Effects Of Doxazosin And Its Enantiomers On Contractile Function And The MRNA Expression Of α₁-adrenoceptor Subtypes In The Muscle Layer Of Animal’s Stomach

Doxazosin, a selective long-acting α1-adrenoceptor antagonist, has beenused as clinical antihypertensive drugs. Since α1-adrenoceptors located in thelower urinary tract play a large role in regulation of urination, selectiveα1-adrenoceptor antagonists including doxazosin are also the current first linetherapy for patients suffering from lower urinary tract symptoms （LUTS） dueto benign prostatic hyperplasia （BPH）. One chiral carbon atom exists in thechemical structure of doxazosin, and despinner of doxazosin （（±）doxazosin）has been used in routine clinical practice until now. Owens et al have alreadycompleted the chromatographic resolution of （±）doxazosin to get2enantiomers of （-）doxazosin and （+）doxazosin. It has been reported that（+）doxazosin and （-）doxazosin may produces converse inotropic action in themouse atrium. In preliminary experiments, we have further observed that（±）doxazosin obviously enhances the contractile response to5-HT in isolatedlongitudinal muscle strips of the rabbit gastric body. Owing to the fact thatdoxazosin is an orally administered agent, it could maintain higherconcentrations in the GI tract before being absorbed into the bloodstream. It isestimated that95%of5-HT is found within the GI tract, and5-HT（1B）,5-HT（1D）and5-HT_2Breceptors are present on the GI smooth muscle being responsiblefor gut motility. On the other hand, there were few studies on the location ofα1-adrenoceptor and its subtypes （α（1A）, α（1B）, α（1D）） in the muscle layer of stomachand it is not clear that the effects of long-term administration of doxazosin onthe expressions of subtype of α1-adrenoceptor in the muscle layer of stomach.Therefore, the aims of the present study were1) to study the roles ofdoxazosin and its enantiomers on modulation of the contractile responses to5-HT in longitudinal muscle strips of the rabbit gastric body and characterize pharmacologically the5-HT receptor subtypes that mediate the5-HT-inducedresponses in longitudinal muscle strips of the rabbit gastric body,2) toevaluate the location of subtypes of α1-adrenoceptor （α（1A）, α（1B）, α（1D）） in themuscle layer of stomach,3) to investigate the effects of long-termadministration of doxazosin on the expression of subtype of α1-adrenoceptorin the muscle layer of stomach.Part Ⅰ Effects of doxazosin on5-HT-induced contractile responses andpharmacological characters of5-HT receptor subtypes that mediate the5-HT-induced responses in longitudinal muscle strips of the rabbit gastricbodyLongitudinal muscle strips of the rabbit gastric body were prepared andan initial resting tension of2g was applied to the preparations that wereallowed to equilibrate for60min. Responses of the preparations to agents wererecorded on a polygraph.1Effects of indomethacin and atropine on the contractile responses to5-HT inthe gastric longitudinal muscle preparationsThe dose-response curves for5-HT did not change significantly in thepreparations pretreated with atropine (1μmol L^-1)（P>0.05）. However, thecontractile responses to5-HT were almost completely inhibited byindomethacin (10μmol L^-1) with a decrease in E_max by95.16±2.01%in thelongitudinal muscle strips of rabbit gastric body（P<0.01）.2Effects of5-HT receptor subtype antagonists on the contractile responses to5-HT in the gastric longitudinal muscle stripsNeither a5-HT3receptor antagonist of ondansetron (3-30μmol L^-1) nor a5-HT_2Breceptor antagonist of yohimbine (1-10μmol L^-1) affected thecontractile responses to5-HT significantly in the longitudinal muscle strips（P>0.05）.A5-HT_2Areceptor selective antagonist of nefazodone (1,10and100nmol L^-1), however, produced a parallel shift to the right of the dose-responsecurves for5-HT without significant changes in the E_max values （P>0.05）. Theslope of the Schild plot for nefazodone was0.982±0.099which was not significantly different from unity （P>0.05, n=8）, indicating a competitiveinhibition by nefazodone （pA₂value=8.643±0.175） in the isolated longitudinalmuscle strips.3Effects of5-HT agonist and motilin receptor agonist of erythromycin to thelongitudinal muscle strips of rabbit gastric bodyA5-HT（1A）agonist (0.1-3μmol L^-1) of urapidil and a motilin receptoragonist of erythromycin (0.1-10μmol L^-1) did not produce obvious contractileresponses in the longitudinal muscle strips of rabbit gastric body.Part Ⅱ mRNA expression of α1-adrenoceptor subtypes （α（1A）, α（1B）, α（1D）） inthe muscle layer of rat stomachThe relative expression of α1-adrenoceptor subtype mRNAs wasanalyzed by real time RT-PCR utilizing SYBR-green fluorescence in themuscle layers of rat cardia, gastric fundus, body and antrum.1The relative expression of α（1A）-adrenoceptor subtype mRNA detected by realtime fluorescence quantitative RT-PCR in the muscle layers of rat cardia,gastric fundus, body and antrumThe expression levels of α（1A）-adrenoceptor subtype mRNA in the musclelayer of rat cardia, gastric fundus, body and antrum were significantly lowerthan that in the rat spleen （P<0.05）. The expression level of α（1A）-adrenoceptorsubtype mRNA in the muscle layers of rat cardia and gastric antrum werelower than that in gastric fundus and body （P<0.05）. The rank order ofα（1A）-adrenoceptor mRNA expression levels was cardia=antrum<fundus=body.2The relative expression of α（1B）-adrenoceptor subtype mRNA detected by realtime fluorescence quantitative RT-PCR in the muscle layers of rat cardia,gastric fundus, body and antrumThe expression levels of α（1B）-adrenoceptor subtype mRNA in the musclelayer of rat cardia, gastric fundus, body and antrum were significantly lowerthan that in the rat spleen （P<0.01），but there were no significantly differencesamong the muscle layer of rat cardia, gastric fundus, body and antrum（P>0.05）.3The relative expression of α（1D）-adrenoceptor subtype mRNA detected by real time fluorescence quantitative RT-PCR in the muscle layers of rat cardia,gastric fundus, body and antrumThe expression levels of α（1D）-adrenoceptor subtype mRNA in the musclelayer of rat cardia, gastric fundus and body were significantly lower than thatin the rat thoracic aorta （P<0.01），but there was not significantly differentbetween the muscle layer of rat gastric antrum and thoracic aorta （P>0.05）.The rank order of α（1D）-adrenoceptor mRNA expression levels was cardia<fundus=body<<antrum.Part Ⅲ Effects of long-term administration of doxazosin and itsenantiomers on the mRNA expressions of α1-adrenoceptor subtypes （α（1A）,α（1B）, α（1D）） in the muscle layer of rat stomachDoxazosin and its enantiomenrs were dissolved in distilled water andwere intragastrically administrated at a dose of8mg/kg once a day for12weeks. Eight hours after the final administration, rats were exsanguinated andtissues were rapidly isolated. The relative expression of α1-adrenoceptorsubtype mRNAs was analyzed by real time RT-PCR utilizing SYBR-greenfluorescence in the muscle layers of rat cardia, gastric fundus, body andantrum in control,（±）DOX,（+）DOX and （-）DOX group.1Effects of long-term administration of doxazosin and its enantiomers on themRNA expressions of α1-adrenoceptor subtypes （α（1A）, α（1B）, α（1D）） in the musclelayer of rat cardiaThe expression levels of α（1A）-adrenoceptor subtype mRNA in the musclelayer of rat cardia in （±）DOX group,（+）DOX group,（-）DOX group weresignificantly higher than that in control group （P<0.01）. And the expressionlevel of α（1A）-adrenoceptor subtype mRNA in the muscle layer of rat cardia in（±）DOX group was higher than that in （+）DOX group and （-）DOX group（P<0.05）.There were no significant differences among the expression levels ofα（1B）-adrenoceptor subtype mRNA in the muscle layer of rat cardia in controlgroup,（±）DOX group,（+）DOX group and （-）DOX group （P>0.05）.The expression levels of α（1D）-adrenoceptor subtype mRNA in the muscle layer of rat cardia in （±）DOX group,（+）DOX group were significantly higherthan that in control group （P<0.01）. However, there were no significantdifferences between that in （-）DOX group and control group （P>0.05）.2Effects of long-term administration of doxazosin and its enantiomers on themRNA expressions of α1-adrenoceptor subtypes （α（1A）, α（1B）, α（1D）） in the musclelayer of rat gastric fundusThe expression levels of α（1A）-adrenoceptor subtype mRNA in the musclelayer of gastric fundus in （±）DOX group,（+）DOX group,（-）DOX group weresignificantly higher than that in control group （P<0.01）.There were no significant differences among the expression levels ofα（1B）-adrenoceptor subtype mRNA in the muscle layer of gastric fundus incontrol group,（±）DOX group,（+）DOX group and （-）DOX group （P>0.05）.The expression levels of α（1D）-adrenoceptor subtype mRNA in the musclelayer of gastric fundus in （±）DOX group,（+）DOX group,（-）DOX group wereslightly lower than that in control group （P<0.05）.3Effects of long-term administration of doxazosin and its enantiomers on themRNA expressions of α1-adrenoceptor subtypes （α（1A）, α（1B）, α（1D）） in the musclelayer of rat gastric bodyThe expression levels of α（1A）-adrenoceptor subtype mRNA in the musclelayer of gastric body in （±）DOX group,（+）DOX group,（-）DOX group weresignificantly higher than that in control group （P<0.01）. And the expressionlevel of α（1A）-adrenoceptor subtype mRNA in the gastric body in （+）DOX groupwas slightly higher than that in （±）DOX group and （-）DOX group （P<0.05）.There were no significant differences among the expression levels ofα（1B）-adrenoceptor subtype mRNA in the muscle layer of gastric body in controlgroup,（±）DOX group,（+）DOX group and （-）DOX group （P>0.05）.The expression levels of α（1D）-adrenoceptor subtype mRNA in the musclelayer of gastric body in （-）DOX group were slightly lower than that in controlgroup （P<0.05）. However, there were no significant differences among theexpression levels of α（1D）-adrenoceptor subtype mRNA in the muscle layer ofgastric body in control group,（±）DOX group,（+）DOX group （P>0.05）. 4Effects of long-term administration of doxazosin and its enantiomers on themRNA expressions of α1-adrenoceptor subtypes （α（1A）, α（1B）, α（1D）） in the musclelayer of rat gastric antrumThe expression levels of α（1A）-adrenoceptor subtype mRNA in the musclelayer of gastric antrum in （±）DOX group,（+）DOX group,（-）DOX group weresignificantly higher than that in control group （P<0.01）. And the expressionlevel of α（1A）-adrenoceptor subtype mRNA in the gastric antrum in （+）DOXgroup was slightly lower than that in （±）DOX group and （-）DOX group（P<0.05）.There were no significant differences among the expression levels of α（1B）and α（1D）-adrenoceptor subtype mRNA in the muscle layer of gastric antrum incontrol group,（±）DOX group,（+）DOX group and （-）DOX group （P>0.05）.ConclusionIn four of clinically used quinazoline-based α1-adrenoceptor antagonists,only doxazosin selectively potentiates the contractile responses to5-HTthrough α1-adrenoceptor-independent mechanism in the rabbit gastriclongitudinal muscle strips without chiral recognition of its enantiomers. Thecontractile responses to5-HT are mediated by5-HT_2Areceptors, and closelyrelated to a synthesis of PGs in the rabbit gastric longitudinal muscle strips.The expression level of α（1A）-adrenoceptor subtype mRNA in the musclelayers of rat cardia and gastric antrum were lower than that in gastric fundusand body. However, there were no significantly differences among the musclelayer of rat cardia, gastric fundus, body and antrum. The expression levels ofα（1D）-adrenoceptor subtype mRNA in the muscle layer of gastric antrum weresignificantly higher than that in rat cardia, gastric fundus and body.α（1A）-Adrenoceptor subtype mRNA was up-regulated in the muscle layerof rat stomach by long-term administration of doxazosin and its enantiomers.While only in the muscle layer of cardia, α（1D）-adrenoceptor subtype mRNAwas slightly up-regulated. And long-term administration of doxazosin and itsenantiomers did not significantly affect the expression level ofα（1B）-adrenoceptor subtype mRNA.