muscle preparations and tension measurement : abomasums from adult bovine 
 of either sex were obtained from a local abattoir .
the mucosal layers were removed by 
 cutting with fine scissors , and smooth muscle tissues were isolated from the fundic region . 

circular muscle strips were incubated with physiological salt solution ( pss ) containing ( in 
 mm ) 136.8 nacl , 5.4 kcl , 2.5 cacl2 , 1.0 mgcl2 , 23.8 nahco3 
 and 5.6 glucose .
the pss was aerated with 95% o2 and 5% co2 to adjust 
 the ph to 7.4 at 37c .
one end of each strip was 
 bound to a glass holder , and the other end was connected by a silk thread to a strain - gauge 
 transducer ( tb-611 t ; nihon kohden , tokyo , japan ) in an organ bath containing pss with a 
 resting tension of 2  g. the muscle strips were equilibrated for 30 min to obtain stable 
 contractility induced by hyperosmotic addition of 65 mm kcl .
when the contractile response 
 induced by 1 m carbachol ( cch ) reached a steady level about 1520 min 
 after addition to a muscle strip , vinpocetine ( type 1 ) , ehna ( type 2 ) , milrinone ( type 3 ) , 
 ro20 - 1724 ( type 4 ) , vardenafil ( type 5 ) , brl-50481 ( type 7 ) or bay736691 ( type 9 ) was added 
 cumulatively .
rt - pcr analysis : total rna was subsequently extracted from the tissues or 
 cells with trizol ( invitrogen japan , tokyo , japan ) , and then , the cells were precipitated 
 with isopropanol and suspended to a concentration of 1 
 g/l in rnase - free distilled water .
briefly , first - strand cdna was synthesized 
 with random 6-mer oligonucleotide primers and primescript rtase ( takara bio , tokyo , japan ) 
 at 30c for 10 min , 42c for 30 min , 95c for 5 min and 4c for 5 min .
pcr amplification 
 using takara ex taq ( takara bio ) was conducted in the presence of the oligonucleotide 
 primers listed in table 1table 1.primer sequences used in the present studytarget geneforward primer sequence ( 5  3)reverse primer sequence ( 5  3)pde4atgcggaggtggagatagaggtaggagacagggcagggatgpde4bcaagttccggtgttcttctcctattcctccatgattcggtctgtpde4cttcaaggtggcagagctaagtgagcatcagtaggtaggtggcaagpde4dtggatgagcaggtggaagagcacaaacgaaaggcatggaapde5catacacacaaacacacacacacactctaccagaagccagagagaacaagapdhttgtgatgggcgtgaaccccctccacgatgccaaa .
the pcr samples were amplified with 36 cycles at 94c for 30 sec , 55c for 45 
 sec and 72c for 30 sec in a thermal cycler ( takara pcr thermal cycler mp , takara bio ) .
the 
 pcr products in each cycle were separated electrophoretically on a 2% agarose gel containing 
 0.1% ethidium bromide .
fas - iii ultraviolet transilluminator ( toyobo , tokyo , japan ) was used 
 for visualizing the fluorescent bands .
assay of cgmp and camp contents : the cgmp and camp contents in the muscle 
 strips were measured by enzyme immunoassay .
after incubation of the strips with ro20 - 1724 or 
 vardenafil for 10 min in the presence of carbachol ( 1 m ) , the strips were 
 rapidly frozen in liquid nitrogen and stored at 80c until homogenized in 6% 
 trichloroacetic acid ( 0.4 ml ) .
the homogenate was centrifuged at 3,000  
 g for 15 min , and the supernatant was washed with 1.5 ml 
 of water - saturated dietyl ether four times ; the cgmp and camp contents of the strips were 
 assayed by using an enzyme immunoassay kit ( ge healthcare , buckinghamshire , u.k . ) .
the cgmp 
 and camp contents were expressed as picomoles per gram wet weight of tissue .
chemicals : the chemicals used in the present study were forskolin , sodium 
 nitroprusside ( snp ) , milrinone , bay73 - 6691 , carbachol ( sigma - aldrich , st .
louis , mo , 
 u.s.a . ) , vinpocetine , erythro-9-(2-hydroxy-3-nonyl ) adeninehcl ( ehna ) , brl-50481 ( biomol 
 research laboratories , plymouth meeting , pa , u.s.a . ) , ro20 - 1724 ( lc laboratories , woburn , 
 ma , u.s.a . ) and vardenafil ( lkt laboratories , st .
statistics : values are expressed as the mean  sem , and ic50 
 values ( the concentration producing 50% relaxation ) were determined by linear regression 
 analysis .
effects of various selective pde inhibitors on cch - induced contraction : 
 when a contractile response induced by 1 m cch reached a steady level 
 about 1520 min after application , each pde inhibitor was added cumulatively .
vinpocetine 
 ( type 1 ) , ehna ( type 2 ) , milrinone ( type 3 ) , ro20 - 1724 ( type 4 ) , vardenafil ( type 5 ) , 
 brl-50481 ( type 7 ) and bay73 - 6691 ( type 9 ) inhibited the cch - induced 
 contraction in a concentration - dependent manner ( fig . 

1.effects of vinpocetine , ehna , milrinone , ro20 - 1724 , vardenafil , brl-50481 and 
 bay73 - 6691 on the contraction induced by 1 m carbachol ( cch ) in the 
 bovine abomasum .
preparations were precontracted with cch , and then , the agents were 
 added cumulatively .
the maximum contractions induced by 1 m cch in 
 the absence of the agents were taken as 100% . each point represents the mean of 4 - 6 
 preparations .
the ic50 and maximum relaxation values for cch - induced contraction are 
 presented in table 2table 2.ic50 and maximum relaxation values for various selective pde 
 inhibitors in the bovine abomasum treated with 1 m carbacholagentic50 ( m)maximum relaxation ( % ) nvinpocetine ( type 1)>10048.0  12.14ehna ( type 2)25.0 ( 15.831.6)80.6  4.36milrinone ( type 3)8.4 ( 4.516.0)76.7  6.35ro201724 ( type 4)4.3 ( 2.57.9)73.9  4.55vardenafil ( type 5)10.0 ( 7.912.6)89.6  4.24brl-50481 ( type 7)79.0 ( 63.1100.0)58.4  2.04bay73 - 6691 ( type 9)25.0 ( 20.032.0)76.8  3.84numbers in parentheses indicate 95% confidence limits .
the maximum relaxation 
 represents the resting tension after washing and was considered to be 100% .. the rank order of the ic50 values was ro20 - 1724 > vardenafil 

moreover , the rank 
 order of maximum relaxation was vardenafil > ehna > milrinone , ro20 - 1724 , bay73 - 6691 
 > brl-50481 > vinpocetine .
effects of vinpocetine , ehna , milrinone , ro20 - 1724 , vardenafil , brl-50481 and 
 bay73 - 6691 on the contraction induced by 1 m carbachol ( cch ) in the 
 bovine abomasum .
preparations were precontracted with cch , and then , the agents were 
 added cumulatively .
the maximum contractions induced by 1 m cch in 
 the absence of the agents were taken as 100% .
the maximum relaxation 
 represents the resting tension after washing and was considered to be 100% .
forskolin , an adenylyl cyclase activator , or snp , a soluble guanylyl cyclase activator , 
 inhibited the cch - induced contraction in a concentration - dependent manner ( data not shown ) . 

the ic50 values for forskolin and snp were 12.9 ( 1015.8 m ) and 
 0.3 ( 0.20.5 m ) m , respectively .
expression of pde4 and 5 in smooth muscle of the bovine abomasum : 
 semquantitative rt - pcr analysis using specific primers for several bovine pde genes was 
 performed in smooth muscle strips of the bovine abomasum to check for the expression of pde4 
 ( a , b , c and d ) and pde5 .
the pcr products were analyzed on 2.0% agarose gel , and pictures of the ethidium 
 bromide - stained gels are shown .
the pcr primers used for this analysis are described 
 in materials and methods . ) .
bovine abomasal smooth muscle expresses pde4b , pde4c , pde4d and pde5 . regarding 
 pde5 ,
two products with different sizes of pde5 were amplified , indicating the existence of 
 two splice variants ( fig .
the pcr products were analyzed on 2.0% agarose gel , and pictures of the ethidium 
 bromide - stained gels are shown .
effects of ro20 - 1724 and vardenafil on cgmp and camp contents : in the 
 presence of cch ( 1 m ) , ro20 - 1724 ( 10 and 100 m ) 
 increased the camp content in a concentration - dependent manner ( fig . 3afig .
3.the effects of ro20 - 1724 ( a ) and vardenafil ( b ) on the camp or cgmp contents of the 
 bovine abomasum .
preparations were precontracted with 1 m cch and 
 were then treated with the agents for 10 min .
* * : significant difference from each respective 
 control , with p<0.05 and p<0.01 , 
 respectively . ) , but did not affect the cgmp content ( data not shown ) .
by contrast , vardenafil ( 30 
 and 100 m ) increased the cgmp content ( fig .
3b ) , but did not affect the camp content ( data not shown ) .
the effects of ro20 - 1724 ( a ) and vardenafil ( b ) on the camp or cgmp contents of the 
 bovine abomasum .
preparations were precontracted with 1 m cch and 
 were then treated with the agents for 10 min .
vertical bars indicate the sem . * and * * : significant difference from each respective 
 control , with p<0.05 and p<0.01 , 
 respectively .
physiological abomasal contractions are regulated by second messengers , such as calcium 
 ion , camp and cgmp . in the bovine abomasum , forskolin and
snp inhibited the muscarinic 
 agonist carbachol ( cch)-induced contraction in a concentration - dependent manner .
these 
 results suggest that the camp / adenyl cyclase pathway and cgmp / guanylyl cyclase pathway are 
 both involved in the relaxation of bovine abomasal smooth muscle .
therefore , we examined the 
 effects of selective pde inhibitors on cch - induced contraction to confirm the role of camp 
 and cgmp in mediation of abomasal contractions .
pde regulates the intracellular concentrations of camp and cgmp and is classified into 11 
 families .
moreover , the pde1 - 5 isozymes have 
 selective inhibitors , and selective inhibitors for 
 pde7 and 9 have also been made recently and commercialized .
it has been shown that the 
 potency of the relaxation induced by these pde inhibitors differed in many smooth muscles 
 including gastrointestinal smooth muscles [ 1 , 12,13,14 , 22 ] . 
 therefore , we investigated the effects of selective pde inhibitors for pde1 - 5 , 7 or 9 on 
 cch - induced contractions of the smooth muscles in the bovine abomasum .
it has been reported that selective inhibitors for pde1 - 5 inhibited agonist - induced contraction dose - dependently , that rolipram 
 ( type 4 ) was most effective in the guinea pig trachea   and that zaprinast ( type 5 ) was most effective in the rat urinary bladder 
  and porcine iris sphincter muscle . on the other hand , selective inhibitors for pde1 - 5 
 also inhibited agonist - induced contraction dose - dependently in gastrointestinal smooth 
 muscle , zaprinast ( type 5 ) was most effective in the guinea pig taenia coli   and ileum , and ro20 - 1724 ( type 4 ) was most effective in the guinea pig gall bladder . in the present study , selective pde inhibitors 
 inhibited cch - induced contraction in the bovine abomasum , dose - dependently , and ro20 - 1724 
 ( type 4 ) and vardenafil ( type 5 ) were more effective than the other pde inhibitors .
since 
 the rank order of inhibitory potency was different by organ , it is suggest that the 
 characterization of pde isozymes differs by organ .
moreover , it is clear that the types 4 
 and 5 inhibitors were more potent than the other types of inhibitors in bovine abomasum .
moreover , it is now recognized that there are four genes ( pde4a - pde4d ) 
 that make up the pde4 family , and pde4 is expressed in a plethora of tissues and cell types 
 and plays a role in a large number of physiological processes . in the present study ,
we 
 showed the expression of pde 4b , 4c and 4d in bovine abomasal smooth muscles by rt - pcr 
 methods .
it has been reported that rolipram ( type 4 ) potently relaxed electrical field 
 stimulation - induced contraction in the cat gastric fundus .
ro20 - 1724 inhibited cch - induced contraction in the bovine abomasum ( in the 
 present study ) and guinea pig gall bladder 
 potently , but poorly in the guinea pig ileum   and 
 taenia coli .
these data suggested that the 
 functional distributions of pde4 isozymes of gastrointestinal smooth muscles differ by 
 organ . in our study , the relaxation effect of vardenafil was more potent than those of the other 
 cgmp - related pde inhibitors , and vardenafil increased the cgmp content .
these data are 
 consistent with the report that vardenafil also inhibited phenylephrine - induced contraction 
 in the rat aorta and resulted in an increase in cgmp content .
moreover , zaprinast , a pde5 inhibitor , relaxed the guinea pig taenia 
 coli and ileum the increases in cgmp content [ 12 , 
 13 ] . in the present study
, we showed the expression 
 of pde5 in bovine abomasal smooth muscles by rt - pcr methods .
the data suggested that the 
 pde5 isozymes have an important role in regulation of motility in gastrointestinal smooth 
 muscles .
pde7 is a camp - specific enzyme , and two pde genes ( pde7a and b ) have been identified .
pde7a 
 is expressed in a number of cell types including muscle cells . in the trachea of ovalbumin - sensitized guinea pig , but not in healthy 
 guinea pig trachea , brl-50481 inhibited histamine - induced contraction . moreover , the inhibitory effects of brl-50481 were poor than those 
 of the other pde inhibitors we used for bovine abomasal smooth muscles .
thus , it is probable 
 that the pde7 expression in smooth muscle is increased by inflammatory disease , but in 
 normal condition , the expression of pde7 is poor and modifies smooth muscle tension less 
 potently than the other pdes in various tissues including the bovine abomasum .
pde9a is the only isoform identified , and pde 9a is expressed in all 
 tissues , except the blood in mammals [ 11 , 20 ] .
similar manner of expression was detected for pde9a 
 and pde5 mrnas in the mouse corpus cavernosum ; however , bay 73 - 6691 on its own did not relax 
 the mouse corpus cavernosum or alter sildenafil ( type 5)-induced relaxation . in bovine abomasums , we did not compare expression of 
 pde5 and 9 ; however , the ic50 for cch - induced contraction of bay73 - 6691 is 2.5 
 times lower than that of vardenafil .
bay 73 - 6691 preferentially inhibits pde9 more than pde 
 5 , over 77 times . .
these data raise the 
 possibility that the pde9 is expressed in smooth muscles including the bovine abomasum . 

however , we infer that the regulation of normal smooth muscle contraction performed by pde9 
 may be less than that of pde5 in bovine abomasum .
it has been reported that the contraction of preparations originating from the abomasal 
 antrum of a healthy cow evoked by electrical stimulation was inhibited by left displaced 
 abomasum ( lda ) , right displaced abomasum ( rda ) or abomasal volvulus ( ad ) .
these 
 data imply that the expression of nitric oxide ( no)-cgmp signals , including the expression 
 of pdes , may increase in the abomasum of lda , rda or av .
however , we have no data indicating 
 the changes in expression of pdes in the bovine abomasum affected by these condition .
a 
 further study is involvement of the changes in the expression of pdes in the bovine 
 displaced abomasum . in conclusion
, selective pde inhibitors inhibited cch - induced contraction in the bovine 
 abomasum . among the pde inhibitors ,
ro20 - 1724 and vardenafil induced more relaxation than 
 the other inhibitors according to the data for the ic50 and maximum relaxation . 

moreover , the results suggest that ro20 - 1724-induced relaxation was correlated with camp and 
 that vardenafil - induced relaxation was correlated with cgmp in the bovine abomasum .
pde4 and 
 pde5 are the enzymes involved in the regulation of relaxation associated with camp and cgmp , 
 respectively , in the bovine abomasum .
 the effects of various 
 selective phosphodiesterase ( pde ) inhibitors on carbachol ( cch)-induced contraction in the 
 bovine abomasum were investigated . various selective pde inhibitors , vinpocetine ( type 1 ) , 
 erythro-9-(2-hydroxy-3-nonyl ) adenine ( ehna , type 2 ) , milrinone ( type 3 ) , ro20 - 1724 ( type 
 4 ) , vardenafil ( type 5 ) , brl-50481 ( type 7 ) and bay73 - 6691 ( type 9 ) , inhibited cch - induced 
 contractions in a concentration - dependent manner . among the pde inhibitors , ro20 - 1724 and 
 
 vardenafil induced more relaxation than the other inhibitors based on the data for the 
 ic50 or maximum relaxation . in smooth muscle of the bovine abomasum 
 , we 
 showed the expression of pde4b , 4c , 4d and 5 by rt - pcr analysis . in the presence of cch 
 , 
 ro20 - 1724 increased the camp content , but not the cgmp content . 
 by contrast , vardenafil 
 increased the cgmp content , but not the camp content . 
 these results suggest that 
 ro20 - 1724-induced relaxation was correlated with camp and that vardenafil - induced 
 relaxation was correlated with cgmp in the bovine abomasum . in conclusion , 
 pde4 and pde5 
 are the enzymes involved in regulation of the relaxation associated with camp and cgmp , 
 respectively , in the bovine abomasum . 
MATERIALS AND METHODS
RESULTS
DISCUSSION