Alpha-adrenergic stimulation activates a calcium-sensitive chloride current in brown fat cells.

Abstract

The first response of brown adipocytes to adrenergic stimulation is a rapid depolarizing conductance increase mediated by 0t-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 significandy 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 I~M 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 ~>10 ~M 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 ICs0s of 3 and 7 t~M, or by higher concentrations of SITS (ICs0 = 170 o~M), DIDS (Its0 = 50 I~M), or 9-anthracene carboxylic acid (ICs0 = 80 I~M). The chloride conductance activated in whole cell by intracellular calcium had the permeability sequence PNo3 >PI >PBr >Po >> P~,par~te, 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, reAddress correspondence to Dr. Pamela Pappone, Division of Biological Sciences, Section of Neurobiology, Physiology, and Behavior, University of California, Davis, CA 95616. 232 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 106 9 1995 quired 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. We conclude that the intracellular calcium increases resulting from adrenergic stimulation in brown fat activate a chloride-selective membrane conductance distinct...

9–Anthracene carboxylic acid is more suitable than DIDS for characterization of calcium-activated chloride current during canine ventricular action potential

“…The half-inhibitory dose of 9-AC on TMEM16A currents in HEK 293 cells that stably expressed human TMEM16A was reported to be 58 μM (Bradley et al 2014). Calcium-sensitive chloride current was reduced by 9-AC with a half-inhibitory dose of 80 μM on brown fat cells (Pappone and Lee 1995). The EC 50 of 9-AC was higher in our study comparing to the other two recently mentioned reports.…”

A Flow-Activated Chloride-Selective Membrane Current in Vascular Endothelial Cells

“…However, this voltage error is Ͻ7 mV for our solutions. 32 The fact that the flow-induced Cl Ϫ current was observed in the presence of the K ϩ channel blocker Ba 2ϩ ( Figure 5) suggests that this current is activated independently by flow and is not a consequence of the activation of the flowsensitive K ϩ channels. Furthermore, Figures 5B and 6 reveal an additional characteristic of the flow-activated current in low external Cl Ϫ solution, a substantial increase in conductance.…”

“…Voltage ramps from this experiment intersected at Ϫ18 mV ( Figure 5D), indicating that the reversal potential of the flow-activated current was near this value and consistent with Cl Ϫ (E Cl ϭϪ36 mV) carrying a significant portion of the current. The reversal potential for the flow-activated current (intersection point of voltage ramps) shifted with E Cl on replacement of external Cl Ϫ with aspartate 31,32 (current reversal potential28 mV; E Cl 31 mV), indicating that the current was largely mediated by Cl Ϫ ions ( Figure 5B). In contrast, the reversal potential of the flow-activated current was, for the most part, insensitive to replacement of external Na ϩ with the impermeant cation NMDG ( Figure 5C; current reversal potentialϭϪ11 mV; E Cl ϭϪ34 mV); the intersection point would have shifted to a more hyperpolarized level if a Na ϩ -selective current were involved.…”

“…These results are consistent with the activation of an anion-selective current. 32 Figure 6 illustrates the net (leak-subtracted) currents elicited by flow in the data of Figure 5. This figure more clearly demonstrates the reversal potentials, and it illustrates that the flow-induced current is altered considerably in low external Cl Ϫ while remaining virtually unchanged in low external Na ϩ .…”

Heterotrimeric G-proteins Activate Cl− Channels through Stimulation of a Cyclooxygenase-dependent Pathway in a Model Liver Cell Line

“…Instead, the dominant conductance exhibited properties more compatible with a Ca 2ϩ -activated Cl Ϫ conductance (I Cl(Ca) ), including outward rectification and slow activation at depolarizing potentials. The I Cl(Ca) was also observed after increasing [Ca 2ϩ ] i in secretory epithelia (42)(43)(44)(45), smooth muscle cells (46), and adipocytes (47). Whereas in these cells the I Cl(Ca) has roles in fluid secretion and response to neurotransmitters, the physiological roles of this current in liver cells are not readily apparent.…”