Long-term induction of a unique Cl- current by endothelin-1 in an epithelial cell line from rat lung: evidence for regulation of cytoplasmic calcium
Norbert Mair, Manfred Frick, Andreas Meraner, Herbert Schramek and Paul Dietl
Department of Physiology, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria
Using conventional microelectrodes, the perforated patch clamp technique and fluorescence microscopy with fura-2, we investigated the relationship between the cell membrane potential, whole-cell currents and the free cytoplasmic Ca2+ concentration ([Ca2+]i) in response to 10 nM endothelin-1 (ET) in a rat respiratory epithelial cell line (L2).
Microelectrode experiments revealed that ET caused an immediate depolarization of the cell membrane potential (Vm) by 25 mV, which was unaffected by Na+ replacement with N-methyl-D-glucamine+ (NMDG+) or by omission of bath Ca2+. In contrast, ET depolarized the cells by 61 mV in the presence of low Cl- (6 mM), resulting in a complete breakdown of Vm.
In perforated patch clamp experiments, the ET-induced whole-cell current (IET) exhibited a slight outward rectification with a reversal potential (Vrev) of -22·7 mV. IET was reduced by 85 % in low Cl- (6 mM), but was unaffected by Ca2+ removal, Na+ replacement with NMDG+, pipette K+ replacement with Cs+ or 1 mM Ni2+ in the bath.
IET was unaffected by (+)-isradipine (100 nM), a specific L-type Ca2+ channel (L-VDCC) blocker. Transient inward Sr2+ currents through L-VDCCs were blocked by ET.
ET induced a biphasic Ca2+ signal, consisting of a 'peak' and a 'plateau' elevation of [Ca2+]i. Simultaneous patch clamp and fura-2 measurements revealed that IET coincided with intracellular Ca2+ release but clearly outlasted the elevation of [Ca2+]i. When the rise of [Ca2+]i was prevented by pretreatment with thapsigargin in a Ca2+-free bath, both activation time and amplitude of IET were reduced. Under these conditions, ET caused a decrease of [Ca2+]i.
The Cl- channel blocker mefenamic acid (MFA) had a dual, concentration-dependent effect on both IET and the ET-induced 'plateau' elevation of [Ca2+]i: an increase at 10 µM, but an almost complete block at 100 µM. The effect of MFA on IET preceded the effect on [Ca2+]i.
The ET-induced 'plateau' [Ca2+]i fell below control values in a low-Cl- (6 mM) solution.
These data indicate an amplifying function of intracellular Ca2+ release on an otherwise Ca2+-independent, unique Cl- current by ET. Moreover, this Cl- current appears to be functionally coupled with dihydropyridine (DHP)-insensitive Ca2+ entry, suggesting a modulatory role for long-lasting effects of ET.
