organophosphorus ester pesticides ( ops ) are mainly used as agricultural and domestic insecticides , leading to increasing numbers of cases of toxic effects on humans and livestock .
ops poisoning can result in neurotoxicity , myocardial damage , microvascular dysfunction , cytotoxicity , respiratory failure , and functional disorder of immune system et al . in human
[ 1 , 2 ] . a key mechanism of intoxication of ops is the irreversible inhibition of acetylcholinesterase ( ache ) and resulting in the accumulation of acetylcholine ( ach ) and overstimulation of its receptors .
however , cholinergic hyperexcitability can not account for all of the neurological , behavioral , and cardiovascular system manifestations arising from exposure to ops .
there is evidence for a wide range of novel targets that are more sensitive than ache to exposure to ops [ 2 , 4 ] .     
it has been reported that some ops altered vascular contractility and exacerbated ops - induced hypertension in acetylcholinesterase - independent manner [ 5 , 6 ] .
the response to vasoactive agents is altered by treatment with paraoxon in hens ischiadic arteries [ 7 , 8 ] .
however , whether paraoxon directly modulates the function of vascular endothelium and/or smooth muscle cells in rabbits has not been examined .
it has been demonstrated that paraoxon blocked gaba , nmda , glycine , nicotinic and muscarinic receptor , and chloride channels in neurons [ 4 , 9 ] .
it has been also reported that paraoxon inhibited ca inflow during the action potential and could downregulate ca - activated k channels leading to a reduction of the afterhyperpolarization and an increase in snail neurons firing [ 10 , 11 ] . however , there are some inconsistent results between calcium homeostasis and paraoxon in nonneuronal cells [ 12 , 13 ] .
thus in this study , we investigated the effect of paraoxon on vascular contractility in rabbits thoracic aortic rings and calcium homeostasis in aortic smooth muscle cells .
acetylcholine ( ach ) , sodium nitroprusside ( snp ) , phenylephrine ( phe ) , potassium chloride ( kcl ) , n - nitro - l - arginine ( l - nna ) , atropine , paraoxon ( pxn ) , verapamil hydrochloride , nicotinamide , ryanodine , tetraethylammonium ( tea - ci ) , hepes , egtazic acid , csoh , csci , mgatp , and tetrodotoxin ( ttx ) were purchased from sigma - aldrich ( st . louis , mo , usa ) , paraoxon was dissolved in ethanol in organ baths experiment and in dmso in measurement of ca current experiment .
the thoracic aortas were dissected from male new zealand rabbits ( 2.0  0.3  kg , supplied by the animal center of south china university ) killed by exsanguination after anesthesia with pentobarbital sodium ( 80  mg / kg , iv ) . the adhering perivascular tissue was removed carefully .
the thoracic aortas were cut into ring segments , 4 to 5  mm in length .
the aortic endothelium was removed by inserting a plastic club into the lumen followed by gentle rubbing in some experiments .
each ring was then placed in a 10  ml organ bath and mounted on two stainless steel hooks , one of which was fastened to the bath and the other connected to a strain gauges for measurement of isometric force .
the organ bath was filled with krebs solution at 37c and bubbled with a mixture of 95% o2 + 5% co2 .
the rings were equilibrated for 60  minutes under a resting tension of 2.0  g and the solution was changed at 20-minute intervals .
the integrity of the endothelium was affirmed by the addition of the endothelium - dependent vasodilator , ach ( 1  m ) , after contractions had been induced by 1  m phe .
this was similarly made with the denuded vessels in order to exclude any endothelial function .
after phe and ach were washed out with krebs solution , the aortic rings were contracted by 50  mm kcl .
after recording the contractile tension of each ring following stimulation with 50  mm kcl , the baths were washed out with krebs solution .
the contractile responses were expressed relative to those measured for the 50  mm kcl - induced contraction in the test experiments [ 14 , 15 ] .
paraoxon was added to the incubation system 30  minutes prior to or during the contraction induced by phe or kcl .
endothelium - denuded thoracic aortic rings were contracted by phe in the presence of snp ( 1  nm ) without paraoxon administration . in some other experiments ,
thoracic aortic contractions were performed in the absence of extracellular ca using ca - free krebs solution containing 1  mm edta .
the concentrations of inhibitors or antagonists used in our study were as follows : l - nna ( no synthase inhibitor ; 30  m ) , verapamil ( l - type voltage - dependent ca channels blocker ; 55  m ) , atropine ( a specific muscarinic receptors antagonist ; 5  m ) , ryanodine ( ryanodine receptors blocker ; 30  m ) , and nicotinamide ( adp - ribosyl cyclase inhibitor ; 6  mm ) .
the thoraotic aortic rings were pretreated with each inhibitor for 20 minutes and then contractions were induced by 1  m phe or 80  mm kcl .
the thoracic aortic rings without endothelium were washed three times with ca - free krebs solution containing 1  mm edta and then exposed to the same buffer for 15  minutes .
the thoracic aortic rings were pretreated with 30  m paraoxon for 30  minutes and then stimulated with kcl ( 80  mm ) . after 30 minutes incubation , cacl2-(10  m to 10  m ) induced contractions were measured for each aortic ring .
the aortic rings were loaded with fura-2 acetoxymethyl ester ( fura-2 am ) through incubation in krebs solution at ph 7.4 , containing 10  m fura-2 am and 0.025% pluronic f-127 at room temperature for 2  hours .
after fura-2  am loading , extracellular fura-2  am was washed off in krebs solution .
then , the aortic rings were longitudinally cut open and pinned onto a block of silicone elastomer , which was fixed onto a base plate of the flow chamber .
after vessel fixing , the flow chamber was placed on an inverted microscope equipped with a spectrophotometer and perfused with krebs solution .
then , the fura-2  am - loaded vessels were visualized through an inverted fluorescence microscope to perform measurements of fura-2  am microfluorometry .
the ratio of the fluorescence intensities at alternating 340  nm ( f340 ) and 380  nm ( f380 ) excitation wavelengths was monitored to measure intracellular calcium concentration ( [ ca]i ) .
the aortic tissues were allowed to recover for 30  minutes and then exposed for 30  minutes to ca - free 80  mm k solution containing 1  mm na2-edta .
subsequently , they were perfused with the same high - k solution supplemented with 0.1 , 0.3 , 1 , and 3  mm cacl2 .
tissue was then washed several times in ca - free 80  mm k   solution until baseline was recovered . after incubating with paraoxon
( 30  m ) for 30  minutes , cumulative perfusion of cacl2 induced a second concentration - dependent increase in [ ca]i .
male new zealand rabbits weighing approximately 2.0  kg were stunned by heavy blow on the head .
five to 10 segments ( 610  mm in length ) of thoracic aortas were quickly dissected free of connective tissue in ice - cold solution containing 4.7  mm kcl , 145  mm nacl , 1.2  mm nah2po4 , 2  mm pyruvic acid , 1.17  mm mgso4 , 0.02  mm edta , 0.1  mm cacl2 , 5  mm d - glucose , and 10  mg / ml bovine serum albumin ( bsa ) .
thoracic aortas were placed in an isolation solution : 4.2  mm kcl , 120  mm nacl , 25  mm nahco3 , 1.2  mm mgcl2 , 0.6  mm kh2po4 , 1  mg / ml bsa , 0.1  mm cacl2 ,   and 11  mm d - glucose , ph 7.4 , when bubbled with 5% co2 and 21% o2 ( at room temperature ) .
the arteries were cut into 4 to 5  mm segments , which were then digested in isolation solution containing papain ( 1.0  mg / ml ) , bsa ( 1  mg / ml ) , and dithiothreitol ( 1  mg / ml ) for 10 minutes at 37c .
the artery segments were then transferred to isolation solution containing collagenaseii ( 0.36 mg / ml ) , 0.1  mm cacl2 , dithiothreitol ( 1  mg / ml ) , and bsa ( 1  mg / ml ) for 10 minutes at 37c .
the tissue was then washed 10 times in ice - cold isolation solution without collagenase and triturated gently with polished pasteur pipettes to separate the individual cells .
freshly isolated cells with characteristic elongated morphology of vascular myocytes were added directly to the chamber and allowed to adhere for 10 to 15 minutes for whole - cell patch - clamp electrophysiology .
isolated aortic smooth muscle cells were placed in the experimental chamber ( 0.4  ml ) mounted on the stage of an inverted microscope ( ix-70 olympus , tokyo , japan ) . after setting to the bottom of chamber
, the cells were superfused with external solution for 15 minutes at a rate of 2~2.5  ml / min at 25c .
transmembrane currents were recorded using an axopatch amplifier ( 200b , axon instruments , usa ) .
patch - clamp pipettes were manufactured from borosilicate glass capillaries ( gc 150t-7.5 , clark electromedical instruments , london , uk ) .
the resistance of the patch pipette was 24  m , when filled with electrode internal solution .  
liquid junction potential between the pipette solution and external solution was corrected after the pipette tipped into the external solution .
after gigaseal formation , the membrane was ruptured with a gentle suction to obtain the whole - cell voltage - clamp configuration . to minimize the duration of capacitive current , membrane capacitance and series resistance were compensated after membrane rupture .
the external solution was changed to na - free solution in which na was replaced by equimolar tetraethylammonium chloride ( tea - ci ) .
na current was also inactivated at the holding potential of 40  mv and blocked by tetrodotoxin ( ttx , 2  10  mol / l ) .
computer - generated voltage or current pulses were programmed using the pclamp 8.0 software ( axon instruments , usa ) .
all experiments were carried out at room temperature ( 22~24c ) [ 17 , 18 ] .
the electrode internal solution for whole - cell recording contained ( in mm ) 3 mgatp , 140 cscl , and 10 hepes , and 10 egtazic acid , and ph was adjusted to 7.2 with csoh .
the external solution was composed of ( in  mm ) 140 tea - cl , 2.0  mgcl2 , 1.5  cacl2 , 10  glucose , 10  hepes , and 0.002  ttx , gassed with 100% o2 , and the ph was adjusted with naoh to 7.3~7.4 [ 17 , 18 ] .
paraoxon was dissolved in dmso and diluted in external solution at the concentrations of 0.3 , 3 , and 30 
statistical comparisons were made using one - way anova plus bonferroni multiple comparison tests . *
the effect of pre - treatment with paraoxon on contraction induced by phe or high concentration of k was investigated in rabbits thoracic aorta .
the maximum contractions of aorta rings induced by 1  m phe were decreased by pretreatment with paraoxon ( 30  m ) , regardless of the presence ( 41.20%  11.05% ) or absence ( 38.87%  8.74% ) of endothelium ( figure 1(a ) ) .
the vasoconstriction of aortic rings induced by 80 mm kcl was also attenuated by pretreatment with paraoxon ( 30  m ) regardless of the presence ( 42.42%  8.57% ) or absence ( 40.09%  6.18% ) of endothelium ( figure 1(b ) ) .
meanwhile , l - nna ( 30  m ) , an inhibitor of no synthesis , had little effect on the inhibitive effect of paraoxon on 1  m phe - induced vasoconstriction , but sodium nitroprusside ( 1  nm ) , a donor of no , significantly attenuated 1  m phe - induced vasoconstriction in endothelium - denuded aortic rings ( figure 1(a ) ) .
furthermore , atropine ( 5  m ) , a specific muscarinic receptors antagonists had also little effect on the inhibitory effect of paraoxon on 1  m phe - induced vasoconstriction in endothelium - denuded thoracic aortic rings ( figure 1(a ) ) . as shown in figure 1(c ) , paraoxon at a concentration of 30  m significantly inhibited 1010  m of concentration phe - induced constriction in thoracic aortic rings without endothelium .
likewise , pretreatment with 30  m paraoxon also significantly attenuated 40100  mm kcl - induced vasoconstriction in endothelium - denuded thoracic aortic rings ( figure 1(d ) ) .   as shown in figure 2(a ) , treatment of paraoxon ( 30  m ) resulted in vasodilation in the thoracic aortic rings without endothelium that had been precontracted by phe ( 1  m ) .
the relaxant effect lasted for 35 minutes after treatment with paraoxon ( 30  m ) .
as well , paraoxon induced vasodilation in thoracic aortic rings without endothelium contracted previously by 80  mm kcl ( figure 2(b ) ) . to investigate the involvement of extracellular ca in the inhibitory effect of paraoxon on vasoconstriction ,
thoracic aortic rings were washed and incubated with ca - free krebs solution . as shown in figures 3(a ) and 3(b ) , a lack of extracellular ca almost completely inhibited phe ( 94.1%  1.6% ) and kcl- ( 78.3%  2.2% ) induced vasoconstriction . under ca - free conditions , paraoxon failed to inhibit vasoconstriction ( figures 3(a ) and 3(b ) ) .
these results showed that the effect of paraoxon on phe- or kcl - induced contraction depended on extracellular ca .
paraoxon did not relax either phe- or kcl - induced vasoconstriction in the presence of 55  m verapamil ( figure 3(c ) ) .
the decrease of vasoconstriction induced by paraoxon was also abolished by verapamil ( figure 3(d ) ) .
ryanodine and nicotinamide , ca - induced ca release pathway blocker , also had no influence on the effect of paraoxon on phe- or kcl - induced contraction ( figure 3(e ) ) .
as shown in figure 3(f ) , to investigate the effect of paraoxon on ca influx - induced vasoconstriction , cacl2 was administrated cumulatively [ 10  m10  m ] to the aortic rings which had been incubated in ca - free krebs solution . to activate ca influx into the smooth muscle cells ,
the aortic rings were stimulated with kcl ( 80  mm ) prior to the cumulative addition of cacl2 .
addition of cacl2 increased the contractile tension of the endothelium - denuded aortic rings in concentration - dependent manner .
the contraction induced by the cumulative addition of cacl2   was attenuated by pre - treatment with paraoxon in endothelium - denuded aortic rings . to investigate whether paraoxon attenuated phe- and kcl - induced vasoconstriction in the thoracic aorta mainly through inhibiting ca influx , the effect of paraoxon on vascular smooth muscle [ ca]i   was measured in thoracic aorta without endothelia .
cacl2 induced a rise in vsm [ ca]i  in ca - free 80  mm k solution , and the first and second concentration - dependent responses were similar .
as shown in figure 4 , changes in vsm [ ca]i  measured as the fluorescence ratio in thoracic aorta in response to cacl2 in 80  mm k solution after treatment with 30  m paraoxon for 30 minutes .
[ ca]i  were significantly attenuated by treatment with paraoxon ( 30  m ) for 30 minutes .
moreover , treatment with 1  m atropine did not influence the paraoxon- ( 30  m ) mediated inhibition of the cacl2-stimulated [ ca]i rise ( figure 4 ) .
l - type calcium current ( ica , l ) in thoracic aortas smooth muscle cells was evoked by a depolarizing step pulse from the holding potential of 40  mv to 0  mv at the frequency of 0.1  hz .
paraoxon ( 0.3 , 3 , and 30  m ) inhibited the peak amplitude of ica , l ( figure 5 ) .
current - voltage ( i - v ) curve of l - type calcium current was obtained by a number of depolarizing step pulses ( 350  ms ) from the holding potential of 40  mv to test potentials between 50  mv and 50  mv .
ica , l was activated at 30 mv and the peak amplitude occurred at the potential of 0  mv .
paraoxon ( 0.3 , 3 , and 30  m ) upshifted the i - v curve ( figure 6(a ) ) , but the current density at test potential of 0  mv was significantly decreased only in paraoxon ( 30  m ) group compared with the control group ( figure 6(b ) ) .
organophosphorus ester pesticides ( ops ) are well acknowledged to have the potential to result in severe , acute toxicity through the phosphorylation of serine residues of acetylcholinesterase ( ache ) and the subsequent accumulation of ach .
however , many researchers have reported additional noncholinesterase actions for ops . it has been shown that ops could interact directly with targets other than acetylcholinesterase [ 20 , 21 ] .   at the cellular level ,
the main targets of ops include receptors , enzymes , ion channels , cell signaling molecules , and cytoskeletal elements et al .
[ 2224 ] . likewise , there is multiple evidence that ops can interact with targets other than ache in the cardiovascular system [ 25 , 26 ] .  
paraoxon is the active metabolite of parathion which is one of the most acutely toxic organophosphorus ester pesticides .
we examined the effect of paraoxon on the vasoconstrictor - induced contraction in rabbits thoracic aortic rings .
the results showed that paraoxon ( 30  m ) attenuated significantly both phe- and kcl - induced aortic contraction regardless of the presence or absence of aortic endothelium .
our findings also showed that l - nna had little effect on the inhibitory effect of paraoxon on phe - induced contraction , while snp inhibited phe - induced contraction in endothelium - denuded aortic rings .
these results suggested that paraoxon attenuated vascular contraction independent of generation of no by the endothelia .
but l - nna markedly increased the response to phe in the presence of aortic endothelium .
this may be explained by the fact that l - nna decreases the production of endothelium - derived relaxing factor , which results in an increase of vascular sensitivity to vasoconstrictor .
meanwhile , atropine had also little effect on the inhibitory effect of paraoxon on phe - induced contraction in endothelium - denuded aortic rings .
phe can bind to -adrenoceptor on the plasma membrane , leading to the activation of phospholipase c , which produces inositol 1- , 4- , 5-triphosphate ( ip3 ) and diacylglycerol ( dg ) .
vascular smooth muscle contraction is then triggered by an increase in [ ca]i induced by ca mobilization from the sarcoplasmic reticulum and a membrane depolarization - stimulated ca influx from the extracellular spaces .
this vasoconstrictor- induced ca inflow from the extracellular spaces is principally mediated by l - type ca channels .
release of ca from sarcoplasmic reticulum is mainly mediated by ip3 receptors and ryanodine receptors , both of which contribute to the transient increase in [ ca]i .
ryanodine receptor is activated by influx of ca from the extracellular spaces , usually called ca - induced ca release . in addition , various types of k channels exist in vascular smooth muscle cell .
the k current hyperpolarizes the vascular smooth muscle cell membrane and prohibits the entry of ca through closing the l - type ca channels , resulting in vasorelaxation .
so high environmental k level leads to membrane depolarization and increases the entry of ca from extracellular spaces .
our results showed that the degree of aortic contraction was significantly decreased in ca - free medium and/ or the presence of the l - type ca channel inhibitor , verapamil .
our findings also showed that ryanodine and nicotinamide , ca - induced ca release pathway blockers , had little effect on the action of paraoxon , but blockade of extracellular ca entry in the absence of extracellular ca or use of verapamil abolished the effect of paraoxon on contraction in rabbits thoracic aorta .
moreover , pre - treatment with paraoxon ( 30  m ) decreased contractions induced by the cumulative addition of cacl2 .
these data suggested that the suppressive effect of paraoxon on vascular contraction was involved in the extracellular ca influx , but not the release of ca from the sarcoplasmic reticulum .  
the results suggested that pre - treatment with paraoxon attenuated significantly the transient increase in [ ca]i in vascular smooth muscle cells of the rabbits thoracic aorta .
ca inflow from the extracellular spaces is mainly mediated by l - type calcium channels .
so we investigated the effect of paraoxon on l - type calcium current ( ica , l ) in isolated thoracic aortas smooth muscle cells of rabbits .
the results showed that paraoxon ( 30  m ) decreased significantly l - type calcium current .
therefore we inferred that paraoxon attenuated vasoconstrictor - induced contraction through inhibiting ca influx via blocking l - type calcium channels in vascular smooth muscle cells of the rabbits .  
calcium homeostasis is a highly controlled process , essential for various cellular functions including the contraction of smooth muscle and skeletal muscle , transmitter release , the cell excitability , and cell death [ 34 , 35 ] .
the interaction of some ops with membrane ca channels has been reported [ 36 , 37 ] .
soman has been shown to decrease calcium entry through voltage - dependent calcium channels in bullfrog peripheral sympathetic neurons .
methyl parathion and malathion inhibit the [ ca]i rise induced by nicotinic agonist in bovine adrenal chromaffin cells .
exposure of sn56 cells to 10  m paraoxon for 18  hours significantly decreases the ca mobilization elicited by carbachol . however , there are some different results about the effects of organophosphates on ca homeostasis .
reported that paraoxon did not have any influence on the [ ca]i in tracheal smooth muscle cells of guinea pigs , whereas sun et al .
found an enhanced ca release and influx mechanisms in presence of paraoxon in the human parotid cell - line hsy .
, we have demonstrated that paraoxon attenuates vasoconstrictor - induced contraction and induces vasodilation through inhibiting ca influx in the rabbits thoracic aorta .
 we investigated the effect of paraoxon on vascular contractility using organ baths in thoracic aortic rings of rabbits and examined the effect of paraoxon on calcium homeostasis using a whole - cell patch - clamp technique in isolated aortic smooth muscle cells of rabbits . 
 the findings show that administration of paraoxon ( 30  m ) attenuated thoracic aorta contraction induced by phenylephrine ( 1  m ) and/or a high k+ 
  environment ( 80  mm ) in both the presence and absence of thoracic aortic endothelium . 
 this inhibitory effect of paraoxon on vasoconstrictor - induced contraction was abolished in the absence of extracellular ca2 + , or in the presence of the ca2 + channel inhibitor , verapamil . 
 but atropine had little effect on the inhibitory effect of paraoxon on phenylephrine - induced contraction . 
 paraoxon also attenuated vascular smooth muscle contraction induced by the cumulative addition of cacl2 
  and attenuated an increase of intracellular ca2 + concentration induced by k+ 
  in vascular smooth muscle cells . 
 moreover , paraoxon ( 30  m ) inhibited significantly l - type calcium current in isolated aortic smooth muscle cells of rabbits . in conclusion , 
 our results demonstrate that paraoxon attenuates vasoconstrictor - induced contraction through inhibiting ca2 + influx in the rabbits thoracic aorta . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion