Li Fangxia, De Godoy Márcio, Rattan Satish
Department of Medicine, Jefferson Medical College, Thomas Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107, USA.
J Pharmacol Exp Ther. 2004 Mar;308(3):1111-20. doi: 10.1124/jpet.103.060145. Epub 2004 Jan 7.
The purpose of the present study was to ascertain the role of adenylate (AC) versus guanylate cyclase (GC) signaling pathways in the internal anal sphincter (IAS) smooth muscle relaxation by beta(1)-, beta(2)-, and beta(3)-adrenoceptor (AR) activation by xamoterol, procaterol, and disodium 5-[(2R)-2-(3-chlorophenyl)-2-hydroxy-ethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), respectively. The above-mentioned agonists produced concentration-dependent relaxation of the smooth muscle strips. Both the selective G(i/o)alpha and G(s)alpha antagonists 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalene trisulfonic acid) (NF 023) and 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF 449), respectively, inhibited the relaxation induced by procaterol. However, only NF 023 inhibited the relaxation induced by xamoterol and CL 316243. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, a soluble GC inhibitor, significantly inhibited the relaxation induced by different agonists. In contrast, the selective AC inhibitor [9-(tetrahydro-2'-furyl)adenine] (SQ 22536) inhibited only the relaxation induced by procaterol. (9R,10S,12S)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT 5720), a cAMP-dependent protein kinase inhibitor, attenuated the relaxation by procaterol, whereas (9S,10R,12R)-2,3,9,10,11,12, hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT 5823), a selective cGMP-dependent protein kinase (PKG) inhibitor, attenuated the relaxation induced by xamoterol and CL 316243. Xamoterol produced significant increase in cGMP levels, whereas only procaterol enhanced the cAMP levels. Western blot analysis confirmed the presence of beta(1), beta(2), and beta(3)-AR subtypes in the IAS. In summary, beta(2)-AR activates both G(s)alpha and G(i/o)alpha-protein subunits and induces relaxation in the rat IAS via both cAMP/cGMP pathways. In contrast, the beta(1)/beta(3)-ARs activation causes the smooth muscle relaxation via G(i/o)alpha-protein subunit/GC/GMP/PKG pathway. These studies are important for the understanding of intracellular mechanisms underlying IAS smooth muscle relaxation and in turn the pathophysiology of certain anorectal motility disorders.
本研究的目的是确定腺苷酸环化酶（AC）与鸟苷酸环化酶（GC）信号通路在氨甲醇、丙卡特罗和5-[(2R)-2-(3-氯苯基)-2-羟基-乙基]氨基)丙基]-1,3-苯并二恶唑-2,2-二羧酸钠（CL 316243）分别激活β(1)-、β(2)-和β(3)-肾上腺素能受体（AR）时，对内括约肌（IAS）平滑肌舒张的作用。上述激动剂使平滑肌条产生浓度依赖性舒张。选择性G(i/o)α和G(s)α拮抗剂8,8'-(羰基双(亚氨基-3,1-亚苯基))双-(1,3,5-萘三磺酸)（NF 023）和4,4',4",4"'-(羰基双(亚氨基-5,1,3-苯三基双(羰基亚氨基)))四苯-1,3-二磺酸（NF 449）分别抑制了丙卡特罗诱导的舒张。然而，只有NF 023抑制了氨甲醇和CL 316243诱导的舒张。1H-[1,2,4]恶二唑并[4,3-a]喹喔啉-1-酮，一种可溶性GC抑制剂，显著抑制了不同激动剂诱导的舒张。相反，选择性AC抑制剂[9-(四氢-2'-呋喃基)腺嘌呤]（SQ 22536）仅抑制了丙卡特罗诱导的舒张。(9R,10S,12S)-2,3,9,10,11,12-六氢-10-羟基-9-甲基-1-氧代-9,12-环氧-1H-二吲哚并[1,2,3-fg: 3',2',1'-kl]吡咯并[3,4-l][1,6]苯并二氮杂卓-10-羧酸己酯（KT 5720），一种cAMP依赖性蛋白激酶抑制剂，减弱了丙卡特罗诱导的舒张，而(9S,10R,12R)-2,3,9,10,11,12-六氢-10-甲氧基-2,9-二甲基-1-氧代-9,12-环氧-1H-二吲哚并[1,2,3-fg:3',2',1'-kl]吡咯并[3,4-I][1,6]苯并二氮杂卓-10-羧酸甲酯（KT 5823），一种选择性cGMP依赖性蛋白激酶（PKG）抑制剂，则减弱了氨甲醇和CL 316243诱导的舒张。氨甲醇使cGMP水平显著升高，而只有丙卡特罗提高了cAMP水平。蛋白质印迹分析证实了IAS中存在β(1)、β(2)和β(3)-AR亚型。总之，β(2)-AR激活G(s)α和G(i/o)α蛋白亚基，并通过cAMP/cGMP两条途径诱导大鼠IAS舒张。相反，β(1)/β(3)-ARs的激活通过G(i/o)α蛋白亚基/GC/GMP/PKG途径导致平滑肌舒张。这些研究对于理解IAS平滑肌舒张的细胞内机制以及某些肛肠动力障碍的病理生理学具有重要意义。
