离体灌注胰管转运系统的电生理研究：基底外侧膜的特性
Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane.
作者信息
Novak I, Greger R
机构信息
Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Federal Republic of Germany.
出版信息
Pflugers Arch. 1988 Jan;411(1):58-68. doi: 10.1007/BF00581647.
In order to study the mechanism of pancreatic HCO3- transport, a perfused preparation of isolated intra- and interlobular ducts (i.d. 20-40 microns) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PDte) in ducts bathed in HCO3(-)-free and HCO3(-)-containing solution was -0.8 and -2.6 mV, respectively. The equivalent short circuit current (Isc) under similar conditions was 26 and 50 microA . cm-2. The specific transepithelial resistance (Rte) was 88 omega cm2. In control solutions the PD across the basolateral membrane (PDbl) was -63 +/- 1 mV (n = 314). Ouabain (3 mmol/l) depolarized PDbl by 4.8 +/- 1.1 mV (n = 6) within less than 10 s. When the bath K+ concentration was increased from 5 to 20 mmol/l, PDbl depolarized by 15.9 +/- 0.9 mV (n = 50). The same K+ concentration step had no effect on PDbl if the ducts were exposed to Ba2+, a K+ channel blocker. Application of Ba2+ (1 mmol/l) alone depolarized PDbl by 26.4 +/- 1.4 mV (n = 19), while another K+ channel blocker TEA+ (50 mmol/l) depolarized PDbl only by 7.7 +/- 2.0 mV (n = 9). Addition of amiloride (1 mmol/l) to the bath caused 3-4 mV depolarization of PDbl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PDbl. An increase in the bath HCO3- concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PDbl by 8.5 +/- 1.0 mV (n = 149). It was investigated whether the effect of HCO3- was due to a Na+-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K+ conductance caused by lowered intracellular pH. Experiments showed that the HCO3- effect was present even when the bath Na+ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO3- effect remained unchanged after Ba2+ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na+ + K+)-ATPase, a Ba2+ sensitive K+ conductance and an amiloride sensitive Na+/H+ antiport. The HCO3- effect on PDbl is most likely due to rheogenic anion exit across the luminal membrane.
为了研究胰腺HCO₃⁻转运机制，开发了一种大鼠胰腺小叶内和小叶间导管（内径20 - 40微米）的离体灌注制备方法。通过电生理技术监测上皮对浴液离子浓度变化和添加转运抑制剂的反应。在本报告中描述了胰腺导管细胞基底外侧膜的一些特性。在无HCO₃⁻和含HCO₃⁻溶液中灌注的导管，其跨上皮电位差（PDte）分别为 - 0.8和 - 2.6 mV。在类似条件下的等效短路电流（Isc）分别为26和50 μA·cm⁻²。跨上皮比电阻（Rte）为88 Ω·cm²。在对照溶液中，基底外侧膜的电位差（PDbl）为 - 63 ± 1 mV（n = 314）。哇巴因（3 mmol/L）在不到10秒内使PDbl去极化4.8 ± 1.1 mV（n = 6）。当浴液K⁺浓度从5 mmol/L增加到20 mmol/L时，PDbl去极化15.9 ± 0.9 mV（n = 50）。如果导管暴露于K⁺通道阻滞剂Ba²⁺，相同的K⁺浓度变化对PDbl无影响。单独应用Ba²⁺（1 mmol/L）使PDbl去极化26.4 ± 1.4 mV（n = 19），而另一种K⁺通道阻滞剂TEA⁺（50 mmol/L）仅使PDbl去极化7.7 ± 2.0 mV（n = 9）。向浴液中添加氨氯地平（1 mmol/L）使PDbl去极化3 - 4 mV。呋塞米（0.1 mmol/L）和SITS（0.1 mmol/L）对PDbl无影响。浴液中HCO₃⁻浓度从0增加到25 mmol/L导致PDbl快速且持续去极化8.5 ± 1.0 mV（n = 149）。研究了HCO₃⁻的作用是由于基底外侧膜上依赖Na⁺的转运机制（离子复合物转运到细胞内会带正电荷），还是由于细胞内pH降低导致K⁺电导降低。实验表明，即使浴液Na⁺浓度降至名义值0 mmol/L，HCO₃⁻的作用仍然存在。同样，向浴液中添加Ba²⁺（5 mmol/L）后，HCO₃⁻的作用保持不变。结果表明，在导管细胞的基底外侧膜上存在一种对哇巴因敏感的（Na⁺ + K⁺）-ATP酶、一种对Ba²⁺敏感的K⁺电导和一种对氨氯地平敏感的Na⁺/H⁺反向转运体。HCO₃⁻对PDbl的作用很可能是由于生电阴离子通过腔面膜排出。
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