Characterization of the apamin- and L-nitroarginine-resistant NANC inhibitory transmission to the circular muscle of guinea-pig colon.

Journal of autonomic pharmacology Pub Date : 1996-06-01
C A Maggi, S Giuliani
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引用次数: 0

Abstract

1. The aim of this study was a pharmacological characterization of the multiple NANC inhibitory transmission systems producing relaxation of the circular muscle of guinea-pig proximal colon. In the presence of atropine (1 microM), guanethidine (3 microM) and of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (0.3 microM) and MEN 10627 (1 microM), respectively, electrical field stimulation (EFS) produced a frequency-dependent (0.1-3 Hz) relaxation. During a cumulative frequency-response curve, the maximal relaxant effect was produced at 3 Hz and approached the maximal relaxation to 1 microM isoprenaline. In the presence of both apamin (0.3 microM) and L-nitroarginine (L-NOARG, 100 microM), EFS failed to evoke relaxation up to 1 Hz; at 1-10 Hz, a slowly developing relaxation ensured which approached 50% of the Emax to isoprenaline. The EFS-evoked NANC relaxation, either in the presence or absence of apamin and L-NOARG, was unaffected by in vitro capsaicin pretreatment (10 microM for 15 min). 2. Three protocols of EFS were developed for further pharmacological analysis: (a) EFS at 1 Hz for 5 s in the presence of L-NOARG, producing a transient fast apamin-sensitive relaxation; (b) EFS at 1 Hz for 5 s in the presence of apamin, producing a transient fast L-NOARG-sensitive relaxation; and (c) EFS at 10 Hz for 5 s in the presence of both apamin and L-NOARG, producing a transient but slowly developing and more sustained relaxation. 3. The neutral endopeptidase inhibitor, thiorphan (1-10 microM), enhanced and prolonged the apamin- and L-NOARG-resistant NANC relaxation produced by EFS at 10 Hz, without affecting that evoked at 1 Hz in the presence of apamin or L-NOARG. The angiotensin converting enzyme inhibitor, captopril (1-10 microM) was without effect. 4. The cAMP analogue inhibitor of protein kinase A, Rp-cAMPs (100-300 microM) significantly reduced and shortened the NANC relaxation produced by 10 Hz EFS in the presence of L-NOARG without affecting that produced by 1 Hz EFS in the presence of apamin or L-NOARG. 5. The inhibitor of sarcoplasmic reticulum Ca-ATPase, cyclopiazonic acid (CPA, 3-10 microM for 60 min) abolished the 1 Hz EFS-induced relaxation in the presence of L-NOARG, and greatly inhibited that produced by 10 Hz EFS in the presence of both apamin and L-NOARG. The relaxation produced by 1 Hz EFS in the presence of apamin was inhibited by about 32% at 10 microM only. 6. Nifedipine (1 microM) did not affect the EFS-induced NANC relaxations. In the presence of nifedipine, tetraethylammonium (TEA, 1 mM) enhanced the 1 Hz EFS-induced relaxation in the presence of L-NOARG (158% of control) and that produced by 10 Hz EFS in the presence of apamin and L-NOARG (215% of control) while that evoked by 1 Hz EFS in the presence of apamin was slightly affected (109% of control). 7. In the presence of atropine, guanethidine, SR 140333 and MEN 10627, bath application of human vasoactive intestinal polypeptide (VIP, 0.1 nM-10 nM) produced a concentration-dependent, slowly developing relaxation of colonic strips. The relaxation to VIP was unaffected by apamin (0.3 microM), L-NOARG (100 microM), nifedipine (1 microM) or nifedipine plus TEA (1 mM); it was inhibited by CPA (10 microM) and Rp-cAMPs (100 microM) and was potentiated by thiorphan (10 microM). 8. The putative VIP receptor antagonist, VIP(10-28) (10 microM) did not affect the VIP-induced relaxation nor the NANC relaxation to 10 Hz EFS in the presence of apamin and L-NOARG. 9. The present findings provide evidence that three distinct NANC inhibitory mechanisms mediate relaxation of the circular muscle of the guinea-pig proximal colon. The first system provides a fast relaxation in response to low frequency of stimulation and may involve the action of a transmitter(s) (possibly ATP) which mobilizes intracellular Ca2+ from sarcoplasmic reticulum leading to the activation of apamin-sensitive K+ channels. The second system likewise provides a fast relaxation of the colon in

抗维生素a和l -硝基精氨酸的NANC在豚鼠结肠环形肌的抑制传递特性。
1. 本研究的目的是对产生豚鼠近端结肠环形肌松弛的多个NANC抑制传递系统进行药理学表征。在阿托品(1微米)、胍二胺(3微米)和速激肽NK1和NK2受体拮抗剂SR 140333(0.3微米)和MEN 10627(1微米)的存在下,电场刺激(EFS)产生频率依赖性(0.1-3赫兹)弛豫。在累积频率响应曲线中,在3 Hz时产生最大松弛效应,接近于1 μ m异丙肾上腺素的最大松弛效应。在同时存在维生素a(0.3微米)和l -硝基精氨酸(L-NOARG, 100微米)的情况下,EFS不能引起高达1 Hz的松弛;在1-10赫兹,一个缓慢发展的松弛确保接近50%的Emax对异肾上腺素。无论是在存在或不存在apamin和L-NOARG的情况下,efs诱发的NANC松弛都不受体外辣椒素预处理(10微米,15分钟)的影响。2. 为了进一步的药理学分析,我们开发了三种EFS方案:(a)在L-NOARG存在的情况下,1 Hz 5 s的EFS产生短暂的快速apamine -sensitive弛缓;(b)在apamin存在的情况下,1 Hz持续5 s的EFS,产生短暂的快速l - noarg敏感弛豫;(c)在apamin和L-NOARG同时存在的情况下,10 Hz持续5 s的EFS,产生短暂但缓慢发展且更持久的松弛。3.中性内肽酶抑制剂硫硫孤儿(1-10微米)增强并延长了10hz时电场产生的抗维生素a和L-NOARG的NANC松弛,而不影响在1hz时产生的抗维生素a或L-NOARG。血管紧张素转换酶抑制剂卡托普利(1-10 μ m)无效果。4. 蛋白激酶A的cAMP类似物抑制剂Rp-cAMPs(100-300微米)显著降低和缩短了10 Hz EFS在L-NOARG存在下产生的NANC松弛,而不影响1 Hz EFS在apamin或L-NOARG存在下产生的NANC松弛。5. 肌浆网ca - atp酶抑制剂环吡唑酸(CPA, 3-10 μ m, 60 min)可消除L-NOARG存在时1 Hz电刺激引起的松弛,并可显著抑制apamin和L-NOARG存在时10 Hz电刺激引起的松弛。仅在10微米时,1赫兹电刺激产生的弛豫就被抑制了约32%。6. 硝苯地平(1微米)不影响efs诱导的NANC松弛。硝苯地平存在时,四乙基铵(TEA, 1 mM)增强了L-NOARG存在时的1 Hz EFS诱导的松弛(对照组的158%)和apamin和L-NOARG存在时的10 Hz EFS产生的松弛(对照组的215%),而在apamin存在时的1 Hz EFS引起的松弛(对照组的109%)受到轻微影响。7. 在阿托品、胍醚、SR 140333和MEN 10627存在的情况下,人血管活性肠多肽(VIP, 0.1 nM-10 nM)浴用可产生浓度依赖性、缓慢发展的结肠条状松弛。apamin (0.3 μ m)、L-NOARG (100 μ m)、硝苯地平(1 μ m)或硝苯地平加TEA (1 μ m)对VIP松弛无影响;CPA (10 μ m)和Rp-cAMPs (100 μ m)对其有抑制作用,硫磷(10 μ m)对其有增强作用。8. 假设的VIP受体拮抗剂VIP(10-28)(10微米)在apamin和L-NOARG存在下不影响VIP诱导的弛豫和NANC弛豫到10 Hz的EFS。9. 目前的研究结果提供了三种不同的NANC抑制机制介导豚鼠近端结肠环状肌松弛的证据。第一个系统在低频刺激下提供快速松弛,可能涉及递质(可能是ATP)的作用,它从肌浆网动员细胞内Ca2+,从而激活阿帕胺敏感的K+通道。第二个系统同样提供了结肠的快速松弛
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