Physiologisches Institut, Albert-Ludwigs-Universität, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Pflugers Arch. 1998 Jul;436(4):538-44. doi: 10.1007/s004240050669.
NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl– channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl– channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl– channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 µmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G m). The membrane capacitance (C m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C m correlated with the increase in G m with a slope of 51 fF/nS. Next the effect of furosemide (500 µmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na+2Cl–K+ uptake and to reduce cell volume but not membrane trafficking of Cl– channels. Furosemide reduced G m slightly (due to the fall in cytosolic Cl– concentration) and C m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC m/ΔG m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 µmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G m or C m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C m changes observed with Stim reflect a minor cell volume increase and an ”unfolding” of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl– channels in these cells.
真鲨科鱼类直肠腺管(RGT)中的 NaCl 分泌需要激活管腔膜中的 Cl–通道。RGT 及其激活机制是外分泌分泌的早期进化范例。相应的 Cl–通道可能类似于鲨鱼中囊性纤维化跨膜电导调节因子(CFTR)的等效物。这些 Cl–通道的激活是通过 cAMP 发生的。有人假设 CFTR 的激活是通过胞吐作用或抑制的内吞作用发生的。为了通过电测量直接检查这个问题,我们对体外灌注的 RGT 进行了全细胞膜片钳分析。用含有 forskolin(10 μmol/L)、二丁酰环腺苷酸(0.5 mmol/L)和腺苷(0.5 mmol/L)的溶液(Stim)刺激 NaCl 分泌。这导致了预期的强烈去极化和膜电导(G m)的增加。通过新设计的双频同步检测器方法测量膜电容(C m)。与刺激相比,C m 显著增加,从 5.00±0.22 增加到 5.17±0.21 pF(n=50)。C m 的增加与 G m 的增加相关,斜率为 51 fF/nS。接下来,在先前刺激的 RGT 中检查了呋塞米(500 μmol/L)的作用。呋塞米应该抑制耦合的 Na+2Cl–K+摄取并减少细胞体积,但不影响 Cl–通道的膜运输。呋塞米略微降低了 G m(由于细胞浆 Cl–浓度下降),并将 C m 降低到 Stim 增加的程度。这两种变化均具有统计学意义,ΔC m/ΔG m 的斜率与 Stim 引起的斜率相似。微管或肌动蛋白的抑制剂(在管内液中加入 10 μmol/L 的秋水仙碱、鬼笔环肽和细胞松弛素 D,并透析>10 min)不会改变细胞电压、G m 或 C m,也不会消除 cAMP 的刺激作用。这些数据表明,用 Stim 观察到的小 C m 变化反映了细胞体积的轻微增加和质膜的“展开”。本数据不支持 cAMP 介导的这些细胞中 Cl–通道激活的胞吐作用/内吞作用假说。