hyperglycemia causes vascular damage with several underlying mechanisms . among them , high glucose levels in the blood may contribute to endothelial dysfunction by mediating abnormal endothelial permeability or an imbalance between endothelium - derived relaxing and contracting factors .
several previous studies also demonstrated that prolonged exposure to high glucose may affect endothelium - dependent vasodilation by stimulating the production of reactive oxygen species ( ros )   or voltage - gated k channel function in coronary vascular smooth muscle cells .
the majority of previous studies demonstrated effects of hyperglycemia on vascular functions with long - term exposure ; however , a few studies have been conducted to determine the effects of high glucose with short - term exposure .
the incubation of high glucose ( 20 mm ) for 2 h induced impairment of bradykinin - induced relaxation in human subcutaneous arteries whereas induced opposite result in human mesenteric arteries ( mas ) .
also , exposure of high glucose ( 44 mm ) for 3h produced the attenuation of phenylephrine ( phe)-induced contraction in rat aortic rings .
free fatty acids ( ffas ) regulate vascular functions , and circulating ffa levels in blood serum are often associated with hyperglycemia .
some studies have demonstrated that excessive levels ffas may cause vascular dysfunction by enhancing oxidative stress or reducing nitric oxide ( no ) production in endothelial cells .
a previous study has suggested that exposure to ffas at both high and physiological concentrations impaired endothelium - dependent relaxation ( edr ) in ma .
however , results from another study showed that ffas did not affect endothelial function or ros generation in the vessels .
conventional in vitro studies of arterial contractility is conducted in physiological salt solutions with glucose only for the metabolic substrates .
considering the importance of vascular functions in the pathophysiology of metabolic diseases , it is requested to have a comprehensive understanding of systemic arterial contractility under physiological conditions . on these backgrounds , we investigate the effects of short - term incubation with high glucose with or without other metabolic substrates on -adrenergic agonist - induced contraction in rat mesenteric arteries ( mas ) and deep femoral arteries ( dfas ) .
experiments were performed with the approval of the institutional animal care and use committee in chung - ang university ( approval no .
adult male sprague - dawley rats ( 240~320 g ) were anesthetized by intraperitoneal injection of pentobarbital sodium ( 60~100 mg / kg ) .
the intestine and proximal hind limbs were removed quickly and placed into normal tyrode ( nt ) solution at 4. the nt solution contained the following ( in mm ) : nacl 140 , kcl 5.4 , 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 10 , nah2po4 0.33 , glucose 10 , cacl2 1.8 , and mgcl2 1 .
the second and third branches of mas and dfas were isolated quickly ( inner diameter : 200~300 m ) and cut into segments 2.5~3 mm in length .
endothelium - intact artery rings were mounted on two 25 m tungsten wires in a dual - wire myograph ( 620 m ; dmt , aarhus , denmark ) .
each myograph chamber was filled with 5 ml of physiological salt solution ( pss ) .
an arterial ring segment was stabilized in pss aerated with 21% o2,/5% co2 ( the remainder n2 ) at 37. the pss solution consisted of the following ( in mm ) : nacl 118 , kcl 4 , mgso4 1 , nahco3 24 , nah2po4 0.44 , glucose 5.6 , and cacl2 1.8 . for each vessel , 80 mm kcl - pss
the contractile responses induced by different concentrations of phenylephrine ( phe ) were normalized to the 80 mm kcl - induced maximum constriction in each vessel .
in addition , edr was measured by the response to 10 m acetylcholine ( ach ) with rings pre - contracted with 10 m phe . the contractile response induced by phe or kcl
was assessed in the presence or absence of high glucose ( 23 mm ) , or with metabolic substrates .
the metabolic substrate compositions were modified based on analyses of the human 's blood sample . the nf solution consisted of the following ( in mm ) : 96 ml pss , oleic acid 200 m , palmitic acid 100 m , linoleic acid 100 m , sodium l - lactate 1 mm , sodium pyruvate 100 m , and carnitine hydrochloride 50 m .
palmitic acid , linoleic acid , and oleic acid were dissolved in 0.1 n naoh .
all drugs and chemicals were purchased from sigma - aldrich ( st . louis , mo , usa ) .
the response to phe is expressed as the percent of the second 80 mm kcl - pss - induced maximum constriction .
half - maximal effective concentrations ( ec50 levels ) were calculated by non - linear regression analyses of the concentration response curves generated with originpro 8.0 software ( originlab , northampton , ma , usa ) .
experiments were performed with the approval of the institutional animal care and use committee in chung - ang university ( approval no .
adult male sprague - dawley rats ( 240~320 g ) were anesthetized by intraperitoneal injection of pentobarbital sodium ( 60~100 mg / kg ) .
the intestine and proximal hind limbs were removed quickly and placed into normal tyrode ( nt ) solution at 4. the nt solution contained the following ( in mm ) : nacl 140 , kcl 5.4 , 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 10 , nah2po4 0.33 , glucose 10 , cacl2 1.8 , and mgcl2 1 .
the second and third branches of mas and dfas were isolated quickly ( inner diameter : 200~300 m ) and cut into segments 2.5~3 mm in length .
endothelium - intact artery rings were mounted on two 25 m tungsten wires in a dual - wire myograph ( 620 m ; dmt , aarhus , denmark ) .
each myograph chamber was filled with 5 ml of physiological salt solution ( pss ) .
an arterial ring segment was stabilized in pss aerated with 21% o2,/5% co2 ( the remainder n2 ) at 37. the pss solution consisted of the following ( in mm ) : nacl 118 , kcl 4 , mgso4 1 , nahco3 24 , nah2po4 0.44 , glucose 5.6 , and cacl2 1.8 . for each vessel , 80 mm kcl - pss
the contractile responses induced by different concentrations of phenylephrine ( phe ) were normalized to the 80 mm kcl - induced maximum constriction in each vessel .
in addition , edr was measured by the response to 10 m acetylcholine ( ach ) with rings pre - contracted with 10 m phe . the contractile response induced by phe or kcl
was assessed in the presence or absence of high glucose ( 23 mm ) , or with metabolic substrates .
the metabolic substrate compositions were modified based on analyses of the human 's blood sample . the nf solution consisted of the following ( in mm ) : 96 ml pss , oleic acid 200 m , palmitic acid 100 m , linoleic acid 100 m , sodium l - lactate 1 mm , sodium pyruvate 100 m , and carnitine hydrochloride 50 m .
palmitic acid , linoleic acid , and oleic acid were dissolved in 0.1 n naoh .
all drugs and chemicals were purchased from sigma - aldrich ( st . louis , mo , usa ) .
is expressed as the percent of the second 80 mm kcl - pss - induced maximum constriction .
half - maximal effective concentrations ( ec50 levels ) were calculated by non - linear regression analyses of the concentration response curves generated with originpro 8.0 software ( originlab , northampton , ma , usa ) .
after confirming 80 mm kcl - induced ( 80k contraction ) and 10 m phe - induced contraction , endothelium - dependent ach - induced vasorelaxation was tested in each vessel .
the tested vessels were either incubated with 23 mm glucose - containing or control pss , and then the concentration - dependent contractile responses to phe were analyzed .
incremental application of phe ( 0.05~10 m ) induced stepwise increase of arterial tones in both mas and dfas ( fig .
the phe- contractions at 2 m and above were more persistent in dfas than mas ; mas showed partial relaxation following peak contractions .
thus , for the comparison between mas and dfas , the peak amplitudes of phe- contraction were analyzed after normalization to 80 k contraction in each vessel .
after 30 min incubation in the presence of high glucose ( 23 mm , hg ) , the amplitudes of phe - contraction became higher in both mas and dfas ( fig .
the ec50 values of phe - contraction in mas were 1.960.37 and 1.220.25 m for control and hg , respectively ( fig .
1a ) , and 2.410.14 and 1.610.34 m for control and hg in dfas ( fig .
1b ) , respectively . to determine the effect of hg on the purely membrane depolarization - dependent contraction , cumulative doses of kcl were applied to both arteries ( fig .
next , we tested whether combined application of hg and saturated fatty acid differently affects the phe- contractions .
when co - incubated with palmitic acid ( 100 m ) , the enhancement of phe - contraction by hg was similarly observed in mas and dfas ( fig .
the ec50 values were : control mas , 1.960.27 m ; high glucose mas , 1.410.33 m ; control dfas , 2.410.19 m ; high glucose dfas , 1.200.05 m .
it has to be noted that the concentrations - dependent phe- contraction data for the control condition were same ones in fig . 1 and fig .
then we tested the effects of nf solution on phe - contractions . a significant reduction of phe ( 1~5 m)-induced contraction was observed when incubating ma rings with nf solution for 30 min ( fig .
interestingly , however , the incubation of dfa rings with nf solution for 30 min produced an increased sensitivity to phe - induced contraction ( fig .
the nf solution caused a significant leftward shift of the phe ( 1 and 2 m ) concentration - response curve ( fig .
the co - incubation with hg in nf solutions did not increase but slightly decreased the sensitivity to phe- contraction in mas ( fig .
ec50 values were 1.960.28 and 3.070.27 m for control and hg with nf in mas , respectively ( fig .
more interestingly , the enhanced phe- contraction of dfas under nf solution became reversed to inhibition smaller than the control level .
significant rightward shifts of the concentration - response curves to phe ( 1 and 2 m in mas , 1 and 10 m in dfas ) were observed in the presence of the combined high glucose and nf solutions ( fig .
the results of the present study showed that only 30 min of incubation in hg enhances the sensitivity to phe - induced contraction in rat mas and dfas .
more interesting finding was that the incubation in nf solution induces opposite responses , decrease and increase of phe - contractions in mas and dfas , respectively .
furthermore , the pro - contractile effect of hg alone converts to an attenuation of phe - contraction in nf solution , especially in dfa .
the effects of hg on phe - induced contraction vary depending on the experimental conditions .
although long term exposure to high glucose inhibits edr in a majority of studies , several studies show conflicting results with regard to the effects of short - term exposure to high glucose .
a previous study using diabetic rats showed decreased sensitivity to phe - induced contraction in blood vessels   whereas another study demonstrated the opposite result .
a more recent study also reported that phe - induced contraction in aortic rings was attenuated by acute exposure to high glucose ( 44 mm ) for 3 h .
in the conventional pss solution , the acute exposure to hg consistently enhanced the phe - contraction in both mas and dfas ( fig .
1 ) . because the high k - induced contractions were not affected by hg , the procontractile effects appears to be more specifically associated with the a - adrenergic signaling pathways or might be mediated by k channel inhibition .
the absence of any change in the high - k contraction also suggests that the endothelial function is not acutely changed by hg for 30 min .
this supposition is supported by a previous finding that the relaxation produced by ach was induced by a 6 but not 2 h incubation in high glucose ( 20 mm ) in human mesenteric and subcutaneous arteries .
several studies demonstrated that impairment of edr augmented phe - induced contraction following prolonged exposure to high glucose .
in addition , a high concentration of glucose induced endothelial dysfunction as shown by decreased ach - induced no - mediated relaxation   and increased production of ros in cultured endothelial cells .
several studies have indicated that high glucose activates nox and induces ros production in endothelial and vascular smooth muscle cells via a protein kinase c - dependent pathway , and that ros interact with endothelial no to reduce no bioactivity .
therefore , a decrease of edr by short - term exposure to high glucose could affect increased sensitivity to phe - induced contraction in mas and dfas .
the glucose concentration we applied in the present study was similar to postprandial glucose levels in type ii diabetic patients .
initially , it was suggested that the augmentation of phe - contraction by the acute hg suggested that the total peripheral resistance under tonic sympathetic tone might induce an increase of blood pressure after some heavy meals .
however , the differential effects of hg on phe - contractions in nf solution imply that the actual influence of glucose uptake on the resistance of circulation system requires careful interpretation . in the present study ,
however , we cautiously assume that the specific composition of the nf solution may reduce sensitivity to phe - induced contraction in mas . at first , we assumed that saturated fatty acids such as palmitic acid might be responsible for the differential responses in nf .
however , it was found that the sensitivity to phe - induced contraction was consistently augmented by co - incubation with palmitic acid and high glucose in both arteries ( fig .
, other components of nf , or more complicated effects of the combined components seem to be responsible for the intriguing influence of nf solution .
previous studies showed that individual free fatty acids have similar effects in mas and dfas , and endothelial cell dysfunction was induced with high and physiological concentrations of both saturated and unsaturated fatty acids .
further investigation is needed to identify these inconsistent effects of nf solutions using various arteries and solution compositions . in conclusion , the results of the current study demonstrated that short - term exposure to a high glucose solution augmented sensitivity to phe - induced contraction in rat mas and dfas .
in addition , this is the first study to examine the effects of nf solution alone and with high glucose .
these findings provide preliminary evidence for identifying the action mechanism of metabolic substrates and high glucose on vascular reactivity .
 hyperglycemia is associated with an increased risk of cardiovascular diseases . 
 it has been demonstrated that chronic exposure to high glucose impaired endothelial functions . however , specific effects of short - term exposure to high glucose on vascular reactivity are controversial . 
 moreover , the combined effects of other metabolic substrates such as free fatty acids ( ffa ) on vascular reactivity remain poorly understood . here 
 we investigate the effects of short - term exposure to high glucose with or without other metabolic substrates including ffas termed  nutrition full  
 ( nf ) solution , on mesenteric ( ma ) and deep femoral arteries ( dfa ) of rats . 
 arterial ring segments were mounted in a double - wire myograph . 
 contraction in response to phenylephrine ( phe ) was determined in control ( 5 mm ) and high glucose ( 23 mm , hg ) environments over a 30 min period . in both arteries , 
 phe - inducedvasocontraction was enhanced by pre - incubation of hg solution . a combined incubation with hg and palmitic acid ( 100 m ) induced similar sensitization of phe - contractions in both arteries . 
 in contrast , high k+-induced contractions were not affected by hg . 
 interestingly , pre - incubation with nf solution decreased phe - induced contraction in ma but increased the contraction in dfa . in nf solution 
 , the hg - induced facilitation of phe - contraction was not observed in ma . 
 furthermore , the phe - induced contraction of dfa was attenuated by hg in nf solution . 
 our results demonstrate that the sensitization of phe - induced arterial contraction by hg is differentially affected by other metabolic substrates . 
 the conversation of skeletal arterial contractility by hg in nf solution requires careful interpretation of the previous in vitro studies where only glucose is included in physiological salt solutions . 
 further studies are required to elucidate the mechanism underlying the inconsistent effect of nf solution on ma and dfa . 
INTRODUCTION
METHODS
Preparation of arterial rings
Tension measurement protocol
Solutions and chemicals
Statistical analysis
RESULTS
DISCUSSION