Effect Of Botulinum Toxin Type A On The Tension Of Isolated Superior Mesenteric Artery In Rat

Objective:The effect and mechanism of botulinum toxin type A(BTX-A)on isolated superior mesenteric arteries(SMA)were investigated in rats;the effect of BTX-A on release of calcitonin gene-related peptide(CGRP)in SMA.The aim of this study is to demonstrate the efficiency of BTX-A to neurotransmission of noncholinergic nerves.Methods:Sprague-Dawley rats weighing 350～500 g were killed by stunnting and exsanguination via the femoral arteries before removal of the SMA.The entire length of each main SMA was dissected free form the adjacent superior mesenteric vein and adipose tissue.A ring segment 2-3 mm long was dissected form the caudal end of one SMA from each animal, two 0.2 mm diameter wires were threaded through the lumen of the vessel segment and put in an organ bath containing 2ml Krebs solution[composed of(in mM)120 NaCl,5.9 KCl,25 NaHCO₃,17.5 dextrose,2.5 CaCl₂,1.2 MgCl₂,1.2 NaH₂PO₄(pH 7.4)],maintained at 37℃and oxygenated with 95%O₂ and 5%CO₂.One wire was attached to a stationary support on the bottom of the bath,whereas the other was attached to an isometric force transducer.After mounting,SMA were maintained under 1g loading tension and left to equilibrate for 60 minutes. then the stable force was resting tone.the viability of the vessels was assessed by measuring the contractile response to 1μM methoxamine.After washing and reverting resting tone,various experiments were done,SMA were subdivided randomly into control groups and BTX-A groups:â‘ BTX-A groups were added directly 10 U/ml(n=8),20 U/ml(n=8),40 U/ml(n=8)of BTX-A into organ bathâ‘¡SMA were precontracted with 100 mM KCl,when the contractile response had reached a stable plateau,BTX-A groups were added 20 U/ml(n=8)of BTX-A into organ bathâ‘¢BTX-A groups were added 10 U/ml(n=5)of BTX-A into organ bath,control groups (n=5)without BTX-A,at the same time,All SMA were added guanethidine(5μM),After 60 min of incubation,SMA were precontracted with 100 mM KCl,then added CGRP(0.5μM)and CGRP(8-37)(1μM),a CGRP receptor antagonist,Subsequently.In some experiments,after precontracted with 100 mM KCl,SMA were added CGRP(8-37)(1μM)directly.All control groups were added 0.5 ml Krebs solution to substitute BTX-A.Results:â‘ 10,20 and 40 U/ml of BTX-A decreased the tension in SMA and respectively 3.70±0.93%,11.58±1.67%and 15.33±1.36%in percentage of reductive amplitude compared with pretreatment.Krebs' solution as control did not affect the tension in SMA in same condition.These inhibitory effects of BTX-A exerted dose-dependent.Phentolamine(2μM),a antagonist of adrenergicα-receptor,subsequently treated the samples incubating with different dosage of BTX-A and Krebs' solution,and further decreased the tension in SMA and respectively shown reductive percentage 23.04±3.82%,20.96±3.04%,18.23±2.44%and 33.10±5.35%.The phentolamine-induced tensional reduction in control group was significantly more than that in BTX-A treated groups(all P＜0.01).â‘¡BTX-A(20 U/ml)inhibited in other experimental test,100 mM KCl-enhanced tensional response in SMA.The percentage of reductive amplitude of the BTX-A was 10.15±0.60%compared with KCl-induce tensional response.Contrastively,Krebs' solution as control did not affect the KCl-enhanced tensional response.Subsequently,an administration of phentolamine(2μM)further reduced the tension in SMA pretreated with BTX-A(26.54±8.92%)or Krebs'solution(32.40±4.69%,P＜0.01 compared with BTX-A treated).â‘¢CGRP(0.5μM)inhibited the contractile response to KCl,the reductive amplitud of the BTX-A group was 0.2560±0.0114g and the control group was 0.2620±0.0356 g. Subsequently,an administration of CGRP(8-37)(1μM)enhanced the tension in SMA pretreated with BTX-A(0.2040±0.0439 g)or Krebs' solution(0.2600±0.0316 g,P＜0.01 compared with BTX-A treated).After precontracted with 100 mM KCl,the tension in SMA pretreated with BTX-A was not changed by CGRP(8-37)(1μM)directly(P＞0.05),however,the tension of control group pretreated without BTX-A was enhanced with 0.0640±0.0241 g.Conclusions:â‘ BTX-A induces the relaxation of vascular smooth muscle,these effects of BTX-A exerted dose-dependent.Phentolamine further decreased the tension in SMA subsequently.The results suggest that BTX-A induces the relaxation of vascular smooth muscle via inhibiting,probably,the release of norepinephrine from sympathetic adrenergic nerves;â‘¡BTX-A inhibited KCl-enhanced tensional response also suggest BTX-A induces the relaxation of vascular smooth muscle;â‘¢Exogenous CGRP inhibited the contractile response to KCl, BTX-A did not effect on exogenous applied CGRP-induced vasodilation,but may inhibit CGRPergic nerve mediated vasodilation.