Pappone P A, Lee S C
Section of Neurobiology, Physiology, and Behavior, University of California, Davis 95616, USA.
J Gen Physiol. 1995 Aug;106(2):231-58. doi: 10.1085/jgp.106.2.231.
The first response of brown adipocytes to adrenergic stimulation is a rapid depolarizing conductance increase mediated by alpha-adrenergic receptors. We used patch recording techniques on cultured brown fat cells from neonatal rats to characterize this conductance. Measurements in perforated patch clamped cells showed that fast depolarizing responses were frequent in cells maintained in culture for 1 d or less, but were seen less often in cells cultured for longer periods. Ion substitution showed that the depolarization was due to a selective increase in membrane chloride permeability. The reversal potential for the depolarizing current in perforated patch clamped cells indicated that intracellular chloride concentrations were significantly higher than expected if chloride were passively distributed. The chloride conductance could be activated by increases in intracellular calcium, either by exposing intact cells to the ionophore A23187 or by using pipette solutions with free calcium levels of 0.2-1.0 microM in whole-cell configuration. The chloride conductance did not increase monotonically with increases in intracellular calcium, and going whole cell with pipette-free calcium concentrations > or = 10 microM rapidly inactivated the current. The chloride currents ran down in whole-cell recordings using intracellular solutions of various compositions, and were absent in excised patches. These findings imply that cytoplasmic factors in addition to intracellular calcium are involved in regulation of the chloride conductance. The chloride currents could be blocked by niflumic acid or flufenamic acid with IC50s of 3 and 7 microM, or by higher concentrations of SITS (IC50 = 170 microM), DIDS (IC50 = 50 microM), or 9-anthracene carboxylic acid (IC50 = 80 microM). The chloride conductance activated in whole cell by intracellular calcium had the permeability sequence PNOS > PI > PBr > PCl >> Paspartate, measured from either reversal potentials or conductances. Instantaneous current-voltage relations for the calcium-activated chloride currents were linear in symmetric chloride solutions. Much of the current was time and voltage independent and active at all membrane potentials between -100 and +100 mV, but an additional component of variable amplitude showed time-dependent activation with depolarization. Volume-sensitive chloride currents were also present in brown fat cells, but differed from the calcium-activated currents in that they responded to cell swelling, required intracellular ATP in whole-cell recordings, showed no sensitivity to intracellular or extracellular calcium levels, and were relatively resistant to block by niflumic and flufenamic acids. (ABSTRACT TRUNCATED AT 400 WORDS)
棕色脂肪细胞对肾上腺素能刺激的第一反应是由α - 肾上腺素能受体介导的快速去极化电导增加。我们采用膜片钳记录技术，对新生大鼠培养的棕色脂肪细胞的这种电导进行了特性分析。对穿孔膜片钳记录的细胞进行测量显示，在培养1天或更短时间的细胞中，快速去极化反应很常见，但在培养时间较长的细胞中则较少见。离子置换表明，去极化是由于膜氯化物通透性的选择性增加。穿孔膜片钳记录细胞中去极化电流的反转电位表明，如果氯化物是被动分布的，细胞内氯化物浓度将显著高于预期。氯化物电导可通过细胞内钙的增加而激活，这可以通过将完整细胞暴露于离子载体A23187，或者在全细胞模式下使用游离钙水平为0.2 - 1.0微摩尔的吸管溶液来实现。氯化物电导并不随细胞内钙的增加而单调增加，并且当吸管中无钙浓度≥10微摩尔时进入全细胞模式会使电流迅速失活。在使用各种成分的细胞内溶液进行的全细胞记录中，氯化物电流逐渐衰减，而在切除的膜片中则不存在。这些发现表明，除了细胞内钙之外，细胞质因子也参与了氯化物电导的调节。氯化物电流可被尼氟酸或氟芬那酸阻断，IC50分别为3和7微摩尔，或者被更高浓度的SITS（IC50 = 170微摩尔）、DIDS（IC50 = 50微摩尔）或9 - 蒽甲酸（IC50 = 80微摩尔）阻断。从反转电位或电导测量可知，细胞内钙在全细胞中激活的氯化物电导具有通透性顺序PNOS > PI > PBr > PCl >> Paspartate。在对称氯化物溶液中，钙激活的氯化物电流的瞬时电流 - 电压关系呈线性。大部分电流与时间和电压无关，并且在 - 100至 + 100毫伏之间的所有膜电位下都有活性，但另一个幅度可变的成分显示出随去极化的时间依赖性激活。容量敏感性氯化物电流也存在于棕色脂肪细胞中，但与钙激活电流不同，它们对细胞肿胀有反应，在全细胞记录中需要细胞内ATP，对细胞内或细胞外钙水平不敏感，并且相对抗尼氟酸和氟芬那酸的阻断。（摘要截于400字）
