Pv) were measured from
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side ports in the cannulas using pressure transducers and were recorded continuously . Pv was kept at 1 mmHg by adjusting the level of the reservoir. Because perfusion rate and Pv were kept
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constant, a change in Pa represented a change in pulmonary vascular resistance. Adequate time was allowed for the isolated lung preparation to stabilize (10 to 15 min).
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Baseline measurements of blood gases and pH, Paw, and exhaled NO (NOe) were obtained.
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Exhaled gas from the isolated lungs was collected for > 3 minutes using a 1 L polyvinyl bag
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connected to the expiratory port of the ventilator. After collecting > 0.5 L of exhaled gas, the bag was removed and the concentration of NO in the exhaled gas was measured immediately offline using chemiluminescence NO analyzer (Siever 270B). The NO analyzer was calibrated daily using nitrogen for zero and a mixture of 248 ppb NO in nitrogen as described in more details elsewhere (Carlin et al 1997, Pedoto et al 1998). Lung ventilation was then switched to a low oxygen bag containing 3%O2, and 5% CO2 in nitrogen to test the effect of hypoxia on pulmonary vascular response and on NO concentration in the exhaled gas. After Pa rose to a new steady level (within 5 to 10 min), measurements were repeated and exhaled gas was collected as above. Ventilation was then switched back to the normoxic gas. From here onward, the control lungs or the LPS treated lungs were assigned to receive either L-arginine or nitro-L-arginine
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(LNA), thus generating 4 groups. Group 1 consisted of controls (n=6) and Group 2 consisted of LPS treated lungs(n=6). Groups 1 and 2 were monitored before and after adding L-arginine. Group 3 was a second control group (n=6) but instead of L-arginine, the lungs received LNA after
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the initial baseline measurements, while Group 4 consisted of LPS treated lungs (n=6) and
received LNA after baseline measurements. L-arginine (10 mg) was added to the blood in the
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perfusion system to increase NO production. This dose of L-arginine consistently increased NO
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production and prevented any spontaneous decline in NOe with time. LNA (2.5 mg added to perfusion system) was used to block NO production. After 10 minutes, a new set of baseline
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measurements were made including NOe, and hypoxic ventilation. During normoxic gas ventilation in the isolated lungs, PO2 was 252±3 mmHg, PCO2 was 39±1 mmHg, and pH was
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7.36±0.01, while during hypoxic gas ventilation PO2 was 34±3 mmHg, PCO2 was 34±3 mmHg,
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2.1 Statistical Analysis:
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and pH was 7.38±0.02.
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Statistical analysis of the results was done using repeated measures analysis of variance (ANOVA) and a paired Student’s t test. All results are given as mean plus or minus SEM. A p value of < 0.05 was considered as being significant. The Tukey post-hoc test was performed for multiple comparisons.
3. Results
In the intact anesthetized animals prior to isolating the lungs, NOe in the control animals was 2.2±0.4 ppb, and in the LPS treated animals, NOe was 131±11 ppb. After preparation of the isolated lungs, NOe in the control lungs was 1.8±0.3 ppb, while in LPS treated lungs, NOe was 99±17 ppb (lower than in intact animals), and usually continued to decline gradually with time
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as illustrated in 5 individual experiments in Fig 1. However, this decline in NOe could be prevented when L-arginine is added to the blood. Fig 2 illustrates the effects of adding L-arginine on NOe. In control lungs, addition of L-arginine affected neither NOe nor the perfusion pressure,
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while in LPS lungs, addition of L-arginine, increased NOe markerdly to 143±27 ppb (p<0.05) with minimal decrease in pressure. These results show that increasing L-arginine concentration
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resulted in a significant increase NO production only in LPS treated lungs. Note that, L-arginine
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concentration in the blood (not measured) was likely to have been half the normal plasma concentration because the blood was diluted with Ringer’s lactate.
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The effects of hypoxia are shown in figs 3, 4 and 5. The 3 figures compare responses to
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hypoxia under conditions where NOe was increased or decreased. The scales in these 3 figures were kept similar to allow comparison among the 3 figures. The baseline values and the hypoxic
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responses in the two control groups preceding addition of L-arginine and LNA, were not
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significantly different, therefore, the values from 12 lungs were combined and are shown in Fig 3. Hypoxia, did not change NOe in control lungs, but decreased it slightly in the LPS treated
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lungs from 111±19 to 90±15 ppb (p<0.05). Hypoxia caused the pulmonary arterial pressure to rise from 9.7±0.5 to 17.4±2.7 mmHg in control lungs, and from 10.5±0.5 to 27.1±2.4 mmHg in the LPS treated lungs. Thus HPV was potentiated in LPS treated lungs, despite the elevated level of NO production. Fig 4 shows the responses to hypoxia after NOe was increased by adding Larginine to the blood. Hypoxia had a minimal effect in controls, but decreased NOe in the LPS treated lungs from 143±27 to 115±22 ppb (p<0.05), but the increase in pressure in the LPS treated lungs was about similar to that in controls. After NOe was reduced with LNA (to 32±7 ppb), hypoxia had no effect on NOe in either the control group or the LPS treated group, however, HPV was potentiated equally in control lungs and in LPS treated lungs (Pa rose by approximately 23 mmHg in both groups), despite the marked difference in NOe levels.
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Therefore, based on the data in these 3 figures, we suggest that potentiation or reduction in NO synthesis, did not affect the difference in HPV between control and LPS treated lungs. These results suggest that hypoxia may affect NO production slightly, but NO levels play a minimal role
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in HPV.
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4. Discussion The main findings from the present study is that HPV was unrelated to the existing level
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of NOe or the change in NOe during hypoxia suggesting that NO production is not an important factor in HPV. Despite the marked difference in NOe in control lungs and LPS treated lungs, LNA
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enhanced HPV equally in these 2 groups, further corroborating the minimal role of NO in HPV. It is possible that LNA may potentiate HPV by mechanisms other than inhibition of NO synthesis.
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4.1. Isolated lung preparation: Rat lungs were ideal for this study because they exhibit a brisk pulmonary hypoxic vasoconstriction (HPV) response and NO production can be reliably
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upregulated with endotoxin. Because this is an isolated lung preparation, any change in NOe
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observed would have to be attributed to a direct effect on cells within the lungs that are capable of producing NO or capable of expressing iNOS. In the lung many cells are capable of doing
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doing so including bronchial epithelia , endothelial cells, pulmonary artery smooth muscle cells ,
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macrophages , or neutrophils (Scumpia et al 20022002, Szabo and Salzman 1997). In isolated lung preparation, vascular response to hypoxia (HPV) can also be measure simultaneously while
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measuring NO production, allowing more precise conclusions about the relationship of NO production to vascular responses. The results lead us to conclude that although NO production maybe diminished slightly by hypoxia, the change in NO levels that are sometimes discernible during hypoxia are too small to have a significant influence on vascular tone or on HPV. We are able to make such conclusions with confidence because of the isolated lung preparation where changes in vascular resistance and NOe can be measured with accuracy, and can be reproduced in the same preparation. We have successfully examined the effects of L-arginine supplement, and the effect of hypoxia on NO production in the lung. The limitation of the isolated lung preparation is that we cannot say which cells are involved but it is likely to involve all cells capable of producing NO and expressing iNOS. The cells are in their natural environment and
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usual milieu and their response would reflect best the response in intact animal. Most NO produced in the lungs diffuses easily into the alveoli and exit in the exhaled gas. A small amount of NO diffuses into the blood and is scavenged by hemoglobin but it is possible to perfuse
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isolated lungs with red blood cells free solutions (Grimminger et al 1995). In the isolated lung preparation, the vascular responses can be identified in terms of arteries or veins using the
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arterial, venous, or double occlusion technique (Hakim et al 1997). The isolated lung provided
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an excellent model to explore the role of NO because the changes in NOe can be measured immediately and repeatedly under different conditions, in control lungs with cNOS or LPS
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treated lungs with iNOS upregulated. This isolated lung model is relatively easy to prepare and
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would allow more firm conclusions about the role of NO.
4.2 Effect of L-arginine: The isolated lung was ideal to test the effect of L-arginine on NO
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production from lung cells by measuring NO production immediately. Clearly any effect would
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have to be due to effect on many cells in the lung. To begin with, we found that NO production
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in the isolated lungs that were perfused with diluted blood (i.e with low L-arginine concentration) could exhibit a lower NO production than in intact animal. When L arginine was added to the blood, NO production in the LPS treated lungs increased subtantially. This was not surprising because the blood was diluted and hence L-arginine concentration was low. In contrast to the LPS treated lungs, NO production in control lungs is very low, and was not affected by addition of L-arginine to the blood. In the intact rat, Wu et al (1999) showed that dietary arginine deficiency, limited NO production by cNOS and iNOS. Our results are in partial agreement and show that in the LPS treated lungs (iNOS present), L-arginine supply may be a limiting factor, but L-arginine does not seem to affect NO production in control lungs (cNOS present). Our conclusions are supported by studies in isolated vessels (Schini and Vanhoutte
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1991, Julou-Schaeffer et al 1990). This information may be relevant to issues related to arginine supplementation in healthy subjects (Alvares et al 2012), in patients (Jabecka et al 2012, Antosova and Starpkova 2013) or in sepsis (Luiking and Deitz 2007, Kalil and Danner 2006). The
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results from this study suggest that arginine supplements would have little effect on NO production in healthy subjects and patients with normal plasma arginine levels. Clearly,
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supplementation with L-arginine can elevate plasma arginine levels, but may not necessarily
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increase NO production. In intact rats, we have found that injection of supplemental L-arginine has minimal effect on NOe in normal or septic animals that have normal l-arginine (unpublished
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observation).
As pointed out in Fig 1, NOe concentration in LPS lungs declined slowly with time during
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the course of the experiment. This is important to remember when performing studies on isolated lungs with iNOS upregulated and NO production is elevated. In contrast, we found that
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NOe concentration in control rat lung remained low and stable. Furthermore, we previously
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found that in isolated rabbit lungs that inherently have an elevated level of NOe due to cNOS
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remained fairly stable (Carlin et al 1997). One explanation for the decline in NOe in LPS treated isolated lungs is the high NO production rate in the presence of a limited supply of L-arginine in the blood.
4.3 Effect of hypoxia: HPV in LPS treated lungs was potentiated or remained as brisk as in controls despite the presence of markedly elevated NO levels. That in itself suggests that HPV is independent of NO levels or the change in NO production during hypoxia. Furthermore, LPS treated lungs showed that indeed, hypoxia caused a small decrease in NO production but such a change in NOe does not justify the magnitude of the HPV. Previous investigators examined the role of NO during HPV by comparing HPV before and after blockade with L-arginine analog and
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reported potentiation in HPV after blocking NO synthesis. This indirect finding lead them to conclude that HPV maybe mediated by NO production (Archer et al 1989, Robertson et al 1990, Persson et al 1990). Most studies agree that NOS inhibition augments HPV including this
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present study (Archer et al 1989, Persson et al 1990, Robertson et al 1990, Rodman et al 1990) but it is not clear if this augmentation in HPV is due to the small reduction in NO synthesis. Fig 5
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shows that HPV after LNA, was indeed potentiated but was similar in the control group as in the
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LPS treated group. In most animal species, basal NO production due to cNOS is very low (Gustafson et al 1991), and blocking its synthesis is not likely to have much influence on
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pulmonary vascular resistance that can be attributed to the small reduction in NO synthesis. Although NOS blockers affect vascular resistance slightly, they have a more impressive effect on
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HPV, but the potentiation in HPV after NOS inhibitors, may be due to a nonspecific effect not related to NO production. Some studies reported attenuation in HPV following treatments with
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endotoxins, and attributed it to upregulation of NO synthesis (Weir et al 1976), while others
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reported potentiation in HPV (Castaneda et al 2001). We find that HPV could become enhanced
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in LPS treated lungs in the presence of high NO. Several investigators, demonstrated a small decrease in NOe during hypoxia, but the decrease was usually very small (less than 5 or 10 ppb), it could hardly be responsible for changes in vascular resistance or explain the magnitude of the HPV. There is little doubt that NOe decreases during hypoxia, especially in LPS treated lungs where it becomes more discernible than in control lungs . The cause of such a decrease is thought to be due to less oxygen being available for NO synthesis process. The study by Hemmingsson and Linnarsson (2009) suggest that hypoxia may affect NOe because of changes in diffusion that leads to less NO emerging in the exhaled gas. Such a finding supports our conclusions and suggests that the HPV and reduction in NOe do not necessarily have a cause and effect relationship. NO inhalation therapy requires NO levels much higher (up 100 ppm) to
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lower pulmonary vascular resistance (Lundin et al 1999, Marriagoni et al 1998). Our results are more consistent with HPV being primarily independent of what happens to NO levels. Thus it appears HPV becomes potentiated whether NO becomes elevated or diminished. One may
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think that the issue is confounded by the fact that LPS treated lungs are sick lungs and may involve complicated events. This may not be so, because healthy rabbit lungs that have
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elevated levels of NOe (much higher than in rat lungs), also exhibit potentiation in HPV after
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inhibition of NO production (Carlin et al 1997) as it did in the LPS treated rats lungs. This leads us to the inescapable conclusion that NO production does not play a significant role in HPV,
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most likely because the changes in NO production are too small to have any influence on pulmonary vascular resistance. The most that can be said is that hypoxia decreases NO
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production, and HPV may be slightly modulated by the presence of NO, but NO does not play a significant role during HPV.
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In conclusion, we find that hypoxic pulmonary vasoconstriction is independent of
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existing NO production or the changes in NO production during hypoxia . Hypoxia may lower NO
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production but such an effect may be unrelated to the intensity of the pressor response. Investigators who report enhancement in hypoxic pulmonary vasoconstrions following NOS inhibition, should explore explanations other than NO production. Clearly, the results form rats lungs should be extrapolated with caution to other animal species.
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exhaled gas as an early marker of lung inflammation in a model of sepsis. Am J Resp Crit Care
Sylvester JT. Shimoda LA, Aaronson PI, Ward JPT. 2012. Hypoxic pulmonary vasoconstriction. Physiological Reviews 92(1), 367-520. Szabo C, Salzman L. 1997. The Physiology and pathophysiology of nitric oxide in the lung. The Pediatric Lung Edited by RW Willmott, Chapter 12, Pages 279-310. Weir EK, Milczoch J, Reeves JT, Grover RF. 1976. Endotoxin and prevention of hypoxic pulmonary vasoconstriction. J Lab Clin Med 68:975-983. Wu G, Flynn NE, Flynn SP, Jolly CA, Davis PK. 1999. Dietary protein or arginine deficiency impairs contstitutive and inducible nitric oxide synthesis by young rats. J Nutr 129, 1347-1354.
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Figure Legends Fig 1: Decline of exhaled nitric oxide in 5 individual isolated lungs from LPS treated rats. The
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solid line indicates the average exhaled nitric oxide in intact animal before preparing the isolated lung.
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Fig 2: Effect of adding L-arginine on exhaled nitric oxide and perfusion pressure in control lungs
us
(n=6) and LPS treated lungs (n=6). Lightly shaded bars are values before adding L-arginine and densely shaded bars are values after adding L-arginine. Asterisks indicate significant difference
an
(p< 0.05)
M
Fig 3: Effect of hypoxia on exhaled nitric oxide and on perfusion pressure in control lungs (n=12) and LPS treated lungs (n=12) during baseline at start of the experiment before L-arginine or LNA
d
was added to the perfusion system. Clear bars are values during normoxia, and shaded bars are
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values during hypoxia. Asterisks indicate significant difference (p< 0.05).
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Fig 4: Effect of hypoxia on exhaled nitric oxide and perfusion pressure in control lungs (n=6) and LPS treated lungs (n=6) after adding L-arginine to enhance NO production. Clear bars are values during normoxia, and shaded bars are values during hypoxia. Asterisks indicate significant difference (p< 0.05).
Fig 5: Effect of hypoxia on exhaled nitric oxide and perfusion pressure in control lungs (n=6) and LPS treated lungs (n=6) after adding nitro-L-arginine to block NO production. Clear bars are values during normoxia, and shaded bars are values during hypoxia. Asterisks indicate significant difference (p< 0.05).
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