Nilius B, Oike M, Zahradnik I, Droogmans G
Department of Physiology, KU Leuven, Belgium.
J Gen Physiol. 1994 May;103(5):787-805. doi: 10.1085/jgp.103.5.787.
We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after approximately 50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular Cl- concentrations was always close to the Cl(-)-equilibrium potential. These currents are therefore mainly carried by Cl-. DIDS only weakly blocked the current (KI = 120 microM), while another Cl(-)-channel blocker, DCDPC (20 microM) was ineffective. We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca2+ independent. A similar current was also activated with iso-osmotic pipette solutions containing 300 mumol/liter GTP gamma S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 mumol/liter) was also ineffective in modulation of this current. The HTS-induced current was completely blocked by 10 mumol/liter pBPB, a PLA2 inhibitor. NDGA (4 mumol/liter) and indomethacin (5 mumol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 mumol/liter) were variable. In 12 out of 40 cells it either directly activated a Cl- current or potentiated the current activated by HTS. The membrane current was decreased at all potentials in 18 cells, and was not affected in 10 cells. The HTS-induced currents may therefore be modulated by cleavage products of PLA2, but not by messengers downstream of arachidonic acid. Loading the cells with a segment of the heat stable protein kinase A inhibitor PKI (5-24) did not prevent activation of the HTS-induced current.(ABSTRACT TRUNCATED AT 400 WORDS)
我们采用全细胞和穿孔膜片钳技术，研究了低渗细胞外溶液（HTS，渗透压为185 mOsm而非290 mOsm）对人脐静脉内皮细胞离子电流的影响。施加HTS后30 - 50秒内这些电流被激活，约50 - 150秒后达到最大值，当细胞重新暴露于正常渗透压时电流完全恢复。在正于 +50 mV的电位下，它们会缓慢失活。用高渗微管溶液（渗透压为377 mOsm而非290 mOsm）刺入内皮细胞时，也会激活同样的电流。利用不同的细胞外和细胞内Cl⁻浓度，这些容积诱导电流的反转电位总是接近Cl⁻平衡电位。因此，这些电流主要由Cl⁻携带。DIDS仅微弱地阻断该电流（KI = 120 μmol/L），而另一种Cl⁻通道阻滞剂DCDPC（20 μmol/L）则无效。我们无法在细胞贴附膜片中记录到单通道活性，但在HTS作用期间总是观察到电流方差增加。根据全细胞电流记录的平均电流 - 方差关系，我们确定单通道电导为1.1 pS。电流的大小和动力学与细胞内钙的伴随变化无关。此外，在存在10 mmol/L细胞内EGTA的情况下，电流仍可被激活，因此与Ca²⁺无关。用含有300 μmol/L GTPγS的等渗微管溶液也可激活类似电流。PLC的阻滞剂新霉素（1 mmol/L）并不能阻止该电流的激活。TPA（4 μmol/L）对该电流的调节也无效。HTS诱导的电流被10 μmol/L的pBPB（一种PLA2抑制剂）完全阻断。然而，脂氧合酶的阻滞剂NDGA（4 μmol/L）和环氧化酶的阻滞剂吲哚美辛（5 μmol/L）并不影响低渗溶液诱导的电流。花生四烯酸（10 μmol/L）的作用则各不相同。在40个细胞中的12个细胞中，它要么直接激活Cl⁻电流，要么增强HTS激活的电流。在18个细胞中，膜电流在所有电位下均降低，而在10个细胞中则不受影响。因此，HTS诱导的电流可能受PLA2裂解产物的调节，但不受花生四烯酸下游信使的调节。用热稳定蛋白激酶A抑制剂PKI（5 - 24）的一段序列加载细胞并不能阻止HTS诱导电流的激活。（摘要截短至400字）
