however , the exact mechanisms of vasoconstriction of pulmonary vessels during hypoxia ( hpv ) have not been identified yet .
many investigators have reported the role of reactive oxygen species ( ros ) in hpv . however , there are conflicting results on whether ros decreases or increases during hpv . furthermore , the contribution of ros in the acute and sustained phases of hpv is still arguable .
superoxide anion ( o2 ) is one of the oxygen free radicals which is supposedly involved in hpv .
therefore , it should be eliminated from the cells by a carrier or an anion channel .
so far , no special carrier is known for o2 . however , the rate of o2 release from the isolated pulmonary rat artery increases during short - term hypoxia in the presence of bicarbonate buffer .
furthermore , inhibition of cl - hco3 exchanger ( anion exchanger , ae ) prevents the release of o2 in rat s pulmonary artery and isolated rabbit lung .
two isoforms of ae , na - dependent , and na - independent were detected in the pulmonary vessels .
na - dependent ae leads hco3 anions to enter the cells in exchange for chloride anions .
the activity of this transporter increases during hypoxia and leads to intracellular alkalinization in the pulmonary smooth muscle cells .
therefore , hco3 may enter into the smooth muscle cells in exchange for o2 instead of chloride anion during hypoxic condition .
some studies have reported the role of extracellular hco3 on the regulation of pulmonary vascular tone .
hypercapnia with normal ph ( corrected by bicarbonate ) is indicated to increase pulmonary artery wall tension in rats . also ,
pulmonary artery pressure increases during hypoxia and hypercapnia with ph adjusted by hco3 in the isolated perfused rabbit lung .
a few studies have addressed the role of ae on the regulation of pulmonary vascular resistance .
dids , acetazolamide ( carbonic anhydrase inhibitor ) , and sodium - free buffer decrease pulmonary vascular tone during hypercapnia with normal ph , which suggests the role of na - dependent ae on the pulmonary vasculature .
moreover , high dose of dids prolongs acute phase and prevents sustained phase of hpv in the isolated rabbit lung , which proposes the different role for ae in acute and sustained phases of hpv .
however , it is not fully clear by which mechanism ae may regulate pulmonary vascular tone , and whether changes in intracellular ph or entrance of hco3 have an important role in hpv
there is a general agreement that nitric oxide ( no ) is involved in the regulation of pulmonary vascular tone .
both in vivo and ex vivo studies have revealed that no production decreased in hpv .
therefore , at least a part of vasoconstriction during hypoxic gas ventilation is in concomitant with the reduction of no production , which could influence superoxide release too .
many studies indicated interactions between o2 and no in the lung during normoxic and hypoxic conditions .
inhibition of each may increase the availability of the other and induce pulmonary vasoconstriction by o2 or pulmonary vasodilatation by no .
therefore , it may be concluded that dids increases no production by preventing o2 release through the smooth muscle cells and thereby decreasing hpv .
however , no study has indicated the interaction between ros and anion exchanger and a possible role of no during ventilation of the lungs with hypoxic gas . with the above background , this study was performed to clarify the interaction of o2 and ae in hpv .
therefore , we added dids as an ae inhibitor and antioxidant trolox , 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid , to the perfusate before hypoxic gas ventilation in the isolated rabbit lung and measured changes in pulmonary artery pressure during the time course of experiments
. furthermore , in order to reveal the effects of trolox or / and dids on no production , we measured no metabolites in the perfusate at above conditions .
experiments were approved by the center of comparative and experimental medicine and the ethical committee of animal care at shiraz university of medical sciences , shiraz , iran .
animals were housed in standard cages under controlled laboratory conditions of temperature , humidity , and 12:12-hour light - dark cycle .
they had free access to water and standard food a few days before starting the experiments .
briefly , 20 male new zealand rabbits with body weight ( bw ) of 2.030.07 kg were anaesthetized by i.v .
administration of ketamine ( 30 - 50 mg / kg bw ) and xylazine ( 6 - 10 mg / kg bw ) and heparinized ( 1500 u / kg bw ) .
the trachea was cannulated and animals were ventilated with room air ( tidal volume : 18.480.58 ml , respiratory rate : 30/min ) .
chest wall was opened and about 40 ml heparinized blood was collected by cardiac puncture .
the blood was then centrifuged at 4 c at 4000 g for 7 minutes and about 20 ml of plasma was stored in ice . then ,
pulmonary artery was cannulated and the lung perfused with 4 c air bubble - free krebs - henseleit solution ( perfusate ) through the pulmonary artery cannula connected to a peristaltic pump with a flow rate of 20 ml / min .
finally , isolated ventilated perfused lung was placed in a temperature - equilibrated housing chamber and freely suspended from a force transducer and recirculation was performed .
meanwhile , the flow rate was slowly increased from 20 to 140 ml / min .
the left atrial pressure and positive end - expiratory pressure ( peep ) were set at 1.5 - 2.5 cm h2o and 2 cm h2o , respectively .
after 20 minutes of steady state period , the perfusate was changed , plasma was added to the perfusate , and the lung was stabilized for additional 20 minutes .
two different gas mixtures were employed in this study ; ( i ) normoxic normocapnic gas : 21% o2 , 5.5% co2 balanced with n2 and ( ii ) hypoxic normocapnic gas : 3% o2 , 5.5% co2 balanced with n2 .
the perfusate used in this study contained in mm : 120 nacl , 1.1 k2hpo4 , 1.3 mg po4 , 4.3 kcl , 2.4 cacl2 , and 13.32 glucose .
ph was adjusted to the normal range of 7.35 - 7.40 with nahco3 in all experimental groups .
after 40 minutes of the steady - state period , lungs were ventilated in a humidified chamber with hypoxic gas for 10 minutes to evaluate lung viability and its responses to short alveolar hypoxia as a routine test procedure .
subsequently , the lungs were randomly divided into four groups , namely control ( hox , n=5 ) , dids ( 200 m , sigma , n=5 ) treated , trolox ( 20 m , sigma , n=5 ) treated , and trolox+dids treated group ( n=5 ) .
trolox and/or dids were solved and added to the perfusate 10 minutes before the onset of the hypoxic gas ventilation .
pulmonary artery pressure , left atrial pressure , airway pressure , and lung weight were continuously registered using a data acquisition system ( power lab , ad instrument , australia ) connected to a pressure and force transducers .
pvr was calculated from pulmonary artery pressure , left atrial pressure , and perfusate flow rate by using ohm equation .
pvr was also calculated to evaluate changes in pvr irrespective of its basal values in the experimental groups .
perfusate samples were taken during steady - state and at 30 minutes in order to measure po2 , pco2 , hco3 , ph , and no metabolites of each group .
perfusate samples were taken from pulmonary venous outflow and were stored at -80c until measurement . after incubating the samples and standards by griess reagent and vanadium chloride in a 96-well plate , the absorbance of samples was detected at 540 nm wavelengths using a microplate reader ( biotek , usa ) and the concentrations of no metabolites were calculated .
the analysis of variance ( anova ) with lsd post hoc test was used for the comparison of means between the experimental groups .
paired t - test was used for comparison of the values in each group at 5 and 30 minutes .
experiments were approved by the center of comparative and experimental medicine and the ethical committee of animal care at shiraz university of medical sciences , shiraz , iran .
animals were housed in standard cages under controlled laboratory conditions of temperature , humidity , and 12:12-hour light - dark cycle .
they had free access to water and standard food a few days before starting the experiments .
briefly , 20 male new zealand rabbits with body weight ( bw ) of 2.030.07 kg were anaesthetized by i.v .
administration of ketamine ( 30 - 50 mg / kg bw ) and xylazine ( 6 - 10 mg / kg bw ) and heparinized ( 1500 u / kg bw ) .
the trachea was cannulated and animals were ventilated with room air ( tidal volume : 18.480.58 ml , respiratory rate : 30/min ) .
chest wall was opened and about 40 ml heparinized blood was collected by cardiac puncture .
the blood was then centrifuged at 4 c at 4000 g for 7 minutes and about 20 ml of plasma was stored in ice . then ,
pulmonary artery was cannulated and the lung perfused with 4 c air bubble - free krebs - henseleit solution ( perfusate ) through the pulmonary artery cannula connected to a peristaltic pump with a flow rate of 20 ml / min .
finally , isolated ventilated perfused lung was placed in a temperature - equilibrated housing chamber and freely suspended from a force transducer and recirculation was performed .
meanwhile , the flow rate was slowly increased from 20 to 140 ml / min .
the left atrial pressure and positive end - expiratory pressure ( peep ) were set at 1.5 - 2.5 cm h2o and 2 cm h2o , respectively .
after 20 minutes of steady state period , the perfusate was changed , plasma was added to the perfusate , and the lung was stabilized for additional 20 minutes .
two different gas mixtures were employed in this study ; ( i ) normoxic normocapnic gas : 21% o2 , 5.5% co2 balanced with n2 and ( ii ) hypoxic normocapnic gas : 3% o2 , 5.5% co2 balanced with n2 .
the perfusate used in this study contained in mm : 120 nacl , 1.1 k2hpo4 , 1.3 mg po4 , 4.3 kcl , 2.4 cacl2 , and 13.32 glucose .
ph was adjusted to the normal range of 7.35 - 7.40 with nahco3 in all experimental groups .
after 40 minutes of the steady - state period , lungs were ventilated in a humidified chamber with hypoxic gas for 10 minutes to evaluate lung viability and its responses to short alveolar hypoxia as a routine test procedure .
subsequently , the lungs were randomly divided into four groups , namely control ( hox , n=5 ) , dids ( 200 m , sigma , n=5 ) treated , trolox ( 20 m , sigma , n=5 ) treated , and trolox+dids treated group ( n=5 ) .
trolox and/or dids were solved and added to the perfusate 10 minutes before the onset of the hypoxic gas ventilation .
pulmonary artery pressure , left atrial pressure , airway pressure , and lung weight were continuously registered using a data acquisition system ( power lab , ad instrument , australia ) connected to a pressure and force transducers .
pvr was calculated from pulmonary artery pressure , left atrial pressure , and perfusate flow rate by using ohm equation .
pvr was also calculated to evaluate changes in pvr irrespective of its basal values in the experimental groups .
perfusate samples were taken during steady - state and at 30 minutes in order to measure po2 , pco2 , hco3 , ph , and no metabolites of each group .
perfusate samples were taken from pulmonary venous outflow and were stored at -80c until measurement . after incubating the samples and standards by griess reagent and vanadium chloride in a 96-well plate , the absorbance of samples was detected at 540 nm wavelengths using a microplate reader ( biotek , usa ) and the concentrations of no metabolites were calculated .
data are presented as meanse . the analysis of variance ( anova ) with lsd post hoc test was used for the comparison of means between the experimental groups .
paired t - test was used for comparison of the values in each group at 5 and 30 minutes .
there was no significant difference between po2 , pco2 , and ph in all hypoxic groups at 30 minutes of the experiments . however , concentration of hco3 in the trolox group was lower compared to the control ( p=0.006 ) and dids ( p=0.05 ) groups . also , hco3 in the trolox+dids group was less than the control ( p=0.001 ) and dids ( p=0.011 ) groups ( table 1 ) .
gas parameters in the perfusate at 30 minutes of hypoxic gas ventilation in the experimental groups data are presented as meanse ( n=5 in each group ) ; p<0.01 in trolox vs. control group ; p<0.01 in trolox vs. dids group ; p<0.05 in trolox+dids vs. control group ; p<0.05 in trolox+dids vs. dids group there was no difference between pvr in the control and dids groups at 5 minutes of the experiments ( p=0.667 ) .
the administration of trolox decreased pvr compared to the control ( p=0.016 ) and dids groups ( p=0.007 ) .
pvr in the trolox+dids group was also lower than the control ( p=0.001 ) and dids ( p<0.001 ) groups .
furthermore , pvr in the trolox+dids group was insignificantly less as compared with the trolox group ( p=0.130 ) ( figure 1 ) .
pulmonary vascular resistance ( pvr ) in trolox - treated and trolox+dids - treated groups were lower than other groups at 5 minutes .
* p<0.01 in trolox vs. control group ; * * p<0.01 in trolox+dids vs. control group ; p<0.01 in trolox vs. dids group ; p<0.001 in trolox+dids vs. dids group .
there was no difference between pvr in the control and dids groups at 30 minutes of the experiments ( p=0.457 ) .
however , pvr decreased in the trolox group compared to the control ( p=0.005 ) and dids ( p=0.020 ) groups . similar reduction in pvr occurred in the trolox+dids group compared to the control ( p=0.005 ) and dids ( p=0.022 ) groups .
there was no variation between pvr in the trolox and trolox+dids groups ( p=0.836 ) ( figure 2 ) .
pulmonary vascular resistance ( pvr ) in trolox - treated and trolox+dids - treated groups were lower than other groups at 30 minutes .
data are presented as meanse ( n=5 in each group ) . * * p<0.01 at each group vs. the control group ; p<0.05 at each group vs. the dids group .
the concentration of no metabolites in the perfusate of the trolox+dids group was significantly higher than the control group at 30 minutes ( p=0.007 ) .
however , there was no significant difference between no metabolites in the dids ( p=0.169 ) and trolox ( p=0.101 ) groups compared to the control group ( figure 3 ) .
concentrations of no metabolites of the perfusate in trolox+dids - treated was more than other groups at 30 minutes .
data are presented as meanse ( n=5 in each group ) . * * p<0.01 in trolox+dids vs. control group .
there was no alteration between pvr in the control group at 30 and 5 minutes ( p=0.286 ) .
furthermore , pvr in the dids group at 30 minutes decreased insignificantly compared to 5 minutes ( p=0.184 ) .
however , pvr decreased significantly at 30 minutes compared to 5 minutes ( p=0.038 ) . also , the reduction of pvr in the trolox+dids group at 30 minutes was significant as compared with 5 minutes ( p=0.013 ) ( figure 4 ) .
pulmonary vascular resistance ( pvr ) in trolox - treated and trolox+dids - treated groups at 30 minutes were lower than their values at 5 minutes .
although hpv has been appreciated for about seven decades , but its mechanism is still controversial . so far ,
various mechanisms are attributed to this physiologic phenomenon , including endothelial derived substances and special characteristic of pulmonary smooth muscle cells .
it has been reported that ros may increase or decrease during the exposure of pulmonary vessels to hypoxic gas , thus leading to hpv .
furthermore , any changes in ros result in no change in opposite direction . since there is no special carrier for o2 release from the pulmonary smooth muscle cells , it may exit through other transporters such as ae .
the aim of this study was to investigate the interaction of o2 and ae in hpv .
in addition , our findings indicate the effect of ae and ros inhibition on no production , as a factor for modulation of hpv .
pco2 , hco3 and ph in the perfusate did not alter throughout the experiments in the hypoxic groups , which confirm stable conditions over the time course of experiments . a similar reduction of po2 in all hypoxic groups verifies identical conditions of experiments between groups .
we tested pulmonary vascular responses to hypoxic gas ventilation immediately after the steady - state period in order to examine the viability of the lungs and found no variation between the results in all groups ( data not shown ) .
therefore , any alteration in hypoxic response of pulmonary vessels after the administration of drugs could be independent of the initial responses of the lungs . furthermore , we calculated pvr in order to rule out the effect of the left atrial pressure and perfusate flow rate on pulmonary artery pressure .
ventilation of the lungs with hypoxic gas induced hpv being higher at 5 minutes and then started to reduce insignificantly until 30 minutes of the experiments .
this data is comparable with data from previous studies showing a biphasic response of hpv in the isolated lung .
the administration of trolox decreased hpv at 5 minutes and entirely prevented it at 30 minutes .
trolox is a water - soluble analogue of vitamin e. it diffuses into both lipid and aqueous compartments and has a high potency for ros scavenging .
the effects of trolox in our experiments confirm the role of ros during acute as well as the sustained phase of hpv , which is in line with previous studies .
however , the role of different ros derivatives ( onoo , o2 , h2o2 , and oh ) on hpv can not be differentiated by trolox because of its wide spectrum antioxidant effects .
the administration of dids had no further effect on hpv compared to the control group .
the data is challenged by other studies postulating that the inhibition of ae or carbonic anhydrase decreases pulmonary vascular tone during hypercapnia .
furthermore , 400 m of dids extends the acute phase of hpv in the isolated rabbit lung whereas dids 200 m has no effect on pulmonary artery pressure compared to the control hypoxic group during 60 minutes of the experiments .
since dids is an inhibitor of ae and chloride channels , it can be assumed that the high doses of did have more effect on hpv via inhibition of both anion transporters and channels .
the expression of na - dependent ae increases the pulmonary artery smooth muscle cell during hypoxia .
the activity of this exchanger leads to the influx of hco3 into the cells in exchange for chloride anion .
inhibition of ae prevents hco3 to enter the cells and lead to intracellular acidosis . however , there are conflicting results with regard to the effect of hpv on intracellular ph .
some researchers have reported intracellular alkalosis and a few studies have indicated intracellular acidosis during hpv . it remains unclear whether changes in ph is a fundamental trigger for the induction of hpv .
the combination of trolox and dids resulted in lower hpv compared with the effect of trolox at 5 minutes even though it was not statistically significant .
however , reduction of hpv in the trolox+dids group compared with the control and dids groups was more than the trolox group compared to the control and dids groups .
o2 anion may exit from the pulmonary smooth muscle cells through ae in the isolated rat pulmonary artery and ischemia / reperfusion model in the isolated rabbit lung .
o2 release in exchange for hco3 may lead to the enhancement of intracellular ph in the pulmonary smooth muscle cells .
overall , one could suggest that dids reduces hpv a fraction more than trolox per se , perhaps by the prevention of intracellular alkalosis .
it seems that the presence of ros is fundamental for the induction of hpv and the activity of ae is necessary for alkalinization during hpv .
however , additional studies with different doses of ros inhibitors and ae inhibitors could specify the contribution of each in hpv .
no metabolites in the perfusate were significantly higher in the trolox+dids group compared to the control group .
inhibition of ros increases no production and the inhibition of ae prevents ros release from the pulmonary artery . each inhibitor insignificantly increased no metabolites of the perfusate in the trolox or dids group .
no is a vasodilator involved in the regulation of pulmonary vascular tone . more production in no may lead to more reduction in hpv in the trolox+dids group .
we did not measure extracellular and intracellular ph , and o2 due to certain limitations in our preparations .
these measurements would help us to confirm our hypothesis with regard to the possible interactions between ros and ae .
in the present study , the interaction between ros and ae in hpv has been assessed .
a possible interaction is found by a mechanism partly linked to no production and availability .
 background : the mechanism of hypoxic pulmonary vasoconstriction ( hpv ) is still debatable . it has been proposed that reactive oxygen species ( ros ) might be involved in hpv . 
 however , there is no special transporter for superoxide anion in the cell membrane and it may release from the cells via anion exchanger . 
 therefore , the aim of this study was to investigate the interaction of ros and anion exchanger in acute hpv.methods:the present study was performed in the isolated rabbit lung . 
 after preparation , the lungs were divided into four hypoxic groups of control , trolox ( antioxidant)-treated , 4,4-diisothiocyanatostilbene-2,2-disulfonic acid ( dids , anion exchanger inhibitor)-treated , and trolox+dids - treated . pulmonary artery pressure , left atrial pressure , and lung weight were continuously registered and pvr was then calculated . 
 po2 , pco2 , hco3- , ph , and no metabolites of the perfusate were measured during steady - state and at the end of experiments ( 30 minutes ) . 
 all data were compared with anova and t - test and significance was considered when p<0.05.results : ventilation of the lungs with hypoxic gas induced hpv in the control group . 
 dids did not have a further effect on hpv compared with the control group . 
 the combination of trolox and dids decreased hpv rather than trolox per se at 5 minutes . 
 furthermore , hpv was abolished in both the trolox and trolox+dids groups at 30 minutes . 
 concentrations of no metabolites in the trolox+dids group were more than other groups.conclusion:the present study indicates a possible interaction between ros and anion exchanger in acute hpv . 
 it also suggests the modulatory effect of no at above condition . 
Introduction
Materials and Methods
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Results
Discussion
Conclusion