in coronary artery bypass grafting surgery ( cabgs ) , the vessels that must be used are internal mammary arteries ( i m a ) and saphenous veins ( sv ) ; however , svs present a greater risk of occlusion . nitric oxide ( no ) could be involved in the improvement of sv graft patency rates [ 2 , 3 ] since no plays a pivotal role in vascular homeostasis [ 46 ] . three isoforms of no synthase ( nos ) exist : neural ( nnos ) , inducible ( inos ) , and endothelial ( enos ) . although nnos was first described in neurons , it is also present in vascular smooth muscle cell ( vsmc ) . we have shown in i m a that extraendothelial no is released from nnos present in vsmc [ 9 , 10 ] . some studies have reported that superoxide ( o2 ) contributes to the   development of hypertension [ 12 , 13 ] . in experimental hypertension , li et al .   have found elevated o2 levels in veins , suggesting that nadph oxidase activity induces hyperreactivity to vasoconstrictors . in agreement with these data , we have observed that enhanced nadph oxidase activity drives o2 production in genetically hypertensive rats . in human i m a , we found that extraendothelial no counter - regulates angiotensin ii ( ang ii ) contractility and that this action is altered in hypertension , probably by an increased oxidative stress and a decreased nnos ability to produce no [ 9 , 10 ] . in addition , in human umbilical endothelial cells , nadh-/nadph - oxidases play a role in o2 induced by ang ii . in sv , alterations of the vascular reactivity , no bioavailability , and oxidative stress could affect sv patency rates . this notion would be supported by some reports that indicated that functional and structural abnormalities of sv result in graft thrombosis , intimal hyperplasia , and occlusion [ 3 , 18 ] .   to the best of our knowledge , despite the fact that hypertension is a major risk factor for coronary disease , the impact of hypertension and the role of oxidative stress in the contractile function of sv grafts have not been studied . the objective of this work was to evaluate the impact of oxidative stress on ang ii and norepinephrine-(ne- ) mediated vascular reactivity and no bioavailability in sv grafts of hypertensive patients ( ht ) . discarded sv segments were obtained from cabgs ( centro modelo de cardiologa , tucumn , argentina ) . to establish the impact of hypertension on oxidative stress , no contents and vascular contractility and strict inclusion criteria in relation to the risk factors were taken into account . patients with diabetes , renal failure , pulmonary disease , peripheral vascular disease in a clinical report , uncontrolled dyslipemia , or active smoking at the time of surgery were not included . to test the influence of hypertension , patients were divided into two groups , hypertensive ( ht ) and normotensive ( nt ) , according to clinical report . after surgery , svs were immediately placed in krebs solution ( mm : nacl 118.3 ; kcl 4.7 ; cacl2 2.5 ; mgso4 1.2 ; kh2po4 1.2 ; nahco3 25 ; glucose 11.1 ; na2edta 0.026 ) , maintained at 4c , and transferred to the laboratory . each vessel was dissected free from connective tissue , and 2 to 5 rings ( 5  mm ) were obtained . rings were mounted between two stainless - steel wires in organ chambers filled with krebs solution , which had been gassed with 95% o2 and 5% co2 ( ph 7.4 ) . one wire was anchored , and the other one was connected to an isometric force transducer ( gould uc2 , usa ) and a recorder ( kipp and zonen bd41 , holland ) . isometric tension was measured under an initial tension of 3  g , which was found to be the optimal tension in which the depolarizing high k solution induced contraction . all preparations were equilibrated and washed every 15 minutes during 120 minutes .   to evaluate endothelial function , a cumulative dose response curve ( cdrc ) to acetylcholine ( ach ) ( 1010  m ) in precontracted ne ( 10  m ) rings was performed . similar ach responses were observed in all rings from the same patient , which is necessary to point out . the absence of endothelium - dependent relaxation was observed in rings from 86.4% of the patients ( nt : 12 ; ht : 26 patients ) . according to the objectives of the present work , only these rings ( n = 38 ) were used . in this regard , to rule out the presence of endothelium , immunohistochemical studies were performed ( see below ) . to test endothelium - independent relaxation , sodium nitroprusside ( snp ) 10 snp induced a nearly complete relaxation ( ht : 99  6% and nt : 107  11% of ne pre - contraction ; p : ns ) . ( 1010  m ) or ne cdrc ( 1010  m ) were performed . the maximal contractile response ( rmax ) and negative log of the molar concentration inducing 50% of the rmax ( pec50 ) were measured from the corresponding cdrc .   to evaluate the effect of no - mediated inhibition on ang ii and ne reactivity , rings were pretreated with n-nitro - l - arginine methyl ester ( l - name : 10  m ) for 30  min . to evaluate the possible role of oxidative stress in vascular reactivity , rings were pretreated with diphenylene iodonium ( dpi : 10  m ) or tempol ( 10  m ) for 30  min . at the end of the experiments , nitrites were measured by the griess reaction , which is frequently used to indirectly measure no contents . we have previously shown that stretching is an ideal condition for in vitro nitrite dosage in isolated human vessels [ 9 , 10 ] . therefore , simultaneous in vitro measurements of vascular reactivity and nitrite release data with respect to tissue were expressed in pmol / mg of tissue . to corroborate an extraendothelial no presence , the possible nos isoforms involved were evaluated by treatment with l - name 10  m , l - name plus l - arginine 10  m , s - methyl - l - thiocitrulline 10  m ( nnos inhibitor ) , or aminoguanidine 10  m ( inos inhibitor ) . because constitutive nos has been described as a ca / calmodulin - dependent enzyme , some rings were incubated in ca - free media ( krebs solution without cacl2 plus egta 3  mm ) . the role of oxidative stress on nitrite contents was evaluated with dpi 10  m or tempol 10  m. no release was evaluated in real time , with the membrane - type no - sensitive electrode ( iso - nop ; wpi , usa ) . this electrode records an electrical current that is directly proportional to the no concentration . the signal was acquired by the apollo 4000 recording system ( wpi , usa ) . after 120 minutes of equilibration , the electrode was stabilized and the baseline of the current became stable . the role of oxidative stress in the no content was evaluated with tempol 10  m. at the end of the experiments , the rings were fixed in buffered formol and stained with hematoxylin and eosin ( h&e ) . immunohistochemistry examination of endothelium was performed with monoclonal cd34 antibodies ( clone : qbend 10 ; bio genex , usa ; dilution : 1/160 ) , anti - enos antibody ( santa cruz , usa , dilution 1/100 ) . in all cases , the absence of an endothelial cell layer was observed by h&e , anti - cd-34 , and anti - enos staining . nnos examination was performed with rabbit anti - nnos antibody ( bd bioscience pharmingen ; dilution : 1  :  100 ) . the positive control for vsmc presence was performed with anti- actin antibodies ( sigma chemical company , usa ) . the anti - nnos antibody - stained area was calculated using a light microscope connected to a video camera and an informatics system ( image j 1.43   , md , usa ) calibrated to correspond to an equivalent m value ( 1  m = 18.400 px ) , and the anti - nnos - stained area / total area ratio was calculated . to evaluate the oxidative state protein carbonyl content , conjugated diene ( cd ) ( the primary product of lipid peroxidation ) , the reduced / oxidized glutathione ( gsh / gssg ) ratio , and thiobarbituric acid ( tba ) reactive substances ( tbars ) were determined . rings were homogenized in ice - cold tris - kcl buffer ( 0.15  m , ph 7.4 ) , precipitated by trichloroacetic acid ( tca : 20% ) , and centrifuged . the pellet was incubated with 2,4-dinitrophenylhydrazine ( dnph : 0.002  g / ml ) , washed with an ethanol - ethylacetate mixture , centrifuged , and redissolved with guanidine hcl / dithiothreitol . protein carbonyl contents were read spectrophotometrically at 370  nm   and expressed as pmol / mg protein . cd was determined spectrophotometrically at 233  nm using a chloroform - methanol mixture   and was correlated with mg of phosphates . , fractions were homogenized in phosphate buffer plus edta 6.3  mm ( ph 7.4 ) , mixed with tca 10% , and centrifuged . the supernatant was incubated with nadph 0.3  mm , dtnb 6  mm , and glutathione reductase 0.077  u ( 10  l ) . to calculate gsh , 20  l of the supernatant was incubated for 15  min with dtnb 6  mm ( 0.1  ml ) . absorbance was read spectrophotometrically at 412  nm and correlated with protein contents , and then the gssg / gsh ratio was calculated . lipid peroxidation was evaluated in sv fractions homogenized in detergent - free buffer by measurement of malondialdehyde contents . the results were expressed as mol tbars / mg of protein , as determined by the lowry 's method . data were expressed as mean  standard error ( se ) . to calculate pec50 for each cdrc , the sigmoid equation of the curve fitting program paired or nonpaired student 's t - test and anova with newman - keuls tests were used when appropriate . figure 1 shows the vasocontractile response to ang ii ( figure 1(a ) ) and ne ( figure 1(b ) ) on sv rings without endothelium and the effect of dpi in this response . ang ii ( 1010  m ) produced a dose - dependent contraction in both nt and ht rings ( figure 1(a ) ) ; however , ht showed higher ang ii rmax values ( table 2 ) . a curve fitness analysis showed no difference in potency ( pec50 ) between nt and ht ( 6.5  0.2 ; n = 7 versus 6.6  0.1 ; n = 6 , resp . ) . in ht , dpi decreased the ang ii dose - dependent contraction ( figure 1(a ) ) and rmax ( table 2 ) , however , this agent did not modify the ang ii response in nt . tempol did not modify the ang ii rmax in ht or nt ( table 2 ) . similar to ang ii , ne ( 1010  m ) produced a dose - dependent contraction in both nt and ht ( figure 1(b ) ) . no difference in the ne pec50 values between nt and ht was found ( 7.6  0.6 ; n = 6 versus 7.1  0.1 ; n = 6 ) . in ht , dpi decreased the ne dose - dependent contraction ( figure 1(a ) ) and rmax ( table 2 ) , however , this agent did not modify the ne response in nt . tempol did not modify the ne rmax in ht or nt ( table 2 ) .   in ht , l - name 10  m did not modify the ang ii rmax , however , it increased the ang ii reactivity in nt ( table 2 ) . l - name did not modify the ne rmax in ht , but it did increase the ne reactivity in nt ( table 2 ) . in contrast to ang ii and ne , the response to kcl 100  mm was similar in ht ( 1115.9  141.7  mg ; n = 22 ) and nt ( 1056.7  193.8  mg ; n = 10 ) . nitrite contents were present in both nt and ht ( 1066.1  86.3  pmol / mg ; n = 11 versus 487.8  51.6 ; n = 23 ; p < 0.01 ) , despite the endothelial absence . moreover , rubbed maneuvers did not blunt the nitrite contents in either nt (  : 5  13% , n = 8 ; p : ns ) or ht (  : 6  12% , l - name significantly decreased nitrites in all cases . no differences between nt and ht in the presence of l - name l - name plus l - arginine did not modify the nitrite contents in ht (  : 11.9  4.2  pmol / mg ; n = 10 ) and nt (  : 15.4  5.2  pmol / mg ; n = 6 ) . similarly to l - name , s - methyl - l - thiocitrulline ( nnos inhibitor ) inhibited nitrites ( 53.6  6.8% ; n = 8 ; p < 0.01 in ht ) . to test whether nitrite release was mediated by nnos ( a ca / calmodulin dependent enzyme ) , ca - free media decreased the nitrite contents in nt (  : 881.6  37.2  pmol / mg ; n = 7 ; p < 0.001 ) and in ht (  : 252.4  43.1  pmol / mg ; n = 6 ; p < 0.01 ) . in any case , aminoguanidine did not modify the nitrite content in nt (  : 54.2  77.4  pmol / mg ; n = 7 ; p : ns ) and in ht (  : 4.1  53.8  pmol / mg ; an increase of nitrites was observed with dpi ( 1024.7  45.4  pmol / mg ; n = 7 ; p < 0.001 ) and tempol ( 1071.2  77.8  pmol / mg ; figure 3 shows typical recorders of direct measurements of no in basal conditions and the effect of tempol in nt ( a ) and ht ( b ) . nt showed higher no values than ht ( 48.8  4.7 na ; n = 6 versus 17.2  0.8 ; n = 7 ; p < 0.001 ) . in ht , tempol 10  m increased no release ( 42.5  2.7  na ; n = 7 ; p < 0.001 ) . in nt , tempol did not increase the no contents ( 49.63.6 na ; n = 6 ; p : ns ) . only in ht , dpi increased no release (  : 24.5  1.8  na ; n = 7 ; p < 0.001 ) . because no was present in sv rings without endothelium and based on previous findings from our laboratory in which we demonstrated the nnos presence in vsmc of i m a rings , immunohistochemistry for nnos was performed in sv . in both nt and ht , the anti - nnos antibody specifically stained vsmc ( figure 4 ) . the anti - nnos - stained area was higher in nt than ht ( 26  3.2% of the wall area ; n = 7 versus 10  0.6 ; n = 7 , resp . ; p < 0.001 ) . no immunoreactive product was observed when samples were prepared without the primary antibody .   in both nt and ht no staining with the anti - enos antibody the levels of protein carbonyl groups were higher in ht than in nt ( 21.7  4.4  pmol / mg protein ; n = 5 versus 3.9  0.8 ; n = 4 ; p < 0.05 ) . determinations of cd showed higher values in ht than in nt ( 0.26  0.012  l / mg phosphates ; n = 6 versus 0.17  0.01 ; n = 6 ; p < 0.05 ) . the levels of gssg were higher in ht than in nt ( 40.3  16.3  mol / mg protein ; n = 7 versus 4.5  1.8 ; n = 7 ; p < 0.05 ) . in addition , ht had elevated gssg / gsh compared to nt ( 21.9  8.6 ; n = 6 versus 3.1  1.4 ; n = 7 ; p < 0.05 ) . finally , tbars levels were higher in ht than in nt ( 2.2  0.6  nmol / mg protein ; n = 6 versus 0.1  0.01 ; n = 8 ; p < 0.01 ) . we studied the contractility response to exogenous agonists in sv rings with endothelial dysfunction and the role of the oxidative stress on this response . few studies have evaluated the vascular reactivity , or the impact of arterial hypertension , in sv . lytle et al .   have indicated that after 5 years of cabgs , the graft patency of sv exceeded that of i m a . however , dashwood has indicated that the patency rate is poor , with a high proportion of patients requiring further surgery . some factors implicated are aging , serum cholesterol , diabetes , harvesting technique , or endothelial integrity . ca - protein kinase c - pathways are implicated in the reactivity of sv grafts . in the present work , we found a hyperreactivity to both ang ii and ne in sv from ht patients , which was reverted by antioxidant agents . in this sense , hyperreactivity to vasoconstrictors has previously been observed in doca - salt hypertensive rats . xu et al .   have observed increased reactivity to ne in mesenteric veins , and li et al . have reported a hyperreactivity to endothelin-1 in the vena cava , which was related to the presence of oxidative stress . in human sv , hypertension stimulates endothelin-1 release , which not only affects vascular reactivity but also increases oxidative stress . in sv , sharif et al . have studied the role of oxidative stress , indicating that n - acetylcysteine does not improve endothelial - dependent relaxation and vsmc function . however , the same authors found that vitamin c improves the endothelial - dependent relaxation . in our vessels , antioxidant agents diminish vascular reactivity , suggesting that oxidative stress is implicated in the hyperreactivity of ht . we also found that the antioxidant agents dpi and tempol increased no release in sv . this observation is in concordance with previous works from our laboratory on i m a [ 9 , 10 ] , in which we proposed that extraendothelial no release is present through nnos . here , the extraendothelial no presence in sv is supported by the observations that l - name and s - methyl - l - thiocitrulline decrease nitrite levels and that nnos is present in vsmc ( figure 4 ) . another finding that support that the source of extraendothelial no is through nnos is the lack of staining with anti - enos antibody . in addition , the nnos presence in sv has previously been demonstrated by webb et al . . this result is in agreement with previous work in i m a . to the best of our knowledge , no comparison of extraendothelial no release in sv between ht and nt has been performed . dpi increased the no contents in ht , suggesting a higher impact of the oxidative stress in ht patients . o2 production has been demonstrated to be higher in veins than in arteries [ 36 , 37 ] . in human sv rings , have observed that 5-series f2-isoprostanes ( compounds produced by the nonenzymatic peroxidation of arachidonic acid ) do not have vasomotor effects , and antoniades et al . o2 has been demonstrated not only to be a no - scavenger and a vasoconstrictor but also to produce direct vascular damage . in this sense , shi et al .   have observed that biological changes in sv grafts are characterized by oxidative stress resulting from higher o2 production and lower superoxide dimutase activity . in our work , the increased oxidative damage in hypertension is supported by the observation that the levels of protein carbonyl groups , cd , gssg , gssg / gsh ; and tbars are increased in ht rings . the present work demonstrates that extraendothelial no counter - regulates contractility in sv used for cabgs . however , this no action could be altered in hypertensive situations , even if there were no other associated risk factors . we suggest two mechanisms ( 1 ) increased oxidative stress and ( 2 ) a decreased ability of nnos to produce no . further studies should be performed to evaluate the implications of these results in sv graft patency rates .