NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR.
NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR.
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发表时间：
2002-03
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通讯作者：
W. Marshall;J. A. Howard;R. Cozzi;E. M. Lynch
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作者：
W. Marshall;J. A. Howard;R. Cozzi;E. M. Lynch
用钙离子载体L(-1)、L(-1)二丁酰环-环腺苷(db-cAMP)和L(-1)3-异丁基-1-甲基黄嘌呤(IBMX)分别刺激适应海水的异种真核幼鱼的后肠。最近喂食的动物(但不是过夜不喂食的动物)的肠袋制剂从未经处理的对照组的液体吸收(+18.9+/-8.30微l cm(-2)h(-1)，N=8)转变为刺激后的净液体分泌(-7.43+/-1.30微l cm(-2)h(-1)，N=8，P<；0.01；Means+/-S.E.M.)，这表明硬骨鱼肠道的分泌能力。放置在Ussing膜室中的后肠片在离子霉素+db-cAMP+IBMX的刺激下出现净氯摄取(+2.245+/-0.633微当量cm(-2)h(-1)，N=7)，并转为净分泌(-3.809+/-1.22微当量cm(-2)h(-1)，N=7，P<；0.01)。在离子霉素+db-cAMP+IBMX处理后，粘膜应用阴离子通道阻滞剂L(-1)二苯胺-2-羧酸(DPC)，可显著降低血清-粘膜单向氯(-)通量(P<；0.001)、净氯(-)通量(P<；0.001)、短路电流(I(Sc)，P<；0.001)和组织电导(G(T)，P<；0.001)；0.001)，而阴离子交换阻断剂L(-1)4，4‘-二异硫氰基-2，2’-二苯乙烯二磺酸(DIDS)无作用。Db-cAMP+IBMX(非离子霉素)刺激可显著增加I(Sc)和G(T)的单向通透量，但不产生净氯(-)分泌。单独的离子霉素引起I(Sc)的一过性增加，但对G(T)没有影响，也没有引起单向或净氯(-)通量的显着变化。离子霉素处理后加入db-cAMP+IBMX可使细胞净分泌Cl(-)，单向通量和G(T)显著增加。免疫细胞化学染色发现囊性纤维化跨膜传导调节因子(CFTR)存在于所有肠细胞的细胞质和基侧膜以及部分细胞的刷缘膜中，而NKCC免疫荧光则显示Na(+)/K(+)/2Cl(-)共转运体存在于细胞质和刷缘膜中。我们得出结论，只有当细胞内Ca(2+)和环磷酸腺苷途径一起被刺激时，硬骨肠才能分泌盐分和液体，而且这种分泌似乎涉及到一群肠细胞顶膜上的CFTR离子通道的激活。
Sections of posterior intestine of the euryhaline killifish Fundulus heteroclitus adapted to sea water were stimulated by the calcium ionophore ionomycin (1 micromol l(-1)) in combination with agents to elevate intracellular cyclic AMP levels, 0.5 mmol l(-1) dibutyryl-cyclic AMP (db-cAMP) with 0.1 mmol l(-1) 3-isobutyl-1-methylxanthine (IBMX). Intestinal bag preparations from recently fed animals (but not from overnight unfed animals) changed from fluid absorption (+18.9+/-8.30 microl cm(-2) h(-1), N=8) in the untreated control period to net fluid secretion after stimulation (-7.43+/-1.30 microl cm(-2) h(-1), N=8, P<0.01; means +/- S.E.M.), indicative of the capacity of teleost intestine to undergo secretion. Posterior intestinal pieces mounted in vitro in Ussing-style membrane chambers showed net Cl(-) uptake (+2.245+/-0.633 microequiv cm(-2) h(-1), N=7) that turned to net secretion following stimulation by ionomycin + db-cAMP + IBMX (-3.809+/-1.22 microequiv cm(-2) h(-1), N=7, P<0.01). Mucosal application of the anion channel blocker 1 mmol l(-1) diphenylamine-2-carboxylate (DPC) after ionomycin + db-cAMP + IBMX treatment significantly reduced serosal-to-mucosal unidirectional Cl(-) flux (P<0.001), net Cl(-) flux (P<0.05), short-circuit current (I(sc), P<0.001) and tissue conductance (G(t), P<0.001), while 0.1 mmol l(-1) 4,4'-diisothiocyano-2,2'-stilbene-disulphonic acid (DIDS, a blocker of anion exchange) was without effect. Stimulation by db-cAMP + IBMX (no ionomycin) significantly increased unidirectional fluxes, I(sc) and G(t) but did not produce net Cl(-) secretion. Ionomycin alone produced a transient increase in I(sc) but had no effect on G(t) and caused no significant changes in unidirectional or net Cl(-) fluxes. Addition of db-cAMP + IBMX after ionomycin treatment produced net secretion of Cl(-) and large increases in unidirectional fluxes and G(t). Cystic fibrosis transmembrane conductance regulator (CFTR) was immunocytochemically localized with a monoclonal mouse antibody to the carboxy terminus and found to be present in the cytoplasm and basolateral membranes of all enterocytes and in the brush-border membrane of some cells, whereas NKCC immunofluorescence, demonstrating the presence of the Na(+)/K(+)/2Cl(-) cotransporter, was present in the cytoplasm and brush-border membrane. We conclude that the teleost intestine is capable of salt and fluid secretion only if intracellular Ca(2+) and cyclic AMP pathways are stimulated together and that this secretion appears to involve activation of CFTR ion channels in the apical membrane of a subpopulation of enterocytes.